Investigations Into the Novel Proteins of Influenza B

Imperial College London

Investigations into the novel proteins of influenza B

Ruth Alice Elderfield

Supervisor: Professor Wendy Barclay

Department of Infectious Diseases

Doctor of Philosophy 2010

Declaration

I confirm that this is my own work, the use of all materials from other sources has been properly acknowledged and that this work has not previously been submitted for a degree at Imperial College London or any other institution

Ruth Elderfield

Acknowledgements

I owe thanks to:

A series of amazing ladies helped me get this far: Sarah Howell, Judith Taylor, Jane Reed, Crystal Baker, Maxine Allen, Alisoun Carey, Wendy Howard, Holly Shelton, Lorian Hartgroves, Kim Roberts, Manuela Mura, Lupita Ayora-Talavera, Elizabeth Townsend and Wendy Barclay.

The past and current members of the ‘Flu group, for their constructive criticism and support.

Guo Zhang, a tirelessly enthusiastic project student.

The BBSRC for funding, Reading University for starting my doctorate and Imperial College for helping me finish it.

Professor Barclay for her tireless support and encouragement, above and beyond the call of duty, then a little bit more.

Liz & Lorian for listening, sympathising and laughing at all the right times.

Mum, Alex & Anne-Marie, Love you, Thank you for everything.

Abstract

Influenza B viruses encode two small proteins, NB and BM2. BM2 is translated from segment 7 mRNAs by a mechanism involving sequence complementarity to the 18S ribosomal subunit. The importance of these complementary sequences was tested in the context of infectious virus using a reverse genetic approach. A series of mutations were introduced into this region of segment 7. Recombinant viruses with disrupted 18S complementarity displayed deficiency in BM2 expression in infected cells.

The BM2 protein is essential for virus replication because its ion channel activity is required during virion entry to the cell. There is also evidence that the cytoplasmic tail of BM2 is involved in viral assembly. A series of amino acid truncations and substitutions in the BM2 cytoplasmic tail were engineered. Recombinant viruses that lacked more than 5 residues at the carboxyl terminus of the protein were not recovered and key residues in the region -5 to -10 were identified.

The influenza B virus RNA segment 6 encodes the neuraminidase protein, NA, as well as NB. NB is a 100 amino acid transmembrane protein with a glycosylated ectodomain. NB is conserved in all natural influenza B virus isolates. Influenza B viruses that lack the NB protein can replicate in cell culture to wild-type levels, However, the deletant viruses showed attenuated growth in complex airway cultures derived from humans and ferrets. In vivo, infected ferrets excreted infectious virus in the nasal wash one day later than for viruses that encode NB. Alterations in the expression of the NA protein were not responsible for the attenuated phenotype shown by NB deletant viruses.

The role of the host cell ESCRT pathway or of the interferon-induced tetherin protein in assembly and release of influenza viruses was assessed. No evidence was found for either host pathway in the replication of influenza viruses.

Table of Contents

Declaration ...... 2 Acknowledgements ...... 3 Abstract ...... 4 Table of Contents ...... 5 Table of Figures ...... 10 List of Tables ...... 14 Chapter 1. Introduction ...... 15 1.1 The Orthomyxoviridae ...... 15 1.2 Influenza B classification ...... 17 1.3 The influenza B replication Cycle ...... 20 1.3.1 Attachment ...... 21 1.3.2 Cell Entry ...... 24 1.3.3 Fusion of the virus and endosomal membranes ...... 25 1.3.4 Viral Uncoating and the Ion Channels ...... 26 1.3.5 Nuclear localisation ...... 27 1.3.6 Non-Structural protein: NS-1 ...... 28 1.3.7 Replication ...... 29 1.3.8 Promoter Structures and compatibility ...... 29 1.3.9 Polymerase interactions ...... 31 1.3.10 mRNA, cRNA and vRNA ...... 32 1.3.11 Dissociation from the nucleosome ...... 33 1.3.12 Export from the nucleus ...... 33 1.3.13 Transport to the Apical membrane ...... 33 1.3.14 Glycoproteins and the Trans Golgi Network (TGN) ...... 34 1.3.15 vRNP’s and the cytoskeleton ...... 34 1.4 ESCRT: Endosomal Sorting Complexes Required for Transport ...... 35 1.5 PA (Polymerase Acid) and assembly ...... 35 1.6 M1(matrix protein), glycoproteins and lipid rafts ...... 36 1.7 Packaging Signals ...... 37 1.8 The Ion Channel Reprise: AM2 and BM2 role in assembly ...... 38 1.9 AM2 in viral dissemination ...... 39 1.10 The actions of neuraminidase ...... 39

1.11 The NB and CM2 Proteins...... 40 1.12 The impact of Influenza B and Vaccine production...... 41 1.13 Animal models in the study of influenza virus ...... 45 1.14 Reverse Genetics ...... 48 1.15 Thesis Aims ...... 51 Chapter 2. Modifications in regions complementary to the 18S ribosomal subunit upstream of the BM2 initiation codon affect protein production ...... 53 2.1 Introduction ...... 53 2.2 Results ...... 62 2.2.1 Design of influenza B virus mutants with altered complementarity to the 18S Ribosome in the segment 7 mRNA...... 62 2.2.2 Analysis of BM2expression from influenza B virus segment 7 mRNA mutants with altered complementarity to the 18S Ribosome...... 63 2.2.3 Analysis of BM2 expression by recombinant influenza B virus mutants with altered complementarity to the 18S Ribosome in the segment 7 mRNA in the virally infected cell...... 66 2.2.4 Phenotype of recombinant viruses with altered BM2 expression...... 70 2.3 Discussion ...... 72 Chapter 3. The reverse genetic manipulation of the BM2 cytoplasmic tail ...... 77 3.1 Introduction: Influenza B virus BM2 protein ...... 77 3.2 Results: ...... 79 3.2.1 The effect of deletion of amino acids from the terminus of its BM2cytoplasmic tail...... 79 3.2.2 Alanine scanning mutagenesis in the last 10 residues of the BM2 cytoplasmic tail reveals a critical role for position- 6(103) and -7(104)...... 87 3.2.3 The influenza B virus tolerates non-conservative changes in the BM2 protein cytoplasmic tail at position -6(103) or -7(104)...... 89 3.2.4 Recombinant influenza B viruses with BM2 amino acid substitutions at threonines and serines in the cytoplasmic tail...... 95 3.2.4 Is it possible to block the release of Virus with a small protein fused to EGFP to act as a dominant negative? ...... 103 3.3 Discussion ...... 106 Chapter 4. The involvement of host proteins in budding of influenza virus...... 109 4.1 Introduction: The ESCRT Pathway ...... 109 4.1.1ESCRT-0 ...... 110 4.1.2 ESCRT-1 ...... 110

4.1.3 ESCRT-II ...... 110 4.1.4 ESCRT-III ...... 110 4.1.5 Alix ...... 111 4.1.6 HD-PTP and ESCRT III ...... 111 4.1.7 Vps4 ...... 111 4.1.8 Ubiquitin ligases ...... 112 4.1.9 ESCRT and viral budding ...... 113 4.2 Results: ...... 114 4.2.1 ESCRT, Influenza and late domains...... 114 4.2.3 Influenza and the ESCRT pathway proteins ...... 120 4.2.4 The effect of overexpression of ESCRT proteins on the recovery of infectious influenza virus from cDNAs...... 121 4.2.5 The effect of over expression of ESCRT dominant negative proteins on virus yield following infection...... 124 4.3 Introduction: Investigating a potential role for tetherin in controlling the budding of Influenza viruses ...... 130 4.4 Results: ...... 133 4.4.1 Tetherin inhibits retrovirus budding in the absence of Vpu ...... 133 4.4.2 The effect of tetherin on release of influenza A virus in the presence or absence of the M2 ion channel protein...... 134 4.4.3 The effect of tetherin on the release of influenza B virus ...... 138 4.5 Discussion ...... 142 Chapter 5. Investigations into the role of the NB protein in the influenza B replication cycle 145 5.1 Introduction ...... 145 5.2 Results: ...... 149 5.21 Influenza B replication can proceed in the absence of the NB protein...... 149 5.22 Influenza B NB deletion and glycosylation mutants display different kinetics in the ferret model...... 155 5.2.3 Influenza replication in the absence of NB is attenuated in Ferret Airway Epithelium cell culture...... 160 5.2.4 Influenza B virus replication in FAE cells is selective for ciliated ferret epithelial cells...... 164 5.2.5 Influenza NB mutants are attenuated in human epithelial cell cultures...... 169 5.2.6 There is no phenotypic advantage observed by ferret passaged glycosylation knock out virus...... 170

5.2.7 The levels of Neuraminidase at the cell surface are unaffected by loss of NB in the context of whole virus...... 171 5.2.8 The levels of neuraminidase activity in viruses with deleted or mutated NB are as wild-type levels ...... 175 5.2.9 The Influenza NB mutants are able to replicate to wild-type levels in NCI-H292 cells ...... 179 5.2.10 The influenza NB mutants display attenuated viral entry into MDCK cells in the presence of ferret mucus ...... 181 5.2.11 Virus entry into MDCK cells is not affected by the presence of re-suspended Porcine Mucus ...... 183 5.2.12 Virus entry into MDCK cells is not affected by the presence of human mucus ...... 183 5.3 Discussion ...... 184 Chapter 6. General Discussion ...... 186 6.1 BM2 Cytoplasmic Tail ...... 186 6.2 BM2 translation and the 18S ribosomal subunit ...... 188 6.3 Tetherin and proteins of the ESCRT pathway’s involvement in the influenza B virus’ replication...... 189 6.4 The function of the NB protein...... 190 6.4.1 Neuraminidase ...... 190 6.4.2 Mucus ...... 191 6.4.3 Other possible roles ...... 192 6.4.4 Protection ...... 192 6.4.5 Auxiliary receptor ...... 192 6.5 General future research into Influenza B ...... 194 Chapter 7. Materials and Methods ...... 196 7.1 Plasmids ...... 196 7.2 Oligonucleotides ...... 200 7.3 Viruses ...... 204 7.4 Solutions ...... 207 7.5 Primary and secondary antibodies ...... 212 7.6 Cell culture ...... 212 7.7 Virus infection ...... 213 7.8 Reverse Genetics ...... 214 7.9 Directigen™ kits ...... 215 7.10 Plaque assay ...... 215

7.11 Haemagglutination assay ...... 215 7.12 Immunohistochemistry: ...... 215 7.12.1FACS ...... 215 7.12.2 En face staining ...... 216 7.13 Neuraminidase assays ...... 217 7.14 Mucus Assays ...... 217 7.15Blue cell Assay ...... 217 7.16 Expression plasmid Construction of NEP and NS-1 ...... 218 7.17 SDS Page gels and Western blotting ...... 219 7.18 BCA (bicinchoninic acid) Assay for protein content ...... 219 7.19 Transfections ...... 220 7.20 PCR amplification ...... 220 7.21 Site directed mutagenesis ...... 220 7.22 vRNA Extraction ...... 221 7.23 Gel Electrophoresis ...... 222 7.24 Transmission Electron microscopy ...... 222 7.25 Sucrose Cushions ...... 222 8. Bibliography ...... 223

Table of Figures

Figure 1: Schematic of influenza B virus cross section...... 17 Figure 2: Genotypes of influenza B viruses...... 19 Figure 3: The Replication cycle of influenza virus...... 20 Figure 4 : Receptor binding sites of Influenza B virus Haemagglutinin compared to Influenza A H3:Structure of B/HK HA...... 23 Figure 5: The influenza B antigenic epitopes...... 23

Figure 6: Structural comparison of the HA2 subunit of influenza B/Hong Kong/8/73 (green) and influenza A H3 (blue) and H5 (white) proteins...... 25 Figure 7: Schematic of the monomers of the AM2, BM2 , CM2 and NB ion channel proteins...... 26 Figure 8: Schematic comparing numerous proposed actions of Influenza A (green) & B (blue) NS-1 proteins...... 28 Figure 9: The predicted cock-screw secondary structures at the termini of the RNA composing the promoter sequences...... 30 Figure 10: Host factors (in boxes) known to be involved in the influenza A replication...... 32 Figure 11: Schematic of gene segments of Influenza A, B and C viruses...... 37 Figure 12: A. The HPA community and hospital infections caused by influenza 2005-2006, Characterised by the Enteric, Respiratory and Neurological virus Laboratory (ERNVL)...... 42 Figure 13: Illustration of the Influenza B reverse genetics system...... 49 Figure 14: A simplified version of the eukaryotic translation initiation pathway...... 55 Figure 15: Schematic illustrating the identical motif regions in Influenza B (putative), RHDV and FCV, where termination-re-initiation events are known to occur...... 57 Figure 16: The tip region of the 18S rRNA in helix 26 displaying complementarity to the motif 1 regions of the caliciviruses and the TURBS motifs of influenza B/Beijing/1/87...... 58 Figure 17: Proposed method of action for the recruitment of the Ribosomal translation machinery to Influenza B (motif 2) and Caliciviruses (motif 1)...... 59 Figure 18: Simplified version of the predicted structures of the influenza B virus segment 7 RNA from Powell et al., 2008...... 61 Figure 19: The segment 7 mRNA detailing the region upstream of the BM2 open reading frame that shows complementary to the 18S rRNA subunit...... 62 Figure 20: BM2 protein expression from viral like mRNAs generated in situ...... 65 Figure 21: Quantitation of BM2 expression from viral like mRNAs generated from plasmids modified in the TURBS region of segment 7...... 66 Figure 22: Relative expression of BM2 from -30 A→G, -36 U→G mutant and wild-type plasmids...... 67 Figure 23: Western blot of BM2 and NP expression by recombinant viruses modified in the TURBS region of segment 7...... 68 Figure 24: Quantitation of BM2 expression in recombinant viruses modified in the TURBS region of segment 7...... 68 Figure 25: Western blot of viruses modified in the putative TURBS of segment 7 Influenza B...... 69 Figure 26: Quantitation of BM2 expression in recombinant viruses modified in the TURBS region of segment 7 relative to expression of the M1 protein...... 70 Figure 27: Multi-cycle growth of recombinant influenza B viruses modified in the TURBS region of segment 7...... 71 Figure 28: Plaque assays of the 5 recombinant viruses modified in the TURBS region of segment 7 and the wild-type version of the B/Beijing/1/87 virus...... 71 Figure 29: M-fold 1: Enlarged image of the structure predictions from Powell et al., the pentanucleotide motif is in blue...... 73

Figure 30: M-fold 2: Enlarged image of the structure predictions from Powell et al., the pentanucleotide motif is in blue...... 74 Figure 31: Schematic illustrating the identical TURBS motif regions in Influenza B and an alternative AUGGGA Motif 1(alt), further upstream in the M1 coding sequence (256-261)...... 75 Figure 32: Schematic of the Influenza B segment 7, highlighting the pentanucleotide stop:start motif (TAATG) that terminates the M1 protein and initiates BM2 protein production...... 78 Figure 33: Representative coding sequence of the BM2 (B/Beijing/76/98-AAU01002.1) and M2 (A/Nanchang/58/93 (H3N2)-ABB79970.1) proteins from the influenza A & B viruses...... 78 Figure 34: Sequence confirmation of BM2-5 Virus...... 81 Figure 35: Western blot of expression in BM2 truncation mutants in 293T cells transfected with 500 ng of the plasmids...... 82 Figure 36: Multi-step growth curve illustrating the attenuation of the recombinant BM2-5 virus...... 83 Figure 37: Multi-step growth curve illustrating the attenuation of the recombinant BM2-5 virus when compared to the Wt virus...... 83 Figure 38: Electron micrograph of the wild-type B/Beijing/1/87...... 84 Figure 39: Morphology of B/Beijing/1/87 virions with truncated BM2-5...... 85 Figure 40: Plaque assay of truncated BM2-5 and BM2 wild-type viruses...... 86 Figure 41: Western blot of wild-type and truncated BM2-5 virus...... 87 Figure 42: Western blot of BM2-6A and -7A expression...... 88 Figure 43: The sequence variation in the BM2 protein amongst the 294 sequences for segment 7 RNA in the NCBI Influenza database (7)...... 89 Figure 44: Western blot to illustrate expression of BM2 mutant proteins with 6Q and 6Y substitutions...... 90 Figure 45: Sequence confirmation of BM2 -8A, -7V and -6Y viruses...... 91 Figure 46: Multi-step growth curve of recombinant viruses containing the valine substitution at position -7 (103) (BM2-7V) and the alanine substitution at position -8 (102) (BM2-8A)...... 92 Figure 47: Multi-step growth curve of recombinant viruses containing the valine substitution at position -7 (103) (BM2-7V) and the alanine substitution at position -8 (102) (BM2-8A)...... 92 Figure 48: Single-step growth curve of wild-type BM2 B/Beijing/1/87(Wt) and the virus with the tyrosine (Y) substitution at position -6 (104) in the BM2 protein...... 93 Figure 49: Transmission electron micrograph images of the B/Beijing/1/87 wild-type and BM2 -6Y viruses...... 94 Figure 50: Blot taken from the Thesis of David Jackson...... 95 Figure 51: Phosphorylation predictions in BM2 cytoplasmic tail...... 96 Figure 52: BM2 protein produced in 5 plasmid transfection with wild-type, the phosphorylation mutant (Ser91) and a double phosphorylation mutant (Ser91 + Thr101)...... 97 Figure 53: Recombinant viruses with alanine substitutions at putative phosphorylation sites in the BM2 protein successfully generated using the 12 plasmid B/Beijing/1/87 reverse genetics system...... 98 Figure 54: Sequence confirmation of phosphorylation mutant viruses...... 98 Figure 55: Multi-step growth curve of recombinant viruses containing the phosphorylation mutants of the BM2 protein serine to alanine substitution at position 91 (Ser91) and threonine to alanine at position 101...... 99 Figure 56: Haemagglutinin Assay of a multi-step growth curve of recombinant viruses containing the phosphorylation mutants of the BM2 protein serine to alanine substitution at position 91 (Ser91) and threonine to alanine at position 101...... 99 Figure 57: Plaque morphology of mutant influenza B viruses altered at residues 91 and 101 in the BM2 cytoplasmic tail...... 100 Figure 58: Western blot of cells infected with the wild-type (Wt), Thr101A and Ser91A viruses...... 101

Figure 59: Transmission electron micrographs of the wild-type B/Beijing/1/87 virions and virus particles formed by the BM2 Ser91A phosphorylation mutant...... 102 Figure 60: Construction of the eGFP fused peptide from the terminal 31 amino acids of the Cytoplasmic tail of BM2...... 104 Figure 61: Expression of the BM2 putative dominant negative protein...... 105 Figure 62: Virus yield in the presence of BM2 putative dominant negative cytoplasmic domain...... 105 Figure 63: The ESCRT pathway...... 113 Figure 64: Sequence alignment of A/Winsconsin/3523/88 (H1N1) and B/Yamagata/1311/2003 segment 7 Matrix protein...... 115 Figure 65: Differential expression patterns of ESCRT dominant negative proteins...... 117 Figure 66: HIV-1 -pseudo-type particle system...... 118 Figure 67: Pseudotype particle yield in the presence of ESCRT dominant negative plasmids...... 120 Figure 69: Viral yield when ESCRT plasmids are incorporated into the reverse genetics system of for B/Beijing/1/87...... 122 Figure 70: Method Two: Transfection and infection...... 123 Figure 71: Viral NP protein and ESCRT dominant negative fluorescence...... 125 Figure 72: Titres of influenza B virus produced from cells transfected with ESCRT dominant negative proteins...... 127 Figure 73: Titres of influenza virus produced from cells transfected with dominant negative ESCRT plasmids...... 128 Figure 74: Predicted structure of tetherin (CD317)...... 131 Figure 75: Retrovirus pseudo-particle release in presence of Tetherin...... 133 Figure 76: Influenza A M2 knockout virus yield from cells stably transfected with tetherin...... 135 Figure 76: Influenza A virus yield from cell stably transfected with tetherin (at high MOI)...... 136 Figure 77: Influenza A virus yield from cell stably transfected with tetherin (Low MOI)...... 137 Figure 78: Influenza A yield from HeLa cells transiently transfected with tetherin...... 138 Figure 79: Influenza B yield from cells stably transfected with Tetherin...... 139 Figure 80: Influenza B yield from cells stably transfected with Tetherin...... 139 Figure 81: Influenza B yield from cells transiently transfected with Tetherin...... 140 Figure 82: Influenza B yield from HeLa cells transiently transfected with tetherin...... 141 Figure 83: Schematic of the coding strategy for the NB and NA proteins of influenza B on segment 6...... 145 Figure 84: Alignment of influenza B virus NB protein amino acid residues 1-28...... 146 Figure 85: Schematic of recombinant viruses altered in segment 6 sequence to disrupt the NB gene...... 150 Figure 86: The 20 amino acid residue differences between the NB sequence of B/Lee/40 and B/Beijing/1/87 highlighted in bold...... 150 Figure 87: Western blots displaying the expression of the NB protein by a panel of recombinant Influenza B viruses...... 151 Figure 88: Transmission electron micrographs of influenza B B/Beijing/1/87 viruses harvested from MDCK cells...... 153 Figure 89: Immunofluorescence to stain for NB protein expression in cell infected with the wild-type or glycosylation (g-1-2) NB protein...... 154 Figure 90: Shedding of recombinant viruses modified in the NB protein in nasal washes of infected ferrets assayed by plaque assay...... 157 Figure 91: Temperature changes in ferrets infected with recombinant viruses modified in the NB protein...... 158 Figure 92 : Variation in weight of ferrets infected with recombinant influenza B viruses that differ in the NB protein...... 159

Figure 93: Western blot of NB expressed in cells infected by viruses harvested from ferrets 4 days post-infection...... 160 Figure 94: Schematic of Ferret Airway Epithelial cultures...... 160 Figure 95: A cross section of a Human Airway Epithelial cells culture...... 161 Figure 96: Replication of recombinant influenza B viruses that differ in NB in Ferret Airway Cell Cultures ...... 162 Figure 97: Differentiated and ciliated ferret airway culture infections with recombinant influenza B viruses...... 163 Figure 98: Expression of different sialic acid linkages on Human Airway Epithelia...... 165 Figure 99: Ferret Airway Cultures with En face staining of influenza B virus infection...... 167 Figure 100: Ferret Airway Cultures stained en face for recombinant influenza B infection...... 168 Figure 101: Replication of recombinant influenza B viruses altered in NB protein in human airway cultures...... 169 Figure 102: Replication of recombinant influenza B viruses altered in NB protein in Human airway culture...... 170 Figure 103: Growth curve of ferret airway culture infection by recombinant influenza B virus altered in NB glycosylation derived by infecting MDCK cells with material obtained pre- or post-ferret infection...... 171 Figure 104: Western blots of NA expression in cells infected by recombinant FLAG-tagged influenza B viruses altered in the NB gene...... 174 Figure 105: Internal and external cell expression of neuraminidase by recombinant influenza B viruses altered in NB...... 174 Figure 106: A fetuin/peanut lectin/HRP sialidase assay (PNA). This requires the binding of fetuin to an assay plate...... 176 Figure 107: Optimisation of the PNA-Fetuin assay for neuraminidase activity...... 178 Figure 108: Neuraminidase activity of recombinant influenza B viruses altered in NB assessed by PNA assay...... 179 Figure 109: A multi-step growth curve of recombinant virus in H292 cells...... 181 Figure 110: Inhibition of cell entry of recombinant influenza B inhibition by ferret mucus...... 182 Figure 111: Inhibition of Recombinant influenza B viruses altered in NB by human mucus...... 184

List of Tables

Table 1: The viral segments and associated proteins of the three different influenza viruses A, B & C. 15 Table 2: Table of Animal models used in influenza research...... 45 Table 3: The influenza B reverse genetics systems currently in use and the papers in which they were published...... 50 Table 4: Culture systems used in the study of influenza viruses...... 51 Table 5: Plaque size of recombinant viruses modified in the TURBS region of segment 7. Plaques were measured for 2 wells per virus and the average and range were expressed in mm...... 72 Table 6 : Table listing recombinant viruses truncated in the cytoplasmic tail of the BM2 protein successfully generated using the 12 plasmid B/Beijing/1/87 reverse genetics system...... 80 Table 7: Comparative plaque sizes of wild-type and BM2-5 recombinant virus...... 86 Table 8: Recombinant viruses with alanine substitutions (positions 100-104) in the cytoplasmic tail of the BM2 protein successfully generated using the 12 plasmid B/Beijing/1/87 reverse genetics system...... 88 Table 9: Recombinant viruses truncated in the cytoplasmic tail of the BM2 protein generated using the 12 plasmid B/Beijing/1/87 reverse genetics system...... 91 Table 10: Comparative plaque sizes of wild-type, Ser91A and Thr101A recombinant virus...... 100 Table 11: Average sizes of the wild-type and putative phosphorylation mutant Ser91A as assessed by TEM...... 103 Table 12: The potential nucleotides for amino acids at positions -6 (A) or -7 (B) in the BM2 protein’s cytoplasmic tail...... 107 Table 13: Influenza B Sequence alignment of Segment 7. Amino acids 85 -120 shown...... 115 Table 14: List of the plasmids expressing dominant negative versions of the ESCRT proteins and viruses experimentally inhibited by their expression...... 116 Table 15: Expression plasmids used to express the tetherin, viral and control proteins in transfected 293T cells...... 132 Table 16: Influenza infection assessed by Directigen A&B kits...... 156 Table 18: Plasmids used in this project ...... 200 Table 19: Oligonucleotides used in this project ...... 203 Table 20: Viruses used in this project ...... 206 Table 21: Solutions used in this project ...... 211 Table 22: Antibodies used in this project ...... 212

Chapter 1. Introduction

1.1 The Orthomyxoviridae

Influenza viruses in humans cause mainly a seasonal acute respiratory tract infection that has a high social and economic impact. They are part of the Orthomyxoviridae, a family of segmented, negative sense, single stranded RNA viruses (227), that replicate by means of an RNA dependant RNA polymerase. This family includes the Thogoto virus, Infectious Salmon Anaemia virus and the Influenza Viruses A, B and C genera.

Influenza viruses have a segmented genome which comprises 8 segments (7 for influenza C) each of them encoding for a different protein. Some of them (segments 2, 7 and 8) encode for more than one protein. Not all of the virally encoded proteins are incorporated into virions, but some are only expressed inside the host cell for aid during the viral replication cycle (Table 1).

Segment Influenza Influenza Influenza Function A B C 1 PB2 PB2 PB2 Polymerase 2 PB1 (PB1- PB1 PB1 Polymerase F2) 3 PA PA P3 Polymerase 4 HA HA HEF Attachment, esterase & fusion (HEF) 5 NP NP NP Nucleoprotein 6 NA NA & NB Viral Release 7 (6) M1 & M2 M1 & M1 & Matrix and ion channel BM2 CM2 proteins 8 (7) NS1 & NS1 & NS1 Control of the immune NEP NEP response & nuclear export

Table 1: The viral segments and associated proteins of the three different influenza viruses A, B & C. Polymerase Basic 1 (PB1), Polymerase Basic 2 (PB2), Polymerase Acidic (PA), Haemagglutinin (HA), Nucleoprotein (NP), Neuraminidase (NA), Matrix Protein (M1), Non-structural protein 1 (NS1) and Nuclear Export Protein (NEP). The numbers in brackets correlate to the last 2 segments of influenza C.

Influenza A viruses’ natural reservoir are wild aquatic birds, to which they are usually restricted in terms of infection and replication. There is a high level of diversity in this virus reflected in the 16 subtypes of HA and 9 subtypes of NA. All these subtypes have been identified in birds and can recombine in diverse combinations. Moreover some of them have the potential to infect and acquire transmissibility within a different species, subsequently establishing a new virus subtype for that species. As a consequence influenza A virus has been found to infect and circulate amongst humans, pigs, horses, dogs and seals, etc... This ‘jumping’ ability is what makes them the most serious threat to public health among all influenza viruses, typified by the current H1N1 triple reassortant strain which has zoonosed by an avian →swine→human route by acquiring gene constellations through the process of segment reassortment (98).

Influenza B has a reduced host range and is found almost exclusively in humans. When the virus was isolated in seals, it was of the same genetic composition as a strain isolated from humans (267). There is no recombination between the A and B viruses, attempts to generate hybrids using viruses were unsuccessful, although by reverse genetics viruses with chimeric NA or HA proteins’ could be produced (82, 131, 132, 187, 200, 346).

Influenza C is restricted to humans and pigs and generally causes a febrile respiratory tract illness to which most adults acquire antibodies early in life, though in children under the age of six, complications can occur (218). As the symptoms are similar to those observed with influenza A & B, diagnosis can missed due to the difficulty in identifying the virus (219).

Estimates of when the three virus genera diverged vary. These range from 4000 years for the divergence from A & B, and 8000 years for influenza C (338), to as recently as 104 years for the divergence of the B from the A viruses (259). The Webster et al., 1992 analysis suggests that the influenza HA lineages diversified between themselves, prior to the divergence of influenza B, as the Influenza B HA is closer the Influenza A H8, H9 and H12 virus lineages, than they are to other influenza A HA’s (373). The time period since the divergence has been sufficient for there notable differences to have evolved between the three viruses in regards to their encoded proteins.

Influenza A, B and C encode genus specific proteins, some of which have only recently been identified. For example influenza B segment 6 codes for the neuraminidase glycoprotein and the unique protein NB whose function is still unknown (316); the influenza A virus’ Polymerase Basic 1 gene on segment 2 also has alternative reading frames believed to encode the novel PB1-F2 and ND40 proteins (40, 385). Influenza C contains the HEF protein which acts as both

16 an attachment protein and an esterase, providing a function analogous to that of the neuraminidase of influenza A or B virus (122). The remaining proteins have the same functions during the virus life-cycle, but are not genetically identical; they differ in the amino acid composition and in the length of coding and non-coding regions.

Figure 1: Schematic of influenza B virus cross section. The 3 polymerases (PB1, PB2 & PA) in conjunction with the nucleoprotein (NP) and viral negative sense RNA (vRNA) compose the ribonucleoprotein complex at the central core of the virion. The nuclear export protein (NEP) is also associated to the vRNP. This core is surrounded by the virus matrix protein (M1), which is in turn surrounded by the membrane bound spike glycoproteins required for virus attachment haemagglutinin (HA) and release, neuraminidase (NA). Within this membrane bound layer is the ion channel protein (BM2) and the protein of unknown function (NB).

1.2 Influenza B classification

Unlike Influenza A, influenza B is not classified into antigenic subtypes by the numerous HA and NA glycoprotein variations. Instead the lineage is defined by comparison of the HA protein to one of two viruses, either B/Yamagata/16/88 or B/Victoria/2/87, phylogenetically known as Yamagata-like or Victoria-like viruses. These viruses evolve slowly, but evade the immune response by a series of systematic insertion, deletion and point mutations with the HA gene (195, 251). These viruses can co-circulated in a single epidemic season allowing recombination

17 events to occur (195, 303, 390), though typically one lineage will predominate during a single influenza season (244).

The level of recombination events can be visualised in the McCullers et al.,2004 (226) paper (Figure 2), which illustrates the recombination of gene segments possible between 31 influenza B viruses over a 24 year period (1979-2003). They took three viruses as their initial base, B/Yamagata/16/88 (lineage II), B/Victoria/2/87 (lineage III) and a virus which appeared to be the nodal virus for the divergence from a B/Russia/69 (lineage I). Excluding the initial 3 genotypes, recombination events allowed the production of an additional 12 constellations, this suggests a greater diversity within the viruses than would be suggested by the Yamagata- like or Victoria-like designation. However, this study also noted that the most divergent polymerase genes had a sequence identity of 94% at the nucleotide level and a 97 to 98% identity at the amino acid level for these 31 viruses, displaying a high level of amino acid conservation between influenza B viruses in the 24 year period.

Figure 2: Genotypes of influenza B viruses. Hatched boxes represent lineage I genes, open boxes represent lineage II genes, and filled boxes represent lineage III genes. From top to bottom, the boxes within each virus diagram represent the lineage of gene segments 1 through 8, which code for PB1, PB2, PA, HA, NP, NA and NB, M1 and M2, and NS1 and NS2. Adapted and reproduced with permission from McCullers et al., 2004, (Copyright © 2004, American Society for Microbiology).

Influenza C viruses can be divided into five lineages (220). A study in Japan isolated 45 strains of influenza C over nine years. The genomic complement of 44 of these 45 strains was distinguishable from the 5 reference strains for the classification lineages. This indicates frequent reassortment of the influenza C viruses (221).

1.3 The influenza B replication Cycle

Figure 3: The Replication cycle of influenza virus. The virus attaches through interactions between the HA protein and the sialic acid receptors of the cell surface. The virus is endocytosed and the acid environs of the endosome alters the HA conformation to allow fusion with the endosomal membrane. The viral ion channel encourages the dissociation of the vRNP complex. The vRNP travels to the nucleus where the polymerases generate mRNA, cRNA and vRNA. The former is used to generate the new viral proteins; these components travel to the cell membrane and assemble into new viral particles. This schematic is not to scale.

Most of the research on influenza virus has centred on type A virus; assumptions have been made that the B virus behaves in a similar manner and interacts with analogous cellular components. Research results obtained by specifically studying influenza B virus will be highlighted in the text.

1.3.1 Attachment

Influenza viruses attach to the surface of the host cell by the interaction between haemagglutinin (HA) and host sialic acid (SA) receptors. The type of sialic acid receptor depends upon the host species and the type of tissue. Human influenza viruses bind predominantly to sialic acids linked to the sugar chain in an α2,6 linkage (found most commonly in the human upper respiratory tract). In contrast avian influenza viruses bind preferentially to the α2,3 linked SA. The ability of the virus to bind to a specific receptor type is one of the factors that define the host range of influenza A virus. In order for the virus to reach the cell surface of the epithelium in the respiratory tract, it has been proposed that the NA viral protein could cleave through the mucus to allow access to the sialic acid receptors (217). The influenza C virus combines the attachment and esterase cleavage function in one HEF (Haemagglutinin, Esterase and Fusion) protein (280).

Influenza B HA predominantly binds to α2,6 receptors, but there are instances (B/Gifu/2/73) where there is a preference for α2,3 (216, 388). The egg adaptation in influenza B appears to occur through the loss of glycosylation of the HA protein at the amino acid position 194 -196, which in turn causes an increased affinity for the α2,3 linked receptors (91).

Both influenza A & B HA’s bind to N-acetylneuraminic linked sialic acids, whereas influenza C’s

HEF protein binds to 9-0-acetylneuraminic acid (Neu5, 9AC2)(280).

The Haemagglutinin protein of both the A & B genera forms a trimer, with an elongated fusion domain, composed of the HA2 coiled-coiled structure and two regions of the HA1 subunit and a globular membrane distal domain which incorporates a receptor binding site (RBS) and a vestigial esterase subdomain (both part of the HA1 subunit). Figure 4A illustrates the influenza B haemagglutinin protein. There are 7 disulphide bridges linking the protein together in influenza B HA protein, five of them are structurally conserved in influenza A HA, an additional disulphide bond in the A genus has no structural counterpart in influenza B (362).

The Haemagglutinin protein possesses glycosylation sites that appear to aid in immune evasion in both virus genera. Influenza B has 7 potential glycosylation sites on the HA1 subunit and 3 on the HA2 subunit. These can vary depending upon antigenic drift.

Research by Wang et al., 2007 (363) investigated the structural basis of the differences between the influenza A and B HA attachment proteins. Using the crystal structure of HA from the B/HongKong/8/73 complexed with either α2,6 or α2,3 receptor analogues, they compared the structures with a human type Influenza A H3 HA (X31) . Essentially they found that despite the low level of sequence identity (~20% in HA1 and ~30% in HA2), they both have a similar fold pattern (73, 92, 113, 114, 310, 323, 330, 331, 372, 374, 378, 382) and possess the sialic acid receptor binding site as a shallow depression at the top of the molecule. Influenza B has a set of 4 aromatic amino acids that form the basis of the pocket and a series of amino acids which provide the hydrogen bonds to the sialic acid receptor, these are labeled in Figure 4b and Figure 5, reproduced from Wang et al., 2007.

The difference between the H3 and the B/HongKong virus HA receptor binding sites are illustrated in the overlay diagram in Figure 4c. The phenylalanine at position 95 gives the receptor binding pocket of the B-HA a different shape to that given by the tyrosine 98 (structurally in the same position) in the H3 protein, thereby altering the orientation of some of the internal amino acids and contributing to making the pocket wider with a lower opening. Interestingly, if the tyrosine 98 is replaced with Phenylalanine in influenza A HA, the mutant virus is unable to agglutinate erythrocytes or to replicate in modified MDCK cells that have reduced levels of sialic acid, indicating changes to the binding affinity of the pocket (213). Within this binding pocket lies the region thought to provide the selectivity between the avian and human receptors for influenza A, specifically amino acids Leu (226) and Ser (228), the structurally conserved counterparts in the influenza B HA are Pro (238) and Ser (240). There is evidence that viruses from the B/Yamagata-like lineage are able to tolerate alterations in the Pro (238) amino acid in monoclonal antibody escape mutants, but it is not known what effects modifying these proteins would have influenza B receptor specificity or host range (13, 14, 362).

For Influenza C the amino acid 285 in the HEF protein appears to be key in receptor binding and a mutational change of Thr184Ile, can increase the affinity to the Neu5,9AC2 receptor (52).

Figure 4 : Receptor binding sites of Influenza B virus Haemagglutinin compared to Influenza A H3:Structure of B/HK HA. (a) The overall structure of B/HK HA with three subunits differently coloured. There is one receptor- binding site in each subunit), highlighted in red. (b) Close-up view of the receptor-binding site of B/HK HA. Important residues forming the receptor-binding site are explicitly drawn, with hydrogen-bonding interactions shown as dashed lines. (c) Comparison of the receptor-binding sites of B/HK (red) and human H3 (grey) HAs. Their corresponding residues are shown in yellow and cyan, respectively. Two large structural shifts between them, at residues Ser-140 and Asp-193, respectively, are indicated by black dashed lines with the distances labelled. The hydrogen-bonding interactions are shown as dashed lines. Wang Q. et.al. PNAS 2007;104:16874-16879 Reprinted with permission, Copyright (2007) National Academy of Sciences, U.S.A.

Figure 5: The influenza B antigenic epitopes. Consisting of the 150-loop (green), the 160-loop (blue), 190-helix (red) and the 120-loop (cyan), with the key amino acids listed. Reprinted with permission from Wang et al., 2008. Journal of Virology, March 2008, p. 3011-3020, Vol. 82, No. 6. Copyright © 2008, American Society for Microbiology.

There are 4 major antigentic epitopes (120-loop, 150-loop, 160-loop and 190-loop) for the influenza B HA protein clustered close to the receptor binding site, which are positively selected for during the constant attempts to evade the immune system. A single amino acid change a position 196 of alanine to threonine was sufficient to cause an epidemic (245, 362).

The Influenza A and B viruses both possess functionally and structurally similar HA proteins, despite the low level of sequence homology. Experimentally the ecto-domain of influenza B can be fused to the influenza A cytoplasmic domain to generate a functional influenza A virus (82). The influenza C HEF protein has only a 12% sequence identity to the influenza A HA protein, yet when artificially introduced by reverse genetics (with influenza A packaging signals), it can functionally replace the HA protein and generate viable viruses (94). Despite these key differences in receptor binding sites and glycosylation patterns affect the immune evasion and host specificity.

1.3.2 Cell Entry

Research with the influenza A virus indicates that once the virus is successfully attached to the appropriate receptor, the virions enter the cell by receptor mediated endocytosis. Approximately 65% of the virus enters via clatherin mediated endocytosis; the remaining 35% use a poorly understood clatherin and caveolin – independent route (304, 321). The clatherin- dependant entry is mediated by Epsin1 as a cargo-specific adaptor for the influenza A virus (39). The pre-early endosomes are sorted; those containing the influenza virus are transported on a ‘fast track’ microtubule dependant network rather than mixing with a static slowly maturing population of early endosomes. This ‘fast track’ pathway is typified by the presence Rab5 and the gradual accumulation of Rab7 endosomal marker (178). Influenza virus endocytosis is reviewed in detail in reference (177). A review of the scientific literature available on cell entry indicates that there is no direct experimental evidence that influenza B uses the same methodology. However, there is no evidence to contrary, especially as both viruses use sialic acid receptors as a means of cell entry and the influenza B ecto-domain can support influenza A replication (82).

1.3.3 Fusion of the virus and endosomal membranes

Fusion of the viral and endosomal membranes is required for the exit of the viral core contents to initiate influenza infection. The endosomal marker Rab5 indicates the acidic nature of the early endosome (178). The acidified early endosome core (~pH 5-5.5) causes a conformational change in the NH2 region of the HA2 subunit which mediates the fusion between viral and endosomal membranes (177, 376). For this fusion to occur, the HA glycoprotein must have been cleaved from its precursor form, HA0, into two subunits by host proteases. This happens upon virus release from the infected cell by trypsin-like enzymes. The confirmation change itself is mediated by ionizable residues that are susceptible to changes in pH, these residues are found at key structural positions capable of causing conformational change in the fusion peptides of A, B and C HA2 and HEF2 subunit proteins respectively (362).

The influenza A and B HA2 subunit, which contains the fusion peptide display only 39% homology in their amino acids, but share a very similar structure as can be observed in Figure 6, comparing the B/Hong Kong/8/73 and the influenza A H3 and H5 sub-types. This also indicates that the function of these proteins at this stage in the replication cycle is conserved.

Figure 6: Structural comparison of the HA2 subunit of influenza B/Hong Kong/8/73 (green) and influenza A H3 (blue) and H5 (white) proteins. Encompassing helix A, B and the fusion peptide. Reproduced with permission from Wang et al., 2008. Journal of Virology, March 2008, p. 3011-3020, Vol. 82, No. 6. Copyright © 2008, American Society for Microbiology.

1.3.4 Viral Uncoating and the Ion Channels

Simultaneously with the low pH triggered HA conformational change, the AM2/BM2 proteins are required to create an ion channel through the viral membrane, resulting in an acidic environment inside the viral particle which induces the dissociation of the vRNP complex from the matrix protein shell (214, 236). As with the Haemagglutinin proteins, these viral proteins share a low level of coding homology but are functionally equivalent.

AM2 and BM2 are encoded on segment seven, CM2 on segment six. These segments also encode the matrix proteins. The M2 proteins are homotetrameric, type III integral membrane proteins. They have a single transmembrane domain which is flanked by a short N- terminal ectodomain and a long C-terminal cytoplasmic tail (279) Figure 7.

Figure 7: Schematic of the monomers of the AM2, BM2 , CM2 and NB ion channel proteins. The lengths of the 3 domains are represented by numbers of amino acids. The glycosylation sites on the N-terminal domains of the CM2 (one)(273) and NB protein (two)(380) are also shown. Modified from illustration in (168).

Initially it was thought that NB was the influenza B functional equivalent of AM2 due to its ion channel abilities (337), but later work has indicated that BM2 is the true functional homologue, BM2 shares only the essential HXXXW motif in the inner membrane spanning residues, that coveys the ion channel abilities of AM2 (270, 279). NB is Na+ permeable at pH 5.5 to 6.5 (337) and CM2 is permeable to Cl- in a voltage-gated manner; neither of them are believed to be involved in the acidification of the virion core (168).

There are crucial differences between BM2 and AM2, in particular, their sensitivity to the drug amantadine and its derivatives. For AM2 the hydrophobic pore lining residues interacts with the amantadine molecule, allowing the formation of a hydrogen bond that would disrupt the normal configuration of crucial His and Trp residues at the ion channel pore opening. The BM2 ion channel escapes this inhibition as its His and Trp residues are inside the pore in close proximity to polar residues that would not allow such a strong bond to form with amantadine (279). These differences explain why influenza B viruses are not susceptible to amantadine.

All three proteins are generated by different coding strategies. Whilst AM2 is translated from an alternatively spliced mRNA, BM2 is coded for by an overlapping stop-start pentanucleotide (UAAUG) in a section of biscistronic RNA. This coding strategy is seen in diverse representations across biology including a mycovirus, retrotransposons and other mammalian viruses (111, 167, 202, 232). A region upstream of this pentanucleotide motif found in influenza B segment 7 is believed to be key in recruiting the 18S ribosomal subunit for successful translation of the BM2 sequence, This TURBS region is dealt with in greater detail in chapter 2 (282, 283).

CM2 generation is even more complicated and involves the generation of a protein (p42), which undergoes proteolytic cleavage in the endoplasmic reticulum to yield CM2 and a rapidly degraded protein (p31) (274).

To summarize, the AM2 and BM2 proteins, whilst possessing little amino acid homology are responsible for the influx of ion to allow the dissociation of the vRNP’s from the matrix protein. CM2 and NB appear not to share this role. Cell lines have recently been established expressing CM2 in an attempt to elucidate what role, if any it has in uncoating (238).

1.3.5 Nuclear localisation

Once released, the vRNPs are imported into the nucleus of the cell through the nuclear pore in an ATP-dependent manner. Even though all four proteins of the influenza A vRNP contain their own nuclear import signals (247), it is the interaction between A-NP and the cell nuclear

27 import factors Importins (karyopherins) α & β that allows the passage of the vRNPs into the nucleus (260). A-NP contains at least 2 nuclear localization signals (NLS) (57, 252), the one at the N-terminal region of A-NP interacts with importin α (361). Whilst protein-protein interactions, RNA binding domains and NLS have been studied intensively for influenza A virus proteins, it is only known that the N-terminus of influenza B-NP is required for nuclear import (332).

1.3.6 Non-Structural protein: NS-1

The NS-1 protein in both the influenza A and B viruses are responsible for countering the innate antiviral responses of the immune system and act as interferon antagonists (364). The numerous host protein interactions made by the NS-1 proteins are summarized in Figure 8. A full review of these complex interactions can be found in Hale et al., 2008. Both the influenza B-NS-1 and A-NS-1 proteins form dimers in infected cells and possess a conserved N-terminal RNA binding domain. However, the C-terminus of B-NS-1 shares little in the way of sequence or functional homology to the C-terminal ‘effector’ domain of the A-NS-1 (364). Unlike A-NS-1, the B-NS-1 does not bind to and therefore does not activate the P13K signalling pathway, an action possibly responsible for the A-NS-1 inhibition of premature apoptosis in infected cells (71).

Figure 8: Schematic comparing numerous proposed actions of Influenza A (green) & B (blue) NS-1 proteins. (Reviewed in Hale et al., 2008)

1.3.7 Replication

Once in the nucleus the influenza polymerase complex has the dual role of generating the mRNA transcripts required for viral proteins production, and the cRNAs that will in turn be converted into new vRNAs.

The viral polymerase is a trimeric complex formed by subunits PB1, PB2 and PA. The PB1 protein’s N-terminal region interacts with the C-terminal region of the PA protein, whilst PB1 C-terminal region links to the N-terminal region of PB2 within the complex (265).

1.3.8 Promoter Structures and compatibility

The promoter for the viral polymerases resides in the highly conserved non-coding regions at the 5’and 3’ ends of each gene segment. These regions are partially complementary so that they align to create a hairpin-loop structure known as the ‘cork-screw’, as depicted in Figure 9. The secondary structure of the influenza B promoter sequences has been previously published as a pan-handle (186, 187). However, by comparison of the prediction by Crow et al., 2004 (53) for influenza A ‘cork-screw’, the influenza B virus and the non-coding nucleotide sequences can be proposed to form a ‘cork-screw’ secondary structure for both the vRNA and cRNA promoters. The influenza C terminal sequences published by Crescenzo-Chaigne et al., 2008 (50) provided the basis for the influenza C prediction.

The internal base pairing generates structures that are remarkably conserved, but does contain nucleotide variations between the A, B & C influenza viruses, Figure 9.

Lee and Seong (1996) performed a mutational analysis of the influenza B promoter sequence. They highlighted the importance of the G nucleotide nine bases from the end of the 3’ sequence (in vRNA) the activity levels decreased to ~5 - 10% of the wild-type confirmation, without being able to explain it through their pan-handle structure. This base is of great importance in the G-C pairing in the cork-screw structure predicted in Figure 9. Nor could they explain why a C to G at 10 nucleotides from the end of the 3’ sequence should increase activity levels to 102% of wild-type, but this is again explained in the cork-screw structure as base pairing of a C with a corresponding G would increase further stabilising the structure (186). Though there is ambiguity at this base and the corresponding base pair partner (Y= C or T and M= A or C). All bases are highlighted in red in Figure 9.

Figure 9: The predicted cock-screw secondary structures at the termini of the RNA composing the promoter sequences. The Influenza A sequences are adapted from a figure in Crow et al., 2004, pink boxes indicate conserved sequence in stalk region. The Influenza B & C promoter sequence secondary structure predictions were inferred by comparing the structure of the Influenza A promoters with the sequences available for B & C (50, 157, 333). ‘W’ is an ambiguity symbol for the A or U that can be in the 6th base of the 5’ vRNA or the 3’cRNA influenza B sequence as this nucleotide is (in respect to the vRNA) an ‘A’ in PB1, PB2, PA, NP and M and a ‘U’ in the HA, NA/NB and NS/NEP segments.(Other ambiguity codes M=C or A, K=G or T, S=G or C, Y =C or U). In the Influenza C vRNA 5’ sequence, the 6th base highlighted in red is a ‘G’ in the NS segment. The stalk regions are from the NS segment of all three virus genera.

Crescenzo-Chaigne et al., found that influenza A polymerases and NP were able to recognise the (+) and (-) sense (vRNA and mRNA respectively) promoter regions of the A, B and C viruses to generate a reporter signal. Whilst the influenza B polymerases and NP were also able to recognise the A and C promoter sequences, the level of signals produced were 10-20 fold less and delayed. The Influenza C polymerases saw no such restriction with influenza B promoters, but again the level of signal from the promoters were reduced and delayed (51). Non- conserved non-coding sequences outside the range of these promoter sequences may play a role in genus: genus polymerase restriction (187) see (Figure 11).

Combinations of the A and B polymerases are unable to function except for the PA of A/WSN combined with influenza B PB2, PB1 and NP, which are able to express a reporter with the HA promoter sequences. Further research by Iwatsuki-Horimoto et al., indicated it could be the polymerase interaction between PA and PB1 that limits the compatibility. As not all segments can be transcribed, the polymerases and promoters from the two virus types are incompatible (155).

1.3.9 Polymerase interactions

Research with the influenza A viruses indicates that once in the nucleus the polymerase subunits have different but interrelated roles. The PB1 protein binds the promoter structures and catalyses the addition of nucleotides to the RNA to cause chain elongation. PA acts as an endonuclease and creates capped primers (taken from the host pre-mRNA) required to generate the viral mRNA (63). The C-terminal region responsible for this action is highly conserved in A, B and C viruses (83). PA provides the link to the host RNA polymerase II (Pol II) through interactions with host hCLE (75, 140), Figure 11.

A conformational change of the polymerase complex at the 5’ binding site of PB1 to the vRNA allows PB2 to bind to the cellular mRNA cap structure (192). Polyadenylation of the viral mRNAs is brought about by the stuttering of the polymerase enzyme caused by steric hindrance on a patch of uridines (201). The so formed mRNA can then be translated into the viral proteins using host ribosomes and translation machinery. The nuclear import of the newly translated PB1-PA proteins as a dimer is aided by Ran-binding protein 5 (RanBP5)(61) and Heat Shock Protein 90 (Hsp90) (234, 243). In the nucleus RAF-2p48 promotes the formation of the NP-RNA complex for new vRNA synthesis (233) as vRNA –NP complexes are aided by the action of Tat-SF-1 (242).

As illustrated by the genomic replication of viral RNAs, Influenza viruses as obligate pathogens utilise and interact with host proteins. A selection of the known interactions are summerised in Figure 10, a few are dealt with in the text, for a comprehensive review please refer to reference (241).

Figure 10: Host factors (in boxes) known to be involved in the influenza A replication. The key viral proteins are M1, NS-1 NEP (NS-2), NP, the polymerases and the viral RNP are represented in schematic form. The function of selected interactions are mentioned in the text, but for full details please see the Nagata et al., review ref (241) from which this figure is adapted.

1.3.10 mRNA, cRNA and vRNA

After an initial round of mRNA transcription and translation, the viral polymerase switches to vRNA replication for production of genomic RNA. vRNA replication requires the synthesis of a positive sense cRNA intermediate which is then copied to produce vRNA. The generation of cRNA does not require capped primers derived from cellular mRNAs. Moreover, compared to mRNA transcripts, cRNAs is a perfect complementary copy of the whole vRNA because they are not terminated at the site of polyadenylation. The mechanism of unprimed viral RNA replication is poorly understood. The process starts with the formation of a dinucleotide, which in turn realigns to the first two nucleotides of the 3’ terminal region of the promoter (62) therefore acting as the primer for subsequent chain elongation. It is assumed that all three influenza viruses use the same methods. NEP proteins of influenza A and B have a role in the regulation of the mRNA, cRNA and vRNA levels, but the mechanism is as yet undefined (26, 295).

1.3.11 Dissociation from the nucleosome

The NP protein contains a nuclear retention signal that allows the accumulation of the NP until sufficient vRNA is accumulated to construct the vRNPs. The newly synthesized vRNPs are attached to the nucleosome through an interaction between NP and histone. M1 and host cell mediators interrupt this interaction and allow export of the viral genome (95, 394).

1.3.12 Export from the nucleus

The influenza A NEP interacts with the H6 domain of the viral M1 protein (144) which in turn binds to the vRNP, either through interaction with the vRNA, nucleoprotein or viral polymerases (343). As NEP has nuclear export signals, the vRNP-M1-NEP complex is then exported through the nuclear pore by interacting with RanGTP and Crm1 (Chromosome Region Maintenance protein 1 or Exportin 1). The interaction of NEP with Crm1 is dispensable, as NP can bind directly to Crm1 (74), but NEP is required to form a ternary export complex with RanGTP (253).

Influenza B forms a vRNP-NEP-M1-Crm1 complex for nuclear export, possible because the NEP is able to interact directly with the vRNPs (152). However, there is a 10 amino acid sequence with 50% identity between the A & B NEPs, potentially acting as the NES, which can interchanged and still support nuclear export (269).

Influenza C NEP also has an NES, and there is evidence to suggest it co-localises with NP and interacts with Crm1 and nucleoporin, therefore provides a similar function in the replication cycle as A-NEP and B-NEP. Indeed, all three NEPs are incorporated into the newly formed respective virions, possibly as a result of their association to vRNP and M1 (152, 166, 293).

1.3.13 Transport to the Apical membrane

The viral components travel to the apical membrane of the cell in order to bud from lipid rafts. The journey of the vRNP complex to the cell membrane is believed to be mediated by M1, either via the trans Golgi network with the glycoproteins or along the cellular cytoskeleton through interactions between M1 and F-actin (5, 247).

1.3.14 Glycoproteins and the trans Golgi network (TGN)

Haemagglutinin and neuraminidase are translated on membrane bound ribosomes, then modified in the endoplasmic reticulum (ER) and transported through the TGN. Their voyage is partnered and chaperoned by AM2/BM2 in order to generate a relatively neutral pH in the late TGN. Acidity at this stage could (in some viral strains) trigger premature conformational changes in HA that would expose the fusion peptide and destabilise the protein. The N- terminal half of BM2 appears to be crucial for this task in influenza B virus infected cells (151). It has been proposed that the CM2 protein could replicate this function (15).

A-HA and AM2 cytoplasmic tails have cysteines that are palmitoylated within the ER (which later can increase membrane association with the lipid rafts, a step important for virus budding) (392). The sorting signals that carry the HA and NA to the apical membrane reside in the ectodomain or transmembrane domain respectively (170, 171, 194).

Even when the apical sorting signals are removed from the HA and the protein accumulates on the basal membrane, budding still occurs on the apical surface of the cell, indicating that HA is not the determinant of the budding location (8).

When the HEF protein is expressed independently of other influenza C viral proteins it displays cell surface transport and expression in all but the C/Johannesburg/1/66 strain. The inability of this strain to reach the cell membrane cannot be mapped to residues in the cytoplasmic tail and this issue remains unresolved (272).

1.3.15 vRNPs and the cytoskeleton

Confocal microscopy has indicated the co-localisation of HA, M1 and NP with the actin cytoskeleton (322). A-NP is able to associate with the cytoskeleton, whilst, in the absence of other cellular and viral components (excluding NP) M1 is not (5). When NP is expressed with M1, it is packaged into virus-like particles (VLPs), despite the absence of vRNA (103).

In influenza C infections, the C-NP also co-localises with the actin cytoskeleton. In persistent infections of C/Ann Arbor/1/50 persistent variant (C/AA-pi), the C-NP is unable to associate with the actin cytoskeleton. The C-NP and C-M1 in these persistent infections are homogenous in their expression pattern, whereas wild-type infections give a typical granular aggregate expression. Persistent infections have lower levels of apoptosis in infected cells. This may be accounted for by variations in the length of the NS-1 proteins of (C/AA-pi). Interestingly, the (C/AA-pi) does not display its persistent phenotype in CEF (chicken embryo fibroblasts) or Vero

(Green African Monkey cells), indicating the presence of different cytoskeleton structures or other cell type specific host factors (119, 209).

Influenza A and C viruses often form viral particles with a filamentous morphology. The cytoskeleton is also involved in the creation of filamentous particles. Simpson-Holley et al., disrupted the production of the filamentous particles, but allowed the generation of spherical particles by adding actin cytoskeleton inhibitors. Whilst the morphology changed, the virus titre did not. The cytoskeleton may be responsible for coordinating the movement of lipid raft domains together in order to form the longer membrane regions required for filamentous particles. Thus in the presence of the drugs, the HA localization was to smaller ring shaped membrane areas, which could represent raft fragments (322). Influenza B viruses have not been reported to form filaments.

1.4 ESCRT: Endosomal Sorting Complexes Required for Transport

The Human Immunodeficiency Virus (HIV-1) and other viruses have used signals provided by the Endosomal sorting pathway to target the virions to the cell membrane; it is possible that the influenza viruses use a similar route. This host pathway will be discussed in detail in the introductory section for results chapter 5.

1.5 PA (Polymerase Acid) and assembly

The PA polymerase subunit may be involved in the assembly of influenza A virus. PA has a P loop-like motif, usually found in proteins involved in binding ATP or GTP. Alterations to the loop inhibit assembly by interfering with the production of the new virus-like particles. Preventing the binding of the polymerase complex to the core of densely stacked array of the vRNAs could also be preventing the movement of the vRNAs to the assembly site (289).

Using a sequence alignment prediction of the corresponding amino acid in the Influenza B PA protein an alanine substitution was equally able to provide polymerase activity, but was unable to support virus generation using the influenza B reverse genetics system (Elderfield and Barclay, unpublished observation).

1.6 M1 (matrix protein), glycoproteins and lipid rafts

Viral budding requires curvature of the membrane at lipid rafts (areas of the cell membrane rich in sphingolipids and cholesterol). Initially it was proposed that M1 alone was able to drive this process based on experiments in COS cells (103). However, HA and NA increase the association of M1 to the membrane, through their cytoplasmic tails (3).

Transient expression of (HA) or (HA & M2) or (HA, NA & M2) proteins can generate VLPs containing HA alone or HA & M2 released from the cell (in the presence of bacterial neuraminidase) or HA, M2 and NA. Small amounts of M2 and NA can be found in particles in the absence of HA, but HA appears to drive budding. M1 alone was unable to drive budding, but when expressed with HA, high levels of M1 were found associated with membrane fractions (37).

Moreover is has been previously established that there is an accumulation of the HA and NA glycoproteins at the lipid rafts. The glycoproteins associate with the cell membrane through their cytoplasmic tails (392). The loss of the cytoplasmic tail affects the morphology of the resultant virions (391).

The palmitoylation of three cysteines in the cytoplasmic tail of HA aids in the association with the lipid rafts, However, this may be strain dependent (38). The clustering of HA in lipid rafts results in budded virion rich in lipids favourable for virus: cell fusion in the next infected cell (341).

ATP hydrolysis is absolutely required for virus budding and release (146). Blocking G protein signaling results in a decrease in viral titre, and inhibitors of the host cell kinase, Casein Kinase 2 CK2, which itself becomes stimulated during the course of infection, inhibit budding (147). Interestingly a known function of CK2 is to phosphorylate the PA polymerase component of the vRNP, which may itself aid in segment sorting and packaging (289).

1.7 Packaging Signals

The influenza virus RNA genomic segments consist of a 5’ and 3’ promoter region, a section of non-conserved non- coding sequence and an internal open reading frame that encodes the protein (see Figure 11). As previously mentioned the highly conserved 5’ and 3’ promoters are required for the generation of mRNA, cRNA and vRNA as part of the viral life-cycle.

Figure 11: Schematic of gene segments of Influenza A, B and C viruses. The range of lengths for non-conserved regions (NCRs) for the individual segments is displayed at the 3’ and 5’ ends for the three genera. The dark blue terminal boxes indicate the conserved non-coding sequence containing the promoter sequence for the three genera. Packaging signals for influenza A are located in the coding and non-coding regions are indicated by the red box. The location of influenza B or C virus packaging signals have not yet been defined.

The influenza A packaging signals have been elucidated for all of its 8 genomic segments. They are located in the coding and non-coding regions of the genes (6, 67, 87, 88, 262, 371). These packaging signals are responsible for the inclusion of all of the segments into the new virus particles. How exactly these signals interact with each other or the polymerases and NP that make up the RNP complex is not yet known. The PB2 segment may be acting as the central link to the other segments in the packaging into new VLPs (239, 258).

Influenza B differs from influenza A in the length of the non-conserved non-coding regions at the terminal ends of the virus vRNA segments. In influenza B they are generally much longer. Since at least part of this segment specific non coding region seems to be required for influenza A vRNA packaging, it is possible that influenza B packaging signals could differ from those of the influenza A virus and perhaps be encompassed entirely within non-coding regions.

1.8 The Ion Channel Reprise: AM2 and BM2 role in assembly

As mentioned previously one of the main functions of AM2 and BM2 is as an ion channel used during the initial uncoating of the virus and as a chaperone for the other glycoproteins, especially HA, through the ER and Golgi (48, 307). A third function appears at the assembly step.

Influenza B has an absolute requirement for BM2. However, it is possible that the 20-50 molecules of BM2 per virion can be decreased some what without any effect on the viability of the virus, at least in tissue culture. If there is a severe reduction in the levels of BM2 in the new virus particles, this results in a lower infectivity to particle ratio (151, 160). In contrast, influenza A viruses that lack AM2 have been rescued (368) although the loss of AM2 severely attenuates the virus replication in both cell culture and mice (156). Large reductions in AM2 production in the infected cells, causes only a 30% reduction in the levels of AM2 in the new virus particles, suggesting that AM2 is selectively incorporated into virions (22).

VLPs that do not encode A or BM2 protein can be created with the complementation of the full-length protein in trans (e.g. a cell line that expresses the M2 protein). However, the complemented virions are unable to express BM2 in the cells they infect virions that are released from cells infected with viruses that do not encode BM2 have decreased levels of NP and M1 (70 and 90% reduction respectively). This indicates BM2 is involved in vRNP incorporation (151). Influenza A viruses also display a reduction in NP when the AM2 protein is absent or the cytoplasmic tail is truncated, suggesting that the AM2 protein may also enhance RNP incorporation into virions (224).

A 28 amino acid deletion of the AM2 cytoplasmic tail caused a four fold reduction in released virus particles and a 1000 fold decrease in the infectivity of the virus that correlated with loss of packaged RNPs. Serial truncation of the cytoplasmic tail of AM2 caused a significant loss in RNP levels if more than 22 carboxy-terminal residues were removed. Interestingly, a change from spherical to filamentous morphology was observed in some of the mutants (156).

BM2 appears to influence the membrane association of M1, though not the distribution of the glycoproteins HA and NA (151). AM2 and BM2 do not associate with lipid rafts but are associated with cholesterol at the periphery of these domains (188), a finding that might explain their relative paucity in virions.

1.9 AM2 in viral dissemination

A fourth role for the AM2 ion channel protein has been suggested by electrophysiology experiments. During the course of an infection lung epithelial cells express an ion channel to pump Na+ ions from the alveolar to the interstitial sides. This transports the ions into the airway and distal lung epithelial cells through the amiloride-sensitive epithelial Na+ channels (ENac) and cation channels. The transport of Na+ ions helps prevent alveolar edema after lung damage. It has been proposed that AM2 expressed at the apical surface of the cell increases the steady state level of reactive oxygen species (ROS) and activates PKC. This action in turn increases the ubiquitination of the ENac receptor and its targeting for destruction to the proteasome. To summarize, the AM2 down regulates the activity of ENac which reduces the level of fluid in the lungs, delays viral clearance and increases damage by the virus (41, 185).

Interestingly A-NS-1 is also implicated in the absorption of Na+ ions and the concurrent decrease in lung fluid (89). Both these reports acknowledge that this is contrary to the overall increase in lung fluid observed in an influenza infection. As yet, no corresponding research has been conducted on influenza B or C.

1.10 The actions of neuraminidase

The viral neuraminidase must cleave the sialic acid linkages on the cell surface in order to prevent the reattachment of the virus to receptors on the membrane of the already infected cell, allowing virus to transmit to naïve cells. Obviously there must be a balance between the attachment protein and the receptor destroying protein, both in terms of the activity level of the desialylating enzyme, the receptor type (α2-6 or α2-3) and the affinity for the receptor of the haemagglutinin (359).

Neuraminidase is a membrane bound glycoprotein encoded on segment 6, the same segment as the NB protein, which with be discussed at length in chapter 5. The neuraminidase tetramer forms a mushroom shaped spike; it has four roughly spherical shaped subunits which are attached centrally to a stalk (2). As mention previously, neuraminidase may also be involved in cleaving through the sialidases on the mucins that protect the respiratory tract (217).

It has also been proposed that neuraminidase may cause a localized immunosupression in the mucosa by desialylation of the hinge region of IgA which allows it to be cleared from the mucosa. This is mediated by the hepatic asialoglycoprotein receptor (ASGPR) and by the desialylation of mucosa residing gamma and delta T cells or IgA producing B cells, which again their encourages clearance from the mucosa, this time by altering the homing signals (17).

There is also evidence that the influenza A and B neuraminidase may activate Transforming Growth Factor β (TGF-β) by cleaving the sialic acid residues on the latency associated peptide (LAP). TGF-β has both an immunosuppressive effect and acts as proinflammatory agent, the latter function promoting apoptosis (312). How key this ability of the neuraminidase is in the viral replication cycle is open to debate as the TGF-β activation is virus strain specific, for example the Hong Kong H5N1 viruses (i.e A/ck/HK/220/97 ) were unable to activate TGF-β whereas other highly pathogenic H5N1 viruses are (i.e A/ck/Scotland/59) in vitro and in vivo (mice) assays (69, 118). Indeed in a human volunteer trial involving an A/Texas/36/91 (H1N1) infection there was no upregulation of TGF-β activity (118).

The importance of neuraminidase is highlighted by the efficacy of the anti-influenza drugs (Tamiflu™ *Oseltamivir+ & Relenza™ *Zanamivir]) acting against the neuraminidase activity by blocking the active site, which inhibits further viral spread. Unfortunately, resistance to the inhibitors has been found in both the A and B viruses, the impact of which is reviewed in Lackenby et al., 2008 (155, 173).

The influenza B neuraminidase has less than 25% sequence identity to the influenza A protein (2). Yet the influenza B protein can functionally replace the type A protein. The type B neuraminidase was supplemented in trans to a influenza A virus lacking a functional neuraminidase and virus was rescued. Interestingly, the influenza B NA segment cannot be packaged into the A virions, possibly due to packaging signals and/or polymerase/promoter incompatibility (99, 155).

The HEF protein of Influenza C provides the esterase function required to allow the release of the virus from the surface of the cell (301).

1.11 The NB and CM2 Proteins.

Research into the actions of the NB protein have been undertaken, but to date its function is unknown. A detailed account of current knowledge of the structure and actions of this protein will be discussed in Chapter 5. The similarity of NB to the CM2 protein in terms of its polylactosaminoglycan modifications may be of interest in terms of the adaptation to the human host (15, 93, 273).

The Respiratory Syncytial Virus (RSV) SH protein also displays polylactosaminoglycan modifications and is also cation permeable (93). Other viruses also display cation channels. The Coronaviridae possesses an E protein, which when deleted in Mouse hepatitis virus (MHV) causes small plaque sizes and attenuated growth, whereas Transmittable gastroenteritis virus

(TGEV) displays an absolute requirement for the E protein. The Severe Acute Respiratory Syndrome (SARS) virus representative of this genus has an E protein which is a cation-selective ion channel (383). Hepatitis C Virus (HCV) p7 (285), 6k (or Trans Frame) protein of alphaviruses (78, 229) , HIV-1 Vpu (76) have also all displayed cation channels. Many of these proteins have been implicated in viral budding, but as yet the mechanisms appear unclear.

1.12 The impact of Influenza B and Vaccine production.

Influenza B causes regular seasonal epidemics in the human population, due to the selective pressure exerted by the acquired immunity, causing antigenic drift and reassortment within the Influenza B strains (226). The Health Protection Agency (HPA) isolate and identify the aetiologic agents of influenza like illness submitted through the Royal College of General Practitioners (RCGP) scheme and other surveillance routes each year in the UK.

Figure 12: A. The HPA community and hospital infections caused by influenza 2005-2006, Characterised by the Enteric, Respiratory and Neurological virus Laboratory (ERNVL). The dark blue bars represent isolates typed as influenza B detections (PCR and isolation). This season saw a peak in influenza B infection which has not been replicated in the following two seasons. B. The HPA community and hospital infections caused by influenza in the 2008-2009 Influenza season to date. The dark blue bars again represent influenza B detections (PCR and isolation). Influenza A was (Red, Pink, Green, Yellow and turquoise) is the predominant source of infections. The pandemic H1N1 is extending beyond the traditional influenza season. (http://www.hpa.org.uk/HPA/Topics/InfectiousDiseases/InfectionsAZ/1191942171484/).

Vaccine strains of Influenza B viruses are selected in biannual meetings after a worldwide surveillance effort in order to predict which virus strain is most likely to be the predominant form in the upcoming influenza season (reviewed in (31). Unlike their Influenza A counterparts, influenza B viruses do not currently have a master donor virus (A/PR8/34) upon whose backbone of internal proteins, the NA and HA surface proteins can be exchanged for those of the predicted predominant strain (104).

The strain chosen must display the correct surface antigens and have the ability to grow well in eggs, the currently preferred method of vaccine production. This requirement is not always met the B/HK/330/2001 strain chosen for the 2001 influenza season did not grow well in eggs and there was difficulty is providing sufficient antigen for vaccine production (129, 158).

It would be helPFUl to create an influenza B virus that had a high yielding backbone, either in eggs or preferably in cell culture. Ideally reverse genetic (discussed in detail later in this chapter) would be of benefit to sidestep the laborious reassortant process of mixing viruses and attempting to isolate the virus with the correct backbone and surface antigen (often referred to as the 6:2 reassortant).

Cell culture would be preferable to eggs as a culture medium. As was mentioned earlier in the attachment section of this chapter, growth in eggs can cause an adaptation in the attachment site of the HA molecule to give a preference for avian receptor binding (363). This would make the vaccine protein less like the virus the recipient would be exposed to in the influenza season and can sometimes result in a significant antigenic mismatch (395). MDCK cells or other mammalian cell lines are more likely to produce viruses with unadapted surface antigens. Cell culture also provides the potential for scaling up the process; systems using entirely MDCK cells (365) or PER.C6 cells (169) have already been established.

The reverse genetics methodology that will be described in section 1.14, has allowed the creation of interesting chimeras, both in terms of mixing A & B viral proteins, but also in terms of creating vaccine strains that contain components of both viruses. Viruses were created that had the influenza A backbone, but expressed the HA of influenza B or the ectodomain of the influenza B HA fused to the transmembrane domain and cytoplasmic tail of the influenza A HA (82). These viruses were able to grow to wild-type levels in cell culture. Similar viruses were attenuated in mice but were able to protect the mice from challenge with an otherwise lethal dose of both influenza A and B viruses (132, 133).

Conversely, should eggs be retained as the culture method of choice, adaptations that promote the growth in eggs could be specifically engineered in a controlled fashion rather than being randomly selected for (198).

A live attenuated influenza virus (LAIV) vaccine backbone has been developed for influenza B viruses. A series of experiments comparing the use LAIV in comparison to inactivated vaccines was conducted in ferrets. The LAIV displayed a clear superiority (141). The master donor for the LAIV’s is Ann Arbor/1/66. This virus displays temperature sensitivity (ts), attenuation (att)

43 and cold adaptation (ca) in a genetically stable manner (326). This attenuated strain was created by repeated serial passage of the wild-type virus in chicken eggs and in primary chicken kidney cells at gradually lower temperatures (205). These phenotypes have been mapped to NP (A114 & H410) PA (M431) for ts; the ts changes plus M1 (Q159 & V183) give the att phenotype (128), cold adaptation again requires all the ts changes plus (T509) also in NP and (R630) in PB2 (42). Interestingly this differs from the explanation for attenuation in the Influenza A analog (A/Ann Arbor/60). Whilst an attenuated phenotype has previously been ascribed to the M1 protein in Influenza A viruses (325) for the LAIV B virus, ca and att maps to the polymerase genes; and M1 was not implicated (49, 325, 339).

1.13 Animal models in the study of influenza virus

In order to understand influenza infection in whole organisms various animal models have been used. Studies have used mice, ferrets, rats, cotton rats, pigs, non-human primates, cats, dogs, guinea pigs, birds and even human volunteers.

Animal Influenza Adapt? Benefits Limitations Genera Mouse A, B & C Yes Low cost Mx-1 gene (BALB/cJCitMoise (B/c) (excl. Well developed A/SnJCitMoise,CBA/CaLacSto HP H5) reagents C57BL/6LacSto &BALB/c) Rat A Yes Low cost Limited data (Brown Norway Developed reagents Fischer-344 Sprague–Dawley) Cotton Rat A, B No Functioning Mx gene Aggressive Symptoms mirror /rarity humans. Developed reagents Ferret A, B No Functioning Mx gene Cost/housing/ Symptoms mirror outbred/limited humans reagents Guinea Pig A No Low cost Limited data Small animal Pig A, C No Highly susceptible. Cost/housing/ Similar airway waste receptors to humans Non-human primate A No Similar to Humans Cost/housing/ (Pigtailed, Rhesus & Useful for vaccine ethics Cynomolgus Macaques) studies Pathology varies Dogs A (H5N1) No Limited data Limited transmission Cats A (H5N1) No Hyaline membrane Limited data

Table 2: Table of Animal models used in influenza research.

The mouse is a convenient small model for the study of influenza virus. Their relative ease of handling, low cost and well defined genealogy makes them a popular tool in biological research. Indeed they have been repeatedly used in influenza research for pathogenesis, vaccine and drug studies. They have the advantage of a wide and diverse set of reagents for dissecting the various immunological responses to infection. For influenza A virus, some well characterized strains that readily infect mice exist such as the A/WSN/33 or the A/PR8/34. However, clinical isolates often require repeated passage in mouse lungs before the virus is able to adapt to the mouse. Highly pathogenic avian influenza is an exception and readily infects and kills mice (9). However, many inbred mouse strains of influenza possess a non- functioning Mx1 gene, this anti-viral GTPase is responsible for innate anti-viral responses limiting the usefulness of the data obtained. Conversely, mice with the fully functional Mx1- gene are highly resistant to influenza infection(115).

Most influenza B viruses do not effectively infect wild-type mice. Palese et al., recently described successful infection of PKR knockout mice with influenza B virus (56). Influenza B/Lee/40 virus has been described to infect mice but the titres recovered are low and symptoms are rare. McCullers et al., have described a mouse adapted variant where a N221S substitution confers lethality to wild- type mice in both B/Memphis/12/97 and B/Yamanashi/166/98 virus influenza infection (225).

Mice do not show the clinical symptoms observed in humans, as such, research groups and industrial researchers alike are increasingly turning to the use of the ferret model to support the mouse data and as a useful tool in its own right (45, 207, 230, 275, 286, 302, 328, 349). Ferrets do display the characteristic nasal discharge, sneezing, watery eyes and fever of an influenza infection (9). The human influenza viruses readily infect ferrets without adaptation. However, ferrets are more expensive than mice, require more careful handling and the reagents to fully investigate the viruses effects in the ferrets are only just being developed. Currently the ferrets are out bred, a limited resource and not necessarily naïve to influenza infection. With increased demand for both reagents and suitable ferrets these drawbacks will be resolved (9). Cotton rats can also be readily infected with influenza virus A and B viruses. As with ferrets their functioning Mx gene allows them to display the innate antiviral immune responses similar to those observed in humans. The reagents to study these responses in the cotton rat are readily available. However, these animals are scarce, due to low demand as they have an aggressive nature that makes them difficult to work with (72, 268). In contrast, to establish an infection in ‘true Rats’, the virus must be passaged at least 11 times in rat lungs

46 before sufficient adaptive changes have occurred. Subsequently these are not yet a popular tool (9, 55).

Guinea pigs have been used in a few transmission studies and can be infected with the influenza virus. Their use for influenza is only in its infancy (197).

Pigs, have recently re-emerged as a popular host for the influenza virus. Indeed they are reputed to have all the necessary receptors for both human and avian viruses; hence the pig is often termed a ‘mixing vessel’. However, pigs are big, expensive and present considerable husbandry considerations. This may limit the number of experiments one is able to perform. Due to their high susceptibility to influenza infection, their serological status must also be established (9). There are no reports of influenza B virus experimental infections in pigs.

Dogs and cats have been tested for there susceptibility to H5N1 infection. Whilst dogs can be infected with the H5N1 influenza viruses they do not appear to transmit the virus to other mammals. The lungs of cats display a Hyaline membrane not observed in any other animal model except humans (101, 354).

Non-human primates are often used for vaccine studies, but are an expensive and emotive animal model choice, despite their similarity to humans. Pigtailed, Rhesus and Cynomolgus Macaques have all been used in the context of influenza research, including efficacy of drugs and vaccines (9).

A major advantage in animal infections as opposed to cell culture is the interactions with the immune system. More complex interactions such as secondary bacterial infections can also be studied important, as they are thought to be responsible for up to 25% of influenza related deaths during seasonal pandemics (112).

An alternative to animal models is the use of primary airway cell cultures. Whilst this does still require the death of an animal or human, the number of experiments that can be conducted can be increased, allowing consistency that animal models cannot provide, different treatments can be used on the cell cultures from one animal, reducing animal to animal variations (important in the out bred ferret populations). Diverse airway cell types and mucus will be present. It may also be possible to introduce secondary bacterial infections into the airway cultures to evaluate any increase in pathology. They will not however, provide information on adaptive immune responses, more information on these systems are provided in chapter 5.

1.14 Reverse Genetics

Recombinant influenza viruses can be created by ‘natural’ reassortment of their 8 gene segments, but the generation of a particular virus is cumbersome as there are 256 possible outcomes and the nucleotide sequence of each segment can only be that of either parent. The creation of a Bunyavirus (a segmented negative sense RNA virus) from plasmids (24), suggested a similar system for the influenza virus was feasible.

In 1999 two groups (84, 254) created an entirely helper virus independent system to generate influenza A virus entirely from cDNA. Both groups generated plasmids representing all 8 segments, which when transfected into Vero or 293T cells (respectively) were able to direct the synthesis of negative sense vRNA from their RNA Pol I promoters. The terminator sequence is generated by a Hepatitis Delta Virus (HDV) ribozyme sequence or the mouse RNA Pol I terminator sequence, the latter is deemed the most efficient (77). The four helper plasmids (expression plasmids that produce the three polymerase proteins and NP) were required for the generation of the RNP complex to transcribe the viral gene segments into mRNA that would give rise to proteins and to replicate them into the genomes of the next virus-like particles.

An equivalent system for the rescue of recombinant influenza B virus (Figure 13), was generated by David Jackson, for his studentship in 2002. He used B/Beijing/87 as a template for his viruses (except for segment 5 viral RNA plasmid which came from B/Lee/40). The Pol I promoter and the HDV ribozyme terminator sequences were used; the helper plasmids used a Cytomegalovirus (CMV) promoter sequence (159).

Figure 13: Illustration of the Influenza B reverse genetics system. The 8 plasmids expressing the viral RNA gene segments and the 4 expression plasmids encoding the 3 polymerase proteins and the nucleoprotein are transfected into 293T cells. The 293T cells are incubated overnight to allow plasmid entry. The 293T cells are then co-cultured with MDCK cells. The plasmids work in concert to produce the viral proteins and the viral genomic complement required to generate virus particles. The newly generated viruses are released from the 293T cells and amplified in the permissive MDCK cells.

At around the same time, Hoffman et al., used an 8 plasmid system to also generate an influenza B recombinant (129). There are currently five influenza B reverse genetics systems in use; they are listed in

Table 3. As of 2007, a reverse genetics system now exists for the study of Influenza C, using C/Johannesburg/1/66 as the base strain (52).

Virus Published B/Lee/40 Hatta & Kawaoka, 2003; Dauber et al., 2004 B/Beijing/1/87 Jackson et al., 2002 B/Yamagata/1/73 Imai et al., 2004 B/Yamanashi/166/98 Hoffmann et al., 2002 B/Ann Arbor/1/66 Wang & Duke et al., 2007

Table 3: The influenza B reverse genetics systems currently in use and the papers in which they were published. The B/Ann Arbor/1/66 system has the canine promoter sequence for growth in MDCK cells.

A current limitation of influenza virus reverse genetics is that it is necessary to introduce up to 12 plasmids into a single cell simultaneously in order to reconstitute all the components required for virus rescue. Many cell types that are highly permissive for plasmid transfection do not efficiently support a full round of virus replication. For example, yields of most strains of influenza A, B or C from highly transfectable 293T cells are very low. To overcome this 293T cells are often co-cultured with a more permissive cell line such as MDCK or CEF. PerC.6 cells (human embryonic retinal cells) allow the transfection and amplification steps to be conducted in the same cell line (169) allaying the need for the co-culture step. Alternatively, alterations have been made to the plasmids themselves, by the insertion of a T7 promoter which allows transcription in a wider range of cell types or by the introduction of the canine promoter with allows for viral generation solely in MDCK cells (60, 365). The latter system has also been adapted for rescue of influenza B viruses. Advances in the manufacture of extended oligonucleotides provides the ability to order the synthesis of whole viral gene segments in a plasmid format, these are ready for incorporation into the reverse genetics system, allowing the rapid generation of tailor-made recombinant viruses, albeit at a cost.

Culture Transfectability Virus Yield Immunofluorescence 293T High Low Medium Human Embryonic kidney (Low adherence) cells MDCK Low High Good Mabin Darby Canine Kidney Cells Vero Medium Medium Good Green African Monkey Cells PerC6 High High No Human Embryonic Retinal (suspension) cells CEF Medium Low Medium Chicken Embryonic Fibroblasts Eggs No High No (adaptive changes)

Table 4: Culture systems used in the study of influenza viruses. Listing relative strengths of each culture medium, in terms of transfectability, the yield of virus from infected/transfected cells and the ease of manipulation for microscopy.

1.15 Thesis Aims

The influenza A and B viruses have proteins that appear to be functionally similar causing many assumptions to be made about the mechanisms of actions of the influenza B proteins based on research into the influenza A proteins. Many of these assumptions may be perfectly valid, but they should be confirmed especially as there are differences between these two viruses, in protein composition (the novel PB1-F2 in influenza A and NB in influenza B), mutation rate and host range which imply subtle alternatives in the mechanisms of action.

Research into the influenza A M2 (AM2) protein’s cytoplasmic tail would indicate it has a role in virus assembly (156, 223).This thesis will endeavor to discover if the Influenza B virus BM2 protein’s cytoplasmic tail also has a similar role in influenza B virus assembly despite the very different amino acid coding of the two proteins.

One of the unique features of influenza B is the mechanism used to initiate translation of the BM2 ion channel protein (134). The recent discovery of TURBS regions upstream of the pentanucleotide (282, 283) has led to questions on how alterations in this sequence can affect BM2 protein production in the context of virus rather than in vitro expression systems and whether such alterations affect virus multi-cycle replication. This project will use recombinant viruses altered in one of the TURB motifs evaluate the corresponding effects on BM2 production and virus fitness.

The virus is an obligate pathogen; it has to adapt normal cellular processes to its own advantage, in order to successfully complete its replication cycle. How the newly synthesized virus genomic complement and structural proteins are transported to the site of viral assembly site on the plasma membrane is not fully understood, though the actin cytoskeleton has been implicated. A method used by other viruses is through interactions with their viral ‘late domains’ and components of the ESCRT pathway. It was plausible that the Influenza viruses may use a similar methodology; chapter 4 will investigate this possibility using a selection of ESCRT proteins.

Tetherin is a host protein that inhibits the release of some budding viruses from the host cells (250), chapter 4 will also investigate if this protein has any role in preventing the release of newly synthesized influenza viruses from host cells.

Finally, the influenza B virus has the novel NB protein, which is not required for virus replication in cell culture, but proffers an advantage to the virus in vivo (116). The function of the protein in the influenza B replication cycle is not currently known. Using a series of recombinant viruses where the NB protein has either been removed or modified, we have investigated if the NB protein proffers an advantage in the ferret, a popular in vivo model for human influenza infection. Through a series of experiments with FAE and HAE (primary ferret and human trachea cell culture systems) we investigated if the any advantage on virus fitness in conferred by NB is maintained in cultures that are representative of the natural sites of infection.

Chapter 2. Modifications in regions complementary to the 18S ribosomal subunit upstream of the BM2 initiation codon affect protein production

2.1 Introduction

The BM2 protein is encoded on Influenza B virus segment 7. This segment initially encodes the M1 matrix protein, the major structural protein of the virus. Then, at a stop:start pentanucleotide motif, UAAUG, translation of the M1 protein is terminated and translation of the BM2 protein is initiated in a -1 reading frame (134). Since the discovery of this unusual coding strategy in influenza B virus by Lamb and co-workers more than 20 years ago, similar re-initiation motifs have been found within genes of several other organisms including the mycovirus Cryphonectria hypovirus 1 (CHV1) (111) and various retrotransposon (1, 108, 109, 167, 202, 231, 232, 340). Whilst these examples all possess the same UAAUG stop-start motif as BM2 (167), an embryonic RNA splicing variant of glutamic acid decarboxylase has a UGAUG motif (340). In addition a number of viruses and retrotransposons including RSV, Pneumovirus of mice (PVM), Feline Calicivirus (FCV) and Rabbit Haemorrhagic Disease Virus (RHDV) and three non-LTR retrotransposon elements RT1, RTAg4, and HidaAg1, have been shown to utilize inversely orientated re-initiation events whereby the initiation codon is immediately upstream of the termination codon, for example AUG(n)UGA. The number of initiation codons and termination codons and the distance between them varies between these examples but the principle that two open reading frames are linked and that translation of the downstream one depends on termination of the upstream one is the same.

The mechanism by which such coupled termination and re-initiation occurs is only just being elucidated. Current knowledge about orthodox translation initiations is as follows: eIF3 is an essential scaffolding component for the initiation of translation. In the case of standard start codons, initiation occurs when free 40S subunits are stimulated to bind to the

Met ternary complex of eIF2, GTP and Met-tRNAi (Initiator methionyl tRNA charged methionine).

The stimulation is provided by eIF1, eIF1A, eIF5 and eIF3. A complex known as 43S PIC (pre- initiation complex) is formed (see Figure 14).

Association of the PIC with the mRNA requires the poly (A) binding protein (PABP) bound to the m7 G-capped 5’ end of the mRNA to be complexed with translation factors eIF4G, eIF4A, eIF4B. After this, there is recruitment of the cap binding protein eIF4E and other components of the eIF4F complex, and the whole complex is renamed 48S PIC.

The 48S PIC can then scan the mRNA for the start codon, a process that requires ATP hydrolysis should a secondary structure be present in the RNA. Once the complex comes into contact with a start codon, which enters the ribosomal P-site, eIF1 is released and the 48S PIC changes conformation so it can no longer scan the RNA. The remaining translation factors (including eIF3) are released when GTP-bound eIF5B stimulates the recruitment of 60S subunit to form the 80S initiation complex and elongation can then occur. The 13 subunits of eIF3 are responsible for many steps in this initiation process including the assembly of the Ternary complex (TC), binding of the TC to the 43S PIC and to the 40S subunit, mRNA recruitment to 43S PIC and the scanning of the mRNA for the start codon. (The initiation complex is reviewed in (125).

Figure 14: A simplified version of the eukaryotic translation initiation pathway. Modified from (125) the blue diamond represents the eIF3 complex, an essential component of the 43S and 48S PICs, but is released prior to the joining of the 60S to create the 80S subunit which acts to elongate the translation.

In order for a second open reading frame to be translated from the same mRNA, a pre- initiation complex must be recruited. A select few eukaryotic genes possess the ability to direct reinitiation of translation; GCN4 in yeast, where the first open reading frame is sufficiently short that the ribosomes and initiation complex can remain attached until they reach start codons for later open reading frames (237); and the ATF4 gene where, under stress conditions in which the translation factor eIF2 has a particular state of phosphorylation, translation is reinitiated (356).

Viral sequences in which internal initiation of translation occurs have been known for many years. The most famous strategy is the IRES (internal ribosomal entry site) which are highly structured RNAs first described in the 5’ non-coding region of the poliovirus genome that can recruit ribosomes directly through their interaction with host factors. It is possible for an internal initiation codon with an IRES sequence close by to recruit an initiation complex as it does not require as many eIFs or the m7G cap for initiation to occur (reviewed in (125). None of the coupled termination:reinitiation viral sequences in the caliciviruses, RSV or influenza B virus are close to an IRES that would allow direct recruitment of the 40S subunit required for transcription.

Rather, the caliciviruses, RHDV and FCV have regions upstream of the initiation codon that have been dubbed TURBS (202). Recently work from Powell et al., (2008) confirmed that influenza B virus segment 7 also contained a TURBS and this accounted for the stop:start mechanism for translation of the BM2 protein. The TURBS regions that have been thus far characterized contain two distinct motifs that each display complementarity to the tip of helix 26 of the 18S ribosomal subunit (202, 231).

The 18S rRNA is the 1874 nucleotide component of the 40S ribosomal small subunit. Figure 15 modified from that in Powell et al., 2008, illustrates the two motifs with complementarity to 18S rRNA that comprise the TURBS in RHDV, FCV and the two stretches of nucleotides spanning nucleotides -171 to -166 and -35 to -30 relative to the AUG codon that Powell at el., have proposed to be TURBS motifs in influenza B segment 7 mRNA.

Figure 15: Schematic illustrating the identical motif regions in Influenza B (putative), RHDV and FCV, where termination-re-initiation events are known to occur. The motif regions compose the TURBS. Modified from Powell et al., 2008 (282). The putative motif regions in the Influenza B segment 7 sequence Motif 1 & 2 identified by Powell et al., Motif 1 can vary in the last base as A or G, hence is represented by R. (VP1 & VP2 of RHDV formerly known as VP60 & VP10).

Figure 16 illustrates regions in the secondary structure of the ribosomal RNA (shown in the 3’- 5’ orientation) (202, 215, 231) aligned with the complementary sequence of motif 1 of the viral mRNAs (shown in 5’-3’ orientation) of RHDV and FCV and mRNA segment 7 of the B/Beijing/1/87 strain of influenza B virus. Whilst in RHDV and FCV it is motif 1 that displays the complementarity to the 18S subunit, both proposed influenza B motifs contain the necessary sequence ‘UGGGA’. Deletion, single and double point mutations in both motifs of RHDV and FCV results in reduction or ablation in expression of the downstream protein, though motif 2 tolerates a greater degree of modification and is less conserved (202, 231).

Figure 16: The tip region of the 18S rRNA in helix 26 displaying complementarity to the motif 1 regions of the caliciviruses and the TURBS motifs of influenza B/Beijing/1/87. The wobble base is a G (blue) in B/Beijing/1/87 for motif 1. The green bases are the 18S sequences complementary to the TURBS bases in the sequence alignment of FCV, RHDV (orange) and influenza B (red). (Modified from figure in (282).

The termination: initiation mechanism directed by a TURBS relies on the translation and termination of the first open reading frame, which allows the ribosome to be in the proximity of the start codon of the second open reading frame. It has been proposed that translation through the motif 1 sequence would cause a remodelling of the RNA, allowing an upstream secondary structure to form which directs the binding of eIF3. eIF3, as previously mentioned, is responsible for stimulating most of the reactions in the initiation pathway, crucially the dissociation of the 60S subunit through its binding to the 40S subunit and binding to the mRNA to allow scanning for start codons. Figure 17 gives a stepwise illustration of the proposed model by which the calicivirus and influenza B virus TURBS interact with the initiation factors and ribosomes (reviewed in (125).

RSV, APV (Avian Pneumonia Virus) and PVM have no sequence similarity to motif 1 of the caliciviruses and it has been proposed they may utilise an alternative pathway (108, 109).

Figure 17: Proposed method of action for the recruitment of the Ribosomal translation machinery to Influenza B (motif 2) and Caliciviruses (motif 1). Modified from Powell et al., 2008 (282). A) The translation complex of the upstream open reading frame with the intact 80S ribosome (40S+60S). B) The possible recruitment of eIF3 which could enhance the dissociation of the 60S subunit, the TURBS region of helix 26 is represented by the yellow star. C) Secondary structure remodelling allowing the presentation of the TURBS region to the 18S component, thus tethering the complex to the viral mRNA.

Recently Powell et al., (282, 283) looked in detail at the BM2 initiation: termination strategy using a dual luciferase reporter system in which a 250 nt insert from segment 7, consisting of 230 nucleotides sequence upstream of the UAAUG and 20nt of sequence downstream of the stop start motif was engineered such that it separated two easily assayable reporter products, renilla and firefly luciferase. The full-length 230 nucleotides incorporated both motif 1 and 2 of the proposed TURBS sequence. Using in vitro rabbit reticulocyte mediated translation they were able to visualise the translation of the downstream reporter that represented BM2 whilst altering the region upstream. They elucidated that the stop codon must not be further than 8 codons from the initiation codon for translation to occur. A series of deletions indicated that 42 nucleotides upstream of the initiation codon were absolutely required; within this sequence

59 there was a region that displayed a degree of sequence similarity to those of the RHDV and FCV’s TURBS motif 1, the section that is complementary to the 18S ribosomal sequence (see Figure 16). In contrast to the RHDV and FCV TURBS motifs, loss of the AUG-distal motif 1 was tolerated for expression of the downstream protein. Changes were made in the inserted influenza B virus segment 7 sequence to vary the complementarity to the 18S region within motif 2 of this putative TURBS. Altering nucleotide position -35 from an A to a G increased the level translation. Bearing in mind that the G-U base pair would still be able to form this is not very surprising. All other alterations that decreased the proposed interaction between the sequence surrounding the influenza B TURBS motif and the ribosome subunit decreased protein expression, lending weight to the idea that this influenza B virus sequence was indeed a TURBS.

Interestingly, they also found that, at least during translation within the rabbit reticulocyte system, altering the BM2 initiation codon from AUG to CUG, GUG, ACG, CUA or UCG did not prevent re-initiation, although the amount of translation was reduced. By a combination of biochemical mapping experiments and computer fold predictions, two possible structures of the RNA in region surrounding the initiation codon for BM2 were presented (Figure 18).

Figure 18: Simplified version of the predicted structures of the influenza B virus segment 7 RNA from Powell et al., 2008. The termination initiation pentanucleotide is highlighted in turquoise, and the putative TURBS motif region is highlighted in pink

Recently a publication from the Kawaoka group (117) described a similar approach to that of Powell but used viral-like RNAs as the context in which the BM2 translation was analysed. This work also concluded that the 45 nucleotides upstream of the BM2 initiation codon were essential for the translation of BM2. However, neither of these studies confirmed that the TURBS motif was actually used during influenza B virus infection. All of the aforementioned work by Powell or Hatta et al., has been carried out either using in vitro translation systems or exogenous expression of mRNAs that contain pieces of influenza B virus sequence. Entering into collaboration with Powell and co-workers, we wanted to discover if alterations in the nucleotide sequence of the region within the essential 45 nt complementary to the 18S tip region (called the TURBS motif from hereon) would affect the level of BM2 protein produced in the context of whole virus.

2.2 Results

2.2.1 Design of influenza B virus mutants with altered complementarity to the 18S Ribosome in the segment 7 mRNA.

The mutations that could be made in the putative BM2 TURBS motif were limited because this region exists within the coding sequence of the matrix protein. Making amino acid changes in the M1 protein could alter the fitness of the virus and prevent virus production independently of any effect on 18S rRNA binding activity of the TURBS RNA. Only the nucleotides at position - 36 and -30 relative to the stop-start motif could be mutated without causing amino acid changes in M1 (Figure 19). The -36 U nucleotide lies one base upstream of the region complementary to the 18S rRNA sequence and the -30 U nucleotide is located within the TURBS motif.

Figure 19: The segment 7 mRNA detailing the region upstream of the BM2 open reading frame that shows complementary to the 18S rRNA subunit. The white block represents the nucleotides encoding the M1 protein, the purple block represent nucleotides encoding the BM2 protein. In the Matrix protein open reading frame, the only two nucleotides that can be altered without altering the coding sequence, are highlighted in red. The encoded amino acids of the matrix protein are in blue. Neither the methionine (M) codon nor the first two nucleotides of the glycine (G) codon or the first nucleotide of the asparagine codon (N) can be exchanged without altering the protein sequence of M1.

Reverse genetics can be used to create viruses altered in the nucleotide sequence of specific segments. Using site directed mutagenesis, point mutations were made in the segment 7 pPolRT plasmids at nucleotides -36 and -30 relative to the BM2 AUG. Nucleotides naturally present at these positions were changed individually to each other nucleotide to create 6 mutants.

2.2.2 Analysis of BM2expression from influenza B virus segment 7 mRNA mutants with altered complementarity to the 18S Ribosome.

The expression of BM2 from the 6 mutated viral-like mRNAs that contained these changes was then tested by transfecting the Pol 1 plasmid into cells that were co-transfected with plasmids that direct expression of PB1, PB2 PA and NP. This resulted in generation of viral like RNAs in situ that would be transcribed to mRNAs by the assembled viral polymerase complex. The level of BM2 protein expression was assessed at 24 hours post transfection (h.p.t.) by Western blot using a polyclonal antibody for BM2 and by assaying for an abundant cell protein, vinculin as a loading control.

BM2 expression was detected following transfection of each mutated plasmid but to different degrees depending on the extent to which the mutation disrupted the complementarity between the viral TURBS motif and the 18S rRNA (Figure 20).

The amount of BM2 translated from the mutant -36 U→C was actually greater than for wild- type segment 7. This is unsurprising as this mutation increases the complementarity between the two sequences; by allowing a G→C base pair in place of a G→U the recruitment of the 18S rRNA to the BM2 sequence is presumably increased. Interestingly, this U to C change is the predominant sequence variation in this region that naturally exists as documented in the NCBI Influenza virus resource database (7). Indeed this variation occurs in ~16% of the influenza B virus strains whose segment 7 RNA sequence has been deposited. Other than B/Lee/40, all early influenza B viruses have U at this nucleotide. The next occurrences of the C were in a proportion of influenza B viruses isolated between 1972 and 1995; then there is a 10 year gap where only strains with U at this position are present until 2006 when the C genotype reappeared. Of the 10 circulating strains in the UK 1972-2002, seven had genotypes available in public databases, only the virus strain (B/HongKong/5/72) which is evident from 1972 -1976 possesses the C genotype (138).

Interestingly, globally there were also 2 strains that had an A genotype at this position both isolated in Nanchang in 1994 or 1996, suggesting a close relationship. Figure 21 shows that the U to A mutation reduces but does not prevent the expression of BM2 protein.

Conversely, mutation at the -30 nucleotide position had a detrimental effect on BM2 expression levels. The -30 A→C mutation resulted in barely detectable levels of BM2 production when compared to wild-type, and the -30 A→U mutation also decreased translation. This indicates that disruption of the U-A base pair proposed between the rRNA and the viral TURBS is deleterious to TURBS function. The -30 A→G that would at least allow some U-G base pairing between the rRNA and viral mRNA was still deleterious at this position compared to wild-type, though supported slightly higher levels of BM2 expression than the other two mutations (Figures 20 and 21). There is no recorded natural variation at this nucleotide in the sequence databases. As these nucleotide changes tested here differ from those used by Powell at al., in the in vitro system (due to limitations imposed by the M1 coding sequence), it is difficult to correlate our two sets of results. Powell et al., had found that changes made at the -31 and -32 positions reduced expression of the luciferase reporter to minimal levels, but at position -35 there was tolerance for one amino acid change (A→G) but not (A→C) suggesting there may be some variability tolerated at the 5’ end of the TURBS.

Taken together these data support the theory that the complementarity of the segment 7 mRNA putative TURBS sequence and the 18S ribosomal sequence enhances protein expression from the downstream AUG codon in the context of an influenza B viral mRNA.

Figure 20: BM2 protein expression from viral like mRNAs generated in situ. 293T cells were transfected with 4 expression plasmids encoding the polymerases and nucleoprotein and the pPolRT segment 7 plasmids. The cells were harvested 24 hours post transfection and proteins separated on SDS PAGE and transferred to nitrocellulose for Western blot. A) Probed against vinculin and BM2 (No Iodoacetamide-(IDOC)) therefore single bands. B) Independent transfection probed against BM2 (RIPA buffer contains Iodoacetamide, therefore double bands). These results are representative of four separate experiments.

1.25

1.00 p= <0.001 0.75

0.50

0.25 Relative Relative Intensity

0.00

wt -36UC -36UA -36UG -30AC -30AU -30AG Mock Plasmid

Figure 21: Quantitation of BM2 expression from viral like mRNAs generated from plasmids modified in the TURBS region of segment 7. The results of Western blots illustrated in Figure 20 were analysed and the level of expression from each mutated segment 7 mRNA were compared with those from wild-type. The data displayed represent 3 independent transfections. Relative Intensities were measured using ImageJ software (as outlined at http://www.lukemiller.org/journal/2007/08/quantifying-western-blots-without.html). The data were analysed by paired Students t test and the reduction in BM2 expression compared to wild-type expression was found to be significant p<0.001 for mutants -36UG, -30AC, -30AU and -30AG.

2.2.3 Analysis of BM2 expression by recombinant influenza B virus mutants with altered complementarity to the 18S Ribosome in the segment 7 mRNA in the virally infected cell.

We next tested whether changing the sequence in this region of viral RNA would make a difference to BM2 translation in the context of virus infection.

The altered plasmids were each incorporated into the 12 plasmid Influenza B reverse genetics system in place of the wild-type control. In cases where mutated plasmids did not give rise to infectious recombinant viruses, two further attempts were made with independently prepared plasmids. In addition, an additional BM2 expression plasmid was supplemented in trans during the transfection in order to help the virus overcome any deficiency in BM2 during the first round of rescue.

All of the plasmids, except the one containing the -30 A→G point mutation, were able to successfully support virus generation in the reverse genetics system. The inability to rescue virus with the -30 A→G mutation is not surprising in light of the results in Figure 20 that show that in the context of a full-length influenza B virus mRNA, the -30 A→G mutation decreased

BM2 expression to less than one quarter of wild-type levels. However, the -30 A→C mutation was rescued even though this mutation decreased BM2 expression even further. Moreover the A→G mutation gives rise to the possibility of a U-G base pair between the rRNA and the viral TURBS but the A →C mutation destroys any possible base pairing interaction with the 18S rRNA at nucleotide -30. In order to clarify this discrepancy the plasmid transfections were repeated once more using the A→G mutated segment 7 plasmid and a longer exposure was applied in order to detect any BM2 that might be generated from this mutant. Figure 22 displays that there was indeed a low level of BM2 protein expression from the A→G mutant, indicating that the plasmid was intact. An additional two attempts to rescue the virus with the plasmid below were undertaken but still no viable virus was recovered. The reason why this mutant in particular was not rescued in the reverse genetics system is not clear.

Relative intensities: 1 0.22 0.85

Figure 22: Relative expression of BM2 from -30 A→G, -36 U→G mutant and wild-type plasmids. Transfection of 293T cells with -30AG and -36UG Pol I RT plasmid and the 4 polymerase plasmids alongside an infection of the wild- type virus, harvested with RIPA buffer (No IDOC) and subjected to SDS-Gel electrophoresis and Western blot. The blot was probed with poly-clonal BM2 antibody and relative intensity of mutanst vs wild-type measured using ImageJ software.

The viruses generated by the reverse genetics system were plaque picked and the stocks were amplified and sequenced to confirm the presence of the point mutations and absence of any extraneous mutation within the segment 7 RNAs.

To observe the levels of BM2 protein produced in the context of whole virus for each mutant virus, we performed Western blot on lysates of infected MDCK cells, probing with anti-BM2 and anti-NP antibodies.

In general these data confirmed the results that had been obtained in transfected cells (Figure 23). Again the -36 U→C mutation resulted in high levels of BM2, at least as high as wild-type (see Figure 23 Figure 24), whereas BM2 translation from the -36 U→A remained similar to wild-type and -36 U→G mutant virus had decreased BM2 when compared to wild-type. This is

67 in line with the observation that the C and A are naturally occurring genotypes at this position but the G does not naturally occur. It is surprising that the mutants altered at position -30 were rescued at all bearing in mind that the mutation of the adenosine nucleotide from the - 30 position severely inhibited BM2 production in the context of infectious virus.

Figure 23: Western blot of BM2 and NP expression by recombinant viruses modified in the TURBS region of segment 7. MDCK cells were infected at an MOI of 1 and incubated for 18 hours then harvested with RIPA buffer (No IDOC), subjected to Western blot and probed with anti-NP & anti-BM2 antibody.

1.00

0.75

0.50

0.25 RelativeIntensity

0.00

-36 UG -36 UA -36 UC -30 AU -30 AC Virus

Figure 24: Quantitation of BM2 expression in recombinant viruses modified in the TURBS region of segment 7. MDCK cells were infected with recombinant viruses at an MOI of 1 and incubated for 18 hours. The cells were harvested with RIPA buffer, subjected to Western blot and probed with anti-NP & anti BM2 antibody. Relative intensities were measured using ImageJ software, the levels of BM2 were normalized against NP protein expression. Significant differences are not illustrated as these samples were only run once precluding a statistical analysis.

The three recombinant viruses with the lowest BM2 expression were subjected to Western blot then probed with a matrix protein antibody. The low expression of the BM2 protein in the viruses with the -36 U→G, -30A→C and -30 A→U modifications were consistent in this independent infection even when normalised to viral M1 expression levels (Figure 25 and 26).

This indicates that the low BM2 expression is not due to a low level of replication or expression of segment 7 RNA in general but is specific for the BM2 open reading frame.

Figure 25: Western blot of viruses modified in the putative TURBS of segment 7 Influenza B. MDCK cells were infected with recombinant viruses at an MOI of 1 and incubated for 18 hours. The cells were harvested with RIPA buffer (with IDOC), subjected to Western blot and probed with anti-M1, anti-BM2 and anti-vinculin antibodies.

1.00

0.75

0.50

0.25 Relative Relative Intensity

0.00 Wt -36 UG -30 AC -30 AU Virus

Figure 26: Quantitation of BM2 expression in recombinant viruses modified in the TURBS region of segment 7 relative to expression of the M1 protein. The Western blot illustrated in Figure 25 was analysed for relative protein expression. Relative intensities were measured using ImageJ software. The levels of BM2 were normalized against M1 protein expression. Significant differences are not illustrated as these samples were only run once precluding a statistical analysis.

2.2.4 Phenotype of recombinant viruses with altered BM2 expression.

Previous research has demonstrated that viruses with decreased levels of BM2 show reduced fitness and an attenuated multi-cycle replication phenotype in cell culture (160).

To test the effects of decreased BM2 resulting from disruption of the TURBS, a multi-cycle growth curve was performed on MDCK cells infected at an MOI of 0.01. The replication of the - 30 A→C mutant virus was severely attenuated, showing a 2 log decrease in titre at early time points. Surprisingly the other mutant in which BM2 expression was barely detectable, the -30 A→U virus did not show an obvious decrease in fitness in this system, even though the virus required two attempts in the reverse genetics system before it could be generated. None of the viruses sequenced displayed any alteration in Motif 1 or the M1 coding sequence (up to 170 nt from the initiation of the matrix protein).

8 Wt 7

) -36 UA

10 6 -36 UC 5 -36 UG 4 -30 AU

3 -30 AC Virusyield

2 PFU/ml (Log 1 0 0 12 24 36 48 60 72 Time in hours

Figure 27: Multi-cycle growth of recombinant influenza B viruses modified in the TURBS region of segment 7. MDCK cells were infected with the recombinant and wild-type viruses at an MOI of 0.01. The viral titres released into supernatants at 12 h.p.i. time points were assessed by titration in MDCK cells by plaque assay. The data points represent triplicate samples.

Figure 28: Plaque assays of the 5 recombinant viruses modified in the TURBS region of segment 7 and the wild- type version of the B/Beijing/1/87 virus. The viruses were plaque picked prior to the growth curve. (well size = 22 mm in diameter.)

Virus Average (mm) Range (mm) Wild-Type 1.80 0.36 – 3.30 -36 U→G 1.80 0.73 – 2.94 -36 U→A 2.16 1.10 – 2.94 -36 U→C 1.69 0.73 – 2.56 -30 A→U 1.39 0.36 – 2.56 -30 A→C 1.21 0.36 – 1.83

Table 5: Plaque size of recombinant viruses modified in the TURBS region of segment 7. Plaques were measured for 2 wells per virus and the average and range were expressed in mm.

Accordingly, the -30A→C virus displayed a reduction in plaque size both assessed by average (1.21 mm) and in range (0.36 – 1.83 mm) compared to wild-type virus (1.80mm) (0.36 – 3.30 mm). Even though all the recombinant viruses were plaque picked prior to performing the growth curve, a mixture of plaque sizes were produced at the 36 hour time point by the - 30A→U virus suggesting that a mixture of viruses may exist with different growth kinetics within this viral population (Figure 28). Interestingly this virus displayed a growth rate greater than wild-type in the growth curve (Figure 27). When sequenced the A to U mutation was maintained in the majority consensus even at the 36 hour time point.

2.3 Discussion

These experiments were designed to test the importance of a proposed TURBS sequence for promoting BM2 translation in the context of infectious virus. The data show that altering the degree of complementarity between the influenza B virus segment 7 RNA putative TURBS motif and the 18S ribosomal subunit helix 26 tip region does indeed affect BM2 expression and when this results in lower BM2 levels in infected cells, this can decrease the fitness of the virus.

These data combined with probing the sequence database of natural virus isolates indicate that the -36 (U) position of the TURBS tolerates variation to a greater degree than the -30 (A) position. Observing the M-fold predictions from Powell et al., 2008 it is possible to see how mutation of -36 U to an alternative base may alter the structure in the stem 2 region of the M- fold 1 predicted sequence. The loss of the -36 U to A pairing that initiates the stem 2 base pairing could prevent the creation of the stem and this in turn could make the pentanucleotide motif more accessible. Independently the TURBS interaction itself could also act to reduce the base pairing of the mRNA sequence again allowing access to the pentanucleotide motif, the possession of a C at this locale would increase the attraction to the 18S rRNA G nucleotide and

72 decrease that of BM2 mRNA A sequence. In regard to the -30 modifications, the A to U alteration would increase length of the stem 2 in the BM2 secondary structure. It is less clear how the A to G or A to C alteration at the -30 position could affect the RNA structure depicted by this two dimensional model (Figure 29).

Figure 29: M-fold 1: Enlarged image of the structure predictions from Powell et al., the pentanucleotide motif is in blue. The complementary motif 2 sequence is pink and the bases altered in these experiments are highlighted in bold. With the addition of the 18S rRNA sequence (Green) the fold structure could be altered. The change of a U to a C at -36 could increase the conformational change.

In the M-fold 2 predicted structure (Figure 30), the TURBS region is exposed, potentially allowing easier access to the tip of the 18S rRNA sequence. Again as this is a 2-D image the potential 3-D interactions cannot be assessed. It may be that by the interactions with the 18S rRNA sequence the ribosome is pulled into closer proximity with the pentanucleotide motif. As with M-fold 1 there is a potential loss of base pairing within the internal mRNA structure,

73 caused by the interaction with the 18S rRNA complementary sequence, but only by 2 base pairs, rather than the potential loss of 6 base pairs in M-fold 1 (indicated in the boxed region in both figures).

The -36 U→C mutation is displayed as its presence increases the affinity for the 18S rRNA sequence and decreases the internal bases pairing in the mRNA secondary structure. Again it is less clear how the -30 modifications disrupt the binding to the 18S rRNA sequence. It is possible that the presence of the addition G nucleotide (-30 A→G) in the mRNA disrupts the correct positioning of the 18S rRNA sequence, shifting all the bases along by one. The three C nucleotides of the 18S rRNA sequence could still bind to 3 G nucleotides and the following UUA sequence could also base pair to the virus mRNA secondary structure. But this shift albeit by only one nucleotide could be sufficient to disrupt TURBS function.

Figure 30: M-fold 2: Enlarged image of the structure predictions from Powell et al., the pentanucleotide motif is in blue. The complementary motif 2 sequence is pink and the bases altered in these experiments are highlighted in bold. The possible interactions of the 18S rRNA ribosomal subunit (Green) with the second mRNA M-fold 2 structure. The -36 U nucleotide is altered to C to display the increased complementarity to the 18S rRNA G nucleotide.

Hatta et al., 2009 confirmed that it is the nucleotide sequence and not the M1 protein that has an effect on BM2 expression. They introduced an expression plasmid of the M1 protein that had no effect on the expression levels of the BM2 reporter (117).They also used sequence that contained the TURBS and the sequence leading up to the pentanucleotide motif in an expression plasmid to drive the production of the mRNA of several foreign genes in the absence of the influenza B polymerase machinery. This demonstrates the potential for this sequence to be used in industrial applications for the enhancing the co-ordinated production of a number of contiguous proteins.

Alternatively, protein sequence permitted, base changes could be made elsewhere in the RNA sequence to affect the opposite side of the base pairing within the secondary structure of the two M-fold predictions to provide genetic evidence for the predicted secondary RNA structures.

Although the minimal RNA sequence to promote BM2 expression extended only to the first proposed TURBS motif in the influenza B segments 7 mRNA, it is noteworthy that the other characterized viruses that utilize this strategy for gene expression have two TURBS regions that both show 18S rRNA complementarity (Figure 16). In their publication Powell et al., report a second possible TURBS sequence at nucleotide 574-579 of the influenza B virus segment 7 mRNA, but they do not test the function of these sequences in promoting BM2 translation.

Figure 31: Schematic illustrating the identical TURBS motif regions in Influenza B and an alternative AUGGGA Motif 1(alt), further upstream in the M1 coding sequence (256-261). Below, two sections of sequence that closely resemble the TURBS motifs, at a distance from the Motif 2 region is more comparable to that in FCV and RHV, yet both have one corrupting base.

There is an additional sequence with 18S rRNA complementarity -AUGGGA- at positions 256- 261 (Figure 31). Hatta et al., did note a 5 fold reduction in BM2 expression when the full length

M1 coding sequence was truncated to just the terminal 186 nucleotides (117) but they did not investigate the role of M1 sequence upstream of the 186 region for TURBS. The distance between motif 1 & 2 in FCV and RHDV is not as long as those in influenza B, indeed there are two sequences or great similarity to the TURBS motif at a comparable distance, yet both differ by one base AUGGGG or AUGGAA, as the terminal A has been highlighted as crucial in this body of research, the addition G may invalidates its abilities as a TURBS, equally the three G’s could be key in locking in the 18S binding.

In future work the importance of these additional regions of 18SrRNA complementarity could be tested by extending the approach employed here. Mutations could be made in the identical motif regions found further upstream from the one implicated in this interaction, at position 256-261 or Motif 1 574-579 as mentioned in the Powell et al., 2008.

On the other hand it may be that influenza B virus mRNA only requires a single TURBS motif and that the sequences noted above are either historical artefacts or are required for the M1 amino acid coding and their TURBS similarity is merely a co-incidence. Or that the initial TURBS sequence at 256-261 is of prime importance and the distance to the Motif 2 is irrelevant due to secondary structures generated in the viral mRNA which brings the two motifs and the translation machinery into close proximity.

Chapter 3. The reverse genetic manipulation of the BM2 cytoplasmic tail

3.1 Introduction: Influenza B virus BM2 protein

Reverse genetics can be used to manipulate the influenza B virus to create mutants that may be altered in some aspect of the virus’ replication cycle. By studying the phenotype of the recombinant virus, the role of the mutated gene may be elucidated.

Previous studies in the laboratory generated mutant recombinant influenza B viruses that were deficient in the amount of BM2 expressed, but a virus lacking BM2 expression altogether could not be rescued (157). This work demonstrated the BM2 protein is essential for the infectivity of the virus. Indeed, the electrophysiology studies of Lamb and Pinto showed that, like the AM2 protein, BM2 is required for the uncoating of the newly endocytosed virion. Because its transmembrane domain can act as an ion channel and acidify the viral core, BM2 mediates the release of the genomic material which is then transported to the nucleus to initiate the production of new virus particles (236, 279).

Interestingly, recent research into the cytoplasmic tail of the AM2 protein suggested that this protein domain plays an additional role during the final stages of virus assembly. Two independent groups have presented data to indicate that without the cytoplasmic tail of M2, influenza A virus did not assemble correctly and was severely attenuated (156, 223).

When the work presented in this thesis was initiated there was no evidence to suggest that the BM2 cytoplasmic tail would behave in the same manner. The AM2 and BM2 proteins share a functional homology in terms of their ion channel capacity, However, other than the ion channel motif there is no amino acid conservation (Figure 33).

Figure 32: Schematic of the Influenza B segment 7, highlighting the pentanucleotide stop:start motif (TAATG) that terminates the M1 protein and initiates BM2 protein production. Below, a representation of the endo, transmembrane and cytoplasmic tail domains of the BM2 protein as a tetramer.

Figure 33: Representative coding sequence of the BM2 (B/Beijing/76/98-AAU01002.1) and M2 (A/Nanchang/58/93 (H3N2)-ABB79970.1) proteins from the influenza A & B viruses. The HXXXW ion channel motif and comparative lengths of the endo, transmembrane and cytoplasmic domains are highlighted.

3.2 Results:

3.2.1 The effect of deletion of amino acids from the terminus of its BM2cytoplasmic tail.

Since it was established that the cytoplasmic tail region of the M2 protein of influenza A virus is important in viral assembly, we hypothesized that that the cytoplasmic tail of BM2 could serve a similar function.

In order to evaluate if terminal regions of the BM2 cytoplasmic tail were necessary for viral replication, a series of viruses with truncated BM2 proteins were created using the B/Beijing/1/87 reverse genetics system (158). The appropriate plasmid containing cDNA for segment 7 that encodes the BM1 and BM2 genes, pPol 1 RT M, was mutated by site directed mutagenesis with the primers listed in table 19. These primers introduced premature stop codons into the coding sequence of the BM2 protein, without affecting the coding sequence of the BM1 matrix protein. Recombinant viruses were rescued using the routine procedure of transfection of 293T cells followed by co culture with permissive MDCK cells. Generally cytopathic effect (CPE) in MDCK cells was observed after several days and indicated rescue of recombinant virus. The cell supernatants containing virus were harvested and plaqued on MDCK cells. Plaque picks were taken and virus grown up from an individual plaque and titred again by plaque assay.

Initially the only virus generated using this method was one lacking a single amino acid from the BM2 cytoplasmic tail, BM2-1. The procedure was modified to incorporate an additional plasmid at the time of transfection that directed full-length BM2 expression to supplement BM2 levels in trans. Using this enhanced methodology, a virus lacking 5 amino acids from the BM2 C terminus, BM2-5, was also generated (Table 6). It can be assumed that the presence of the full-length BM2 protein in the 293T cells during the first round of infection allows the assembly of virions under what would otherwise be limiting conditions. Then, because MDCK cells are more permissive, virus that encodes a truncated BM2 protein and is mildly attenuated is amplified. Nonetheless, despite this modification to the rescue procedure, viruses lacking more than 5 amino acids from the BM2 C terminus were not rescued (Table 6). This indicates that some or all of the amino acids between positions -6 and -10 play an essential role in the virus replication cycle, or that the length of the BM2 cytoplasmic tail is required to be at least 105 amino acids.

Table 6 : Table listing recombinant viruses truncated in the cytoplasmic tail of the BM2 protein successfully generated using the 12 plasmid B/Beijing/1/87 reverse genetics system. Full-length BM2 protein was supplemented in trans by cotransfection of an expression plasmid. Premature stop codons were introduced in the segment 7 pPol 1 RT plasmid to progressively remove the terminal amino acids from the cytoplasmic tail. The presence of viable virus (+) was confirmed by haemagglutination and plaque assay.

The RNA extraction and sequencing of cDNA confirmed that the segment 7 RNA sequence of the recombinant virus BM2-5 was identical to that of the plasmids used for rescue and that the premature stop codon was maintained as can be seen in Figure 34.

Figure 34: Sequence confirmation of BM2-5 Virus. A. cDNA derived from vRNA extracted from the recombinant viruses generated using the influenza B reverse genetics system within this chapter. Each mutation will be discussed in detail later in the text. B. Chromatogram of the BM2-5 virus with a premature stop codon replacing the 5th amino acid from the end of the cytoplasmic tail of BM2, position 105 and the wild-type virus. The red boxes indicate the stop codons. The premature stop codon is maintained in the BM2-5 virus.

In order to demonstrate that the pPol 1 RT plasmids that did not give rise to rescued virus were intact, and that the truncated BM2 proteins they encode were expressed, these plasmids were transfected along with 4 other plasmids that direct expression of the influenza B virus polymerase, into 293T cells. The reconstituted viral polymerase should direct amplification and expression of the segment 7 gene products M1 and BM2 from the viral RNA transcribed from the Pol1 RT plasmids in the cells in situ by polymerase I.

Relative

intensity: 1 0.9 0.7 1

Figure 35: Western blot of expression in BM2 truncation mutants in 293T cells transfected with 500 ng of the plasmids. The cells were incubated for 24 hours prior to lysis with RIPA buffer (with IDOC). Electrophoresed cell lysates were transferred to PVDF (Polyvinylidene Fluoride) membranes and probed for the BM2 protein. All three plasmids were able to generate the BM2 protein despite being unable to generate recombinant virus. Differences in sizes between the truncated mutants were not resolved on this blot. Relative intensities of each mutant as compared to wild-type were determined using ImageJ software.

BM2 protein expression was observed following transfection of any of the pPol 1 RT constructs (figure 35).

Relative expression of the BM2-6 and BM2-7 was less than the Wt plasmid, but the drop was unlikely to have been sufficient to abrogate virus production.

The efficient rescue of the BM2-1 virus indicated that it was not affected in replication by the single amino acid truncation and indeed in early studies no defects were observed for this virus (data not shown), so work was focussed on the other recombinant BM2-5. As the BM2-5 virus was not generated using reverse genetics unless the full-length BM2 was expressed in the transfected cells in trans, it was possible that the recovered virus with the truncated protein would be attenuated. A multi-cycle growth curve was conducted on MDCK cells infected at an MOI of 0.01. BM2-5 virus replication was attenuated by more than 10 fold. According to a two- way Anova there was a significant difference between the growth kinetics of the two viruses p=<0.001.

9 Wt 8 BM2-5

) 7 10 6 5 4

PFU/ml (log 3 2 1 0 10 20 30 40 50 60 70 80 Time in Hours

Figure 36: Multi-step growth curve illustrating the attenuation of the recombinant BM2-5 virus. A multi-cycle growth curve were performed by infecting MDCK cells at an MOI of 0.01. Data points represent triplicate samples. The released virus was titred by plaque assay in MDCK cells. A two-way Anova was used to assess the difference between the two viruses across different time-points.

256 Wt 128

BM2-5 )

2 64 32 16 8

HA titre (Log 4 2 1 0 10 20 30 40 50 60 70 80 Time in Hours

Figure 37: Multi-step growth curve illustrating the attenuation of the recombinant BM2-5 virus when compared to the Wt virus. MDCK cells were infected at an MOI of 0.01, the time points were titred by HA assay. The data points represent triplicate samples. According to two-way Anova, there was no statistically significant difference between the titre of the two viruses.

Research on the Influenza A virus indicated the lack of a cytoplasmic tail on M2 protein can prevent the inclusion of the viral genome in new budding particles, generating vesicles composed of the matrix protein and viral glycoproteins, but incapable of replication (156, 223, 224). In the HA assay these empty vesicles would still give a positive result because the assay

83 relies of the ability of the haemagglutinin protein present on assembled virions to bind to the sialic acid receptors on the surface of red blood cells. Statistically there is no difference between the viruses by HA assay. By comparing the statistical data in Figure 36 Figure 37 it can be suggested that amino acids from the end of the BM2 cytoplasmic tail affect the infectivity of the virus particles released from infected cells, rather than the number of particles. However, visually, there does also appear to be a difference in the number of particles produced.

As well as producing virus particles which lack the viral genomes, influenza A viruses that have AM2 proteins with defective cytoplasmic tails have a heterogeneous morphology (156, 223, 224). To discover if the influenza B viruses with the truncated BM2 protein also display this heterogeneous morphology, supernatant from an infection in MDCK cells/viral reverse genetics rescue was passed through a 30% sucrose cushion to concentrate the virus and remove cell debris. The virions were then visualized by negative staining in the electron microscope (Figure 38 and 39).

Figure 38: Electron micrograph of the wild-type B/Beijing/1/87. The images here are of increased magnification of the same image, but are representative of multiple fields of vision. The viruses were concentrated through a 30% sucrose cushion prior to processing for visualisation by transmission electron microscopy. The virions were relatively uniform in shape and predominantly of a similar size ~117 x 123nm. There was one outlier not included in average ~ 60x60nm. They had the characteristic fuzzy outer ring of surface glycoproteins.

Figure 39: Morphology of B/Beijing/1/87 virions with truncated BM2-5. Viruses were concentrated through a 30% sucrose cushion prior to processing for visualisation by transmission electron microscopy. The virions were of dissimilar sizes large (green) and small (blue) and there were vesicle like amorphous structures (red arrow). The two putative viruses in the bottom Left Hand image ~89 x 93nm are a inset from the image above. The distinctive fuzzy outer ring of surface glycoproteins is less apparent. The viruses (purple arrows) in the bottom Right Hand image have the more typical outer ring and size (102x117 and 109x125 nm) The BM2-5 virus TEM pictures are typical of two separate rescue and TEM experiments.

The wild-type influenza B virus is relatively uniform in its spherical appearance and size, with the characteristic outer ring of surface glycoproteins clearly visible. This contrasts with the amorphous shapes displayed by virus with the BM2-5 truncated cytoplasmic tail where there is a wide variety of shapes and sizes. The outer ring of surface glycoproteins is not as apparent in some of the putative mutant virus virions as in the wild-type virus. The amorphous shapes were not found in the wild-type samples process nor in a sample of cell supernatant from mock-infected cells, but they were observed in viruses discussed later in this chapter that also have defects in the cytoplasmic tail.

Attenuated viruses often generate smaller plaques when titrated in a standard agar plaque assay. The BM2-5 virus generated smaller plaque sizes (~1.04 nm) that the wild-type (~1.61 nm) Figure 40.

Figure 40: Plaque assay of truncated BM2-5 and BM2 wild-type viruses. These plaques were generated during a growth curve to assess viral kinetics. The supernatant was serially diluted and used to inoculate confluent monolayers of MDCK cells. After an one hour incubation, the inoculum was removed an replaced with a media and agar overlay and the cultures were inverted and incubated for 5 days. The agar plugs are then removed and the cells stained with crystal violet, prior to image capture with a scanner. The predominantly smaller plaque sizes are evident in the BM2-5 wells. The Wt virus displays heterogeneity in plaque size, but the plaques are predominantly larger.

Virus Average (mm) Range (mm) Wild-type 1.61 0.24-2.93 BM2-5 1.04 0.24-1.7

Table 7: Comparative plaque sizes of wild-type and BM2-5 recombinant virus. Average and range of plaque size diameter of viruses on a 22 mm diameter well. From plaque assay in Figure 40.

To confirm that the BM2 protein expressed from BM2-5 virus was indeed smaller in size than the wild-type virus, infected cell lysates were separated by SDS page and the viral proteins analysed by Western blot for BM1 and BM2. In cells infected with BM2-5 virus, the truncated BM2 protein was readily visualised Figure 41.

Relative Intensities: 1 1.1

Figure 41: Western blot of wild-type and truncated BM2-5 virus. MDCK cells were infected at an equal MOI, with wild-type and truncated BM2 virus, incubated for 24 hours, and lysed with RIPA buffer (with IDOC). The proteins were separated on SDS PAGE and transferred to PVDF. The blot was probed with antibodies to the Matrix protein M1 and to the BM2 protein. The virus lysate containing the truncated BM2 protein has a smaller molecular weight than the full-length form. The relative intensities were calculated using Image J software and the BM2 signal was normalized to the M1 protein.

3.2.2 Alanine scanning mutagenesis in the last 10 residues of the BM2 cytoplasmic tail reveals a critical role for position- 6(103) and - 7(104).

As the BM2 protein could not be truncated by more than 6 amino acids before virus viability was lost, either the residues around position -6 (103) or the total length of the BM2 cytoplasmic tail must be critical. In order to distinguish between these two possibilities, a series of pPol1 M plasmids were created in order to attempt the rescue of recombinant viruses with single point alanine substitutions at positions from -6 to -10 of the BM2 cytoplasmic tail. When the BM2 protein was supplemented in trans during the rescue procedure, recombinant viruses with substitutions at positions -8, -9 and -10 were recovered whereas in 3 rescue attempts it was not possible to generate viruses with substitutions at positions -6 and -7 (Table 8).

Table 8: Recombinant viruses with alanine substitutions (positions 100-104) in the cytoplasmic tail of the BM2 protein successfully generated using the 12 plasmid B/Beijing/1/87 reverse genetics system. BM2 supplemented in trans. Viable (+) viruses were assessed by plaque and haemagglutination assay.

As before the integrity of the BM2 proteins encoded by the plasmids that did not give rise to rescued virus was checked by co-transfection with the influenza B virus polymerase helper plasmids and analysis of the expressed BM2 proteins. The -6A and -7A BM2 proteins were readily detected in the transfected 293T cells (Figure 42).

Relative Intensity: 0.53 0.8 1

Figure 42: Western blot of BM2-6A and -7A expression. 293T cells were transfected with the pPol 1 RT plasmids with the -6A and -7A amino acid substitutions and the 4 expression plasmids that reconstitute the viral polymerase and the nucleoprotein, driving the expression of the BM2 proteins. After 24 hour incubation the cells were lysed with RIPA buffer (with IDOC) and subjected to SDS-PAGE gel electrophoresis and the proteins transferred to PVDF prior to being probed with a rabbit polyclonal anti-BM2 antibody. Relative intensities determined using ImageJ software.

The BM2-6A plasmid does express the BM2 protein at a lower level despite it being transfected at the same concentration as the BM2-7A and the wild-type virus. This may have affected the ability of the BM2-6A plasmid to generate virus. Multiple independent preparations of plasmids with the BM2-6A genotype were used within the reverse genetics system in an attempt to rescue this virus; none were successful so this was not a result of a single plasmid anomaly. The alanine substitution work was conducted with the help of a project student Guo Zhang.

3.2.3 The influenza B virus tolerates non-conservative changes in the BM2 protein cytoplasmic tail at position -6(103) or -7(104).

A sequence alignment of the 294 influenza B virus segment 7 genes sequences available in the NCBI Influenza Resource virus database illustrates a high level of conservation at certain amino acid residues in the BM2 cytoplasmic tail.

Figure 43: The sequence variation in the BM2 protein amongst the 294 sequences for segment 7 RNA in the NCBI Influenza database (7). The nonessential terminal region is highlighted by the purple box, the region that has been altered in amino acid sequence is highlighted in green, the possible phosphorylation sites are highlighted with red boxes.

All 294 sequences listed had a leucine (L) at position -7 (103) and all but 2 viruses had the - 6(104) position conserved as glutamic acid (E). Thus it may be that the virus absolutely requires leucine (L) at position -7 (103), but can in certain context tolerate other residues such as Asparagine (N) or Lysine (K) at position -6 (104). Both the N and K are polar hydrophilic amino acids, whereas alanine (A) used in the scanning mutagenesis above is a non-polar mildly hydrophobic amino acid. In order to test whether any other changes might be tolerated at the -6 position, pPol 1 RT M plasmids were mutated for rescue of viruses with either of two alternate substitutions at the -6 position, Q or Y. These were chosen because Glutamate (Q) is

89 a polar neutral amino acid with a hydrophobicity in the same range as the glutamic acid and therefore thought the most likely substitution to be tolerated, and Tyrosine (Y) is also a polar neutral amino acid with a higher hydrophobicity, but not as high as that of the alanine. The mutated pPol 1 RT M plasmids were capable of generating the BM2 protein in a 5 plasmid transfection (Figure 44).

Relative Intensity: 1 0.80 0.99

Figure 44: Western blot to illustrate expression of BM2 mutant proteins with 6Q and 6Y substitutions. 293T cells were transfected with the pPol 1 RT plasmids encoding proteins with the -6Q and -6Y amino acid substitutions and the 4 expression plasmids that reconstitute the viral polymerase and the nucleoprotein, driving the expression of the BM2 proteins. After 24 hour incubation the cells were lysed with RIPA buffer (no IDOC) and subjected to SDS- PAGE gel electrophoresis and the proteins transferred to PVDF prior to being probed with a rabbit polyclonal anti- BM2 antibody. Relative intensity was calculated using ImageJ software comparing each mutant protein to the wild- type expression level.

Interestingly, the reverse genetics co-culture initially gave a negative result in haemagglutinin and plaque assay indicating that a virus containing the E104Y (6Y) substitution at position -6 was not rescued. However, the supernatant from the co-cultured cells was blindly passaged onto new MDCK cells in the presence of additional trypsin and after several days incubation sufficient virus was generated to give a positive plaque assay result. Whilst the E104Q (6Q) virus could also be generated in this way, its level of attenuation was greater than that of the E104Y (6Y) virus. Indeed, although CPE was observed in MDCK cells, it proved impossible to amplify stocks of this virus to a titre sufficient to set up a growth curve, despite being treated in the same manner as the E104Y(6Y) virus.

To test whether any other conservative amino acid substitutions would be tolerated at the - 7(103) position, plasmids were generated to attempt the rescue of a recombinant virus in which the BM2 protein contained the L103V substitution. In this case valine was chosen as a non polar neutral amino acid with a high hydrophilic value, in other words very similar in

90 nature to the normal leucine. Indeed a recombinant virus containing this amino acid substitution in BM2 was readily generated.

Table 9: Recombinant viruses truncated in the cytoplasmic tail of the BM2 protein generated using the 12 plasmid B/Beijing/1/87 reverse genetics system. BM2 wild-type protein was supplemented in trans. These viruses were generated by alternative amino acid substitutions at the -6(104) and -7(103) positions in the BM2 protein in the pPol 1 RT segment 7 plasmid. The -/+ symbol for the -6Q virus is an indication of the evidence of CPE but the inability to generate viral stocks to measureable levels.

The mutations introduced into the pPol 1 RT plasmid were maintained in the viable virus as can be seen in the sequence data generated from the extracted vRNA (Figure 45).

Figure 45: Sequence confirmation of BM2 -8A, -7V and -6Y viruses. vRNA was extracted from the recombinant viruses generated by reverse genetics, cDNA was generated and sequenced. The alanine substitution at position -8 (102) encoded by GTT to GCT switch, the valine substitution at position -7 (103) encoded by TTG to GTC switch and the tyrosine substitution at position -6 (104) encoded by GAG to TAC switch. The mutations have been successfully maintained as can be seen in the green boxes GCT=Alanine, GTC=Valine & TAC=Tyrosine. There is no trace for the BM2-6Q virus as amplified stocks were not generated.

The viruses altered at position -7V(103) in the BM2 protein were not attenuated and grew as well as or better than the viruses with the BM2 8A or wild-type BM2 protein. In a two-way Anova the growth of the 7V (103) virus was fitter than the wild-type in a statistically significant manner. Indicating that the -7/103 position could play an important role in virion assembly.

9 Wt 8 BM2-7V ) 7 10 BM2-8A 6 5 4

PFU/ml(Log 3 2 1 0 10 20 30 40 50 60 70 80 Time in Hours

Figure 46: Multi-step growth curve of recombinant viruses containing the valine substitution at position -7 (103) (BM2-7V) and the alanine substitution at position -8 (102) (BM2-8A). MDCK cells were infected at an MOI of 0.01. The titre at multiple time points was assessed by plaque assay on MDCK cells. The data points represent triplicate samples. p=<0.05 for BM2-7V vs wild-type.

256 Wt 128

BM2-7V )

2 64 BM2-8A 32 16 8

HA HA titre (Log 4 2 1 0 10 20 30 40 50 60 70 80 Time in Hours

Figure 47: Multi-step growth curve of recombinant viruses containing the valine substitution at position -7 (103) (BM2-7V) and the alanine substitution at position -8 (102) (BM2-8A). MDCK cells were infected at an MOI of 0.01. The titre at each time point was assessed by haemagglutination assay. (Partner data to Figure 46). The differences are not statistically significant. The data points represent triplicate samples.

The difference between numbers of released virions in these growth curves was not significant when measured by haemagglutination assay (Figure 47).

8 Wt 7 BM2-6Y

6 )

10 5

4 (Log 3

Virus yield PFU/ml 2 1 0 6 12 18 24 Time in hours

Figure 48: Single-step growth curve of wild-type BM2 B/Beijing/1/87(Wt) and the virus with the tyrosine (Y) substitution at position -6 (104) in the BM2 protein. The monolayer of MDCK cells was infected at an MOI of 0.1 and the viruses produced were harvested at 6, 16 and 24 hours. In a Two-way Anova, there was a statistically significant difference, p-value =0.02. The data points represent a duplicate experiment with triplicate samples.

The BM2-6Y virus displayed an attenuated phenotype in a multi-cycle growth curve analysis, but the difference when compared to the wild-type virus was surprisingly slight bearing in mind the difficulties in rescue of the virus. The virus used for this experiment was from a stock that had been passaged 4 times; these include initial virus generation from the 12 plasmid reverse genetics system, growth in a small volume of MDCK cells with high levels of trypsin, plaque assay of these small volumes and plaque picking before growth in a larger stock flask. Considering the markedly better growth in the stock flask it is possible some compensatory adaptation may have occurred elsewhere in the viral genome, but this was not pursued further here.

In order to assess if the BM2-6Y mutation resulted in formation of amorphous virus particles, transmission electron microscopy was utilised. Whilst the BM2-6Y virus was at an undetectable level in the original reverse genetics co-culture, this supernatant was chosen as it would be the least likely to display compensatory mutations. Virus like particles were visible by TEM, but very few particles were resolved. The BM2-6Y virus (top panel of Figure 49) did not display as dark a ring of glycoproteins as the wild-type virus (bottom panel of Figure 49), but it was not possible to say if this was typical of the BM2-6Y morphology due to the low virus count. The

93 other shapes that were visible in the BM2-6Y sample appeared as empty vesicles (data not show).

Figure 49: Transmission electron micrograph images of the B/Beijing/1/87 wild-type and BM2 -6Y viruses. The MDCK grown viruses were passed through a 30% sucrose cushion to remove the majority of the cell debris prior to processing for electron microscopy. The BM2 -6Y virus was from the original rescue stock, the viral titre was very low and only a very small number of viruses were visible. Sizes: wild-type (121x121nm) BM2 -6Y (103x95nm).

3.2.4 Recombinant influenza B viruses with BM2 amino acid substitutions at threonines and serines in the cytoplasmic tail.

It has been suggested in the literature that the BM2 protein of the B/Yakagama/73 virus is phosphorylated (261, 366). This theory had originally been proposed because there were two forms of BM2 protein of different mobility detected in infected cells by Western blot. Indeed, in our own studies using Western blots to detect BM2 in infected cell lysates, a second higher mobility species was evident (see for example Figure 41 where iodoacetamide was incorporated into the RIPA buffer to maintain the protein integrity). Indeed on well resolved SDS –PAGE gels it is possible to resolve six species (monomer, dimer and tetramer forms of the virus in potentially phosphorylated and unphosphorylated states).

Figure 50: Blot taken from the Thesis of David Jackson. The virus has been concentrated through a 30% sucrose cushion to remove cell debris. The Brec is wild-type influenza B virus, S9T, F20L and C11S are virions modified in the transmembrane domain of the BM2 protein. Monomer (12M&17M), Dimer (12M&17D) and Tetramer (12T&17T). 12= 12kDa, 17= 17kDa (157).

However, the amino acid (s) that bore the phopshorylation modification was not identified. The potential residues within the terminal regions of the BM2 cytoplasmic tail that could be substrates for phosphorylation (addition of PO4 groups) by cellular kinases were predicted using the NetPhos software (19). Serine 54, serine 59 and serine 91 gave the highest context prediction scores for phosphorylation. The Serine 59 is not conserved (Figure 43) so was not

95 investigated further. Rather, bearing in mind the previous work described in this chapter concerning the C terminal region of BM2, interest was directed at the serine at position 91. In addition, although threonine at residue 101 was highly conserved it was not likely to be an important phosphorylation site because the context score was comparatively low and work described above in section showed that Thr101 could be replaced by alanine with no adverse effect on virus replication, nor loss the of the banding pattern in the Western blot (Figure 58).

Figure 51: Phosphorylation predictions in BM2 cytoplasmic tail. The BM2 protein was subjected to analysis using the NetPhos2.0 phosphorylation prediction software (19) in order to predict the possible sites of phosphorylation in the terminal portions of the cytoplasmic tail.

To investigate the importance of serine 91 in virus replication, the pPol 1 RT M plasmid was mutated to allow the rescue of a virus in which it was mutated to alanine. An additional plasmid was created that contained both the Ser91A and the Thr101A mutations.

The 5 plasmid transfection of the mutated segment 7 plasmids and the 4 ‘helper’ polymerase and NP expression plasmids generated for the Ser91A a product that was expressed less abundantly than Wt BM2 protein and for which only the lower band was visible. Transfection of the plasmid encoding the double mutant did not generate any BM2 reactive bands at all.

Relative 1 0.17 0

Intensities

Figure 52: BM2 protein produced in 5 plasmid transfection with wild-type, the phosphorylation mutant (Ser91) and a double phosphorylation mutant (Ser91 + Thr101). Lysates of 293T cells in RIPA buffer (with IDOC) were harvested 24 hours after transfection and separated by SDS PAGE. After transfer, blots were probed with anti-BM2 polyclonal rabbit antiserum and developed with anti-rabbit HRP conjugate and ECL reagent. Relative densities were calculated using ImageJ software and are expressed compared to expression of wild-type BM2 protein.

The lack of any BM2 product for the double mutant Ser91A / Thr101A protein may be a result of the loss of phosphorylation affecting protein stability or it may be that altering these two amino acids to alanine affected the protein structure to such a degree that the antigenic epitopes are no longer recognised by the BM2 antibody or the protein is unable to assemble correctly and is thus rapidly degraded.

As there is apparently only a single BM2 antibody reactive band for the Ser91A protein, one might assume that the phosphorylation has been lost by the removal of the serine. However, the level of expression for the mutant was low compared to the wild-type and it cannot be excluded at this stage that the second higher molecular weight species is not present in low amounts (Figure 52).

Using the procedure described above a recombinant virus was rescued that bore the Ser91A mutation. The presence of the mutation was confirmed by sequencing viral RNA and, following plaque purification and amplification of a viral stock, a multi-cycle growth assay was performed. Transfection of the pPol 1 RT M plasmid containing the Ser91A / Thr101A double mutation did not give rise to viable virus, despite attempts with different preparations of plasmids containing the same mutation.

Figure 53: Recombinant viruses with alanine substitutions at putative phosphorylation sites in the BM2 protein successfully generated using the 12 plasmid B/Beijing/1/87 reverse genetics system. BM2 supplemented in trans. The potential phosphorylation sites in the BM2 cytoplasmic tail were mutated in the pPol 1 RT segment 7 plasmid. Viable mutants Ser91A and Thr101A and the non-viable plasmid construct Ser91A & Thr101A

Figure 54: Sequence confirmation of phosphorylation mutant viruses. Sequence chromatographs of viral RNA extracted from the recombinant viruses generated by reverse genetics to introduce alanines to replace the serine at position 91 and threonine at position 101 in the influenza B BM2 cytoplasmic tail. The altered nucleotide sequence is highlighted by a green box, GCT & GCA encode Alanine.

The substitution at the Ser91A position to alanine in the BM2 cytoplasmic tail resulted in attenuation of viral titres by up to 100 fold at 40 hours post-infection, statistical analysis (two- way Anova) indicated this was significant p=<0.01 (Figure 55).

9 Wt 8 Ser91 ) 7 10 Thr101(-9A) 6 5 4

3 PFU/ml(Log 2 1 0 10 20 30 40 50 60 70 80 Time in Hours

Figure 55: Multi-step growth curve of recombinant viruses containing the phosphorylation mutants of the BM2 protein serine to alanine substitution at position 91 (Ser91) and threonine to alanine at position 101. MDCK cells were infected at an MOI of 0.01. The titre at multiple time points was assessed by plaque assay on MDCK cells (Two- way Anova p=<0.01). The data points represent triplicate samples.

512 Wt 256 Ser91

) 128 2 64 Thr101(-9A) 32 16 8

HA HA Titre (Log 4 2 1 0 10 20 30 40 50 60 70 80 Time in Hours

Figure 56: Haemagglutinin Assay of a multi-step growth curve of recombinant viruses containing the phosphorylation mutants of the BM2 protein serine to alanine substitution at position 91 (Ser91) and threonine to alanine at position 101. MDCK cells were infected at an MOI of 0.01. Titre was assessed by haemagglutination assay with red blood cells. Two-way Anova p=>0.05. The data points represent triplicate samples.

The plaque sizes for the Ser91A were heterogeneous: Most of the plaques were small but about 1 in 5 were large. Although the wild-type virus also displayed a heterogeneous plaque morphology, the majority of plaques were large. Similarly The Thr101A mutant showed mainly large plaques with a few small plaques.

Figure 57: Plaque morphology of mutant influenza B viruses altered at residues 91 and 101 in the BM2 cytoplasmic tail. Plaques formed on MDCK cells from viral supernatant taken from the figure 55 growth curve. The Wt and Ser91A display heterogeneous plaque sizes, the Thr101A plaques are more homogenous.

Virus Average (mm) Range (mm) Wild-type 1.68 0.39-3.14 Ser91A 1.44 0.69-2.75 Thr101A 2.01 0.71-3.19

Table 10: Comparative plaque sizes of wild-type, Ser91A and Thr101A recombinant virus. Average and range of plaque sizes of viruses on a 22 mm diameter well. From plaque assay in Figure 57.

A Western blot to detect the BM2 protein expressed following virus infection of in MDCK cells Figure 58 clearly displays the presence of the double band of BM2 protein in the Ser91A and the Thr101A mutant virus cell lysates. This indicates that these substitutions do not affect the production of the BM2 17kDa subspecies that potentially represents the phosphorylated form of the protein.

100

Figure 58: Western blot of cells infected with the wild-type (Wt), Thr101A and Ser91A viruses. The cells were harvested after 24 hours and lysed with RIPA buffer containing iodoacetamide. Probed with antibodies for the loading control Vinculin (Vinc), the nucleoprotein (NP), the matrix protein (M1) and BM2 protein.

The BM2 Ser91A mutation may affect the virion morphology if the BM2 protein is unable to assemble correctly and perform its predicted pinching off and genomic inclusion steps efficiently. TEM of the virions of the BM2 Ser91A virus revealed virus particles with a similar morphology to wild-type, but also particles that were larger or smaller than those present in the wild-type samples. Interestingly there were also amorphous vesicles not visualised in the wild-type virus samples that may be virus particles lacking genomic segments, or lacking the haemagglutinin and neuraminidase surface glycoproteins.

101

Figure 59: Transmission electron micrographs of the wild-type B/Beijing/1/87 virions and virus particles formed by the BM2 Ser91A phosphorylation mutant. The Wt virus displayed a consistently spherical morphology with predominantly dark staining patterns with the external fuzzy ring pattern of the surface glycoproteins and all virions observed were of a similar size. The BM2 Ser91A virus preparation also contained spherical virions, but in addition there were a selection of amorphous vesicles (red arrows) whose sizes displayed a degree of variability.

102

Virus Average* Range (nm) (nm) Wild-type 120x135 116x121 – 127x152 BM2 Ser91A 101x129 79x130 -130x139

Table 11: Average sizes of the wild-type and putative phosphorylation mutant Ser91A as assessed by TEM. Note the amorphous vesicles lacking the typical dark glycoprotein ring were not used in this assessment of virion size *n=5.

3.2.4 Is it possible to block the release of Virus with a small protein fused to Green Fluorescent Protein (EGFP) to act as a dominant negative?

There is evidence that individual protein domains can sometimes exert a dominant negative on the normal function of the protein in the cell (212, 353). When these small peptides are expressed in excess, they may block the function of the full-length protein by interacting with the normal substrate or intracellular component preventing the functionally active protein from fulfilling is function. Examples of such proteins can be found in chapter # which deals with tetherin and the ESCRT pathway.

The work described herein and that of Imai et al., published whilst this project was in progress suggests that the cytoplasmic tail region of the BM2 protein is required for influenza B virus replication. We hypothesized that this was because the cytoplasmic tail domain of BM2 interacts with either a viral or a host cell partner protein and that over expression of a fragment of the cytoplasmic tail region may exert a dominant negative protein and impair viral replication, most likely by blocking the late phase assembly step. Moreover using this BM2 fragment as bait may have enabled the identification of the BM2-interacting partner.

By a series of overlapping amplifications using the polymerase chain reaction, the EGFP protein and an in –frame stretch of nucleotides encoding the terminal 31 amino acids of the BM2 protein (which includes the BM2 Ser91, BM2-6 (103) and BM2-7 (104) amino acids) were incorporated into a pCAGGs expression plasmid.

103

Figure 60: Construction of the eGFP fused peptide from the terminal 31 amino acids of the Cytoplasmic tail of BM2. (A) Primers flanking the eGFP (enhanced EGFP) and BM2 cytoplasmic tail region used to generate amplified sequence of the eGFP and the 31 amino acids of the BM2 protein flanked by overlapping sequence of the other protein at one end and a restriction site at the other. (B) A second amplification to fuse together the two nucleotide sequences, flanked by restriction sites (Not1 & Mlu1). (C) The construct incorporated into the pCAGGs expression plasmid using the Not1 and MluI restriction sites. The amino acid sequence from the BM2 protein from positions 79 to 109 are highlighted in the white text.

This fusion protein was designed to include both the region believed to be required for the effective viral budding and inclusion of the viral genome (150).The EGFP-BM2 tail fusion protein was clearly expressed in transfected cells (Figure 61) but its over expression did not affect the release of virus to any greater degree than the plasmids expressing the cherry red fluorescent protein or untransfected cells(-ZAN) (Figure 62). Inhibition of virus release from infected cells was clearly visible in virally infected cells treated with zanamivir (One-way Anova gives a statistically significant knockdown compared to untreated and any of the wells transfected with the fluorescent proteins, p=>0.0001). However, as discussed in later chapters where dominant negative ESCRT pathway proteins have been expressed from plasmids, many cells in such experimental systems are infected that do not express the dominant negative protein and therefore it is very difficult to detect virus inhibition using this approach.

104

Figure 61: Expression of the BM2 putative dominant negative protein. 293T cells were transfected with 500 ng of the expression plasmids EGFP-BM2, eGFP or Cherry Red, incubated for 24 hours prior to image capture of the BM2 terminal 31 amino acids-green fluorescent fusion protein (DN4), the eGFP protein and the cherry red fluorescent protein.

3500 3000 2500 2000

1500 PFU/ml

Virus Yield 1000 500 0

DN4 eGFP +ZAN -ZAN

Cherry Red Expression Plasmid/Drug treatment

Figure 62: Virus yield in the presence of BM2 putative dominant negative cytoplasmic domain. The 293T cells were transfected with 500 ng of the putative BM2 dominant negative cytoplasmic tail region fused to eGFP (DN4), fluorescent eGFP or cherry red control plasmids. The cells were incubated overnight prior to infection of the cells with influenza B virus at an MOI of 3. Zanamivir was added to one set of wells. The experiment wasperformed in triplicate.

105

3.3 Discussion

By altering the length of the BM2 cytoplasmic tail by introducing a series of premature stop codons it was possible to ascertain the minimal length of the tail that would successfully support the replication of a viable virus. Then by series of alanine substitutions in the extreme tail terminus it was shown that the codons -6 (104) and -7(103) could not be substituted for alanines without abrogation of virus viability. Alternative substitutions allowed the generation of a virus that was a fit as the wild-type form, -7V and or highly attenuated forms, -6Y and -6Q. Thus these amino acids may be crucial for the BM2 protein to interact with a host protein, the BM1 Matrix protein or the ribonucleoprotein complex as is the case for the influenza A virus (156, 223, 224).

An alternative theory suggests that cis acting RNA viral packaging signals could be disrupted by the mutations introduced. Initially it was proposed that the packaging signals that sorted the correct influenza RNA segments into new virions were all located in the extreme UTR regions of the segments. However, more recent research by several groups (6, 88, 149, 193, 343, 371) has identified packaging signals that span both the coding and the non-coding regions of the influenza A virus genome. Further to this, computer modelling by Gog et al., (102) indicated that for influenza A, these signals could be strain-specific.

An analysis of the nucleotide sequences for all the viable mutants that were altered at position -6 in the BM2 cytoplasmic tail indicated that all the codons contained a central A nucleotide, whereas the alanine codon present in the mutant that was not rescued contained a central Cytosine instead (Table 12). At position -7, the alanine substitution that did not give rise to viable virus required two changes to the nucleotide sequence in this region, whereas the mutant that was rescued with a leucine to valine switch had only one nucleotide difference from wild-type virus.

Though the studies on influenza A would suggest that RNA packaging signals involve multiple points on the genomic segments, and small point mutations tend to affect the amount of virus produced rather that completely ablating virus replication (6, 88, 149, 193, 343, 371), the fact that in general lower titres are recovered from the influenza B virus reverse genetics system may mean that disruption of packaging signals in this context preclude virus rescue.

At position -6 the glutamic acid itself is fairly well conserved as there is only one instance of either the Asparagine or the Lysine out of 294 full-length sequences in the NCBI Influenza

106 database, suggesting that it is not simply the loss of the Adenosine that causes the conservation of this amino acid.

Table 12: The potential nucleotides for amino acids at positions -6 (A) or -7 (B) in the BM2 protein’s cytoplasmic tail. The amino acids highlighted in purple are naturally occurring and the artificially introduced amino acid substitutions are highlighted in blue. Variations in the second nucleotide of the coding sequence of the amino acids, caused by the substitutions are highlighted in green.

Imai et al., (150) have also shown that the loss of the amino acids beyond 104 of the cytoplasmic tail of the BM2 virus, resulted in severely attenuated virus. They also showed that this was due to a dramatic reduction in the membrane association of the matrix protein and that this caused deformed vesicles to be released, many of which lacked the ribonucleoprotein complex. Interestingly the research from Imai et al., did not conclude that the -6 and -7 positions were important and they found that for the virus strain B/Yamagata/1/73 alanines could be substituted for the natural residues in these positions. They also concluded that the length of the tail; and not the identity of specific amino acids at the terminal region was of predominant importance. Interestingly they did indicate that the region 86-97 could not support alanine substitutions without attenuating the virus and they also suggested loss of

107 phosphorylation may be the cause of the attenuation. The Ser91A substitution described in this thesis falls within this region. Thus taken together although the general findings of our work corroborate those published by Imai et al,. we have found important subtype strain specific difference in the amino acid deletions and substitutions that are tolerated in the cytoplasmic tail region of the BM2 protein. This may imply that the interaction of this region is with another viral component that also varies with strains rather than with a cell component that would be conserved between our work and that by Imai et al.,.

We did not succeed in defining the site for post-translational modification of BM2 protein that gives rise to the second 17 kDa protein band. Further work in this area could look at other potential phosphorylation sites located further upstream on the cytoplasmic tail specifically the Ser75, Threonine 69 or Serine 54 independently and in conjunction with the Ser91A (Figure 43). Alternatively, other post-translational modifications could be made to the BM2 protein that account for the six species observed in the Western blots.

It was of note that all of the mutants we generated by the reverse genetics technique that contained amino acid changes in BM2 showed a more diverse population of virions that did wild-type virus when visualized by the TEM. This may imply that the exact sequence of the BM2 tail can tolerate some substitution in tissue culture but that the efficiency of formation perfect virions is compromised to an extent that such substitutions would not be enriched under natural selection conditions. In future TEM work relative proportions of NA and HA glycoproteins could be assessed (with immunogold staining) for the different recombinant viruses, which could also elucidate whether the vesicles seen in the BM2-5 and Ser91A TEM images contain any viral components.

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Chapter 4. The involvement of host proteins in budding of influenza virus.

4.1 Introduction: The ESCRT Pathway

At the late stages of influenza virus infection, the newly synthesized viral proteins and genetic material are transported to the apical surface of the plasma membrane where they concentrate within the cholesterol rich areas known as lipid rafts from which the virus buds. Currently undefined viral components must interact with host components in order to be correctly transported and to mediate budding. The Endosomal Sorting Complexes Required for Transport (ESCRT) pathway has been implicated in the transport and budding from infected cells of viruses from many families (reviewed in (36).

The ESCRT machinery is responsible for the ubiquitin-dependent endosomal sorting of membrane proteins and their delivery to the lysosome or TGN for destruction or for the recycling to the cell surface. The limiting membrane of the endosome is deformed by the ESCRT machinery to form multivesicular bodies (MVB) or multivesicular endosomes (MVE) which are then able to fuse with lysosomes which can then degrade the contents with proteases; they are also responsible for the abscission of these deformed membranes to the intraluminal vesicles (Reviewed in 36, 148, 288, 324).

The ESCRT complexes represent 10 out of the 18 class E protein components of the MVB sorting machinery. Four sequential complexes have been indentified: ESCRT 0, I, II & III. These have been extensively studied in a yeast system. Homologous proteins have also been identified in mammals. The nomenclature can be confusing as the proteins have the prefix Vps in yeast and a wider variety of names in the mammalian homologues.

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4.1.1ESCRT-0

The initial step in the pathway is facilitated by the ability of ESCRT-0 to bind to the Endosomal lipid phosphatidylinositol 3- phosphate [PtdIns(3)P], linking ESCRT-0 to the membrane, through the Hrs (hepatocyte growth factor regulated tyrosine kinase substrate) subunit, which together with STAM (signal transducing adaptor molecules) and in human cells Eps15b, are also capable of binding ubiquitin through their UIM (ubiquitin interacting motif) or for Hrs, DUIM (double ubiquitin interacting motif) (81, 127). The membrane proteins (the cargo for sorting) that have been ubiquitinated by ubiquitin ligases can therefore be bound by this complex. The C- terminal portions of Hrs has a clathrin box motif, allowing an association which concentrates the ubiquitinated cargo to the clatherin dense regions of the Endosomal membrane (324) and a P(S/T)XP motif that can interact with the next ESCRT pathway member ESCRT-I (308).

4.1.2 ESCRT-1

The ESCRT-I complex is composed of four subunits; Tsg101 (Tumour Suppressor gene 101), Mvb12 (multivesicular body sorting factor), Vps37 and Vps28. The binding of Tsg101 to the ESCRT-0 complex allows the recruitment of these proteins to the endosomal membrane. The ubiquitinated cargo is passed from the ESCRT-0 to the ESCRT-I complex sequentially, where it is bound by its UEV (ubiquitin conjugating enzyme E2 variant) domain of Tsg101 and the IIe44 residue on ubiquitin (148).

The ESCRT-I complex the passes the ubiquitinated cargo to ESCRT-II, though the exact mechanism is unknown. The ESCRT-I and II complexes are transiently recruited to the MVB after they assemble in the cytoplasm (58, 288).

4.1.3 ESCRT-II

The ESCRT-II complex is able to bind to the endosomal membrane through its GLUE domain on EAP45/ Vps36 and the 3-phosphorylated phosphoinostitides (3-PPI) in the membrane. This GLUE domain can also bind ubiquitin and therefore the cargo from ESCRT-I. Interestingly for HIV-1 budding, ESCRT –II is not required, but for Avian Sarcoma virus, there is no budding without ESCRT –II (183, 204, 276).

4.1.4 ESCRT-III

ESCRT-III assembles on the endosome membrane, unlike the other soluble ESCRT complexes. It is believed that the chains that make up ESCRT-III polymerize into a protein lattice on the cellular membranes. This lattice could be responsible for the deformation and subsequent

110 invagination of the membrane that eventually forms the MVBs (reviewed in (184). It is the most conserved of the complexes within the pathway (189) and it is believed to be a contributor to the abscission mechanism during cell division (309, 329). CHMP4/Vps32 subunits are able to bind to the Alix protein (which can also interact with the Tsg101 protein of ESCRT-I). At the other end of the chain, the CHMP2/Vps2 protein is able to link to Vps4.

4.1.5 Alix

The Alix (ALG-2 (apoptosis linked gene 2) interacting protein X) or AIP1 is localized to endosomal membranes and is able to bind ESCRT-I and ESCRT-III. HIV-1 virions are unable to bud in its absence when the virus’ gag protein PTAP late domains access to Tsg101 is also blocked (30, 212, 334, 358, 375). Alix is composed of three regions the Bro-1 domain (1-359), V domain (360-702) and the C-terminal Proline-rich domain (PRD) (703-868). It is the PRD domain that interacts with Tsg101, and is involved in the negative regulation of the Cbi E3 ubiquitin ligase through CIN85. Alix also has a role in apoptosis, over expression induces apoptosis and use of a dominant negative which lacks the Bro1 domain has an anti-apoptotic effect (206, 263, 348, 357).

4.1.6 HD-PTP and ESCRT III

Interestingly a recent paper by Doyotte et al., 2008 has suggested that the Bro1 domain of HD- PTP/PTPN23 (His domain phosphotyrosine phosphatase/His-Domain/Type N23 protein tyrosine phosphatase) is responsible for the interaction of CHMP4 of ESCRT III and MVB formation rather than that of Alix (65). The requisite Bro1 domains can also be found in Brox and Rhophilin proteins (281). Indeed different over-expressed Bro1 domains have been able to rescue viral budding for HIV-1 mutants that lack the late domain motifs through interactions between the CHMP4 binding site of the Bro1 domain and the N-terminal region of the viral nucleocapsid domain in the Gag protein (68).

4.1.7 Vps4

The assembly and disassembly of the ESCRT-III complex during membrane invagination and MVB formation requires the action of the ATPase Vps4, a type 1 AAA (ATPase Associated with a variety of cellular Actions)(377). The type 1 AAA subfamily of the P loop ATPases as implied by their name are involved in a variety of cellular actions including DNA replication, nuclear- cytoplasmic transport, organelle biogenesis, vesicular trafficking, protein degradation and oncogenic transformation (182). The oligomers characteristically form stacked rings with a central cavity, the ATP binding is believed to aid in oligomer formation and stability, which can

111 be enhanced by the actions of accessory proteins. The energy released by the ATP can be used to remodel the target proteins (377). The dodecamer oligomer that compromises Vps4 is critical for the disassembly of ESCRT-III. The oligomerization process is triggered by ESCRT-III or Vta-1/LIP5, the latter in concert with ATP is also responsible for keeping the structure stable in vivo. ESCRT-III was originally deemed to be essential for Vps4 complex formation, ATP hydrolysis and disassembly through its interactions with the MIT domain of Vps4, however, with the discovery of Vta-1 the oligomerization process is less clear (58, 313).

A functionally inactive form of Vps4 has a mutation in the AAA domain (E223Q). It is able to bind ATP and form oligomers, but is locked and unable to dissociate, effectively blocking the disassembly of ESCRT III (182).

4.1.8 Ubiquitin ligases

Prior to incorporation into the ESCRT pathway proteins require ubiquitination, this function can be provided by one of the 500 members of the E3 family of ubiquitin ligases. WWP1 (WW domain containing E3 ubiquitin protein ligase 1) (AIP5) can act as a scaffold between the E2- thioester complex and the target protein in the E1 (ubiquitin activation enzyme), E2 (Ubiquitin conjugating enzyme) - E3 ubiquitin transfer reaction. The WWP1 is composed of 4 tandem WW domains and a C-terminal HECT (homologous to the E6-associated protein carboxyl terminus) domain. The large N-lobe of the HECT domain is responsible for E2 binding; a flexible linker connects to the C-lobe which contains the active site, a cysteine responsible for the transfer of the ubiquitin to the target protein. Responsibility for target protein specificity is mediated by a region upstream of the HECT domain (162). It is worth noting that WWP1 is not the only ubiquitin ligase that interacts with the late domains and is implicated in viral release. Indeed, Nedd4, LDI-1, 2, BUL1, WWP2 and Itch can also affect viral budding (18).

It has been proposed that the ESCRT-0, I & II soluble components of the pathway sequentially interact to pass the ubiquitinated cargo to the membrane bound ESCRT-III, these then disengage and the ESCRT-III and VPS4 component form the invaginations and perform the pinching off steps.

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Figure 63: The ESCRT pathway. ESCRT-0, I & II are soluble in the cytoplasm prior to recruitment to the membrane, ESCRT III is directly assembled on the membrane and may no longer require the interactions of the other ESCRTs once the cargo has been transferred. WWP1, Alix, Tsg101 and Vps4 for which plasmids that express dominant negative mutants are available are highlighted in green.

4.1.9 ESCRT and viral budding

The involvement of this host cell pathway in viral budding was first demonstrated for retroviruses (85). Retroviruses with mutations in their late domains showed accumulation of virus at the plasma membrane, but the viruses were not able to bud off. Retroviruses hijack the ESCRT system by using their late domains (regions of the genome that are expressed late in the viral life-cycle)(comprehensively reviewed in reference (36). The ubiquitinated form of the gag protein of HIV-1 is able to interact with ESCRT- I through two late domains; PTAP provides the link to TSG101 (this late domain shares some homology to the Hrs of ESCRT-0) and indirectly through the Alix protein and the LYPX(n)L late domain of the gag protein (18, 97). The interaction with Alix also provides a link to ESCRT-III (334) which in turn interacts with the Vps4 ATPase that then aids viral budding. The Alix protein is important for the budding of EIAV, but plays a secondary role in the budding of HIV-1 where the Tsg101 PTAP domain has the predominant role.

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Other viruses that interact with the ESCRT pathway include Mason-Pfizer monkey virus, Moloney murine leukaemia virus, Human T cell leukaemia virus I, Prototypic foamy virus, Vesicular stomatitis virus, Rabies and Ebola viruses (36). Therefore it was not unreasonable to hypothesize that this pathway could also be hijacked by the influenza viruses and indeed a ‘YRKL’ late domain had been identified within the matrix protein of influenza A virus that was proposed to associate with the VPS28 ESCRT protein (143).

4.2 Results:

4.2.1 ESCRT, Influenza and late domains

However, there is some doubt about the results from the work by Hui et al., 2006 that implicated Vps28 with influenza budding (143) and the manuscript that described the late domain interactions has been retracted. Moreover a sequence alignment of the influenza A virus M1 gene shows that the proposed YRKL late domain motif is not absolutely conserved amongst the 262 non identical full-length matrix protein sequences at the Influenza Virus Resource (7). Instead there are examples of YKKL [31], YTKL [1] and CRKL [2] indeed it is worth noting that the incidence of YKKL is approximately 50% when all the 387 sequences are included in the alignment.

This late domain sequence is also not conserved between the influenza A and B M1 proteins but the R and K residues are maintained (Figure 64 and Table 13). The sequence alignment of the M1 proteins of type A and B of influenza illustrates only a low level of sequence identity (30%) indicating that they may differ in conformation when folded, so a late domain motif, if present, could be found in a different region of the influenza matrix protein.

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Figure 64: Sequence alignment of A/Winsconsin/3523/88 (H1N1) and B/Yamagata/1311/2003 segment 7 Matrix protein. The alignment was generated using the Blast2seq program (BLASTP 2.2.15 [Oct-15-2006]) with the Blosum62 matrix default settings. The putative late domain is highlighted in red.

The region of influenza B M1 protein that aligns with the influenza A M1 putative late domain motif displays a degree of conservation amongst influenza B viruses. In a sequence alignment of the 47 non identical influenza B sequences at the Influenza B virus resource (7), 2 strains contained K at position 101 exemplified by AAD29172 (B/Norway/1/84) and 6 strains a K at position 105 ABK51169 (B/Taiwan/16/2004), indicating this region in influenza B M1 protein is not absolutely conserved, but that substitutions were conservative.

Consensus G M G T T A T K K K G L I L A E R K M R R C V S F H E A F E I A E G H E AAK95902 ...... Wt AAD29172 ...... K ...... K101 ABK51169 ...... K ...... K105

Table 13: Influenza B Sequence alignment of Segment 7. Amino acids 85 -120 shown. The region of the influenza B gene that aligns with the influenza A putative late domain is highlighted in red.

Despite the doubt cast on the work by Hui et al., 2006 it is still plausible that the influenza viruses may use a portion of the ESCRT pathway during their assembly process. To address this and to attempt to identify which part of the ESCRT complex might be involved in the influenza virus replication cycle, the effect of disruption of the pathway by over expression of dominant negative mutants of the ESCRT family on the efficiency of influenza virus budding was assessed.

A selection of plasmids that direct expression of ESCRT pathway dominant negative mutants were provided by Dr. Greg Towers & Dr. Juan Martin-Serrano (Table 14). These plasmids direct

115 the expression of wild-type or mutant proteins fused to Yellow Fluorescent Protein (YFP) or EGFP. The proteins expressed included full-length VPS4 (an essential ATPase, lack of which results in the accumulation of ESCRT III components in endosomal storage compartments), WWP1 ΔHect (the ubiquitin ligase lacking the HECT domain), Alix (lacking the Bro1 domain, the link between ESCRT-I & ESCRT III) and a mutant TSG101 (a component of ESCRT-II that associates to late domains and VPS28) (for details of the mutations see Table 14). If influenza viruses use the ESCRT pathway, disrupting this pathway by over expression of the dominant negative mutants should reduce viral yields.

Protein Plasmid Virus inhibited Tsg101 1-157 pCR3.1 -Contains the PTAP HIV-1, Mason-Pfizer monkey virus, (Tumour binding domain , lacks the C- Moloney murine leukemia virus, Susceptibility Gene terminal region required for HTLV-1, Prototypic Foamy Virus, Ebola 101) HIV-1 budding (212) fused to virus (20, 46, 47, 107, 139, 211, 271, (Vps23) EGFP. Unable to bind Vps28. 314, 350, 351, 384). Alix(AIP1) 170-869 pCR3.1 -Lacks a section of the HIV-1*, Equine infectious anemia Bro-1 domain that binds to virus, Moloney murine leukemia virus CHMP4 of ESCRT III- Fused to (64, 97, 121, 153, 179, 287, 306, 314, YFP. 350, 352). WWP1 ΔHect The WWP1 ubiquitin ligase HTLV-1 (360). lacking the HECT domain. Fused to YFP. Vps4 ATPase fused to YFP HIV-1, Mason-Pfizer monkey virus, Rous sarcoma virus, Prototypic Foamy Virus (27, 54, 105, 107, 120, 163, 164, 212, 228, 271, 311, 334, 335, 355, 358). Vps4 E223Q ATPase lacking catalytic HIV-1, Mason-Pfizer monkey virus, function fused to YFP Rous sarcoma virus, Prototypic Foamy Virus EXN-YFP pCR3.1 YFP -A control plasmid N/A that expresses YFP

Table 14: List of the plasmids expressing dominant negative versions of the ESCRT proteins and viruses experimentally inhibited by their expression. (Virus inhibition data from (36) *HIV-1 release is blocked by the Alix dominant negative when the Tsg101 PTAP late domain is removed.

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Expression of the dominant negative ESCRT proteins was readily visualized because they contain C-terminal EGFP or YFP tags. All the plasmids were successfully expressed in 293T cells (Figure 65). Although the amount of plasmid transfected was equal, the expression levels and the localisation of the protein varied. For example the TSG101, Vps4 E223Q fusion proteins and EXN-YFP were expressed throughout the cell, whereas the Vps4 (Wt) and Alix fusion proteins were localized in aggregates within the cells. The WWP1 ΔHect plasmid produced a protein with a diffuse expression pattern, with some localisation to the membrane (Figure 65). The cells remained adhered to the tissue culture dish and remained intact, suggesting that these fusion proteins were not acutely toxic.

Figure 65: Differential expression patterns of ESCRT dominant negative proteins. 293T cells transfected with 500 ng of the ESCRT protein expression plasmids, incubated for 24 hours. The EGFP or YFP (both visualized as a green fluorescence) indicate the presence and localisation of the ESCRT proteins. Vps4 (Wt) is the full-length form of this protein fused to EGFP. EXN-YFP is the control plasmid containing unfused YFP gene. The remaining proteins are the mutant forms as listed in Table 14. The images were all taken at x20 magnification. The cells were intact and adhered to the culture dish.

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In order to confirm that the ESCRT plasmids which produce the modified proteins tagged to YFP or EGFP were able to exert a dominant negative effect in our hands, a retrovirus particle budding system was used. This system generates pseudo-type HIV-1 particles. These are composed of a fused HIV-1 gag-pol protein, the influenza HA (H5) protein and a EGFP reporter (Figure 66 and (10, 222, 246). The gag –pol protein is able to selectively incorporate the EGFP gene into the particles produced because it contains the HIV-1 psi packaging signal. Therefore EGFP is expressed when the particles enter the reporter cells. Once produced the particles are capable of only a single round of infection. The cell entry in this case is facilitated by the H5 HA glycoprotein. This system was generously provided by Dr. Nigel Temperton (UCL).

Figure 66: HIV-1 -pseudo-type particle system. A. pCMV HIV-1 Gag-Pol (contains all the HIV-1 genes except env and nef) B. The influenza A Haemagglutinin (H5) glycoprotein, facilitates particle entry. C. Transfer vector, HIV-1 based contains 350 bp of the gag protein. CMV, cytomegalovirus immediate-early promoter; ψ, packaging sequence; LTR, long terminal repeat. D. Schematic of the pseudo-particle system, the 293T cells transfected with the HA, gag-pol fusion and eGFP reporter plasmids, pseudo-particle release and entry into fresh 293T cells to generate eGFP expression.

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The amount of EGFP positive cells in each case were compared to that when the wild-type Vps4 construct was expressed since it was reasoned that this unmanipulated protein would not adversely affect viral budding. Retrovirus (HIV-1) budding is known to be inhibited by dominant negative forms of Tsg101 and the ATPase defective version of Vps4. This inhibition was confirmed in our hands, with a significant reduction pseudo-particle formation in the presence of either the Tsg101 or Vps4 E223Q (p=<0.05) when compared to budding in the presence of the wild-type Vps4 construct (Figure 67).

Whilst WWP1 ΔHect has been reported to reduce the levels of HTLV-1 released (121), it had no significant effect on the HIV-1 pseudo-particles when compared to the wild-type Vps4 construct (p=>0.05). Surprisingly, the HIV-1 pseudo-particle release was also decreased in the presence of the control EXN-YFP protein, albeit to a lesser level than seen for Tsg101 or Vps4 E223Q dominant negative ESCRT constructs. This and later results in this chapter may indicate that the EXN-YFP protein product may be toxic to the cell.

Interestingly the pseudotype particle formation was increased in the presence of the Alix construct. As previously mentioned, Alix can inhibit HIV-1 particle release, but the effects are often small and rely on other late domain motifs being absent or blocked. Indeed two factors mediate in the increased release of pseudo-particles seen here, apoptosis and ubiquitination.

Firstly, Alix lacking the Bro-1 domain, as is the case for this dominant negative, can inhibit apoptosis (206, 263, 348, 357) allowing the particles time to be released from the cell prior to the programmed cell death. Secondly, to enter the ESCRT pathway the ubiquitinated proteins must be recognized, but during the ESCRT transport process the proteins are deubiqutinated, the late domains potentially acting as a source of recruitment to deubiquitination enzymes (210). The increase in particles released that was observed in our hands could relate to Alix’s ability to deubiquitinate, as is observed in its involvement with EGRF endocytosis. This deubiquitination can be blocked by Src binding to the Bro-1 domain, which is lacking in the dominant negative fusion protein used in this research (for more details on this process please see (199, 263). The HIV-1 gag protein used in these pseudo-particles can be inhibited by ubiquitination (210) and the presence of this over-expressed form of Alix may increase the deubiquitination of the gag protein and the thus proportional increase in the amount of HIV-1 gag-pol based particles released.

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p=<0.05 p=<0.05

) 10

(log 1 GFPcells/ml

Alix HECT Tsg101  Vps4(wt) EXN-YFP Vps4 E223Q No Gag-Pol WWP1 Transfected plasmid

Figure 67: Pseudotype particle yield in the presence of ESCRT dominant negative plasmids. 293T cells were transfected with the 3 plasmids containing HIV-1 gap-pol fusion protein, influenza HA (H5), incorporating a EGFP reporter and co-transfected with one of the ESCRT plasmids. No Gag-Pol sample lacks the fused gag-pol envelope protein. The cells were incubated at 37°C overnight, the media changed, the cells treated with neuraminidase. After overnight incubation the supernatant was transferred to untransfected 293T cells, which were then incubated overnight at 37°C. The eGFP positive cells were counted as a measure of relative pseudo-particle titre (number of green cells/ml of supernatant transferred to reporter cells). The cells turn green only in the presence of a pseudo- particle which is capable of only one round of infection. Significance was assessed using a one way Anova with Tukey’s & Bonferroni’s multiple comparison post tests. This experiment was performed in triplicate.

The results clearly demonstrate effects of over-expression of these mutant proteins on the assembly of HIV-1.

4.2.3 Influenza and the ESCRT pathway proteins

Two experimental approaches were used to assess the levels of influenza virus production in the presence or absence of ESCRT pathway plasmids expressing the dominant negative forms of the proteins. The first method involved incorporating the ESCRT plasmids into the viral 12 plasmid-based rescue system, then quantifying the levels of virus produced by plaque assay.

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The second method involved transfection of the cells with the dominant negative plasmids, then infection with virus.

4.2.4 The effect of overexpression of ESCRT proteins on the recovery of infectious influenza virus from cDNAs.

Each ESCRT plasmid was included in concert with the plasmids required to create recombinant influenza B virus. The efficiency of virus rescue was compared with the unmodified recombinant virus rescue protocol. This method has the advantage that both the viral proteins and the dominant negative host cell proteins are manufactured in the cell at the same time. Thus, the dominant negative mutants will not have the opportunity to disrupt virus entry into the cell and any effects that are seen should be due to interaction of the ESCRT proteins in the assembly pathway.

The disadvantages of this method include the variable nature of the twelve plasmid- based influenza virus rescue system itself, making detection of small effects on rescue efficiency difficult. Secondly, the production of the viral proteins in such a system will be at different levels than during normal infection when gene expression is controlled by the virus. In addition, the dominant negative mutants may not accumulate quickly enough to affect virus production before the virus inhibits host cell driven gene expression, and shuts off expression of non viral proteins including those encoded by the dominant negative ESCRT plasmids.

In these experiments virus rescue was performed in 293T cells, and each dominant negative plasmid or empty vector was included as a 13th plasmid in the transfection mix in an equal quantity to the plasmids used in viral rescues. Day 1 after transfection the 293T cells were co- cultured with more permissive MDCK cells and supernatant was collected on day 4 and day 5 of the co-culture and plaque assays performed in MDCK cells were used to quantify the number of viruses that had been produced (Figure 68).

Two time points were used to assess viral yield in an attempt to balance a sufficiently high titre of virus that would allow measurement against catching the replication at an early enough time point to detect differences in yield before the effects of the dominant negative proteins were masked by amplification in the permissive MDCK cells where ESCRT proteins were not being expressed. In the influenza B reverse genetics system small comets of CPE are visible on day 5 or 6, with the cultures harvested on day 7.

The yield of recombinant influenza B virus (after co-transfection of empty vector) from the co- cultured cells was ~106 PFU/ml on day 4 and this increased to around 107 PFU/ml by day 5. This

121 yield was decreased by approximately 2 logs in the presence of Vps4 E223Q. This would indicate that the Vps4 ATPase function has a role to play in the budding of the influenza B virus particles. A similar decrease was also seen in the presence of WWPI ΔHECT when titres were measured on day 4 after transfection but not on day 5. Moreover, surprisingly, co-expression of unfused YFP protein resulted in decreased levels of recovered virus on day 4, but this decrease was not evident by day 5. However, in a two-way Anova, overall there was no significant difference between the co-cultures with irrespective of the plasmids they possessed. In Bonferroni post tests comparing yields between co-culture pairs, there were again no significant differences on day 4. However, by day 5, Vps4 and Vps4 E223Q co-cultures displayed p=values of p=<0.01, but this positive result must also take into account the (p=<0.05) difference observed between Vps4 and WWP1 dHect and the control plasmids containing the fluorescent protein (EXN-YFP). Indicating that the Vps4E223Q plasmid is unable to block influenza B egress any better than the expressed fluorescent protein alone.

p=<0.05

p=<0.01 8 Tsg101 7 Alix 6 Vps4 (wt)

) 5 Vps4E223Q 10 4 WWP1HECT (log EXN-YFP 3 Empty plasmid

Virusyield PFU/ml 2 1 Day 4 Day 5

Figure 68: Viral yield when ESCRT plasmids are incorporated into the reverse genetics system of for B/Beijing/1/87. Transfection of 12 pPol 1 RT plasmids incorporating viral proteins & ESCRT plasmid transfected into 293T cells, followed by co-culture in MDCK cells. Viral titre assessed by plaque assay on Day 4 and 5 of co-culture step. This data represents two independent experiments. A two-way Anova of day 4 data indicated no significant difference, whereas day 5 data indicated a significant difference between Vps4 (Wt) and Vps4 E223Q.

The rescue procedure includes an amplification step in MDCK cells and this may blur the results by overly amplifying smaller titres of released virus. Although it would be prudent to omit this step, taking supernatant directly from the transfected cells gave a viral yield that was too low to be reliable. In attempts to overcome this, expression plasmids for all of the

122 influenza B virus structural proteins were included in the transfection because it was hoped that the overall viral yield would be increased sufficiently to increase the robustness of the results. Whilst viral production was increased 10 fold using this strategy , it was still too low at only 10-20 plaques per rescue and this number was not affected by the co-expression of any of the dominant negative mutants (data not shown).

Figure 69: Method Two: Transfection and infection. The 293T cells were transfected with the ESCRT plasmids, and incubated overnight to allow the expression of the ESCRT-YFP/EGFP fusion proteins. The monolayer of cells were infected with virus, incubated to allow viral replication. The supernatant was then harvested and titrated on the MDCK cells to assess relative yield of viruses.

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4.2.5 The effect of over expression of ESCRT dominant negative proteins on virus yield following infection.

An alternative strategy involved transfection of the 293T cells with the dominant negative plasmids, then the following day, infecting the cells with the B/Beijing/1/87 virus (Figure 69) to assess yields of virus.

To confirm that the dominant negative plasmids had indeed been expressed in cells that were actually infected with influenza B virus, an immunohistochemical stain for influenza B virus NP protein was employed. The cell monolayer was then analysed for co-expression of the eGFP or YFP tag and the NP protein. An MOI of 10 (as measured in MDCK cells) was used to infect the cells; the fact that a significant proportion of the 293T cells were not positive for nucleoprotein (Figure 70) was surprising and indicated that 293T cells were not efficiently infected by the Influenza B virus.

In these overlaid images displaying the brightfield and fluorescence view, the majority of the cells are either transfected (shown as green) or infected (shown as blue). However, it is possible to see clear evidence of infection and transfection within the same cell (examples of which are encircled in red). This experimental work must be assessed with this limitation in mind. Though there is sufficient co-staining to indicate that the virus was able to enter the cells in which the dominant negative proteins were expressed, it was not possible to deduce whether virus was released from these cells. Importantly there were cells which had only been infected, (staining blue but not green) from which virus could be released independent of the effect of the proteins that may exert a dominant negative effect.

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Figure 70: Viral NP protein and ESCRT dominant negative fluorescence. 293T cells transfected with the ESCRT dominant negative plasmids, then infected with influenza B virus at An MOI of 10. The fluorescence indicates the expression of the plasmids and the blue the presence of the NP protein. The red circles enclose cells both transfected and infected. The yellow circles indicate cells that have only been transfected.

The transfected cells were infected at either an MOI of 1 or 10. Variable levels of virus were used in order to elucidate if lower levels of virus would be more sensitive to the blocking effects of the ESCRT proteins; higher levels were used to increase ratio of cells that were both transfected and infected.

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4 3.5x10 4 2.5x104 5 1.8x104 2.5x10 1.5x104 2x104 1.4x104 4

) 3 10

2 (Log

1 Virus yield PFU/ml

Alix Vps4 HECT Mock Tsg101  EXN-YFP No Plasmid Vps4 E223QWWPI ESCRT Plasmids

7 6 1.3x106 5 1.1x10 5 6.1x10 5 9.1x10 5 6.4x10 3.5x10 5 6 3.3x10 2.8x105

) 10 4

(Log 3

Virus yield PFU/ml 2

Alix Vps4 HECT Mock Tsg101  EXN-YFP Vps4E223Q No Plasmid WWP1 Tsg101 & Alix Transfected Plasmid

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Figure 71: Titres of influenza B virus produced from cells transfected with ESCRT dominant negative proteins. The cells were transfected with 500 ng of dominant negative ESCRT plasmids, then infected with B/Beijing/1/87 at an MOI of 1 (A) and an MOI of 10 (B) then incubated overnight. The supernatant was harvested, incubated in the presence of trypsin for 1 hour and then titrated on MDCK cells by plaque assay. This experiment was performed in triplicate.

At an MOI of 1 the cells were successfully infected and virus was released at measurable levels. Whilst the expression of the Vps4 E223Q gave the lowest average viral yield (1.5x104 PFU/ml), none of the viral yields were statistically significant from one another and importantly from the yield in absence of ESCRT expression in a one-way Anova test. However, the overlap of the transfected and infected cells may not have been at levels sufficient to illustrate any difference that may exist (Figure 71A). Even so in experiments when more than 50% of the cells were expressing the dominant negative proteins, there was no difference in the levels of virus produced.

Increasing the MOI to 10 (Figure 71B) did increase the levels of virus released, presumably because a larger number of cells were originally infected. Again the wells transfected with the Vps4 E223Q plasmid yielded the lowest viral titre (2.8x105 PFU/ml) but again the difference in yield was not statistically significant when compared to the viral yield from the other transfected and infected cells.

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7 6 1.3x106 5 1.1x10 5 6.1x10 5 9.1x10 5 6.4x10 3.5x10 5 6 3.3x10 2.8x105

) 10 4

(Log 3

Virus yield PFU/ml 2

Alix Vps4 HECT Mock Tsg101  EXN-YFP Vps4E223Q No Plasmid WWP1 Tsg101 & Alix Transfected Plasmid

Figure 72: Titres of influenza virus produced from cells transfected with dominant negative ESCRT plasmids. The cells were transfected with 500 ng of the ESCRT plasmids and then infected with B/Beijing/1/87 at An MOI of 10, and incubated overnight. The supernatant was harvested, incubated in the presence of trypsin for 1 hour and then titrated on MDCK cells by plaque assay. In a one-way Anova test, there was no significant difference observed between the sample titres. This experiment was run in duplicate.

It was possible that sampling an earlier time point may have revealed a difference in viral yield due to the influence of the ESCRT plasmids. To address this, attention was focused upon the Vps4 wild-type and the ATPase inactive form Vps4 E223Q as the Vps4 protein is a crucial final endpoint in the ESCRT pathway. The cells were transfected with 500 ng of the plasmid, then infected with an MOI of 3. Supernatant was harvested at 6, 12 and 24 hours, and then titrated by plaque assay. There was no difference observed statistically or visually in the level of virus produced irrespective of the plasmid that was transfected (data not shown).

The single –step growth curve experiment methodology was also used to assess the effects, if any, of these ESCRT plasmids on the yield of the Influenza A virus. A strain of influenza A virus was chosen known as A/WSN/V virus (an Influenza A virus with a characteristic WSN backbone except for segment 7 which has been modified to be that of A/Victoria/ 3/75 which is also used later in this chapter as a control virus) because it was able to replicate in the absence of trypsin, therefore is less likely to disrupt the monolayer of 293T cells that are trypsin sensitive.

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Again there was no difference observed in virus yield irrespective of the plasmid transfected (Data not shown).

In summary, two methodologies have been attempted to observe the role of the ESCRT pathway in the egress of the influenza virus from infected cells. There was no evidence to support the notion that either of the main genera of the orthomxyoviruses utilise the ESCRT pathway for virus egress.

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4.3 Introduction: Investigating a potential role for tetherin in controlling the budding of Influenza viruses

Recent work by Neil et a., l showed that an interferon-induced protein named either Bone Marrow Stromal cell antigen 2 (BST2) or tetherin could block the release of HIV-1 from host cells (249). The HIV-1-encoded protein Vpu appears to be able to overcome this effect. Tetherin is expressed in HeLa cells to a sufficiently high degree that, in order for HIV-1 to bud from the plasma membrane, the virally encoded Vpu protein must be present. In contrast, tetherin expression in 293T, Cos-7, HOS and HT1080 cells is far lower and in these cells the action of Vpu is dispensable (248-250). In order to identify the protein responsible for this host-mediated effect, Neil et al., 2007 searched for a protein that was up-regulated in the presence of IFN-α and was either secreted or membrane bound. By comparing gene expression in different cell types and under different conditions using a microarray analysis, ~10 potential candidates that displayed a pattern of expression corresponding to the Vpu related host protein were identified. A bioinformatic analysis narrowed that down to tetherin. Indeed tetherin was up-regulated 20 fold in HeLa cells in comparison to HOS cells and 20 fold in the presence of IFN-α.

HIV-1-like virus particles which lacked the expression of the Vpu protein were unable to bud from the cell membrane of 293T cells transiently expressing the tetherin protein, whereas in the presence of Vpu, budding was efficient. As 293T cells are usually able to support the budding of HIV-1, this provided compelling evidence that the tetherin protein was a player in viral budding. SiRNA experiments to knock down the expression of tetherin saw an increase in the yield of the HIV-1-Vpu knockout virus particles (249).

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Figure 73: Predicted structure of tetherin (CD317). Modified from Neil et al., 2007,2008 and Rollason et al., 2007. Illustrating the transmembrane cytoplasmic tail, the coiled-coiled extracellular domain and the GPI membrane anchor which is lacking in the ΔGPI tetherin expression plasmid.

Tetherin, also known as BST2, HM1.24 or CD317, is encoded on chromosome 19p13.2 and has been implicated in regulation of growth and development in B cells (106, 154, 264). A schematic diagram of the tetherin protein (Figure 73) displays the amino terminal cytoplasmic tail, the membrane spanning region, a predicted coiled-coiled structure in the extracellular domain and a putative carboxyl – terminal – glycosyl phosphatidylinositol (GPI) membrane anchor. The GPI anchor localises the protein to lipid rafts on the apical surface of polarised cells (172). Interactions of tetherin with the adapter proteins AP2 and AP1 are essential protein cycling between the apical surface and a pool in the trans Golgi network. This process requires internalisation through clatherin-mediated endocytosis (300). The C –terminal truncation of the tetherin protein, effecting the loss of the GPI anchor, abrogated the protein’s ability to inhibit budding of the Vpu knockout virus (250).

Tetherin inhibits the budding of some lentiviruses, Spumaretroviruses, Alpha, Beta and Deltaretroviruses and Filoviruses (161). In common with the influenza virus, viruses such as Ebola and HIV-1, bud from cholesterol rich lipid rafts (11, 255). Thus it was plausible that influenza may also be controlled by the action of tetherin. This might be particularly apparent

131 in the absence of a viral protein that usually overcomes such inhibition. Interestingly, it has previously been suggested that the HIV-1 Vpu protein may share functional homology with the influenza virus ion channel proteins, M2 or BM2. For example it has been demonstrated the transmembrane domain of M2 can functionally replace that equivalent region of Vpu during the generation of HIV-1 based VLPs (135).

The K5 protein from Kaposi sarcoma-associated herpes virus is also able to reduced the levels of tetherin when over expressed and so likely represents another Vpu functional homologue (161).

We were kindly given a series of tetherin and Vpu plasmids and a 293T tetherin expressing stable cell line by Stuart Neil (King’s College London).

Plasmid Function pCR3.1-HA. Expression plasmid with the full-length tetherin Tetherin protein fused to a HA tag protein. pCR3.1-HA. An expression plasmid with a truncated version Tetherin of the tetherin protein where the GPI anchor has ΔGPI been removed, again with a HA tag protein. pCR3.1-HA. An expression plasmid that encodes the HIV-1 Vpu Vpu protein. pCR3.1. An expression plasmid which encodes the red Cherry Red fluorescent protein. p.cDNA.M2 An expression plasmid that encodes the Influenza A ion channel protein M2 pcDNA.BM2 An expression plasmids that encodes the Influenza B ion channel Protein BM2 pcDNA.HA An expression plasmid that encodes the influenza B Haemagglutinin protein.

Table 15: Expression plasmids used to express the tetherin, viral and control proteins in transfected 293T cells.

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4.4 Results:

4.4.1 Tetherin inhibits retrovirus budding in the absence of Vpu

To confirm that the tetherin expressed following transfection of the plasmids was able to inhibit the release of HIV-1 particles as described in Neil et al.,2007, retrovirus pseudo- particles were again used to measure HIV-1 particle release in the presence of these expression plasmids. The 239T or 293FT cells were successfully transfected with the expression plasmids. The tetherin and M2 plasmids were co-transfected with a plasmid expressing cherry red as a transfection control. At the time of these experiments there was no suitable antibody for the detection of tetherin.

p=<0.001 3

p=<0.01

) 10

2 (Log

GFP GFP cells/ml 1

GPI  Tetherin GPI +Vpu EXN-YFP  Cherry Red Tetherin + Vpu Plasmids

Figure 74: Retrovirus pseudo-particle release in presence of Tetherin. 293T cells transfected with the plasmids required to generate pseudo-types and the Tetherin, ΔGPI, +/- VPU and the cherry red & EXN -YFP control plasmids. The cells were incubated at 37°C overnight, the media was changed to remove any untransfected plasmids, and incubation continued for a further 24 hours. Cells were then treated with neuraminidase, the supernatant was removed and transferred to fresh 293T cells, then incubated at 4°C for 30 mins to allow the attachment of the particles, prior to incubation at 37°C overnight before the number of EGFP positive 293T cells were counted as a measure of the relative titre of pseudo-particles This experiment was performed in triplicate.

There was a significant knockdown of pseudo-particles produced in the presence of the full- length tetherin (p= <0.001) or to a lesser extent a tetherin lacking the GPI anchor (p= <0.01) compared to particle release after transfection of the EXN control plasmid (Figure 74). In the

133 presence of full-length tetherin, the Vpu protein was able to rescue particle release. The knockdown caused by the ΔGPI anchor defective tetherin may be a result of this defective protein joining in a dimer formation with a correctly expressed tetherin protein native to the 293T cells, as it has been proposed that the active tetherin is in a dimer complex (264). Interestingly this phenotype was not rescued by Vpu expression.

The data indicated that these proteins when expressed in 293T cells were capable of inhibiting the release of pseudo-type particles composed of the HIV-1 gag protein.

4.4.2 The effect of tetherin on release of influenza A virus in the presence or absence of the M2 ion channel protein.

Whilst the work in this thesis has largely been concerned with influenza B virus, the initial work to analyse the effect of tetherin in influenza budding was performed with influenza A virus. This was because there was available within the laboratory a recombinant influenza A virus that did not express the M2 protein. This virus had been made by rescue in an MDCK cell line that constitutively expressed the WSN/33 M2 protein (344) and indeed the virus could only form plaques on these cells and did not undergo multi-cycle replication in unmodified MDCK cells (Elleman and Barclay unpublished (34, 342, 367-370). We hypothesized that, given the suggestion of homology between M2 and Vpu, such a virus would behave like a Vpu deficient HIV-1 particle and be more sensitive to the levels of tetherin expressed in cells than would a wild-type influenza virus. The cells were transfected with the tetherin plasmid with and without Vpu or M2 expression plasmids. The cells were then infected with virus, and incubated over night; the supernatants were harvested and subjected to a haemagglutination assay to assess virion release.

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Figure 75: Influenza A M2 knockout virus yield from cells stably transfected with tetherin. Virus (WSN/M2KO) yield from 293T cells stably expressing tetherin (+) and naïve (-) 293T cells transfected with cherry red and/or M2 and VPU, after 24 hour incubation the cells were infected with the WSN/M2KO virus. HA titre was measured as the M2 stable cell lines did not support plaque assays at this time. This is a composite of two experiments both run in duplicate.

Release of the WSN M2KO virus was low (Figure 75). This is in line with previous reports (223, 224, 278, 342, 368) and does indeed suggest that M2 has a role to play in virus assembly or release. However, the low yield was not complemented by over expression of the M2 protein, except in the cell line that stably expresses the tetherin protein. During the use of the tetherin expressing stable cell lines it was observed that the cells had increased transfection efficiency than the naïve 293T cells, an observation confirmed in personal communication with their creator Stuart Neil. This could explain the increased yield from the tetherin cell line transfected with expressed M2 protein. It is not clear why the M2 did not enhance the virus yield in the naïve 293T cells.

There was no decrease in virus production in the cells stably expressing tetherin compared to the naïve cells that were co-transfected with the cherry red plasmid. The presence of the Vpu did not affect virus production in either the stable cell line or the naïve cells. These results indicate that tetherin does not inhibit influenza virus release even in the absence of an M2 ion channel protein.

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Next, a virus with full-length AM2 gene was also assayed for the effect of tetherin on virus release (Figure 76).

) 10 4

Virusyield PFU/ml(Log 1

0 + - + - + - Cherry Red Vpu M2

Figure 76: Influenza A virus yield from cell stably transfected with tetherin (at high MOI). WSN/V virus yield measured by haemagglutination assay (A) and plaque assay (B) from 293T cells stably expressing tetherin (+) and tetherin naïve (-) 293T cells transfected with cherry red and/or M2 and VPU prior to infection with WSN/V. This experiment was performed in triplicate.

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The WSN/V virus initial inoculum was higher than that for the WSN/M2KO virus, and the virus yield was proportionally higher (Figure 76A). There was no statistically significant decrease in the levels of WSN/V virus produced when tetherin was over expressed in the 293T cells, nor was there any marked increase in virus production provided by the in trans expression of the M2 or the Vpu ion channels.

As the WSN/V virus is capable of replicating in MDCK cells due to its fully functioning M2 protein, the supernatant harvested from the previous experiment can be subjected to plaque assay as a finer measure of viral titre. There was a decrease in the infectious titres of virus released from the cells stably expressing the tetherin with either cherry red or Vpu although the decrease was very slight. Moreover, for the cells transfected with the M2 protein, more infectious virus was released in the presence of tetherin than in its absence. However, none of these differences are statistically significant (Figure 76B).

It was possible that small but significant differences in virus release exerted by the transiently transfected plasmids might be overwhelmed by excess virus. Replicate experiments were set up using 1:16 and 1:24 dilutions of the original inoculum, these were performed in duplicate. However, even at these low doses there was no difference in the number of virions released from cells that expressed tetherin in comparison with those that did not regardless of co- expression of Vpu or AM2.

Figure 77: Influenza A virus yield from cell stably transfected with tetherin (Low MOI). WSN/V virus yield measured by haemagglutination assay from 293T cells stably expressing tetherin (+) and tetherin naïve (-) 293T cells transfected with cherry red and/or M2 and VPU prior to infection with WSN/V at (A) 1:16 and (B) 1:24 dilution of the inoculum used in Figure 76. These experiments were run in triplicate.

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A single-step growth curve was undertaken with the WSN/V virus to assess whether tetherin affected viral release earlier in the infection cycle. There was no indication that tetherin expression could inhibit influenza A release (Data not shown).

Tetherin expression is naturally high in HeLa cells; HIV-1 is unable to bud from these cells in the absence of the viral Vpu protein (249, 250). However, as can be seen in Figure 78 expression of Vpu or M2 has no effect on viral budding from HeLa cells even when the levels of tetherin expression are transiently increased by the transfection of the tetherin expression plasmid.

Figure 78: Influenza A yield from HeLa cells transiently transfected with tetherin. HeLa cells transiently transfected with Tetherin (+) or Cherry Red (-), then co-transfected with Vpu, M2 and additional Cherry Red expression plasmids as a control. Incubated for 24 hours prior to infection with WSN/V virus, a further overnight incubation prior to harvest and assessment of viral titre by Haemagglutination assay. This experiment was run in duplicate.

Taken together these results indicate that tetherin has no effect on the release of the Influenza A virus.

4.4.3 The effect of tetherin on the release of influenza B virus

Next, the effect of tetherin on release of wild-type influenza B virus was tested. Interestingly less influenza B virus was released from cells that stably expressed tetherin (Cherry red +), and budding was recovered by the supplement of Vpu or HA in trans (Figure 79), although these differences were not statistically significant difference (One-way Anova). The HA plasmid was used in this experiment as recent work by Chen et al., 2007 (37) indicated that the Haemagglutinin protein is responsible for influenza virus budding, and it was possible that over

138 expression of this protein may overcome any impediment (if any) caused by the over expression of the tetherin protein.

Figure 79: Influenza B yield from cells stably transfected with Tetherin. B/ V/87 virus yield from 293T cells stably transfected with a tetherin expression plasmid. These were further transiently transfected with cherry red, VPU and HA expression plasmids. Incubated for 24 hours, then infected with virus at an MOI >10, a further 24 hour incubation prior to harvest and assessment of viral titre by haemagglutination assay. This experiment was run in duplicate.

5 )

10 4 (log 3

Virus Yield PFU/ml 2

1 + - + - + - Cherry Red Vpu HA

Figure 80: Influenza B yield from cells stably transfected with Tetherin. Virus yield from stably transfected tetherin protein 293T cells (+) and transfection naive 293T (-) cells. These cells are then transiently transfected with Cherry Red, Vpu and HA plasmids. Then after a 24 hour incubation, infected with influenza B V/87 virus. This experiment was run in duplicate.

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This experiment was repeated and the titre of influenza B virus released was measured by infectious plaque assay.

293T cells were transfected with plasmids to express tetherin, the tetherin ΔGPI inactive mutant or cherry red prior to infection with B/Beijing/1/87. The cells were incubated overnight in the absence of trypsin. The supernatant was then harvested, incubated with trypsin to convert the virions to an infectious form, subjected to plaque assay in order to assess the levels of virus produced (Figure 81). There was a reduction in the level of virus produced from the cells containing the tetherin plasmid of less than a log, However, the inactive tetherin Δ GPI protein displays an almost equal knockdown of virus, therefore it would appear from these results that tetherin does not inhibit the release of influenza virus. Unless we assume, as previously argued that the tetherin Δ GPI protein is able to form a dimer with fully functioning tetherin. Together they could be responsible for the reduction in influenza B observed. However, whilst the yield from the cells transfected with the cherry red plasmid is higher at 5x104 PFU/ml, this difference is not significant in a one-way Anova or in any statistical post tests.

4 4 4

5 2.67x10 3x10 5x10 )

10 4

1 VirusPFU/ml (log 0

Tetherin Cherry Red GPI Tetherin  Transfected Plasmids

Figure 81: Influenza B yield from cells transiently transfected with Tetherin. B/Beijing/1/87 virus yield from 293T cells transiently transfected with tetherin, tetherin Δ GPI and cherry red, incubated for 24 hours, prior to infection with the influenza B virus. Viral titre was assessed by plaque assay. This experiment was performed in triplicate.

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Clinical isolates of influenza B viruses were often unable to grow in HeLa cells (in one study 70% influenza B viruses grew in MDCK cells compared to 40% in HeLa cells) (389). One explanation for this would be that HeLa cells possess a factor that blocks the release of influenza B viruses; the high level of tetherin expressed in these cells indicated this protein could be a candidate. However, influenza B Beijing virus was released from HeLa cells in our hands, and there was no change in the levels of influenza B released from HeLa cells when either the control cherry red, Vpu and HA proteins were transiently expressed. Nor was any difference observed when the tetherin protein expression was increased transiently by transfection of the tetherin plasmid (Figure 82).

Figure 82: Influenza B yield from HeLa cells transiently transfected with tetherin. HeLa cells transfected with Tetherin (+) or Cherry Red (-), then co-transfected with Vpu, M2 and additional Cherry Red expression plasmids as a control. Incubated for 24 hours prior to infection with B V/87 virus, a further overnight incubation prior to harvest and assessment of viral titre by Haemagglutination assay. This experiment was run in duplicate.

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4.5 Discussion

Several groups have attempted to discern the involvement (if any) of the ESCRT proteins within the late stages of the influenza viral replication cycle, to date none have found a role for this pathway (25, 36). Our investigations would support the conclusion that there is no involvement of the Tsg101 and Alix components of the pathway, though the latter could be replaced by other proteins that possess a Bro-1 late domain. Nor is there evidence that the WWP1 ubiquitin ligase is involved, though this role could be replaced the other ubiquitin ligases that have been implicated in interactions with viral late domains (Nedd4, LDI-1, 2, BUL1, WWP2 and Itch) (18) or one that has not yet been elucidated to have an interaction with late domain motifs. The small difference in the level of virus produced when the ATPase inactive form of VPS4 E223Q is used instead of the ATPase active form would indicate that an ATPase may be involved in influenza B virus particle release. However, were it feasible, a stable cell line expressing the defective dominant negative would be a better method of measuring any viral knockdown as the number of cells expressing the dominant negative form of the protein and also being infected would be higher, therefore any variations in virus levels produced would be more readily detected.

The effect of the Vps4 ATPase activity could be independent of the ESCRT pathway function as we know that the budding of influenza is an ATP hydrolysis dependant process (146) though in this Hui & Nayak research paper they also indicated through the use of inhibitors that the ATPase dependent Na+/K+ ATPase ion pump and ubiquitin ligases (through the use of proteosome inhibitors that deplete the levels of free ubiquitin) have no effect on the budding of influenza A WSN virus. They argue instead that the ATP dependence could instead be due to its contribution to membrane viscosity at the cholesterol rich lipid rafts.

The work carried out by Hui et al., 2006 on the viral late domain in the M1 protein has been retracted (145). This is mainly due to the discovery that many of the supposed mutant viruses described in the manuscript were in fact contaminated with the wild-type viruses. However, it has been suggested that the associations with components of the ESCRT pathway specifically Vps28 and M1 do exist, even if the evidence of the YRKL being a late domain on the influenza M1 protein is less conclusive than originally thought. The possible association of the Vps28 with the influenza B virus’ M1 protein has been tested using co-immunoprecipitation experiments; However, they have been unsuccessful, communication with another laboratory

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(Paul Digard, Cambridge) indicated an in-house generated antibody rather than the commercial version available was required to detect the influenza A virus M1:Vps28 interaction. So it is still possible that this component of the ESCRT pathway may be involved in viral budding. This is surprising as Vps28 is a component of the ESCRT I and is complexed to Tsg101 and it curious that the Tsg101 dominant negative did not hamper the actions of Vps28 in supporting influenza assembly.

There are quite a few alternative host pathways and proteins that could now be investigated to see if they are responsible for the assembly and budding steps of the Influenza A and B viruses:

Cdc42 is a member of the Rho family of GTPases (reviewed by Ridley, 2006); they act upon the membrane-associated actin cytoskeleton. Cdc42 in particular can stimulate the production of lamellipodia (broad sheet-like protrusions with branching actin filaments) and filopodia (finger- like protrusions with parallel bundles of actin filaments) from the plasma membrane.

Cdc42 is know to contribute to actin mediated vesicle transport between the Golgi apparatus and ER, whilst also regulating endocytosis and exocytosis; both the transport and regulation requires interaction with N-WASP (Wiskott-Aldrich syndrome proteins) an actin nucleator which assembles the new filaments from actin monomers. In order to assemble the actin filaments N-WASP activates a stable complex of seven proteins, the Arp2/3 complex through a C-terminal verprolin-cofilin-acidic domain (VCA), which is exposed to the Arp2/3 complex through the binding of Cdc42 (294).

As previously mentioned the M1 influenza A protein has been co-immunoprecipated with Cdc42, indicating that this protein may also have a role in viral component transport and budding. Interestingly a small molecule inhibitor secramine is capable of preventing Cdc42’s association with the plasma membrane. It would be of interest to see if this is also capable of shutting down viral budding late in the virus life-cycle were it possible to source this compound (294).

An alternative pathway that could be used by in influenza virus is a Vps4 independent apical endosome recycling system (ARE) used by RSV Utley et al., 2008 (353) investigated the interaction of the Rab11-FIP2 and the RSV virus whilst budding. A dominant negative form of the Rab11-FIP2 which lacks the N-terminal C2 domain which acts to bind lipid membranes is able to block the budding of RSV and cause the retention of the RSV virus. As with influenza

143 this negative sense single stranded RNA virus buds from the apical surface of respiratory tract epithelial polarized cells (386, 393).

The Rab11 sub family of small GTPases are known to be involved in the recycling system of polarised cells, they are localised to the apical section of epithelial cells, both near the centrosome and beneath the apical plasma membrane (33). The Rab11 family in turn interacts with the actin motor protein myosin Vb and the functionally named Rab11-Family interacting proteins (Rab11-FIP’s) (66).

It would be useful to obtain or generate the dominant negative form of the Rab11-FIP2 protein and confirm if it is able to block the release of the influenza virus. The ratios of viral proteins in the absence and presence of the dominant negative or after RNAi work to knock down Rab11- FIP2 or other steps in the ARE pathway could be used. A variation on the plans initially suggested for the ESCRT and tetherin studies, had there been any initial decrease in viral production.

Alternatively as suggested in the Chen et al., review(36), it is possible that budding of the virus could be independent of host machinery other than interactions with the actin cytoskeleton (with the exception of the final pinching off step). They base this theory on computer modelling work of lipid microdomains by Reynwar et al., 2007 (292) and research into VSV by Solon et al., 2005 (327). The concentration of the viral proteins at the lipid rafts on the plasma membrane could themselves cause the curvature of the membrane causing spontaneous vesicular formation, with the terminal joining of the particle possibly being caused by the BM2 or AM2 proteins’ interaction with a host protein yet to be defined.

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Chapter 5. Investigations into the role of the NB protein in the influenza B replication cycle

5.1 Introduction

Whilst the Orthomyxoviridae Influenza A, B and C have many homologous proteins, they also display novel proteins. Influenza A has PB1-F2 and ND40, small proteins encoded in an alternative reading frame of the PB1 gene (100, 385). Influenza C encodes the HEF protein which contains both the neuraminidase and haemagglutinin functions (123). The influenza B virus segment 6 of encodes the neuraminidase protein and the NB protein from a single mRNA species separated by 4 residues with an overlapping reading frame (316) (Figure 83). This chapter will focus on attempts to find the function of the NB protein.

Figure 83: Schematic of the coding strategy for the NB and NA proteins of influenza B on segment 6. The green ATG indicates the NB protein initiation codon for translation, 4 nucleotides later the blue ATG initiates the neuraminidase protein (NA) translation.

An alignment within the NCBI Influenza Virus Resource database of the 464 full-length segment 6 sequences illustrate that the NB protein is conserved. Where there does appear to be an NB knockout sequence, due to the absence of the primary methionine that initiates the encoding of the NB , closer inspection reveals that there is no untranslated sequence 5’ of the neuraminidase initiation codon. This would suggest that these viruses were not sequenced for full-length segment 6, but just for the coding sequence of the neuraminidase, therefore these sequences should not be considered NB knockout viruses.

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Protein Virus 1 3 5 7 9 28 AAU00997 (B/Beijing/76/98) MNNATFNYTNVNPISHIRGSVIITICVS AAU94754 (B/Nashville/45/91) MNNATFNYTNVNSIFHIRGSVIITICVS AAU94774 (B/Memphis/28/96) MNNATFNYTNINPISHIRGSVIITICVS AAN39740 (B/HongKong/70/1996) MNNATFNYTNVNPISHVRGSVVITICVS AAN39742 (B/Sichuan/281/96) MNNATFNYTNVNPISHIRGSTVITICVS AAN39746 (B/Taiwan/217/97) MNNATFNYTNVNPISHIRGSVVITICVS AAN39756 (B/Texas/1/2000) MNNATFNYTNVNPIPHIRGSVIITICVS AAN39770 (B/Canada/464/2001) MNNATFNYTNINPISHIRGSVIITICVS AAN39784 (B/Oman/16296/2001) MNNATFNYTNVNPISHIRGSAVITICVS AAN39786 (B/Malaysia/83077/2001) MNNATFNYTNVNPISHIRGSVVITICVS AAN39788 (B/HongKong/1351/2002) MNNATFNYTNVNPISHIRGSIIITICVS AAN39796 (B/Beijing/243/97) MNNATFNYTNVNPISHIRGSTVITICVS AAN39798 (B/Guangdong/5/94) MNNATFNYTNVNPISHIRGSVVITICVS AAN39800 (B/Sichuan/379/99) MNNATLNYTNVNPIPHIRGSVIIAICVS AAO38877 (B/Hong Kong/330/2001) MNNATLNYTNINPISHIRGSVIITICVS AAV85966 (B/Houston/1/91) MNNATFNYTNVNSISHIRGSVIITICVS AAV85967 (B/Nashville/48/91) MNNATFNYTNVNSIFHIRGSVIITICVS AAV85973 (B/Memphis/5/93) MDNATFNYTNVNPISHIRGSVIITICVS ABC70924 (B/Nepal/1120/2005) MNNATFNYTNVNPISHIRGSIIITICVS AAU94800 (B/Memphis/8/99) MNNATFNYTNVNPISHIRGSVVITICVS AAU94802 (B/Nanchang/1/00) MNNATFNYTNVNPISHIRGSTVITICVS AAU94804 (B/Maryland/1/01) MNNATLNYTNVNPIPHIRGSVIITICVS AAV28230 (B/Finland/162/03) MNNATFNYTNVNPISHIRGSIIIIICVS AAV28232 (B/Finland/164/2003) MNNATFNYTNVNPISHIRGSIIITIYVS AAV28234 (B/Finland/190/2003) MNNATFNYTNVNPISHIRGSIIITIXVS AAV28236 (B/Finland/191/2003) MNNATFNYTNANPISHIRGSIIITIYVS AAK95904 (B/Shangdong/7/97) MNNATFNYTNVNPISHIRGSIVITICVS ABG85170 (B/Lee/40) MNNATFNCTNINPITHIRGSIIITICVS ABL77027 (B/Paris/549/1999) MNNATFNYTNVNPIPHIRGSVIITICVS ABC70978 (B/Arizona/135/2005) MNNATFNYTNVNPISHIRGSVIITICIS ABN50440 (B/Georgia/09/2005) MNNATFNYTNVNPISHIRGSVIITICVS ABN50451 (B/Singapore/35/1998) MNNATFNYTNVNPISHIRWSVIITICVS ABL76334 (B/Temple/B9/1999) MNNATLNYTNVNPISHIRGSVIITICVS ABL76829 (B/Hong Kong/553a/2003) MNNATFNYTNVNPISHIRGSIIITICVS ABN50550 (B/Iowa/03/2002) MNNATFNYTNINPISHIRGSIIITICVS ABN50561 (B/Taiwan/1484/2001) MNNATFNYTNVNPISHIRGSTVITICVS ABN50660 (B/New York/24/1993) MNNATFNYTNANPISHIRGSVIITICVS ABN50715 (B/Singapore/11/1994) MNNATFNYTNVNPISHIRGSAIITICVS ABL77280 (B/Singapore/04/1991) MNNATFNYTNVNPISHIRGSVTITICVS ACA96621 (B/Miss/UR06-0345/2007) MNNATINHTNVNPISHIRGSIIITICVS ACA65012 (B/Tenesse/UR06-0431/207) MNNATFNYTNVNPISNIRGSIIITICVS ACA33370 (B/Oregon/01/2007) MNNATFNYTNVNPISHIRGSIIITVCVS ACH86199 (B/Vienna/23/2007) MNNATFNYTNVNLISHIRGSVIITICVS BAB32604 (B/Nagoya/20/99) MNNATFNYTNVNPISHIRGSTVITICVS BAB32610 (B/Chiba/447/98) MNNATFNYTNVNPISHIRGSTIITICVS CAH04537 (B/Sichuan/379/99) MNNATLNYTNVNLIPHIRGSVIITICVS CAD11630 (B/Quebec/3/01) MNNATFNYTNVNPISHIGGSVIITICVS CAH04555 (B/Trento/3/02) MNNATFNCTNVNPISHIRGSVIITICVS

Figure 84: Alignment of influenza B virus NB protein amino acid residues 1-28. Of the 494 possible sequences only the viruses that vary in sequence between 1-28 amino acids are displayed and duplications in the amino acid sequence have been removed. Residues highlighted in green are the two asparagines which are post-translationally modified by glycosylation. The residues highlighted in blue are the two threonines required for the post–translation modification of the asparagines to occur. The residues highlighted in yellow are variations from the AAU00997 (B/Beijing/76/98) anchor sequence.

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NB is a 100 amino acid type III integral membrane protein that has a C in and N out orientation and is found abundantly at the infected cell surface (316, 317, 379). The N-terminal 18 amino acids are exposed at the surface of the cell membrane and contain two glycosylation sites at amino acids N3 and N7 that are post-translationally modified by glycosylation, through an N-X- T motif. An alignment within the NCBI Influenza Virus Resource database of the 464 full-length sequences demonstrates a conservation of these amino acid residues (7) (Figure 84). However, some nucleotide sequences displayed in the NCBI Influenza Virus database display only the sequence data for the neuraminidase protein, omitting the initiation codon for NB. There is one instance (B/Finland/607/98) where the threonine at amino acid residue 9 is replaced by an alanine, negating the N-X-T motif required for glycosylation. A BLAST of this stretch of nucleotide sequence in the non-redundant nucleotide database (nr/nt) suggests that this is an isolated instance and it would be worth re-sequencing segment 6 from this virus to confirm the variation.

There are 4 NB species produced as a result of this glycosylation, these can vary in size between 12 -60 kDa depending on the level of modification. The post-translational modifications can be single or double glycosylations and through the addition of multiple Galβ1 4GlcNA β1 3 side chains to the asparagine residues, polylactosaminoglycan modifications (379, 381). It has been proposed that the glycosylation can function as internal cell transport signalling mechanism, but when the glycosylation is inhibited by the addition of tunicamycin, cell surface transport of the NB still occurs (381).

Further NB species are evident depending upon the cell culture substrate used to generate the virus. Brassard et al., suggested that a smaller NBc (NB12) had been cleaved so as to remove the N-terminus and associated lactosaminoglycan side chains, reducing the size to 10 kDa. As there were two species of this cleaved form in eggs but only one in MDCK grown virus, it was suggested that the cleavage was facilitated by proteases found in the allantoic fluid. This cleaved form of NB was only found in the virions, not in the host cells (23). The smaller NB12 and larger NB polylactosaminoglycan modified form are both incorporated into new virions, there are an estimated 15-100 copies per virion (16, 23).

The upstream NB protein is produced at a roughly equivalent level to the NA protein on the overlapping reading frame, though there are virus strain variations in the levels of NB produced and its electrophoretic mobility (315, 316). The stability of the NB protein also varies depending upon the host cell used; HeLa cells display a half life of 1 hour, CV1 and MDCK cells produce a protein with a half life of 4-5 hours (381).

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Shimbo et al., 1996 confirmed the seminal finding by Pinto and Lamb that the M2 protein of Influenza A had an intrinsic ion channel capacity (318) but were unable to prove a similar function for the influenza B NB protein. They did find evidence of that NB could act as a stimulus for endogenous Cl- ion conductance in oocytes (319). Sunstrom et al., 1996 resuspended the purified NB protein in artificial lipid bilayers dividing NaCl solutions of uneven concentrations. They were able to state that at physiological pH the channels were selectively cation permeable, but found no evidence of proton permeability. Amantidine used at concentration 100x of that required to block M2 activity, was able to reduce but not stop the conductance (337). Fischer et al., 2000 & 2001 reconstituted the membrane bound portion of NB with peptides generated from the transmembrane portion of the protein. These were reconstituted by solid-phase synthesis then suspended on planer lipid bilayers. They proposed that fluctuations seen in the conductance levels could be directly related to the level of oligomerisation of the NB protein (80). Amantidine is capable of blocking this activity in a dose dependant manner (79). Using a series of alanine substitutions in this transmembrane region Premkumar et al., 2004 were able to pin point the gating activity to serine 20, and when this residue was substituted, the gate was permanently open (284). But as BM2 appears to be the functional homologue of the influenza A M2 protein selective ion channel protein (379), the purpose of NB’s ion channel facilities is not as yet understood.

Interestingly, the CM2 ion channel of influenza C is selectively permeable to Cl-, but also appears to have Na+ triggered proton conductance at similar levels to those observed in the NB protein (15). CM2 also possesses a single polylactosaminoglycan modification and C-in and N- out orientation (273) suggesting it could share some functional homology with the NB protein.

A study using a recombinant NB knockout virus from a B/Lee/40 backbone indicated viral replication could proceed in the absence of NB in MDCK cells, but when the same virus was used in a mouse model, attenuation was observed (116).

Subsequently, it has been posited that the NB protein initiation codon was conserved to allow the transcription or translation of the neuraminidase protein at the correct levels (380). This theory is supported by a decrease in transcription of NA when the NB initiation codon was removed or altered in position. This research However, was conducted in vitro, in recombinant constructs containing the NB/NA DNA of B/Lee/40 in CV1 cells (380).

In order to assess if the NB protein provides any selective advantage to the virus during the replication cycle, we introduced both NB knockout and viruses altered in the glycosylation sites

148 of the NB protein into the ferret. The ferret is accepted to be a closer animal model of clinical human infection than the mouse. In addition we tested the recombinant viruses in human and ferret airway cultures. We postulated that the polylactosaminoglycan modification of the NB protein might have an effect on the virus’ ability to move through the protective mucus of the respiratory system.

5.2 Results:

5.21 Influenza B replication can proceed in the absence of the NB protein.

Reverse genetics had previously been used in the laboratory by former PhD student, David Jackson to generate a set of recombinant viruses altered in the NB gene (157). One virus was generated by the removal of seven bases including the primary initiation codon and bases adjacent to the initiation codon for the neuraminidase gene (ΔATG). A virus with an NB protein of just 5 amino acids (Δ5) was made by the introduction of a premature stop codon. This generated an NB knockout without altering the amino acid sequence for the neuraminidase protein (Figure 85). As previously mention NB is a highly glycosylated protein, we postulated that the glycosylation may have a role in the protein function, a view supported by the conservation of this region (Figure 84). In virus g-1-2, the glycosylation in the N-terminal region was inhibited by converting two threonine amino acids at positions 5 and 9 to alanine. This disrupts the N-x-T motifs and prevents the glycosylation of the two asparagine residues. These substitutions in the NB coding sequence result in an isoleucine to methionine change at residue 6 in the coding sequence of the NA protein (Figure 85).

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Figure 85: Schematic of recombinant viruses altered in segment 6 sequence to disrupt the NB gene. The green ATG initiates NB coding and the blue ATG initiates NA coding. The red TAG represents the premature stop codon introduced into the Δ5 truncation mutant. The Δ5 transcript produces a truncated NB protein, with a full unaltered NA protein. The ---- represents the deletion of the NB initiation codon and sequence upstream of the NA initiation codon. The ΔATG does not allow translation of any NB protein, but does allow full unaltered NA protein. g-1-2 mutation to remove the glycosylation sites from the NB protein and cause an amino acid change Isoleucine to methionine (I6M) in the NA protein coding sequence. The underlined codons have been altered to remove the glycosylation from the NB protein, the amino acid changes this causes are represented by a red cross on the schematic.

Implicit in their creation using the B/Beijing /1/87 reverse genetics system, the viruses containing the knockout, truncated and unglycosylated mutant NB protein were able to grow and replicate in cell culture as has been described previously for the B/Lee/40 reverse genetics viruses that also lack NB (116) despite the differences between the two proteins (Figure 86).

1-18 19-40 41-100 B/Lee/40: MNNATFNCTNINPITHIRGSIIITICVSLIVILIVFGCIAKIFINKNNCTNNVIRVHKRIKCPDCEPFCNKRDDISTP RAGVDIPSFILPGLNFSEGTPN

MNNATFNYTNVNPISHIRGSVIITICVSFTVILTVFGYIAKIFINKNNCTNNDIGLRERIKCSGCEPLCNKRDDIS SPRTGVDIPSFILPGLNLSESTPN B/Beijing/1/87 Figure 86: The 20 amino acid residue differences between the NB sequence of B/Lee/40 and B/Beijing/1/87 highlighted in bold. 1-18 residues are the N-terminal region (underlined), 19-40 the hydrophobic transmembrane domain and the 41-100 the cytoplasmic region (underlined). The sites relevant to glycosylation are in green (asparagine) and blue (threonine).

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A Western blot analysis of the NB protein for the modified viruses clearly illustrated the phenotypic effects of the mutations introduced and confirmed that there was no reversion to the wild-type genotype (Figure 87). The wild-type virus displayed the unglycosylated 15kDa NB band, a band for doubly glycosylated form at 18 kDa and a large smear where the polylactosaminoglycan modifications increase the protein’s size. The lane in which the unglycosylated (g-1-2) virus was loaded contains a predominant band at ~ 15 kDa, a smaller fainter band appears above this, but there is no polylactosaminoglycan smear. There are no NB antibody reactive bands visible in cells infected with viruses where the protein has either been truncated (Δ5) or deleted (ΔATG).

R.I: 1 0.96 0.94 1.3 1 1.1 1.1 1.3

Figure 87: Western blots displaying the expression of the NB protein by a panel of recombinant Influenza B viruses. A) A549 & B) MDCK cells were infected with each of the viruses generated by reverse genetics; the A549 infected cells were harvested with cell culture lysis reagent (CCL – Promega) after 12 hours and the MDCK cells were harvested after an overnight infected using RIPA buffer. Both lysates were then run on a 15% SDS page gel. The NB protein was detected using an anti NB anti-body. Vinculin (A) or BM2 (B) were used as a loading controls. Relative intensities of each NB15 band normalized for the loading controls have been calculated using ImageJ software and expressed relative to the wild-type virus.

As NB is a surface glycoprotein, it is possible that loss of the NB protein or alteration of NB post-translational modifications could alter the size and shape of the virions produced. In order to assess this the viruses were grown in MDCK cells, filtered to remove cell debris and then passed through a 30% sucrose cushion to concentrate the virus and remove any further debris. They were subjected to processing for TEM in order to visualise the individual virions (Figure 88).

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152

Virus Average (nm) Range (nm) Wild-type 120x135 116x121 – 127x152 Δ5 84x189 37x37 - 135x135 ΔATG 100x117 63x63 – 200x183 g-1-2 93x101 91x98 – 91x106

Figure 88: Transmission electron micrographs of influenza B B/Beijing/1/87 viruses harvested from MDCK cells. (A) Virus with the NB protein deleted through truncation (Δ5). (B) Virus with NB deleted (ΔATG). (C) Virus which lacks the glycosylation sites (g-1-2) in the NB protein. Particles proposed to be smaller virus-like particles are highlighted by a blue arrow; those larger are highlighted by a green arrow. The images denoted by ii & iii are insets of the first screen capture. The table (D) gives the diameters of the different viruses (all but the g-1-2 are an average of 10 viruses, the g-1-2 is an average of only 3 due to the low virus load in this preparation)

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The wild-type virus (Figure 49) was uniformly spherical, except when in close packed formation where the shapes are distorted by the packing density of the virions. The characteristic dark ring pattern of the surface glycoproteins was clearly seen. There was a minimal degree of variation in size, though there were smaller particles, the predominant form was 120x135 nm in diameter.

The truncated Δ5 NB virus also displayed the typical spherical morphology and the characteristic ring of membrane glycoproteins as seen in the wild-type viruses. The truncation mutant appeared to display a higher proportion of smaller particles as are highlighted by the blue arrows (Figure 88A i, ii & iii ) though these smaller particles are not the predominant form. The viruses that lack the initiation codon for the NB protein (ΔATG) also displayed the smaller (blue arrows) and larger (green arrows) virus like particles (Figure 88B i, ii, iii), though the typical size was similar to the wild-type virus particle.

The viruses with the substitutions to remove the glycosylation sites in the NB protein (g-1-2) were also spherical in shape (Figure 88C).

However, the average particle size appeared smaller at ~93-101nm, possibly due to the loss of the polylactosaminoglycan modifications.

Previous work indicated that cell surface transport of the NB protein was impaired by loss of the glycosylation sites (Figure 89). This too may account for the smaller particles as the NB protein is be lacking in virions and is also not adorned with the high molecular weight sugar.

Figure 89: Immunofluorescence to stain for NB protein expression in cell infected with the wild-type or glycosylation (g-1-2) NB protein. Images kindly provided by Prof.Wendy Barclay.

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5.22 Influenza B NB deletion and glycosylation mutants display different kinetics in the ferret model.

Hatta et al., 2003 (116) found that viruses that lacked the NB protein were attenuated in the mouse model. However, they could not exclude that the phenotype was due to decreased activity of the neuraminidase gene. The mouse model is not the best animal model for studying human strains of influenza virus, and many unadapted influenza viruses replicate poorly in mice. Ferrets however, have been used as a model for influenza infection since at least 1934 (336) and viruses do not require adaptation before replication in ferrets. The symptoms in ferrets are similar to those observed in humans, namely nasal discharge, anorexia, watery eyes, otologic (ear) symptoms and fever (9).The ferret trachea and lungs have sialic acid (α2-6 or α2-3) receptor distributions similar to those found in the human respiratory tract (165, 256). In ferrets, symptoms of infection for influenza A typically last for 7-10 days, with a peak of infection 48 hours after infection, but viral shedding has been reported 6 d.p.i. (9). Seasonal influenza, such as influenza B, usually causes only a mild respiratory disease in ferrets (277).

We sought to test whether the B/Beijing/1/87 NB knockout viruses were attenuated in the ferret.

Weight matched ferrets female ferrets (age unknown) n=2 were infected intranasally with 4x105 PFU in 200 µl PBS. The ferrets infected with the truncation (Δ5) mutant were between 5- 8% heavier at the time of infection than those infected with the wild-type and g-1-2 viruses. Virus was collected daily by washing the nasal passages of the ferrets with 2 ml of PBS. The ferret handling was kindly conducted by the two members of the laboratory with home office licences for work with animals, Dr. Kim Roberts and Prof. Wendy Barclay, processing of the nasal washes was performed by Ruth Elderfield.

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Day Wt Wt(2) Δ5 Δ5(2) g-1-2 g-1-2(2)

1 + + - - + -

2 + + + + + +

3 + + + + + +

4 + + + + + +

5 + + + + + +

6 + + + + + +

7 + - - - - -

------8

Table 16: Influenza infection assessed by Directigen A&B kits. Pairs off ferrets were infected with 4x105 PFU in 200 µl PBS with each virus type: wild-type, Δ5, ΔATG & g-1-2 viruses. The nasal wash was harvested daily and tested for the presence of viral infection. BD Directigen™ kits were used to assess the presence of viral proteins (matrix and NP) in the nasal turbinate, positive (+), negative (-) until no viral proteins were detected.

BD Directigen™ A & B kits were used to assess the presence of viral antigen (Table 16), in the nasal wash. The antigen indicative of virus replication was detected 1 d.p.i in both the ferrets inoculated with the wild-type virus and in one of the ferrets inoculated with the virus harbouring the glycosylation mutation in the NB protein. There was a lag of one day before viral proteins were detected in the nasal wash of ferrets inoculated with the virus containing the NB deletion. No virus could be detected in any of the ferrets 8 d.p.i. Though one of the ferrets with a wild-type infection on 7 d.p.i. still tested positive for viral protein, the other wild- type infected ferrets and ferrets infected with the viruses with the Δ5 or g-1-2 mutant NB proteins all tested negative at this time. BD Directigen™ kits are a blunt tool and do not give levels of virus or distinguish between viral proteins and actively replicating virus. Although BD Directigen™ kits detection display a darker hue when detecting viral antigen during the peak of the infection than at 1 d.p.i. and 6 d.p.i, it is not clear how this relates to shedding of infectious virus. A far more accurate measure of viral load can be assessed by plaque assay on MDCK cells

The nasal turbinates were centrifuged to separate out ferret cells which were stored in RNA protect (Qiagen) for later analysis once suitable ferret cytokine assays are developed. The remaining supernatants were subjected to plaque assay on the day of harvest to prevent viral loss during the freeze-thaw process.

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Influenza virus infection in vivo typically has a biphasic curve of infectious viral shedding (32) and this was observed for all three influenza B viruses infecting the ferrets (Figure 90). The titre of virus produced in the nasal turbinate for the truncated protein (Δ5) was over a log lower at 1 d.p.i. than that of the wild-type virus, although the following day the peak titre shed by animal infected with the Δ5 virus exceeded that of the wild-type virus (8.1x104 vs 5.5x103 PFU/ml respectively). The viruses with NB protein that lacks the glycosylation sites (g-1-2) was attenuated for the first peak of infection, but achieved wild-type levels of virus by 3 & 4 d.p.i. This indicates that the NB protein may confer an advantage to virus in the respiratory tract early in infection. The lower viral titres for both the viruses with the mutated NB proteins mirror the results seen with the BD Directigen™ immunoassay. Using a two-way Anova there was a statistically significant difference between each of the groups (p=0.002). The post tests indicate that the difference is generated in variance between the virus types i.e. Wt vs Δ5 or Wt vs g-1-2 or Δ5 vs g-1-2, rather than within the virus types (i.e. Wt vs Wt). The variance occurs at day 1, 2 and 4.

6 wt wt

) 5

10 5 4 5 g-1-2

3 g-1-2 Virusyield

PFU/ml (log 2

1 0 1 2 3 4 5 6 Time in Days

Figure 90: Shedding of recombinant viruses modified in the NB protein in nasal washes of infected ferrets assayed by plaque assay. Weight matched ferrets were infected in duplicate with 4.5x105 PFU of the wild-type (Wt), NB truncation (Δ5) and glycosylation mutant (g-1-2) viruses. The nasal passages of the ferrets were washed daily with 2ml of PBS. The viral titres of the nasal washes were assessed daily by plaque assay on MDCK cells.

The weights and subcutaneous body temperatures of the ferrets were taken daily to assess the clinical effects of virus infection. Influenza B tends to be a mild disease in ferrets (277), indeed there was no significant difference in the temperatures of the ferrets that were infected with

157 the wild-type or the truncated and glycosylation mutant viruses (Figure 91). Nonetheless, small increases in temperature were observed during the infection that coincided with the peak days of virus shedding and were greatest for those ferrets infected with wild-type virus. Thus, the ferrets infected with the wild-type virus had an increased temperature at 1 d.p.i. (39.3°C & 39.1°C); as did the ferrets infected with the glycosylation mutant (38.6°C & 35.9°C, the latter ferret exhibiting a consistently lower temperature than the other animals.) whereas the temperature of the ferrets infected with the Δ5 NB virus peaked on the 2 d.p.i. (38.7°C & 38.7°C.)

105.0 wt 5 102.5 g-1-2

100.0

Temperature 97.5 as a %as of ofDay infection -3 -2 -1 0 1 2 3 4 5 6 7 Day post infection

Figure 91: Temperature changes in ferrets infected with recombinant viruses modified in the NB protein. Temperature readings of the ferrets infected with 4.5x105 PFU the viruses with wild-type (Wt), truncated (Δ5) or glycosylation (g-1-2) mutant NB proteins were taken daily, pre- and post-infection. The readings were expressed as a % of the temperature taken on the day of viral inoculation. The readings represent two ferrets per virus.

The body weight of the infected ferrets was monitored daily. Ferrets infected with wild-type virus lost weight on 2 d.p.i., the day after their peak of virus shedding and increased temperature. One of the animals experienced 8% weight loss. In contrast, none of the other ferrets lost weight. Indeed, the ferrets infected with the virus containing the deleted Δ5 NB protein gained weight during the course of the viral infection. However, the difference in weight variation was not statistically significant between animals infected with the wild-type of the other viruses. When compared to pre-infection weights, there was also no significant difference. This is typical of influenza B ferret infections which are not severe enough to cause any significant weight change (Figure 92).

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110 wt 5 105 g-1-2

100

Weight as Weight % as ofof day infection -3 -2 -1 0 1 2 3 4 5 6 7 Days post infection

Figure 92 : Variation in weight of ferrets infected with recombinant influenza B viruses that differ in the NB protein. The Ferrets inoculated with 4.5x105 PFU wild-type, Δ5 and g-1-2 viruses in duplicate. The ferrets were weighed daily pre- and post-infection. The weight change is expressed as a percentage of the ferret's weight on the day of inoculation. These readings are an average of the weights of the two ferrets inoculated per virus.

Because we were surprised by the increase in virus shedding from ferrets infected with mutant virus at day 4 (Figure 90), the possibility that reversion of the NB mutants had occurred in vivo was assessed. Plaque picks of virus shed 4 days post-infection were taken. This virus was amplified MDCK cells, the lysate of which was run on a SDS Page gel, the PVDF membrane was probed for the matrix and NB proteins.

All the shed viruses retained their original phenotype (Figure 93). This suggests that the advantage proffered by the NB protein early in infection does not exert a strong selection once the infection is established. Alternatively, reversion may not be observed so quickly, for example the PB2 protein exerts a strong effect on the efficiency of virus replication in mice. Viruses with PB2 residue 627 as E are only weakly amplified and do not cause disease. The 627 E→K reversion in mice was observed at between 5-15 passages (190).

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Figure 93: Western blot of NB expressed in cells infected by viruses harvested from ferrets 4 days post-infection. Nasal wash was harvested from the ferrets infected with wild-type (Wt), the glycosylation defective (g-1-2) and truncated (Δ5) NB protein viruses 4 days post-infection and virus was plaqued on MDCK cells. Isolated plaques were picked and grown up on MDCK cells. 24 hours after infection the cells were lysed with RIPA buffer and the lysates analyzed by Western blot for M1 (upper panel) and NB (Lower Panel).

5.2.3 Influenza replication in the absence of NB is attenuated in Ferret Airway Epithelium cell culture.

Figure 94: Schematic of Ferret Airway Epithelial cultures. There is a permeable interface between liquid and cell layers. The cells themselves differentiate into ciliated and non-ciliated cells, which include goblet cells. These cultures have the capacity to produce a mucus layer on the apical surface.

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Ferret Airway Epithelia cell cultures (Figure 94) were constructed from freshly harvested ferret tracheal cells. The trachea was treated with pronase, trypsin and antibiotics to dislodge the cells and prevent the carry over of infectious contaminants. The cells were harvested and further treated with DNAse I and the fibroblasts were removed. The remaining cells were seeded onto collagen treated transwell plates, which were immersed in media both apically and basally. Once the cells formed a suitable air liquid interface (ALI) that is able to restrict the flow of liquid from the basal level, the media was altered to promote the differentiation of the cells to ones which resemble those found in the respiratory tract. Ideally the cells are multi- layered and columnar, with the surface layer differentiating into ciliated cells (Figure 95). The ferret airway cultures used in these experiments did differentiate and produced cilia, but visual inspection of the intact culture by light microscopy indicated that the multi-layer columnar cells were thinner than those observed in the human version seen in the cross section in Figure 95. As the cells were able to produce mucus typical of a respiratory tract, the apical surface was washed with PBS to prevent a mucus build-up that could restrict the cell interface with the air.

Figure 95: A cross section of a Human Airway Epithelial cells culture. (Courtesy of Dr. Holly Shelton, Imperial College London)

Initial attempts to generate ferret airway cultures (FAE) resulted in monolayer cultures that possessed a very low level of ciliated cells. However, these were used to check that the cultures were permissive to viral infection and if there were any difference in growth kinetics between the wild-type and a virus lacking the NB protein (ΔATG) in this complex cell system.

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The cultures were found to be permissive to infection by influenza B virus and displayed a clear difference in growth kinetics between the viruses with and without the NB protein that were significant by a two-way Anova p=0.003. At 36, 48 and 60 hours post-infection, the p-values are <0.001, <0.001 and <0.01 respectively (Figure 96).

7 wt 6

) ATG

10 5 4 3

Virus Yield 2 PFU/ML(Log 1 0 0 12 24 36 48 60 72 84 96 108 Time in Hours

Figure 96: Replication of recombinant influenza B viruses that differ in NB in Ferret Airway Cell Cultures: The FAE were infected at an MOI of 1 with wild-type (Wt) (in duplicate) or NB deletion mutant (ΔATG) (in triplicate) recombinant viruses. The cultures were washed with PBS prior to infection, then washed with PBS post-infection. The viruses produced during cell culture replication were harvested by washing the cultures with 200 ul PBS, the viral titre was assessed on MDCK cells.

Next the panel of viruses altered in the NB protein were used to infect ferret airway epithelial cell cultures with a higher proportion of ciliated cells and mucus. These cells were also permissive to virus infection. Two separate infections of the same batch ferret airway cultures were performed; the initial set as soon as the majority of the surfaces cells appeared to be fully ciliated (Figure 97A), the second set 9 days later (Figure 97B).

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6 wt 5

) 5 10 4 ATG g-1-2 3

Virus yield PFU/ml (Log 1

0 0 12 24 36 48 60 72 84 96 108 Time in Hours

7 wt 6

) 5

10 5 ATG 4 g-1-2 3

Virus yield 2 PFU/ml (Log 1 0 0 12 24 36 48 60 72 84 Time in hours

Figure 97: Differentiated and ciliated ferret airway culture infections with recombinant influenza B viruses. The FAE cultures were washed with PBS then infected at an MOI of 1, the cells were incubated for an hour at 33˚C, then washed with PBS and incubated at 33˚C. Time points were taken at 12 hour intervals by the addition of 200 µl PBS and incubation for 30 minutes. The harvested virus was stored at -80˚C for later plaque assay titration. A) Freshly differentiated cultures (in triplicate) (B) Cultures incubated for a further week, in duplicate.

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The wild-type virus replicated to a higher level than any of the 3 viruses modified in the NB protein. In a two-way Anova there was a significance difference between the replication of the viruses in the first experiment (Figure 97A) p=0.0001. The ΔATG virus and the g-1-2 virus showed approximately 2 logs decrease in replication. The Δ5 virus growth kinetics were less attenuated, though statistically the difference was significant at the 72 hour time point when compared to the wild-type (Figure 97A). In the second set of ciliated FAE cell cultures (Figure 97B), a clear difference between wild-type virus and all of the viruses with altered or deleted NB was observed 36 hours post-infection, which became significant in post test by the 60-72 hour time points (<0.05 -<0.001 respectively) though overall in a two-way Anova there was no significant difference.

The FAE cell cultures are more representative of a respiratory tract than a confluent monolayer of MDCK cells, where all of the cells are permissive to both virus infection and replication. Not all the cells in the FAE culture are permissive to viral entry and some cell types may not yield high titre of virus. On the other hand, viral diffusion through the culture may be aided by the action of cilia. During growth in MDCK cells, the virus is allowed to accumulate in the cultures, re-infect and multiply at each round of infection, where each cell is permissive, in turn multiplying any small difference.

As attenuation occurs in cell culture as well as in the ferrets there is an indication that the attenuation is independent of an acquired immune response. Attenuation could be due to the NB protein’s interaction with the innate immune response or an alternative host defence mechanism.

5.2.4 Influenza B virus replication in FAE cells is selective for ciliated ferret epithelial cells.

Influenza B viruses like those of the influenza A genera, attach to the host cell through interactions with the host cell sialic acid moieties. The structures of these sialic acids can vary in their internal linkages and differently linked sialic acids can be found in different cell types and in different locations in vivo (345).

Sialic acids are negatively charged nine carbon monosaccharides. Often they are found on glycans linked to glycoproteins and glycolipids on the cell surface. Humans and other mammals usually contain the N-acetylneuraminic acid form. Indeed humans are unable to synthesize N- glycolylneuramininc acid (Neu5Gc) and instead digest it from food (reviewed in (257).

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These sialic acids are then added to sugar residues on the terminal sugar in a chain. The linkage facilitated by sialytransferases to galactose is of particular interest to influenza researchers. If the carbon used for the attachment is carbon 3 it gives rise to α2-3 sialic acid receptors or if it is carbon 6 then α2-6 receptors are produced. The former are predominant in birds, the latter in the upper airways of humans. Consequently avian influenza viruses have haemagglutinin attachment glycoprotein that attach to the α2-3 linked sialic acids and human and porcine viruses HA’s bind to the α2-6 sialic acids (299).

The receptor binding preference of a virus for sialic acids is further complicated by the type of sugar that the sialic acid is bound to, some viruses such as the H3N2 cannot bind when the sugar is changed from galatose to glucose (90). In humans the α2-6 sialytransferases can only attach the SA residue to type II glycan chains (Galβ1-4GlcNAc-R) reviewed in (257). The distribution of sialic acid in human airway epithelial cell cultures can be demonstrated using lectins that attach specifically to sialic acids linked in either conformation (Figure 98)

Figure 98: Expression of different sialic acid linkages on Human Airway Epithelia. Sambucus Nigra (SNA) lectin (green),or Maackia amurensis (MAA) lectin(red)were used in conjunction with an antibody to α-tubulin (purple) to stain a cross section of Human Airway Epithelia cell cultures. Picture courtesy of Dr. Holly Shelton (Imperial College London). The Green staining highlights the α-2,6 sialic acid linkages; the red staining, the α-2,3 linkages and the purple highlights the ciliated cells.

In humans, ciliated cells can express both α-2,6 and α-2,3 linkages, whereas non-ciliated cells express predominantly α-2,6 SA. It was possible to use -tubulin to distinguish ciliated cells in

165

HAE cultures (Figure 98). Attempts to distinguish the sialic acids present on the FAE cultures proved unsuccessful, possibly due to the processing during the paraformaldehyde fixation process.

Enface staining of the FAE cell cultures after infection indicated clustered foci of influenza B infection predominately around sites that were counterstained with -tubulin (red dots). This suggests preferential infection of cells that are ciliated (Figure 99A&B).

The immunofluorescence images clearly display an increase in the number of cells expressing NP protein (green) staining as the course of the infection progresses with time (Figure 99A-C). Later in infection (Figure 99C), the staining for the -tubulin is decreased, presumably because the ciliated cells have been ablated by viral infection (though the mechanical action of the repeated PBS washes used to harvest the virus at the time points may also have resulted in some ciliated cell loss). Despite the decrease in ciliated cells, there was a high level of green staining representing the viral nucleoprotein, this would indicate that the virus also infected cells that were not ciliated at this point.

Cross sections of the infected FAE cultures have not yet proved amenable to immunofluorescence staining, again possibly due to issues around the cross sectional processing of the samples, so the exact tropism of influenza B viruses in the FAE cultures awaits confirmation

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Figure 99: Ferret Airway Cultures with En face staining of influenza B virus infection. FAE cell cultures infected at an MOI of 1 with the wild-type B/Beijing/1/87 virus, were incubated for A&D) 12, B) 48 & C) 120 h.p.i. At these times points cells were fixed in 4% (w/v) paraformaldehyde, and permeabilised with 2.5% (v/v) Triton-X. Viral antigen indicative of replication was stained with -NP antibody, and cilliated cells were identified by staining with -tubulin antibody. Each primary antibody was detected using, Alexa fluor 488 (green) or 647 respectively (red). D) Enlarged and cropped version of the merged images at 12 hours post-infection, indicating the predominant co- localisation of the NP viral antigen with the cells that have also stained positive for cilia.

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The viruses altered in the NB protein also infected cells that counterstained with the α-tubulin antibody, again indicating a preference to infect cells that are ciliated (Figure 100). The bright red dots indicative of the α-tubulin staining were also depleted in these cultures by later time points, again indicating a loss of the ciliated cells (data not shown).

Figure 100: Ferret Airway Cultures stained en face for recombinant influenza B infection. FAE cell cultures were infected at an MOI of 1 with the B/Beijing/1/87 viruses with modified NB proteins Δ5, ΔATG and g-1-2. These cultures were incubated for 12 hours before fixation in 4% (w/v) paraformaldehyde, permeabilisation with 2.5% (v/v) Triton-X. Viral antigen wasstained with -NP antibody and cilia were identified with -tubulin antibody. The primary antibodies were detected with, Alexa fluor 488 (green) or 647 respectively (red).

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5.2.5 Influenza NB mutants are attenuated in human epithelial cell cultures.

In collaboration with Margaret Scull and Dr. Ray Pickles from the University of North Carolina at Chapel Hill, a series of infections were conducted with the wild-type and ΔATG NB virus on Human Airway Epithelial Cell cultures. Initial experiments (Figure 101) indicated that the virus lacking the NB protein was substantially attenuated when compared to the wild-type virus irrespective of whether the infections were performed at 32°C or 37°C. Repeated experiments on cultures derived from different individual’s tracheal tissue and using different aliquots of the virus, displayed a decreased (p=0.001) (Figure 102) or non- existent levels of attenuation (data not shown).

Figure 101: Replication of recombinant influenza B viruses altered in NB protein in human airway cultures. The cultures were incubated at 32°C or 37°C. Time points were taken at 2, 24, 48, 72 and 96 hours. The cultures were washed with PBS prior to infection, and then washed with PBS post-infection. The viruses produced were harvested by washing the cultures with 200 ul PBS. The viral yield was assessed on MDCK cells. This work was conducted by Margaret Scull.

169

9 wt 8 ATG ) 7 10 6 5 4

3 Virusyield

PFU/ml (log 2 1 0 0 12 24 36 48 60 72 Time in Hours

Figure 102: Replication of recombinant influenza B viruses altered in NB protein in Human airway culture. Three human airway cultures per virus were infected with wild-type influenza B virus or the ΔATG deletion mutant. Time points were taken at 2, 6, 12, 24, 48 and 72 hours. The cultures were washed with PBS prior to infection, then washed with PBS post-infection. The viruses produced were harvested by washing the cultures with 200ul PBS, the viral yield was assessed on MDCK cells. The samples are in triplicate. This work was conducted by Margaret Scull.

5.2.6 There is no phenotypic advantage observed by ferret passaged glycosylation knock out virus.

In the ferret model of infection, the virus containing the glycosylation mutant NB protein (g-1- 2) was initially attenuated when compared to the wild-type virus, but attained the same titre as the wild-type virus later in infection. Although we showed that the NB mutation had not reverted (Figure 93), a compensatory mutation may have occurred elsewhere in the viral genome that may have allowed the mutant virus to replicate to wild-type levels by day four of the ferret infection. In order to assess if there was any change in the growth kinetics of this virus, plaque picked viruses for the glycosylation mutant from 4 d.p.i. were amplified in MDCK cells and subjected to a growth curve in FAE cell cultures alongside a preparation of the glycosylation mutant that had not been passaged in the ferret.

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7 g-1-2 6

) Ferret g-1-2

10 5 4 3

Virusyield 2 PFU/ml (Log 1 0 16 40 64 88 112 136 Hours post infection

Figure 103: Growth curve of ferret airway culture infection by recombinant influenza B virus altered in NB glycosylation derived by infecting MDCK cells with material obtained pre- or post-ferret infection. The plaque picked virus from the g-1-2 ferret experiment (Figure 90) on 4 d.p.i. was amplified on MDCK cells then subjected to a multi-step growth curve on FAE cultures at an MOI of 1 alongside non-ferret passaged g-1-2 NB mutant virus. The data points represent duplicate samples.

The ferret passaged glycosylation mutant did not display any growth advantage when compared to the glycosylation mutant that had be grown solely in MDCK cells (Figure 103). This would infer that there was no compensatory mutation that occurred during replication in the ferret that increased the fitness of this virus at 4 d.p.i.

5.2.7 The levels of Neuraminidase at the cell surface are unaffected by loss of NB in the context of whole virus

Previous studies into the effects of altering the NB protein have suggested that attenuation observed in vivo could be due to changes in NB translation that inadvertently leads to reduced neuraminidase protein production (116, 380). The NB coding sequence primary methionine is 4 bases upstream of the neuraminidase coding region, and it has been postulated that by removing the primary initiation codon in this mRNA the ribosomes would bind less well to the initiation complex causing less neuraminidase to be produced. Indeed in a series of experiments with SV40 transcribed mRNA derived from influenza B virus segment 6 sequences, the NA levels were reduced to 43% of the wild-type by the removal of the NB initiator methionine (380).

To assess the levels of neuraminidase produced in the context of the mutations that had been engineered to the set of influenza B viruses, a further series of reverse genetics was used to

171 create viruses that contained FLAG tags in the stalk region (nt174-203/aa42-49) of the neuraminidase protein. The introduction of the FLAG epitope (-DYKDDDDK-) into the stalk caused the NB protein to be truncated by 53 amino acids, due to a premature stop codon in the NB. But as these experiments were intended to measure the effects of the sequence alterations on the NA gene expression and not on NB, this strategy was pursued.

The Barclay research group was already in possession of FLAG-tagged Δ5 and ΔATG NB viruses (157), but the glycosylation tagged virus was made specifically for this project.

In Western blot analysis the FLAG-tagged viruses expressed the FLAG (-DYKDDDDK-) motif, but the anti-FLAG antibody showed cross reactivity with cell proteins. Nonetheless, for infected cell lysates, generated from a variety of cell types, a unique band of 52 kDa was evident that represented the NA protein containing the inserted FLAG epitope (Figure 104A&B).

Relative intensities have been calculated from the Western blots for the wild-type, Δ5 and ΔATG FLAG-tagged viruses (normalised to levels of NP) (Figure 104C). The level of expression of the FLAG–tag is higher in the lysates from the Δ5 and ΔATG viruses than those from the wild- type virus. The difference though visible, is not significant (>0.05) according to a one-way Anova. This allows the inference that the respective levels of neuraminidase translated in infected cells are not significantly altered by the modifications in the sequence upstream of the NA initiation codon.

The presence of the FLAG-tag in the g-1-2-FLAG recombinant virus was confirmed by Western blot (Figure 104B).

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2.0

1.5

1.0

0.5 Relative Relative Intensities

0.0 Wt 5 ATG FLAG virus

173

Figure 104: Western blots of NA expression in cells infected by recombinant FLAG-tagged influenza B viruses altered in the NB gene. A) MDCK, 293T and A549 cells were infected with the wild-type, the Δ5 and the ΔATG – FLAG-tagged viruses (At an MOI of 3). B) MDCK cells infected with wild-type and the g-1-2 –FLAG-tagged viruses (unknown MOI). These were incubated overnight, lysed with RIPA buffer and subjected to SDS-PAGE Western blot analysis prior to probing with the (A&B) α-FLAG and (A) α-NP antibodies. The FLAG antibody has high background, but can be observed in all cell types but not in the Mock infected cells. C. Relative intensities of the FLAG epitope of the recombinant wild-type, Δ5, ΔATG viruses normalised by NP relative intensity. (Results given represent 2 blots of 3 cell types.)

In order to assess whether the mutations in NB affect the transport of NA to the cell surface FACS analysis was used.

In FACS analysis, the MDCK cells displayed high background fluorescence with the anti-FLAG antibody. The background FACS signal was minimal for Vero (Green African Monkey) cells. The FLAG-tagged viruses were used to infect Vero cells at an MOI of 1. The cells were then incubated overnight to allow one round of infection. They were fixed with 4% (w/v) paraformaldehyde, and aliquots were either permeabilised to assay for total NA expression or left intact to assess cell surface expression prior to being blocked with Bovine Serum Album (BSA) and subjected to anti-FLAG or anti-NP antibodies (Figure 105).

100 wt 5 75 ATG g-1-2

50 % MFI

0 External NA Internal NA

Figure 105: Internal and external cell expression of neuraminidase by recombinant influenza B viruses altered in NB. Vero cells were infected with an equal MOI of FLAG-tagged virus and incubated overnight. The cells were fixed and subdivided to allow aliquots to be permeabilised to measure internal NA and NP levels or left intact to measure external NA. The cells were treated with -FLAG and -NP antibodies, whose binding was detected with a FITC conjugate. The cells were subjected to FACS analysis, the NA signal was normalized to the NP signal in regards to the Mean Fluorescent Intensity (MFI). The experiment was run in triplicate, with separate infections and staining for each replicate.

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The MFI of the FLAG tag in cells infected with the wild-type and NB mutant viruses was used as a measure for the level of internal (permeabilised Vero cells) and external neuraminidase (cells with fully intact membranes). The MFI values were normalised against NP levels to ensure the level measured were proportional to the amount of virus protein made. There was no statistically significant difference between the MFI intensities of the external (p=0.998) or internal (p=0.896) samples for any of the viruses tested.

Williams et al., 1989 have reported a decrease in NA production when the NB initiation codon is removed (though the 4 bases downstream were not removed) (380). The discrepancy here could be due to strain to strain variations; William et al., used B/Lee/40 segment 6 sequence whereas this research was conducted on B/Beijing/1/87 (315). Host cell type can also be responsible for variations in expression of the 2 proteins translated from the same mRNAs. However, our work using the FLAG-tagged viruses to measure the NA expression by FACS analysis indicates there was no significant difference in NA levels that would explain the attenuation of mutant virus in vivo or in FAE.

5.2.8 The levels of neuraminidase activity in viruses with deleted or mutated NB are as wild-type levels

Although the levels of neuraminidase expression were equivalent for each of the mutant viruses, it was important to check if the activity levels of the NA protein were affected by the introduced mutations. The mutant of greatest concern was the NB protein altered in glycosylation, as the codon sequence of the neuraminidase protein was also affected by this mutation (Figure 85). There was also the possibility that the NA could work synergistically with the NB protein and changes to NB glycosylation or expression may affect NA activity in the context of virus infection.

A method of measuring NA activity was utilised that involved a lectin binding assay that would be permitted following NA activity to desialylate a fetuin (sialylated) substrate (Figure 106). This method was originally described by Lambre et al., and has been used as a measure for influenza neuraminidase activity (180, 181, 296). Initial experiments were conducted using serial dilutions of bacterial neuraminidase (Sigma) to optimize the assay conditions.

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Figure 106: A fetuin/peanut lectin/HRP sialidase assay (PNA). This requires the binding of fetuin to an assay plate. The neuraminidase cleaves the sialic acid residues on the surface of the fetuin, the peanut lectin is able to bind to the exposed sugar residues. The peanut lectin can be purchased with a HRP conjugate, which when exposed to a suitable substrate Tetramethylbenzidine (TMB) gives a blue colour. Exposure to acid fixes the reaction and allows the optical density to be measured at 450 nm. Bacterial neuraminidase was included as a positive control.

Using a 96 well assay plate , varying dilutions of fetuin (substrate) and PNA-HRP (lectin conjugate) were cross titred against a serial dilution of bacterial neuraminidase. As the 50 µg/ml dilution of fetuin with a 1 µg/ml dilution of PNA-HRP (highlighted by parenthesis) gave a good range across the bacterial neuraminidase dilutions, these conditions were selected (Figure 107).

176

0.3

0.2

OD 0.1

0.0 0 250 500 750 1000 Bacterial Neuraminidase

400

300

200

OD as OD a %as of 100 wild-type virus

0 wt E116A(198%) E116D(19%) Virus

177

Figure 107: Optimisation of the PNA-Fetuin assay for neuraminidase activity. A binding assay plate was incubated overnight with 50, 25, 12.5 or 6.25 µg/ml fetuin. 2-fold dilutions of stock bacterial neuraminidase were added; the plate was incubated at 37°C for 1 hour. Two dilutions of PNA-HRP were passed across the relevant rows, 2 µg/ml and 1 µg/ml, incubated for a further hour, incubated with TMB reagent, incubated until a blue colour developed, then inactivated with acid. The optical density was measured at 405 nm. B. Neuraminidase standards assessed by PNA assay. Fetuin/PNA-HRP assay with 2-fold dilutions of stock bacterial neuraminidase (0.01 U/µl). PNA/HRP 1 µg/ml, Fetuin 50 µg/ml processed as per Figure 106. Measured at 405 nm. In duplicate. C. Neuraminidase activity of recombinant influenza B viruses altered in NA assessed by PNA assay. 5x104 virus per well were added to a 50µg/ml fetuin plates, incubated overnight prior to processing as per Figure 106. The viruses used were wild-type (Wt), E116A and E116D, which display (198%) & (19%) of the neuraminidase activity of the wild-type virus in NA- STAR assay. The samples were in duplicate.

The readings were neuraminidase dose dependant (Figure 107) with a linear range from 16- 125 2-fold dilutions of the 0.01U/µl stock solution. To validate the assay and to show it was capable of detecting difference in viral NA activity, two B/Beijing/1/87 based recombinant viruses modified by point mutations in the neuraminidase protein at amino acid 116 were chosen. These mutations had initially been detected in influenza B viruses with resistance to neuraminidase inhibitors. and were known to affect NA enzyme activity, as previously described (157). E116A had a 198% increase in NA activity when compared to wild-type virus measured in the NA-STAR assay (157). In this PNA assay the activity increase was 350% when compared to wild-type (p=0.022). Conversely, the E116D mutant’s neuraminidase activity was 19% of wild-type in the NA-STAR assay (157), whilst using the PNA methodology, the recombinant virus displayed an NA activity of 45% compared to wild-type virus (p=0.025), indicating that the peanut lectin assay may be less sensitive to differences at the lower levels of activity (Figure 107). The Δ5 NB and g-1-2 recombinant viruses showed NA activity similar to that of the wild-type virus, indicating that there was no difference in NA activity conferred by these mutations.

When the wild-type, NB truncation (Δ5), knockout (ΔATG) and glycosylation defective viruses were all tested using the PNA assay (Figure 108), none of the mutant viruses were compromised in NA activity. In fact, all 3 mutants shared somewhat increased NA enzyme activity. The ΔATG NB virus has the highest level of neuraminidase activity across the panel with on average ~101% higher activity than the wild-type virus (p=0.02). This would suggest that any compromise in their replication fitness in vitro or in vivo was not a result of a loss of NA potency.

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225 200 175 150 125 100

75 ODas a% of

Wild-Typevirus 50 25 0 wt 5 ATG g-1-2 Virus

Figure 108: Neuraminidase activity of recombinant influenza B viruses altered in NB assessed by PNA assay. 106 wild-type (Wt), truncated (Δ5), knockout (ΔATG) and glycosylation defective (g-1-2) NB virus particles were added to a 96-well medium binding plate coated in fetuin. The plates were incubated overnight at 33°C and then processed as in Figure 106. The optical density after addition o f TMB substrate was measured at 450nm.The reactions were run in duplicate on two plates.

5.2.9 The Influenza NB mutants are able to replicate to wild-type levels in NCI-H292 cells

One noteable feature of the airway and animal models that showed significant effects of NB deletion, was their ability to produce mucus. Human Airway mucus consists of an aqueous solution consisting of a mix of lipids, glycol-conjugates, proteins, electrolytes, enzymes, anti- enzymes, oxidents, antioxidents, exogenous bacterial products, endogenous antibacterial secretions, cell and plasma derived mediators, DNA and cell debris. The mucins are tightly packed in cytoplasmic granules, highly concentrated levels of Ca2+ allow a hundred fold increases in secreted mucus in a matter of tens of milliseconds. Mucins are described in great detail in a review by Rogers et al., 2007 (297, 298).

To act as a barrier to infection the mucus forms layers; the top layer is watery below and dense above, with a thin layer of surfactant between. The mucus has viscoelastic properties conveyed by the high molecular weight mucin glycoproteins. The mucin proteins themselves form 2% of the airway mucus and are produced by the goblet cells in the epithelium and sero mucus glands in the submucosa. These are long thread like complex glyconjugates, with linear peptide backbone (apomucin), produced by the muc genes that have hundreds of

179 carbohydrates side chains are O-linked and additional N-Linked glycans. The main core are tandemly repeated serine-rich and or threonine rich regions are unique in size and sequence for each mucin which allows for a diverse range of glycoproteins. Mucins can be membrane associated (with a hydrophobic domain anchor) or secreted (intracellular secretrory granules that are released at the apical surface of the cell).The mucin fibres can be 3-10 nm in diameter and 10-40 mDa in size (320). There are aqueous spaces between the fibres that can allow the passage of molecules up to 100 nm, though the effective viscosity increases for molecules of 200-500 nm causing a 4-6 fold reduction in passage (174-176).

There are 20 mucin genes, 9 of which are expressed in the human respiratory tract (MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC7, MUC8, MUC11 and MUC13.)

The levels of mucus within the ferret or airway models could have an effect on the ability of the virus to pass through this protective barrier to the surface of the cell. To test the capacity of the wild-type and the mutant viruses to pass through mucus layers, a variety of biological systems were used; H292 cells that are able to secrete some mucins, mucus harvested from FAE cultures, human mucus and re-suspended porcine stomach mucins.

H292 cells are able to produce MUC1, MUC4 and low levels of MUC5 and MUC5AC and secrete high molecular weight glycoproteins which have N-acetylglycosamine and/or sialic acid appendages (12). Early work with H292 cells highlighted their low permissiveness to influenza infection (124). It was possible that the low degree of permissiveness may accentuate any attenuation caused by the lack of NB protein.

180

wt 8 5 g-1-2 7

) 6 10 5 4

3 Virusyield

2 PFU/ml (Log 1 0 24 48 72 Time in Hours

Figure 109: A multi-step growth curve of recombinant virus in H292 cells. Cells were infected at an MOI of 1. Wild- type, truncated (Δ5) or glycosylation defective (g-1-2) NB mutant viruses were used. Time points were taken at 24 hour intervals and the viral titres produced were assessed by plaque assay on MDCK cells. The data points represent triplicate samples.

An MOI of 1 was used to infect H292 cells to generate a growth curve (Figure 109). The wild- type, truncated NB and glycosylation defective NB protein viruses all successfully replicated in the H292 cells at this titre. However, the NB truncation and glycosylation mutant viruses displayed no attenuation when compared to the wild-type; despite the H292 cells ability to express selected mucins. It was notable that the levels of mucus visible in the FAE cells were not apparent in the H292 cells. It was possible that a wider range of mucins or higher ratio of mucus to media affects NB mutated virus growth in FAE cells.

5.2.10 The influenza NB mutants display attenuated viral entry into MDCK cells in the presence of ferret mucus

We postulated that the mucus visibly observed in the ferret airway cultures could be inhibiting the spread of the viruses that lacked the NB proteins. As mucus accumulates on the surface of the FAE cell cultures it was possible to remove this mucus and use it in conjunction with cells that do not naturally produce mucus, such as MDCK cells. Thus, the mucus was harvested from ferret airway cell cultures by washing with PBS (implicit in this, the mucus was partially diluted by PBS). The panel of viruses were incubated either with the mucus/PBS/serum free media suspension or with serum free media alone, for one hour. The inoculum was then added to the surface of the MDCK cells for a further hour, when the inoculum was removed and the

181 cells were then washed with PBS at pH 5.5. After incubation overnight in serum free media, the supernatant was harvested and subject to HA assay to assess the titre of released virus. This methodology acts as an indirect measure of virus cell entry in the presence of mucus since output virus will depend on efficiency of entry. To test whether the presence of intact sialic acid was necessary for any inhibitory effect so the mucus, it was pre-incubated with bacterial neuraminidase before addition to virus.

1024 Serum Free

512 Mucus )

2 256 128 64 32 16

Virus yield 8

HA Units (Log 4 2 1 - + - + - + - + NA (-/+) wt 5 ATG g-1-2 Virus

Figure 110: Inhibition of cell entry of recombinant influenza B inhibition by ferret mucus. The panel of recombinant influenza B viruses was incubated in the presence of 1:5 mucus (from ferret airway epithelia cell cultures) or serum free media prior to inoculation of MDCK cells at an MOI of 10. After incubation for 1 hour at 4˚C, the inoculum was removed and the cells washed with PBS, PBS pH 5.5 then PBS, then incubated overnight in serum free media. The level of virus released was assessed by Haemagglutination assay. The + symbol indicates the pre- incubation of the mucus with neuraminidase. The samples are in duplicate.

As seen in Figure 110, the titre of released viruses that lacked NB, Δ5, ΔATG and g-1-2 mutants were all lower in the presence of mucus (white bars) than in the presence of serum free media (black bars). The effect was most marked for the g-1-2 glycosylation mutant. This trend was not observed to the same degree in the wild-type virus, though there was some knockdown in virus produced when the infection was performed in the presence of mucus. When the mucus were pre-treated with exogenous NA (indicated by the + symbol in Figure 110), this treatment abrogated the inhibitory effect. For example, replication of g-1-2 virus was equal in presence

182 of NA treated mucus as it was after incubation with serum free medium, whereas in the absence of NA treatment mucus inhibited virus replication 5-fold.

5.2.11 Virus entry into MDCK cells is not affected by the presence of re-suspended Porcine Mucus

An alternative source of mucus was obtained in the form of Porcine Stomach Mucin Type II & Type III (Sigma Aldrich M2378 & M1778). These mucins are provided in a powdered form and can be re-suspended in a buffer solution (50 mM Sodium Phosphate at pH 7 and 150 mM Sodium Chloride) overnight at 4°C forming a viscous liquid. This suspension was used in a similar MDCK mucus barrier assay with the wild-type and NB deletion and glycosylation mutants. No variation was observed in the replication levels of the viruses pre-incubated in the mucins and those pre-incubated in PBS (data not shown).

These suspensions contain mucins from the stomach and but none of the other additives that would be found in human airway mucus. As mention previously, 9 mucins are expressed in the human respiratory tract (MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC7, MUC8, MUC11 and MUC13.) These overlap only with two gastric mucins MUC1, MUC5AC with the additional MUC6 also expressed in the stomach (290). The lack of inhibition observed by the viruses pre- incubated in the resuspended stomach mucins may be due to the absence of the other mucin proteins or the other components of respiratory mucus.

5.2.12 Virus entry into MDCK cells is not affected by the presence of human mucus

Small samples of aspirated human mucus were kindly gifted by the Respiratory Medicine department at Imperial College. These mucus samples were from control or asthmatic patients, therefore would be an accurate representation of the barrier viruses would have to penetrate in order to infect susceptible cells in the human airway.

Viruses were again pre-incubated in the human mucus samples prior to inoculation onto the MDCK cells. The viral titres released were assessed by haemagglutination assays.

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64 PBS 32

) Mucus 2 16

4 Virusyield

HAUnits (Log 2

1 wt 5 ATG g-1-2 Virus

Figure 111: Inhibition of Recombinant influenza B viruses altered in NB by human mucus. The wild-type (Wt), truncation (Δ5), knockout (ΔATG) and glycosylation defective NB protein viruses were pre-incubated in human mucus prior to infection on MDCK cells. The cells were incubated in serum free media overnight. The supernatant harvested was subjected to a haemagglutination assay in order to assess viral titre. The data points represent duplicate samples.

The pre-incubation of the panel of viruses in human mucus caused a decrease in yield for all of the viruses irrespective of the mutations introduced into the NB protein, when measured by the haemagglutination assay. The virus levels used in this experiment were lower than those used previously due to low levels of human mucus available, this explains the lower levels of virus released as compared with data shown in Figure 110. There was no statistically significant difference between any of the viruses or between the different treatment types.

5.3 Discussion

The loss of the NB protein from influenza B virus results in attenuation or virus replication under multi-cycle condition in animal models and in ferret airway cultures.

We postulated two theories as to why this may occur; firstly the cell types that the virus is able to bind to and replicate in whilst in the respiratory tract could be affected by the presence or absence of the NB membrane glycoprotein. To this end we attempted to visualise which cells the viruses, with and without the NB modifications, were able to bind. The en face staining of the FAE cell cultures with the α-tubulin and an antibody directed against NP indicated that the all of the viruses had a preference for the cells that were either stained with the α-tubulin or those in the immediate proximity in early time points in the infection process. Later in the course of infection the NP was detected in a wider range of cells and those cells

184 counterstained with the α-tubulin were reduced in number. The increase in wild-type virus levels compared to the viruses modified in the NB protein in the FAE cell cultures was most apparent at these later time points. There could be a correlation between these two observations. Frustratingly attempts to confirm the cell types infected were unsuccessful, possibly due to problems with histological processing of the samples.

Secondly, as there is a high level of polylactosaminoglycan modification of the NB protein, a modification also found within mucus layers, it was proposed that this could allow easier transit of the virus through the protective mucus barrier to the host cells. In order to evaluate this theory we pre-treated the virus with various forms of mucus, prior to infection of MDCK cells. The only mucus that gave results to robustly support this theory was the mucus harvested from the FAE cultures. The human mucus obtained was noted to be somewhat dilute and this may explain the lack of inhibition on virus entry that it exerted. Alternatively it has previously been reported that deep freezing and thawing can alter mucus structure and therefore viscosity (176). The human sample used had been thawed from -80°C on at least two occasions. Experimentally reconstituted mucins may have been less effective at inhibiting virus because these also display markedly different viscoelastic properties compared to freshly harvested mucin (176). Ideally, an experiment where the levels of mucus are allowed to accumulate in the FAE cell cultures during the course of an infection by not washing the FAE cultures prior to the initial infection, would help to determine whether NB has a role in countering the secreted airway mucus. Variation in mucus secretion between different HAE cultures derived from different individual could also explain the variability in attenuation of NB mutant viruses in that system. Unfortunately it was not possible to assess this retrospectively.

The differences observed between viruses that have or lack NB are not due to changes in NA expression. The caveat to this is that the PNA assay was relatively insensitive. It would be useful to assay these viruses with the widely accepted MuNANA assay to validate the results obtained with the PNA assay, before completely ruling out the effects of the neuraminidase on viral replication.

The ferret is becoming increasingly popular as a model for influenza infection, as a direct result of this more reagents to measure cytokine changes during the course of an infection will become available. Samples have been preserved in order to measure these differences. Indeed, our own laboratory has become more sophisticated in its ability to measure the effects on the ferret as the infection proceeds since these initial experiments were conducted.

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Chapter 6. General Discussion

Assumptions are made about the replication cycle of the influenza B virus based on research of Influenza A. The latter is understandably researched in greater detail due to its ability to cause a global pandemic. The similarities between the two viruses are indicative of a shared evolution, but there are subtle differences in the actions of their proteins that lead to large alterations in aspects such as host range and mutation rates.

The viral proteins themselves, whilst having a functional homology, are diverse in their amino acid composition. The polymerases are most conserved, PB1 displays a 60% homology between the A & B subtypes (373). The HA2 subunits of influenza A and B virus displays only 39% homology in its amino acids, but share a remarkably similar structure

The AM2 and BM2 proteins, which share only 14% amino acid similarity and are generated by different coding strategies, appear to display the same function. Therefore assumptions about influenza B virus’s replication cycle based on influenza A virus research should be confirmed through experimental work.

6.1 BM2 Cytoplasmic Tail

Our work was initiated to test a role for the BM2 cytoplasmic tail in viral assembly because at that time a similar role had been elucidated for the influenza A virus M2 protein. Lack of the M2 protein resulted in the loss of ribonucleoprotein packaged into virions (151, 160). During the course of our investigations, further publications were released regarding in the role in assembly of the Influenza A M2 cytoplasmic tail (156, 223, 224) and also demonstrating the requirement of the tail region of the BM2 protein for influenza B virus (150).

The aim of our research was to discover if the BM2 protein’s cytoplasmic tail shared the same assembly function as its AM2 counterpart. By generating a series of protein truncations at the cytoplasmic tail region of the BM2 protein, we found that virus could not be successfully generated if more than the terminal 5 amino acids had been removed. Indeed the virus truncated by 5 residues displayed an attenuated phenotype typified by smaller plaque sizes and a slower growth rate when compared to a wild-type virus.

Alanine substitution mutagenesis indicated that the residues at 103 and 104 in the cytoplasmic tail were important. Alanine substitutions at positions -6 (104) & -7(103) were unable to

186 support virus production and virus with glutamic acid at -6 (104) virus could not be passaged, Thus in our hands the very viruses that may have revealed critical functions for this region of BM2 were very difficult to grow and work with. Therefore we did not pursue the effects of these mutations on, for example the localisation of virus assembly at the cell membrane or the interactions of BM2 with the RNPs. Work by Imai et al., 2008 (150) indicated the BM2 cytoplasmic tail region affects both the membrane localisation and the association of the M1 protein to the membrane. They were able to deduce this from mutant viruses that lacked BM2 or were deficient in its function only because they could propagate those viruses to high titre in their BM2 expressing cell line, and then use those ‘pseudo typed’ virions to infect normal MDCK cells to study replication in the absence of BM2. Despite efforts we were unable to generate such a line ourselves. Should it prove possible later to generate stable cell lines for the BM2 protein within our laboratory, it would be of interested to generate the viruses with the alanine a position -6(104) and -7(103) using the reverse genetics system with the wild-type BM2 protein being substituted in trans, enabling further studies such as membrane association studies, morphology and virion composition. Overall these studies illustrate a problem with the reverse genetic approach taken in isolation: viruses that can be rescued may not be revealing about gene function since what ever has been altered is rather essential at least in the culture systems used. Viruses that would be the most revealing about gene function may not be rescued unless there is some way to complement the lost function in trans.

The development of a BM2 expressing cell line would also allow the generation of viruses sporting other modifications such as those that may affect the phosphorylation of the virus. The phosphorylated state of this protein was initially suggested by Odagiri et al.,1999 by way of an explanation of the double banding observed in some strains of the influenza B BM2 proteins (261).

Whilst we were able to generate single point mutations at potential phosphorylation sites, T101 and S91, double mutants could not be generated under our conditions but may have been possible with BM2 complementation. Ideally analysis of the BM2 protein phosphorylation status would be advanced from the simple 1-D Western blot/banding patterns method used in this project comparing patterns of viruses that lacked the modification sites with wild-type. In addition, confirmation by 2D-gel electrophoresis and/or mass spectrometry should be considered. As the putative phosphorylation states are proposed to be strain specific (261, 366), it would be worthwhile to look a range of BM2 proteins from different strains, to look for adaptations that may have appeared in the BM2 protein itself, or

187 in other viral proteins (specifically M1 and NP) that remove the requirement for the phosphorylation sites or those that have promoted the requirement for phosphorylation.

In the discussion section of the BM2 cytoplasmic tail chapter it was suggested that some point mutations that altered the BM2 amino acid sequence may have abrogated viability due to an affect on the nucleotide specific packaging signals of the virus particle. These cis-acting motifs allow the selective incorporation of influenza A virus segments into new virions, (6, 96, 102, 193, 203, 343, 371, 391), some may be strain specific (102, 149). Locating the packaging signals in influenza B virus has not yet been achieved but could be, in addition to of academic interest, of benefit during vaccine production, especially should reverse genetics be used to generate a vaccine strain with surface spike glycoproteins from a different origin from the backbone virus strain perhaps even mixing influenza A and B viruses to make a single vaccine strain with dual immunogenicity, similar to that created by Muster et al., 1991, where the non-coding regions of the influenza A NA gene were replaced by those of the influenza B NS, producing a viable but attenuated virus (240). Or alternatively the modification of the packaging signals in the gene segments of the donor glycoproteins could allow for quicker construction of the virus at the point of assembly, therefore producing a fitter virus.

6.2 BM2 translation and the 18S ribosomal subunit

The Pentanucleotide motif used to initiate the production of the BM2 protein is not a strategy utilized by other types of influenza viruses. Indeed to date it has only been found in a mycovirus CHV1, the 5 non-LTR (long terminal repeat) retrotransposons and possibly the mouse embryonic RNA splicing variant of glutamic acid decarboxylase (- UGAUG-)(111, 167, 340).

Research by Powell et al., (282, 283) indicated the presence of TURBS upstream of BM2 in the M1 coding sequence. These TURBS regions are complementary to a stretch of sequence is the 18S ribosomal subunit responsible for the translation of the BM2 mRNA in proteins. We collaborated with Powell et al., to generate viruses modified in one of the proposed TURBS regions and found that these mutations had a clear effect on BM2 production the fitness of the virus as a whole. Analysis presented here found additional TURBS sites in some strains of the influenza B virus. Future research could compare the effects of engineering these TURBS motifs into segment 7 mRNA of B/Beijing/1/87. It may be possible to rescue the -30A→G mutant effectively removing the deleterious effects of the downstream TURBS motif.

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Looking at the predicted secondary structures of the viral mRNA, it would be a natural extension to create compensatory mutations in the complementary strands of the secondary structure; many of these However, would affect the M1 protein coding sequence. The 5 plasmid transfection assay may prove a better method to analyse these modifications, rather than trying to incorporate them into whole virus using the reverse genetics system, as small changes in the M1 protein may skew the viral fitness beyond the effects caused by altering translation of the BM2 protein.

Investigations into the evolution of the TURBS motifs within the influenza B viruses might reveal something about the influenza virus’ evolution, for example by plotting if the number of potential TURBS sequences increase, decrease or stabilize on an evolutionary clock. It could be of interest to see if the influenza A virus strains have vestigial TURBS, or if requirement for the TURBS evolved after the influenza A and B viruses diverged.

The apparent independent evolution of a possible TURBS sequence in the CHV1 transcripts would indicate a functional role for the TURBS motifs. This supports the case for their presence in the sequence of segment 7 of influenza B and their functional role in the transcription of the BM2 protein. The presence of the pentanucleotide sequence in the 5 retrotransposons non- LTR is a particular interest as retrotransposons are considered to be an evolutionary ancestor of the retroviruses. The fact that a further 3 retrortransposons possess the -AUGA- stop:start complex, but 17 identified elements do not possess either the pentanucleotide or –AUGA- could argue an independent evolution of this trsnacription mechanisms (167).

6.3 Tetherin and proteins of the ESCRT pathway’s involvement in the influenza B virus’ replication.

As previously stated, assumptions are often made that influenza B will use the same methods as influenza A in order to replicate, and that its proteins possess the same functions. These assumptions are occasionally stretched further: If one virus uses a seemingly tailor-made system of leaving a host cell it would appear logical that other viruses would use the same system. Indeed, for the ESCRT pathway this has proven to be the case for an ever expanding list of viruses (LIST TABLE), albeit their entrance into the pathway can vary depending on motifs within viral protein domains and their ESCRT interacting partners (HIV-1 and Tsg101 or RSV and Vps4)

The results from this thesis and from a number of other laboratories would indicate that this pathway is not however, utilized by the influenza viruses. Our research was hampered by the

189 need to transfect the host cell prior to viral infection, and that host cell line itself displaying a low level of permissiveness to viral infection, ensuring that the yield would be low and any differences could potentially be masked by the untransfected cells. It would be of benefit to create an inducible stable cell line with these plasmids that produce proteins composed of a fused fluorescent protein and an inactive form of the protein, which together have a dominant negative effect, blocking viral production.

With the Tetherin protein we had the benefit of a stable cell line and all indications were that this protein was also not involved in influenza A or B viruses release from the cell surface. There are alternative promising candidates for host proteins that control influenza budding. Using the assumption that influenza may use as pathway utilised by other viruses, Rab-F11, is, one of the strongest. Indeed towards the end of the study period for this thesis we requested reagents to test the role of this complex in influenza budding but they were not forthcoming. However, another contender could be cellular serine-threonine protein kinase D (PKD), implicated in the Golgi-to-membrane transport of Herpes Simplex Virus type1 Capsids. This route is traditionally basolateral, but in neurons, HSV-1 buds from the apical membrane and thus at least in one system shows the same polarity as influenza (291).

6.4 The function of the NB protein.

The novel NB protein of influenza B virus has no known functional homologue in the A form of the virus. Characteristics of the protein have been described previously; these include its ecto, transmembrane and cytoplasmic domains; its polylactosaminoglycan modifications and the debateable ion channel activity (16, 23, 80, 284, 337, 379, 381). Surprisingly, this protein is not required for successful virus replication in cell culture, though it does seem to proffer an advantage in vivo in both mice (116) and ferrets (chapter 5).

This project was able to confirm that recombinant viruses modified in the NB protein, either through deletion of the NB protein or by the removal of NB’s glycosylation sites, were attenuated in the ferret. Recombinant virus (with the deleted or modified NB) infections of the airway cultures generated from human or ferret trachea, also displayed attenuation.

6.4.1 Neuraminidase

Some groups have suggested that the sole function of the NB protein is to regulate the amount of neuraminidase produced or transported (380). That would appear at odds with membrane spanning structure of the NB that indicates a distinctive role. To discover if the levels of neuraminidase were affected by the alterations to the NB gene we conducted a series of

190 experiments measuring both the NA expression levels (using FLAG-tagged viruses) and the activity of the NA protein, both indicated that the levels of NA produced were no lower in the mutated recombinant viruses than that containing the wild-type form.

6.4.2 Mucus

We postulated that the highly glycosylated NB protein may ease the passage of the virus through the similarly glycosylated mucin fibres. Previous experiments by Olmsted et al., 2001 investigated the ability of Norwalk virus (38 nm), Human Papilloma virus (HPV) (55 nm) and Herpes virus (180 nm) to move through human cervical mucus. Both the Norwalk virus and the HPV were able to move through the mucus as rapidly as they were able to move through a saline solution, whereas the herpes virus’ movement was reduced 100-1000 fold (266). This contrasts with later work by Lai et al., 2007,2009 who were able to move larger nanoparticles (200-500 nm) through mucus with only a 4-6 fold reduction (174). It is possible that some intrinsic property of the herpes virus retarded its flow in the viscous mucus, indeed the herpes virus appeared to co-localised to the mucin fibres (266). The influenza virus varies in morphology, influenza A viruses can be spherical (50-120 nm in diameter) or filamentous (20nm in diameter and 200-300 nm in length) when initially isolated, influenza B, spherical, whereas influenza C is predominantly filamentous. It would be interesting to investigate the ability of influenza viruses (A & B) to move through fresh mucus using the FRAP (Fluorescent Recovery After Photobleaching) and MIP (Multi Image Photography), the methods used for Norwalk virus, HPV and Herpes Virus. The modified and deleted NB recombinant viruses could also be subjected to FRAP or MIP observations in mucus for comparison with wild-type virus.

It could also be of interest to compare the movement of the different viruses through mucus from a variety of animals. Human and dog mucus share similar properties (vicocosity- the gels resistance to flow, and Elasticity – the ability of the gel to recover its original shape, collectively know as rheology), but interestingly ferret or rat mucus are less similar in these parameters; the viscosity of rat nasal mucus is 10-fold less than human nasal mucus (176, 347). The human adapted influenza B virus may show a preference for movement through human mucus.

Because of the presence of the BM2 protein in the influenza B virus and its ability to work as an ion channel, the gated selective permeability of NB for cations (337) has mostly been disregarded in this study. But changes in ionic concentrations can alter the viscosity of the mucus, thus increasing the ionic concentrations decreases the viscosity (176). It is unknown as to whether a gradient exists between the virus core and the external environment sufficient to cause localised changes that could alter mucus viscosity and ease virus transit, or whether

191 expression of the NB protein on the apical surface of the cell could have a similar function, but this is an area that could be investigated.

In a similar vein, the NB protein could be involved in amiloride-sensitive epithelial Na+ channel (ENac) regulation as has been postulated for the influenza A M2 ion channel (185). Possibly in conjugation with, or independent of, the action of the BM2 ion channel protein.

Interestingly despite having some natural glycosylation consensus motifs in its ectodomain, the influenza A M2 protein, unlike NB is not usually glycosylated. However, Holsinger et al., 1994 created a M2 protein with two mutations A30T and a valine at any position between 26-29 and this modified M2 protein became glycosylated and displayed polylactosaminoglycan modification (130). If this M2 is viable as a recombinant virus, it could prove interesting to see if it affects virus transit in mucus or any of the other options discussed below.

6.4.3 Other possible roles

6.4.4 Protection

As NB is a surface glycoprotein with extensive and potentially bulky modifications, it is possible that the NB protein may have a protective role, for example aiding the longevity of the virus. This would be of advantage when the virus is attempting to transmit between hosts and is required to survive in the environment. Transmission experiments recently established in the influenza laboratory would be a useful measure of this capacity. Alternatively one could test the effects on wild-type or NB deleted viruses of simple exposure to environmental conditions prior to infection of cell cultures or airway cultures.

6.4.5 Auxiliary receptor

Nucleolin is a multifunctional protein expressed abundantly in the nucleolus. It is involved in the regulation of RNA binding proteins, ribosome biogenesis, transport between the nucleus and cytoplasm and interactions with various RNA/DNA/Protein targets and is transported to the cell surface through a trans-Golgi-ER route (126, 235). Interestingly is it also thought to be an additional cell surface receptor for HIV-1 (28, 208), Coxsackie B virus (59) and HPIV-3 (21). Despite its lack of a traditional transmembrane structure it has been reported on the surface of cell membranes. Glycosylation is crucial for the cell surface expression of nucleolin, and is localisation appears to be dependant upon an actin substructure (137, 196).

HPIV-3 is a respiratory disease that predominantly uses sialic acid receptors for initial attachment to the cell surface, then heparan sulphate and nucleolin for viral entry. Like the

192 influenza viruses, HPIV-3 buds from the apical plasma membrane, the region of polarised human lung epithelial A549 cells that is enriched in nucleolin (21).

What is most interesting is that nucleolin appears to have binding affinity for the polylactosaminoglycan modification: Thus nucleolin expression on the external surfaces of macrophages conveys upon them the ability to bind the CD43 protein when it is capped with polylactosaminoglycans on the oxidised cell surfaces (70, 126).

It is possible that nucleolin expressed on the apical region of the airway cells could interact with the influenza B virus through the polylactosaminoglycan modified NB protein, acting as a co-receptor to the sialic acid moieties. In cell culture there may be sufficient sialic acid receptors that any advantage proffered by the auxiliary receptor is negligible, but when in the harsher environs of the animal model or ferret airway culture system, the additional affinity provided by the receptor could proffer an advantage. On the other hand, the presence of the polylactosaminoglycan would However, also proffer a recognition point for macrophages, presumably aiding in viral destruction and clearance. If this were the case, the viruses lacking the polylactosaminoglycan (either g-1-2 or the NB deletions) would be more virulent in vivo, an outcome not observed in the ferret experiments.

In order to test the hypothesis that the NB interacts with nucleolin as a secondary receptor, it should be possible to reduce the levels of sialic acid on the MDCK or A549 cells (by pre- treatment with neuraminidase or culture in the presence of lectins (142)), then infect with the wild-type and recombinant NB modified virus and observe there growth kinetics in the more stringent environment. Co-immunoprecipitation experiments could be used to probe for an interaction between NB and nucleolin. Midkine, a cytokine able to block HIV-1 entry in cell culture by binding with low affinity to nucleolin (29, 136, 305) may be able to block/reduce influenza B replication. It would also be interesting to stain FAE and HAE cell cultures for nucleolin on the cell surface; it is possible that the nucleolin provides an additional cell selection criterion for entry of influenza B infection. Antibodies to nucleolin could be used to see if is possible to block viral infection in the airway cells. A similar methodology was successful for discerning the role of nucleolin in HPIV-3 cell entry (21).

Nucleolin is a shuttling protein that moves around the cell between nucleolus and plasma membrane. It may be involved in the transport of NB to the surface of the cell. Indeed when the glycosylation sites, therefore the polylactosaminoglycan modifications, were removed we observed loss of cell surface transport of the NB.

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Other viruses possess proteins with polylactosaminoglycan modifications; Lactate dehydrogenase-elevating virus (LDV) of mice’s VP-3P ectodomain displays these modifications, in fact the presence of 2 or 3 chains conveys the virus presence or lack of neurovirulence (43, 44, 191). Bluetongue virus’ non-structural proteins NS3 and NS3A have single asparagine glycosylation site on their extracellular domains, this site is reported to have polylactosaminoglycan modifications. The NS3 protein is implicated in intracellular transport in connection with the ESCRT pathway (through Tsg101 and a late domain in the N terminus). This is proposed to allow non-lytic virus budding prior to the eventual lytic release of the majority of the virus particles (35, 387). There has been no function assigned to the polylactosaminoglycan modification. Neither of these two viruses provides any explanations for the mechanism of action for the NB protein.

The Human respiratory syncytial virus (RSV) SH protein has also been shown to display a polylactosaminoglycan modification (4). SH has been implicated in preventing TNF-α signalling of NF-κB, which in turn inhibits apoptosis (86). Interestingly, this protein forms a pentamer that becomes a cation selective ion channel (93). The similarity between the two proteins (surface protein, respiratory virus, polylactosaminoglycan modifications, and cation selective ion channel) would imply a similar function. It would be worthwhile to test the TNF-α induction of influenza B viruses with and without the NB protein and similarly to define the SH proteins ability to move through mucus or associate with nucleolin.

6.5 General future research into Influenza B

Influenza B virus causes a comparatively mild disease. The antigenic drift and reassortments between different lineages causes a slow positive selective pressure on its main antigenic epitopes. The presumption has been made that the virus originated in birds and transferred to a human host anywhere from 4000 to 104 years ago (259, 338, 373). At this point, the virus would be under a high level of selective pressure in order to adapt to the new host. It is interesting to speculate on how many of the characteristics of the influenza B virus are due to host adaptation and how many are elements of the ancestral virus? These elements could have been lost in the reservoir of influenza A viruses circulating in birds, a more successful dominant strain eradicating viruses with the traits that we now observe in influenza B & C. Yet redundant features still remain, both the influenza A and B HA proteins have vestigial esterase domains, the fully functioning form is still found in the Influenza C HEF protein (362). Alternatively did the influenza C virus develop the esterase function therefore found the NA protein disposable, effectively removing an epitope for immune recognition? Using influenza A

194 reverse genetics viruses have been created with the influenza C HEF protein instead of NA and HA (94). There is no doubt that fundamental changes occur as the virus transfers from an avian to a human host. As avian influenza viruses adapt to human hosts they decrease the number of CpG dinucleotides they possess, this correlates with the higher number of CpG dinucleotides in birds than in humans. Influenza B has half the number of CpG dinucleotides as the highest number observed avian influenza A viruses (110).

For purely academic reasons it would be interesting to observe what adaptations would the influenza B virus undergo with repeated passage in an avian host? Is the virus capable of replication in an avian host or even a swine? Would the influenza B virus develop characteristics typical of the A viruses or would the NB protein be maintained? Would Influenza B slow adaptation in the human host eventually push it into having milder and milder symptoms as are found in influenza C, or will the adaptations push the virus into a more virulent form?

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Chapter 7. Materials and Methods

7.1 Plasmids

Segment Protein Vector Comments Source 7 BM2 pPolRT Rescue plasmid containing the This project B/Beijing/1/87 virus segment 7 cDNA. Deletion mutant 5 aa residues from the end of BM2 protein, shortens cytoplasmic tail 7 BM2 pPolRT Rescue plasmid containing the This project B/Beijing/1/87 virus segment 7 cDNA. Deletion mutant 6 aa residues from the end of BM2 protein, shortens cytoplasmic tail 7 BM2 pPolRT Rescue plasmid containing the This project B/Beijing/1/87 virus segment 7 cDNA. Deletion mutant 7 aa residues from the end of BM2 protein, shortens cytoplasmic tail 7 BM2 pPolRT Rescue plasmid containing the This project B/Beijing/1/87 virus segment 7 cDNA. Deletion mutant 8 aa residues from the end of BM2 protein, shortens cytoplasmic tail 7 BM2 pPolRT Rescue plasmid containing the This project B/Beijing/1/87 virus segment 7 cDNA. Deletion mutant 9 aa residues from the end of BM2 protein, shortens cytoplasmic tail 7 BM2 pPolRT Rescue plasmid containing the This project B/Beijing/1/87 virus segment 7 cDNA. Deletion mutant 10 aa residues from the end of BM2 protein, shortens cytoplasmic tail 7 BM2 pPolRT Rescue plasmid containing the This project/Guo B/Beijing/1/87 virus segment 7 cDNA. Zhang Alanine substitution at 6 aa residues from end of BM2, within cytoplasmic tail 7 BM2 pPolRT Rescue plasmid containing the This project/Guo B/Beijing/1/87 virus segment 7 cDNA. Zhang Alanine substitution at 7aa residues from end of BM2, within cytoplasmic tail 7 BM2 pPolRT Rescue plasmid containing the This project/Guo B/Beijing/1/87 virus segment 7 cDNA. Zhang Alanine substitution at 8 aa residues from end of BM2, within cytoplasmic tail 7 BM2 pPolRT Rescue plasmid containing the This project/Guo B/Beijing/1/87 virus segment 7 cDNA. Zhang Alanine substitution at 9 aa residues from end of BM2, within cytoplasmic tail

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7 BM2 pPolRT Rescue plasmid containing the This project B/Beijing/1/87 virus segment 7 cDNA. Lysine substitution at 6 aa residues from end of BM2, within cytoplasmic tail 7 BM2 pPolRT Rescue plasmid containing the This project B/Beijing/1/87 virus segment 7 cDNA. Q substitution at 6 aa residues from end of BM2, within cytoplasmic tail 7 BM2 pPolRT Rescue plasmid containing the This project B/Beijing/1/87 virus segment 7 cDNA. Valine substitution at 7aa residues from end of BM2, within cytoplasmic tail 7 BM2 pPolRT Rescue plasmid containing the This project B/Beijing/1/87 virus segment 7 cDNA. Substitution of serine to alanine at position 91 to remove phosphorylation site 7 BM2 pPolRT Rescue plasmid containing the This project B/Beijing/1/87 virus segment 7 cDNA. Substitution of Threonine to alanine at position 69 to remove phosphorylation site 7 BM2 pPolRT Rescue plasmid containing the This project B/Beijing/1/87 virus segment 7 cDNA. Substitution of Threonine to alanine at position 101 to remove phosphorylation site 3 PA pPolRT Rescue plasmid containing the This project B/Beijing/1/87 virus segment 3 cDNA with a point substitution in PA polymerase protein to block virus assembly 3 PA pCIP Expression plasmid containing This project B/Panama/45/90 virus segment 3 Point substitution to block virus assembly but retain polymerase activity 7 M pPolRT Rescue plasmid containing the This project B/Beijing/1/87 virus segment 7 cDNA. Mouse adaptation of Influenza caused by substitution of asparagine for serine 6 NB pPolRT Rescue plasmid containing the David Jackson B/Beijing/1/87 virus segment 6 cDNA. To introduce NB Glycosylation knockout mutant - both sites 6 NB pPolRT Rescue plasmid containing the David Jackson B/Beijing/1/87 virus segment 6 cDNA. To introduce NB glycosylation knockout mutant - T9A site only 6 NB pPolRT Rescue plasmid containing the David Jackson/This B/Beijing/1/87 virus segment 6 cDNA. NB project Glycosylation knockout mutant - both sites - FLAG-TAGGED in NA stalk region 6 NB pPolRT Rescue plasmid containing the David Jackson/This B/Beijing/1/87 virus segment 6 cDNA. NB project Glycosylation knockout mutant - one site only FLAG-TAGGED in NA stalk region

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7 M pPolRT Rescue plasmid containing the Mike Powell B/Beijing/1/87 virus segment 7 cDNA. BM2 translation mutant in segment 7 Subsitution of adenine for uracil/thymine 30 bases upstream of BM2 AUG 7 M pPolRT Rescue plasmid containing the Mike Powell B/Beijing/1/87 virus segment 7 cDNA. BM2 translation mutant in segment 7 Subsitution of adenine for cytosine 30 bases upstream of BM2 AUG 7 M pPolRT Rescue plasmid containing the Mike Powell B/Beijing/1/87 virus segment 7 cDNA. BM2 translation mutant in segment 7 Subsitution of adenine for guanine 30 bases upstream of BM2 AUG 7 M pPolRT Rescue plasmid containing the Mike Powell B/Beijing/1/87 virus segment 7 cDNA. BM2 translation mutant in segment 7 Subsitution of uracil/thymine for guanine 36 bases upstream of BM2 AUG 7 M pPolRT Rescue plasmid containing the Mike Powell B/Beijing/1/87 virus segment 7 cDNA. BM2 translation mutant in segment 7 Subsitution of uracil/thymine for cytosine 36 bases upstream of BM2 AUG 7 M pPolRT Rescue plasmid containing the Mike Powell B/Beijing/1/87 virus segment 7 cDNA.BM2 translation mutant in segment 7 Subsitution of uracil/thymine for adenine 36 bases upstream of BM2 AUG N/A Tsg101 pCR3.1 Expression plasmid containing coding Juan Martin- sequence of Tsg101 1-157 Serrano (Tumour Susceptibility Gene 101)(Vps23).Contains the PTAP binding domain , lacks the C-terminal region required for HIV-1 budding (38)Fused to EGFP N/A Alix pCR3.1 Expression plasmid containing coding Juan Martin- sequence of ALIX(AIP1) 170-869. Lacks a Serrano section of the Bro-1 domain that binds to CHMP4 of ESCRT III- Fused to YFP. N/A Vps4 pCR3.1 Expression plasmid containing coding Juan Martin- sequence of Vps4, ATPase fused to YFP Serrano N/A Vps4 pCR3.1 Expression plasmid containing coding Juan Martin- sequence of Vps4 E223Q, ATPase lacking Serrano catalytic function fused to YFP N/A WWPI pCR3.1 Expression plasmid containing coding Juan Martin- sequence of WWP1 Δhect, The WWP1 Serrano ubiquitin ligase lacking the HECT domain. Fused to YFP. N/A YFP pCR3.1 Expression plasmid containing coding Juan Martin- sequence of YFP -A control plasmid that Serrano expresses YFP

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N/A Tetherin pCR3.1/HA Expression plasmid transiently Stuart Neil expressed. Fully functioning Tetherin protein N/A Tetherin pCR3.1/HA Expression plasmid transiently Stuart Neil expressed. Tetherin protein with GPI anchor removed. C-terminal 19 amino acids N/A Tetherin LHCX-based Stably expressed in cell line. Fully Stuart Neil retroviral functioning Tetherin protein N/A Cherry Red pCR3.1/HA Expression plasmid producing Cherry Red Stuart Neil flourescent protein N/A eGFP Mammalian expression vector which Wendy Barclay expresses the enhanced green fluorescent protein (EGFP) 2 PB1 pPolRT Rescue plasmid containing the David Jackson B/Beijing/1/87 virus segment 2 cDNA 1 PB2 pPolRT Rescue plasmid containing the David Jackson B/Beijing/1/87 virus segment 1 cDNA 3 PA pPolRT Rescue plasmid containing the David Jackson B/Beijing/1/87 virus segment 3 cDNA 5 NP pPolRT Rescue plasmid containing the B/Lee/40 David Jackson virus segment 5 cDNA 4 HA pPolRT Rescue plasmid containing the Andrew Cadman B/Beijing/1/87 virus segment 4 cDNA 6 NA/NB pPolRT Rescue plasmid containing the Andrew Cadman B/Beijing/1/87 virus segment 6 cDNA 7 M1/BM2 pPolRT Rescue plasmid containing the David Jackson B/Beijing/1/87 virus segment 7 cDNA 8 NS1/NEP pPolRT Rescue plasmid containing the David Jackson B/Beijing/1/87 virus segment 8 cDNA 2 PB1 pCIP Rescue 'helper' expression plasmid David Jackson containing the B/Panama/45/90 virus segment 2 cDNA 1 PB2 pCIP Rescue 'helper' expression plasmid David Jackson containing the B/Panama/45/90 virus segment 1 cDNA 3 PA pCIP Rescue 'helper' expression plasmid David Jackson containing the B/Panama/45/90 virus segment 3 cDNA 5 NP pCIP Rescue 'helper' expression plasmid David Jackson containing the B/Panama/45/90 virus segment 5 cDNA 7 BM2 pcDNA Mammalian expression vector which Wendy Barclay expresses the BM2 protein of B/Lee/40 7 M1 Mammalian expression vector which Wendy Barclay expresses the Influenza B Matrix protein 4 HA Mammalian expression vector which Wendy Barclay expresses the Influenza B haemagglutinin protein 6 NA Mammalian expression vector which Wendy Barclay expresses the influenza B Neuraminidase protein

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8 NS pCAGGs Mammalian expression vector which This project expresses theNS protein of B/Beijing/1/87 virus segment 8 cDNA 8 NEP pCAGGs Mammalian expression vector which This project expresses theNEP protein of B/Beijing/1/87 virus segment 8 cDNA 7 M2 pcDNA Mammalian expression vector which Wendy Barclay expresses the M2 protein of influenza A Table 17: Plasmids used in this project

7.2 Oligonucleotides

Name Nucleotide sequence 5'→3' Purpose BM2-5 F GAAACAGTTTTGGAGTAAGAAGAATTGCATTAAACCC Mutagenesis primer to introduce premature stop codon into BM2 protein sequence 5 amino acids from the end of the cytoplasmic tail BM2-5 R GGGTTTAATGCAATTCTTCTTACTCCAAAACTGTTTC Mutagenesis primer to introduce premature stop codon into BM2 protein sequence 5 amino acids from the end of the cytoplasmic tail BM2-6 F GTGAAACAGTTTTGTAGGTAGAAGAATTGCATTAAAC Mutagenesis primer to introduce premature stop codon into BM2 protein sequence 6 amino acids from the end of the cytoplasmic tail BM2-6 R GTTTAATGCAATTCTTCTACCTACAAAACTGTTTCAC Mutagenesis primer to introduce premature stop codon into BM2 protein sequence 6 amino acids from the end of the cytoplasmic tail BM2 -7 F GGTGAAACAGTTTAGGAGGTAGAAGAATTGCATTAA Mutagenesis primer to AC introduce premature stop codon into BM2 protein sequence 7 amino acids from the end of the cytoplasmic tail BM2 -7 R GTTTAATGCAATTCTTCTACCTCCTAAACTGTTTCACC Mutagenesis primer to introduce premature stop codon into BM2 protein sequence 7 amino acids from the end of the cytoplasmic tail BM2 -8 F ATGGGTGAAACATAATTGGAGGTAGAAGAATTGC Mutagenesis primer to introduce premature stop codon into BM2 protein sequence 8 amino acids from the end of the cytoplasmic tail BM2 -8 R GCAATTCTTCTACCTCCAATTATGTTTCACCCAT Mutagenesis primer to introduce premature stop codon into BM2 protein sequence 8 amino acids from the end of the cytoplasmic tail

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BM2 -9 F GATAATAAAAATGGGTGAATAAGTTTTGGAGGTAGAA Mutagenesis primer to GAATTG introduce premature stop codon into BM2 protein sequence 9 amino acids from the end of the cytoplasmic tail BM2 -9 R CAATTCTTCTACCTCCAAAACTTATTCACCCATTTTTATT Mutagenesis primer to ATC introduce premature stop codon into BM2 protein sequence 9 amino acids from the end of the cytoplasmic tail BM2-10 F GAGATAATAAAAATGGGTTAAACAGTTTTGGAGGTAG Mutagenesis primer to AAG introduce premature stop codon into BM2 protein sequence 10 amino acids from the end of the cytoplasmic tail BM2-10 R CTTCTACCTCCAAAACTGTTTAACCCATTTTTATTATCTC Mutagenesis primer to introduce premature stop codon into BM2 protein sequence 10 amino acids from the end of the cytoplasmic tail B/PA(503,50 GGTCTGGCGGTTAAAGCAGCATCTCATCTGAGGGGA Mutagenesis primer to alter 4) F GATACTG amino acids 503 and 504 in the PA gene sequence from ### to alanine B/PA(503,50 CAGTATCTCCCCTCAGATGAGATGCTGCTTTAACCGCC Mutagenesis primer to alter 4) R AGACC amino acids 503 and 504 in the PA gene sequence from ### to alanine NS1splice F GCTACTGATGATCTTACCGTTGAGGATGAAG Mutagenesis primer to remove the NS-1 splice site from segment 8 NS1splice R CTTCATCCTCAACGGTAAGATCATCAGTAGC Mutagenesis primer to remove the NS-1 splice site from segment 8 5'NEP(Not1) TATGCGGCCGCATGGCGGACAACATGACCA Primer to amplify the NEP F CAACACAAATTGAGTGGAGGATGAAGAAGAT protein from segment 8, GGCCATCGG including 5' Not1 restriction site (red), first section of NEP protein (blue), and 5' sequence of second section of NEP sequence (black) 3'NEP(Mlu1) ACGCGTTATTCATAAGCACTGCCTGCTGTACACTTC Primer to amplify the NEP R protein from segment 8, including 3' Mlu1 restriction site (red), 3' NEP sequence (black) 5'NS1(Not1) TATGCGGCCGCATGGCGGACAACATGACCACAACA Primer to amplify NS-1 protein F CAAATTGAG and introduce a Not1 restriction site (red) 3'NS1(Mlu1) ACGCGTTATCTAATTGTCTCTCTCTTCTGGTGA Primer to amplify NS-1 protein R and introduce a Mlu1 restriction site (red) Thr69 F CCAAGCCAAAGAAgCAATGAAGGAAGTACTCTCTGAC Mutagenesis primer to introduce alanine into BM2 protein sequence at position 69 in place of threonine

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Thr69 R GTCAGAGAGTACTTCCTTCATTGCTTCTTTGGCTTGG Mutagenesis primer to introduce alanine into BM2 protein sequence at position 69 in place of threonine Ser91 F GTAATTGAGGGACTTgCTGCTGAAGAGATAATAAAAAT Mutagenesis primer to GG introduce alanine into BM2 protein sequence at position 75 in place of Serine Ser91 R CCATTTTTATTATCTCTTCAGCAGCAAGTCCCTCAATTAC Mutagenesis primer to introduce alanine into BM2 protein sequence at position 75 in place of Serine Thr101 F GATAATAAAAATGGGTGAAgCAGTTTTGGAGGTAGAA Mutagenesis primer to GAATTGC introduce alanine into BM2 protein sequence at position 101 in place of threonine Thr101 R GCAATTCTTCTACCTCCAAAACTGCTTCACCCATTTTTATT Mutagenesis primer to ATC introduce alanine into BM2 protein sequence at position 101 in place of threonine BM2-6A F GTGAAACAGTTTTGGCTGTAGAAGAATTGCATTAAAC Mutagenesis primer to introduce alanine into BM2 protein sequence 6 amino acids from the end of the cytoplasmic tail BM2-6A R GTTTAATGCAATTCTTCTACAGCCAAAACTGTTTCAC Mutagenesis primer to introduce alanine into BM2 protein sequence 6 amino acids from the end of the cytoplasmic tail BM2-7A F GGTGAAACAGTTGCTGAGGTAGAAGAATTGCATTAA Mutagenesis primer to AC introduce alanine into BM2 protein sequence 7 amino acids from the end of the cytoplasmic tail BM2-7A R GTTTAATGCAATTCTTCTACCTCAGCAACTGTTTCACC Mutagenesis primer to introduce alanine into BM2 protein sequence 7 amino acids from the end of the cytoplasmic tail BM2 -8A F ATGGGTGAAACAGCTTTGGAGGTAGAAGAATTGC Mutagenesis primer to introduce alanine into BM2 protein sequence 8 amino acids from the end of the cytoplasmic tail BM2 -8A R GCAATTCTTCTACCTCCAAAGCTGTTTCACCCAT Mutagenesis primer to introduce alanine into BM2 protein sequence 8 amino acids from the end of the cytoplasmic tail BM2 -9A F GATAATAAAAATGGGTGAAGCTGTTTTGGAGGTAGAA Mutagenesis primer to GAATTG introduce alanine into BM2 protein sequence 9 amino acids from the end of the cytoplasmic tail

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BM2 -9A R CAATTCTTCTACCTCCAAAACAGCTTCACCCATTTTTATTA Mutagenesis primer to TC introduce alanine into BM2 protein sequence 9 amino acids from the end of the cytoplasmic tail BM2-10A F GAGATAATAAAAATGGGTGCTACAGTTTTGGAGGTAG Mutagenesis primer to AAG introduce alanine into BM2 protein sequence 10 amino acids from the end of the cytoplasmic tail BM2-10A R CTTCTACCTCCAAAACTGTAGCACCCATTTTTATTATCTC Mutagenesis primer to introduce alanine into BM2 protein sequence 10 amino acids from the end of the cytoplasmic tail EGFP_Not1_ GCGGCCGCTATCGATCGTATATGGTGAGCAA Primer to amplify the EGFP PVU1)F GGGCGAGGAGC protien (green) including the Not1 and PVU1 restriction sites (red) EGFP_(PVU1 CGATCGCTTGTACAGCTCGTCCATGCCGAG Primer to amplify the EGFP )R protien (green) including the PVU1 restriction sites (red) DN_4_EGFP CGAGCTGTACAAGCGATCGCACATAGTAATTGAGGGAC Primer to amplify the last 31 _F: amino acids of the BM2 protein, includes EGFP overlap sequence DN_4_EGFP ATAACGCGTTTAATGCAATTCTTCTACCTCCAAAACTG Primer to amplify the last 31 _R: amino acids of the BM2 protein, includes EGFP overlap sequence BM2 Hind AATGCTTTATATGCTCGAACCATTTCAGATTCTTTCAA Primer to amplify the BM2 III_F TTTG coding sequence and introduce a 5' HindIII restriction site BM2 Xba1_R TCTAGAATATTAATGCAATTCTTCTACCTCCAAAACTG Primer to amplify the BM2 coding sequence and introduce a 3' Xba1 restriction site M1_N221S F CTGGGGGCAAGTCAAAAGTCTGGGGAAGGAATTGC Mutagenesis primer substitute a serine for an asparagine at position 221 in the Matrix protein M1_N221S R GCAATTCCTTCCCCAGACTTTTGACTTGCCC Mutagenesis primer substitute a serine for an asparagine at CCAG position 221 in the Matrix protein Table 18: Oligonucleotides used in this project

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7.3 Viruses

Virus Name Donor Virus Segment Protein Comments Source altered Altered Wild-Type B/Beijing/1/87 None None Reverse Genetics David virus generated Jackson/ using plasmids Andrew containing Cadman - B/Beijing/1/87 gene Plasmids segments, except for segment 5 sourced from B/Lee/40 BM2-5 B/Beijing/1/87 7 BM2 Virus with last 5 bases This removed from the Project cytoplasmic tail of the BM2 protien BM2-6A B/Beijing/1/87 7 BM2 Alanine substitution at This 6 aa residues from end Project of BM2, within cytoplasmic tail BM2-7A B/Beijing/1/87 7 BM2 Alanine substitution at This 7aa residues from end Project of BM2, within cytoplasmic tail BM2 -8A B/Beijing/1/87 7 BM2 Alanine substitution at This 8 aa residues from end Project of BM2, within cytoplasmic tail BM2 -9A B/Beijing/1/87 7 BM2 Alanine substitution at This 9 aa residues from end Project of BM2, within cytoplasmic tail BM2 6Y B/Beijing/1/87 7 BM2 Lysine substitution at This 6 aa residues from end Project of BM2, within cytoplasmic tail BM2 6Q B/Beijing/1/87 7 BM2 Q substitution at 6 aa This residues from end of Project BM2, within cytoplasmic tail BM2-7V B/Beijing/1/87 7 BM2 Valine substitution at This 7aa residues from end Project of BM2, within cytoplasmic tail BM2 Ser91 B/Beijing/1/87 7 BM2 Alanine substitution at This Ser91 position, Project removal of phosphorylation site BM2 Thr69 B/Beijing/1/87 7 BM2 Alanine substitution at This Thr69 position, Project removal of phosphorylation site BM2 Thr101 B/Beijing/1/87 7 BM2 Alanine substitution at This Thr101 position, Project removal of phosphorylation site

204 del5 B/Beijing/1/87 6 NB NB protein deletion, David the protein has been Jackson reduced to 5 amino acids by the insertion of premature stop codon delATG B/Beijing/1/87 6 NB NB protein deletion, David by removal of Jackson initiation codon and following 4 amino acids G-1-2 B/Beijing/1/87 6 NB Removal of David glycosylation site by Jackson the removal of the threonines at 5 & 9, thus disrupting the N- X-T motif in NB protein B/Beijing/87/FLAG B/Beijing/1/87 6 NB/NA Insertion of FLAG David motif in Stalk region of Jackson the neuraminidase gene del5 FLAG B/Beijing/1/87 6 NB/NA Insertion of FLAG David motif in Stalk region of Jackson the neuraminidase gene of segment 6 which also has insertion of premature stop codon delATG FLAG B/Beijing/1/87 6 NB/NA Insertion of FLAG David motif in Stalk region of Jackson the neuraminidase gene, which also contains deletion of NB initiation codon G-1-2 FLAG B/Beijing/1/87 6 NB/NA Insertion of FLAG This motif in Stalk region of Project the neuraminidase gene, which has also been modified to remove glycosylation sites G-- FLAG B/Beijing/1/87 6 NB/NA Insertion of FLAG This motif in Stalk region of Project the neuraminidase gene, modified to remove the first glycosylation site of the NB protien -30 A-U B/Beijing/1/87 7 BM2 BM2 translation This mutant in segment 7 Project Subsitution of adenine for uracil/thymine 30 bases upstream of BM2 AUG

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-30 A-C B/Beijing/1/87 7 BM2 BM2 translation This mutant in segment 7 Project Subsitution of adenine for cytosine 30 bases upstream of BM2 AUG -30 A-G B/Beijing/1/87 7 BM2 BM2 translation This mutant in segment 7 Project Subsitution of adenine for guanine 30 bases upstream of BM2 AUG -36 U-G B/Beijing/1/87 7 BM2 BM2 translation This mutant in segment 7 Project Subsitution of uracil/thymine for guanine 36 bases upstream of BM2 AUG -36 U-C B/Beijing/1/87 7 BM2 BM2 translation This mutant in segment 7 Project Subsitution of uracil/thymine for cytosine 36 bases upstream of BM2 AUG -36 U-A B/Beijing/1/87 7 BM2 BM2 translation This mutant in segment 7 Project Subsitution of uracil/thymine for adenine 36 bases upstream of BM2 AUG Wsn/V WSN/33 & 7 Matrix Influenza A virus with Wendy Victoria the segment 7 of Barclay Influenza A/Victoria M2KO WSN/33 & 7 M2 Influenza A virus with Wendy Victoria the M2 protein Barclay expression removed E116A B/Beijing/1/87 6 NA Influenza B Virus with David mutation to induce Jackson NAI resistance and increase NA activity E116D B/Beijing/1/87 6 NA Influenza B Virus with David mutation to induce Jackson NAI resistance and reduce NA activity Table 19: Viruses used in this project

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7.4 Solutions

Solution Recipe Use

Buffers

RIPA Buffer 100 mM NaCl Lysis of cells for Western blot (50 mM iodoacetamide) analysis/SDS -PAGE 1% Nonidet-P40 0.1% SDS 0.5% Sodium deoxycholate 20 mM Tris-HCl, pH 7.5

SDS loading buffer (2x) 45% Glycerol Western blot/SDS-PAGE 6.5% SDS sample loading buffer 15% 1 M Tris pH 6.8

ddH2O

SDS Running buffer (x10) #1 30.25 g Tris-base Buffer for gel electrophoresis 144 g glycine in Western blot (wet system) 10 g SDS

ddH2O to 1 litre

SDS Running buffer#2 25 mM Tris base Buffer for gel electrophoresis 250 mM Glycine in Western blot (semi-dry 0.1% SDS system) pH 8.3

Western blot Transfer Buffer #1 5.85 g Tris-base Buffer for transfer of proteins 29 g glycine to PVDF membrane in Western 1 g SDS blot (wet system) 200 ml methanol

ddH2O

Western blot Transfer Buffer #2 0.037 % (w/v) SDS Buffer for transfer of proteins 48 mM Tris to PVDF membrane in Western 39 mM Glycine blot (semi-dry system) 20% (v/v) Ethanol added just before use

Western blot Wash Buffer PBS Buffer for washing membrane 0.1% Tween 20 during antibody staining of proteins in Western blots analysis

DNA-Loading Buffer (6x) 0.25% Bromophenol blue Buffer used to load DNA 40% (w/v)sucrose in TAE samples in gel electrophoresis

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TAE Buffer 40 mM Tris acetate pH 8.0 Buffer for making and running 1 mM EDTA agarose gel for DNA separation

PBN PBS Blocking buffer used in 0.5% BSA immunofluorescence 0.02% Na azide experiments

Western blot antibody diluents 5% Marvel, Blocking solution for Western 0.1% Tween 20 blot and antibody diluents PBS

PNA assay wash buffer 0.5% Tween 20 in PBS Buffer used to wash the Peanut lectin fetuin neuraminidase activity assay plates

PNA-HRP 1/500 dilution of PNA- HRP (of Peanut lectin-HRP used in 1 MG/ ml stock) in 1% BSA/PBS PNA-fetuin neuraminidase (Sigma-Aldrich) activity assay plates

Fetuin NaCarbonate/bicarbonate Fetuin used in PNA-fetuin buffer pH 9.6 (Sigma-Aldrich neuraminidase activity assay capsules) 50 ug/ml Fetuin plates (Sigma-Aldrich)

Virus diluent 3% FCS (Bio Sera International) Resuspension of virus from 0.1% Pen/Strep (Gibco) plaque picks or sucrose PBS cushion

Sucrose Cushion

PBS++ 1 litre PBS + 1 ml 1 M NaCl2 Ferret Airway Wash buffer

1:1 mix of methanol acetone

4% (w/v) paraformaldehyde

FAE Permeabilisation 2.5% Triton-X in PBS FAE Permablisation

Poly-L-Lysine 1:20 dilution poly-L-Lysine in Attaching 293T cell to base of ddH2O culture dish/slide for virus infection/immunofluorescence x-gal 10% X-gal in DMF Blue cell Assay

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10xCN Buffer 3 mM Potassium Ferricyanide Blue cell Assay 3 mM Potassium Ferrocyanide 1 mM MgCl2 PBS

X-gal Substrate 8.75 ml PBS Blue cell Assay substrate 1 ml CN buffer 0.25 ml 10% X-gal

Culture Media

LB (Luria-Bertani Broth) 1% tryptone Growth of E.Coli for plasmid 0.5% yeast extract pro duction 0.5% NaCl 0.1% glucose

ddH20

LA (Luria-Bertani Agar) 1% tryptone Growth of E.Coli for plasmid 0.5% yeast extract selection 0.5% NaCl 0.1% glucose 1% Difco agar(oxoid)

ddH20

SOC 2% tryptone Growth of E.Coli for plasmid 0.5% Yeast extract selection 10 mM NaCl 2.5 mM KCl

10 mM MgCl2

10 mM MgSO4.7H2O 20 mM glucose

NZY+ broth 1% NZ amine (casein Growth of E.Coli for plasmid hydrolysate) production 0.5% yeast extract 0.5% NaCl

1.5% 1 M MgCl2

1.5% 1 M MgSO4 1% 1 M glucose ddH2O

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Serum-Free DMEM Dulbecco's modified Eagle's Cell culture media for virus medium (Life Technologies) infection/propagation. 1% glutamine (200mM) 2% non-essential amino acids 1% penicillin-streptomycin (5000 IU/ml; 5000 µl) Sodium Pyruvate

10% DMEM Dulbecco's modified Eagle's Cell culture media for MDCK, medium(Life Technologies) 293T, A549 cell growth 1% glutamine (200 mM) 2% non-essential amino acids 1% penicillin-streptomycin (5000 IU/ml; 5000 µl) Sodium Pyruvate 10% Foetal calf serum (FCS)

RPMI Gibco H-292 cells

Plaque assay overlay media 50 ml 10x EMEM Media for MDCK cells for 14 ml 7.5% fraction V BSA titration of viruses by plaque 5 ml 100x L-glutamine assay. 10 ml 7.5% NaHCO3 5 ml 1 M HEPES 2.5 ml 1% dextran 5 ml 10x penicillin/streptomycin

ddH2O up to 350ml -store in 17.5 ml aliquots, add 7.5 ml 2% oxoid agar/aliquot

F12+ 500 ml Ham’s F12 For Isolation of cells from (Invitrogen,21765-029) Ferret trachea 5 ml Antibiotic/Antimycotic Solution (100X) (Chemicon, CNT-ABM)

BEGM 500 ml BEGM For growth and maintenance BEGM bullet supplements of ferret airway cells (Chemicon, CNT-23) 5 ml Antibiotic/Antimycotic (100X) (Chemicon, CNT-ABM) 360 µl BPE (Sigma-Aldrich- Aldrich, P1476-25)

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BEGM+10% (ALI) 500 ml BEGM For growth and maintenance BEGM bullet supplements of ferret airway cells (Chemicon, CNT-23) 5 ml Antibiotic/Antimycotic (100X) (Chemicon, CNT-ABM) 10% (50 ml) FBS 360 µl BPE (Sigma-Aldrich- Aldrich, P1476-25)

Human Placental collagen Human placenta collagen Coating used of transwells of (Sigma-Aldrich-Aldrich C7521). ferret airway cultures 10x stock solution: o 10 mg Collagen o 20 ml dH2O o 40 µl concentrated Acetic Acid (Glacial acetic acid).

Table 20: Solutions used in this project

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7.5 Primary and secondary antibodies

Protein 1˚ antibody 1˚ antibody 2˚ antibody 2˚ antibody dilution dilution

NP Mouse monoclonal anti-NP 1/500 Goat anti-Mouse 1/10000 HRP

Goat -mouse - gal 1/500

Goat -mouse 1/500 Texas Red

BM2 Rabbit anti-BM2 1/4000 Goat anti-Rabbit 1/4000 HRP 1/500 Goat -rabbit FITC

M1 Mouse monoclonal anti-M1 1/1000 Goat anti-Mouse 1/10000 HRP

NB Mouse monoclonal anti-NB 1/1000 Goat anti-Mouse 1/10000 HRP

HA Monoclonal anti-HA 1/500 Anti-mouse FITC 1/500 VINC Goat polyclonal antibody raised 1/1000 Anti-Goat HRP 1/1000 against vinculin β-tubulin 1/40 1/500 FLAG Mouse Monoclonal ANTI-FLAG® 1/1000 Anti-Mouse FITC 1/1000 M2 VPS28 Goat polyclonal anti- VPS28 1/1000 Donkey anti-Goat 1/10000 IgG HRP

Table 21: Antibodies used in this project

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7.6 Cell culture

7.6.1 MDCK, Vero, A549 & 293T cells (Madin-Darby canine kidney cells; American Type Culture Collection [ATCC]), Green African Monkey cells & Human Embryonic Kidney cells, were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen) containing 2% non- essential amino acids (Invitrogen), 1% glutamine (200 mM) and 10% (v/v) foetal calf serum (FCS) (Bio-Whittaker Europe). PBS, EDTA and trypsin was used to split cells when confluent. Human Embryonic kidney cells (293T) cells were cultured as the MDCK’s except for the addition of pyruvate to the DMEM and division at 70% confluency. MDCK-M2 and tetherin stables were treated with Geneticin (Gibco/Invitrogen) to maintain the stable expression plasmid.

7.6.2 NCI-H292 The cells were cultured in RPMI (Gibco/Invitrogen) + 10% FBS and antibiotics at 37˚C until required for virus infection

7.6.3 FAE’s The Ferret Airway epithelial cells were generated by removing the trachea of a post mortem ferret within 1 hour of death into a solution of ice cold Ham’s F12 (Invitrogen, 21765- 029). The trachea was incubated for 18 hours in a solution of 3 mg/ml pronase (Roche) in Ham’ F12+ solution on a rocking incubator at 4˚C. 1 ml of FBS was used to inactivate the pronase. Serial washing of the trachea in 10% FBS in the Ham’s F12+ solution in fresh tubes was used to remove the cells lining the trachea. The wash solution was pooled and the cells harvested by centrifugation at 400 g at 4˚C. The cell pellet was resuspended in 2 ml of F12+ containing 0.5 mg/ml crude pancreatic DNase I & 10 mg/ml BSA. After 10mins on ice at 4˚C the cells were pelleted by centrifugation at 400 g at 4˚C, then resuspended in BEGM & Supplements & Antibiotic/Antimycotic Solution & BPE & 10% FBS (BEGM+10%) and added to fibroblast selective dishes (BD Biosciences, 353802) after a 4 hour incubation the non adherent cells were collected, centrifuged as before and resuspended in the BEGM +10% solution prior to seeding on collagen coated transwell supports. The cells were incubated at 37˚C at 5% CO2 .

The cells were allowed to adhere and proliferate before the media was altered to force the cells to differentiate into air liquid interface cell layers (ALI).

7.7 Virus infection

Viruses were plaqued on MDCK cells in triplicate prior to use in any growth curves.

7.7.1 MDCK & H292 cells: The cells were washed with PBS and infected with an MOI of 0.01 or 0.1 for growth curves and an MOI of 1 for the Mucus assays. The cells were incubated at 33˚C

213 and 5% CO2 for 1 hour, the inoculum was removed, the cells were washed with PBS or PBS pH5 and incubated in with serum free media and trypsin as required at 34˚C or 37˚C and 5% CO2.

7.7.2 Airway cells: The cells were washed with 0.5 ml of PBS for 10 min. The cells were then infected at an MOI of 1 for the FAE’s and 0.1 for the HAE’s. The inoculum was removed after 1 hour and time points were taken by the addition of 100 µl of PBS incubation for 30 min then the removal of the PBS which was then assayed for viral titre.

7.7.3 Ferrets: Weight matched ferrets were inoculated intranasally in duplicate with 4x105 PFU of virus in PBS. Nasal washes were conducted daily with 1 ml of PBS. The aspirated PBS was tested daily for vial proteins using a BD directigen™ A&B kits (BD Diagnostic systems) and subjected to a plaque assay to assess viral titre.

7.7.4 Growth of virus stocks: MDCK cells grown to confluency in 75 cm2 flasks were infected with virus at a low multiplicity of infection (MOI) 0.001 in a low volume of serum free media for one hour at 33˚C. On removal of the initial inoculum, 12mls of serum free DMEM and 1 µg/ml of trypsin (TRTPCK-Worthington) were added to the flask which was incubated until virus cytopathic effect (CPE) was observed. The virus was harvested by removal and centrifugation of the supernatant at 2500 RPM for 10 minutes to pellet cell debris. After confirmation of viral presence by haemagglutination assay and assessment of quantity through plaque assay, the virus was stored in aliquots at -80˚C.

7.8 Reverse Genetics

Two hours prior to transfection 293T cells that have been grown to 70-80% confluency on 12 well plates were incubated in 3% FCS serum/DMEM. To create the transfection mix, 200 µl of serum free DMEM was placed into a 7 ml bijou, to this a 3:1 ratio of Fugene 6™ (Roche) to µg of plasmid DNA was added to the media (20 µl in a standard rescue), gentle mixing was required. After 5 minutes of incubation, a bulk mix of plasmid DNA was added (500 ng of all pPolRT plasmids representing all 8 genomic segments and 500 ng of PB1 & PB2 helper pCIP plasmids, 200 ng of PA and 1µg of helper NP). After a gentle swirling of the mix, it was incubated for 20 mins prior to its addition, dropwise, to the 293T cells. The cells were incubated overnight at 34˚C at 5% CO2.

A fully confluent 75 cm2 flask of MDCK cells was used to co-culture four viral rescues. The MDCK cells were washed in PBS:EDTA and trypsinised, resuspended and centrifuged for 5 minutes at 700RPM. The media was removed and the cells resuspened to a volume of 20 mls of 10% FCS DMEM with pyruvate. 4 mls of the MDCK cell suspension was added to 25 cm2

214 falcon culture flasks. The transfected 293T cells were washed gently with PBS and treated with a 1:5 dilution of trypsin, prior to resuspension with 1 ml of the MDCK suspension. The transfected 293T & MDCK cells were then transferred to the appropriately labelled 25 cm2 falcon culture flasks containing the 4mls of MDCK cells. The flasks were incubated for 6 hours to allow the cells to adhere to the growth surface of the flasks. After the incubation the serum containing media was removed and replaced with 5 mls of serum free media with 2 µg/ml of porcine trypsin. The cells were incubated for 3 to 5 days until viral cytopathic effect was observed.

7.9 Directigen™ kits

Directigen™ BD Diagnostic Flu A + B System kits were used to access the presence of viral infection in the ferrets nasal turbinates according to the manufacturers instructions.

7.10 Plaque assay

Culture plates were seeded with MDCK cells to form a confluent monolayer and incubated overnight. The growth media was removed from the cells and washed with PBS. The virus was diluted 10-fold in serum free media. 200 ul dilutions of the virus were added to each well. The cells were incubated at 33°C for 1 hour. 1 ml of overlay containing 2% plaque agarose and trypsin was added to each well. After the inoculum was removed. The overlay was allowed to dry, the plates were then stored upside down for 3-5 days to allow for plaque formation. The overlay was then hooked off, prior to the addition of crystal violet. The plaques were then counted.

7.11 Haemagglutination assay

50 µl of 0.5% Turkey or Chicken erythrocytes (in PBS) were added to 50 µl of virus serially diluted in PBS in a V bottomed 96 well plate. These were incubated for 1 hour on ice, the presence of virus was confirmed by a cloudy mixture indicating haemagglutination, absence by the collection of the erythrocytes at the base of the plate.

7.12 Immunohistochemistry:

7.12.1FACS

Vero cells were infected at an MOI of 1, incubated for 1 hour at 34°C for 1 hour, the inoculum removed and replaced with serum-free media. The cells were then incubated for 16 hours, prior to trypsinination, and fixation with 4% (w/v) paraformaldehyde. The cells were divided into aliquots 2 of which were permeabilised by 0.02% triton-X. The cells were the blocked with

215

0.5% BSA in PBS for 1 hour, then labelled with anti-FLAG, or anti NB antibodies, after repeated washing a FITC secondary antibody was used and washed repeatedly. The cells were then counted on a 4 colour Becton Dickinson FACSCalibur analyser and the results analysed with Summit 4.3 software. The data were expressed as mean fluorescence intensity of the anti- FLAG antibody in proportion to the number of cells infected.

7.12.2 En face staining

FAE cells: were washed with PBS and 4% (w/v) paraformaldehyde & HEPES was added to the apical and basal layers and incubated overnight at 4C. The cells are then washed with PBS. The cells are then blocked with 0.5% BSA overnight. The cells were then stained with anti NP and anti -tubulin antibodies, washed repeatedly then secondary alexi fluor 488 and 647 were used respectively.

293T, MDCK and H292 cells: 24 hours prior to use, cover slips were washed in 100% ethanol, allowed to dry, placed in a well of a 6 well plate, covered in a 1:10 dilution of Poly-L-Lysine (Sigma-Aldrich) and incubated at room temperature for 10 minutes. The poly-L-lysine solution were removed add the cover slips washed 3 times in PBS. A 1:4 split of cells was then added to the cover slips and left to adhere for 24 hours.

The cells are transfected with plasmids or infected with virus as previously described.

At a suitable time point the cells were washed with PBS then fixed with either ice cold 50:50 Methanol:Acetone, or 4% paraformaldehyde. The latter requiring permeabilisation of the cell membrane with 2% triton-X(Sigma-Aldrich) should the protein of interest be inside the cell rather than on the membrane.

The cells are then blocked with 5% BSA in PBS with 0.02% sodium azide (Sigma-Aldrich) for 1 hour. The cells were then washed with PBS. The correct dilution of primary antibody (against the protein of interest) in the 5% BSA/PBS solution was added to the cells and incubated for 1 hour. The cells were washed 3 times in PBS, prior to the addition of the secondary antibody (directed against the primary antibody and conjugated to a suitable reporter), again at the correct dilution. The cells were incubated for another hour, washed 3 times with PBS. The cover slips were then removed from the 6 well plates and inverted onto a microscope slide with molviol at the interface. Confocal microscope was used to observe fluorescent dyes to ascertain presence and location of protein.

216

7.13 Neuraminidase assays

Grienier Medium binding plates were coated with 100 µl of 25 ng/ml of fetuin (Sigma-Aldrich) in 0.1M carbonate/bicarbonate buffer (Sigma-Aldrich). The plates were washed with PBS/Tween, incubated with the appropriate viruses and bacterial neuraminidase standards either for 1 hr or overnight depending upon the viral titre. The virus & neuraminidase standards were removed and the plate washed with PBS/tween. The plates were then incubated with 100 µl of 1:1000 peanut lectin conjugated to HRP (Sigma-Aldrich) in 0.5% BSA for 1 hr at 37˚C. After repeated washing with PBS/TWEEN, 50µl of TMB solution was added to the plates which were the incubated until colour develops. The reaction was stopped by the addition of HCl then the absorbance measured at 405 nm.

7.14 Mucus Assays

Mucus was harvested from the surface of the ferret airway cultures. The viruses were incubated in a 1:5 ratio with the mucus, titres being adjusted with serum free media. The MDCK’s were washed with PBS, then incubated with the mucus/virus solution or serum free media/virus solution for 1 hour. The inoculum was then removed at the cells washed with PBS, PBS pH5 and PBS. The cells were then incubated overnight; the substrate was removed and assayed for viral titre by plaque assay or haemagglutination assay. The cells were fixed with 4% (w/v) paraformaldehyde for processing through a blue cell assay.

7.15Blue cell Assay

Cells infected or transfected with viral proteins were left for the appropriate amount of time dependant up viral type (usually overnight), washed with PBS, then fixed with methanol:acetone (if fluorescent proteins are involved 4% paraformaldehyde was used and the cells must be permeabilised with 0.2% Triton-X, as the methanol:acetone leaches out the fluorescent colouring) the cells were washed with PBS to remove any trace of the fixative agent and blocked for 1 hour with 0.5% BSA in PBS. Then incubated for 1 hour with anti-virus protein (anti-NP), washed 3 times with PBS, then incubated with anti 1˚ Antibody conjugated to anti-β-Galatasidase, washed 3 times with PBS, then incubated with X-Gal for 4 to 12 hours. The X-Gal buffer was removed and blue can be observed in the cells positive for virus.

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7.16 Expression plasmid Construction of NEP and NS-1

Using the NS-1 & NEP (segment 8) pPolRT plasmid as template, the NS-1 fragment was amplified using the polymerase chain reaction and the appropriate flanking primers, which also contained Not1 and Mlu1 restriction sites: *5’NS(Not1): TATGCGGCCGCATGGCGGACAACATGACCACAACACAAATTGAG 3’NS1(Mlu1): ACGCGTTATCTAATTGTCTCTCTCTTCTGGTGA.] Site directed mutagenesis was then carried out to remove the splicing site required that would normally generate the NEP fragment: [NS1splice: GCTACTGATGATCTTACCGTTGAGGATGAAG & the reverse and complemented form.]

Using the same template an extended primer was used that contained the entire 5’ region of the NEP protein’s nucleotide sequence, plus the section of sequence where the NEP protein splices back into the NEP sequence, completely omitting the NS-1 sequence (see Error! eference source not found.) Again the flanking primers contained the Not1 and Mlu1 restriction sites:

*5’NEP(Not1) TATGCGGCCGCATGGCGGACAACATGACCACAACACAAATTGAGTGGAGGATGAAGAAGATGGCCAT CGG

& 3’NEP(Mlu1) ACGCGTTATCATAAGCACTGCCTGCCTGCTGTACACTTC+ .

NEP NEP

NS-1

NEP

NS-1

Splice acceptor site removed

218

These fragments, after gel purification were then inserted into a pCR®2.1-TOPO vector (Invitrogen) according to the TOPO TA Cloning ® manual and then as before cultures were grown up and the DNA extracted using the Qiagen Spin Mini-prep® kit. The NS-1 and NEP fragments were then ligated into a pCAGGs expression vector using the Not 1 and Mlu1 restriction enzymes (a CIP step was not included) using T4 ligase.

7.17 SDS Page gels and Western blotting

SDS pages gels, in association with Western blotting, allow the separation and identification of specific viral proteins. Virus was grown on a suitable host cell (MDCK, A549, HEK293T, PER.C6™, H292). Once ready for harvesting, the cells were washed with PBS, then treated with RIPA lysis buffer (100 mM NaCl, 50mM Iodoacetamide, 1% Nonidet-P40, 0.1% SDS, 0.5% Sodium deoxycholate, 20 mM Tris-HCl, pH 7.5) for 10 minutes or until the cells no longer adhered to the growth vessel. The lysate was then centrifuged to remove cell debris, the remaining supernatant was mixed in equal amounts with a gel loading buffer containing DTT; this was boiled prior to loading onto a 15% polyacrylamide gel with a suitable ladder to measure the size of the protein. After applying 160 V current to the gel for 1-2 hours, the proteins were transferred to a pre-prepared PVDF membrane (Immobilon- Millipore) at 250 mAmps for 1hour. The membrane was then blotted overnight in 5% Marvel or horse serum in PBS, treated with the appropriate antibody to the viral protein, washed repeatedly with PBS/ 0.05% Tween 20. The membrane was treated with a secondary antibody conjugated to horse radish peroxidase directed against the primary antibody, after incubation and washing with PBS/0.05% Tween 20, the membranes were treated with an enhanced chemiluminescence (ECL) Western blotting detection reagent (Amersham Biosciences). On exposure to Hyperfilm ECL chemiluminescent film (Amersham Biosciences), the protein size was assessed by comparison to the ladder. Quantification was undertaken using Image J software using the method outlined at http://www.lukemiller.org/journal/2007/08/quantifying-western-blots- without.html.

7.18 BCA (bicinchoninic acid) Assay for protein content

The Thermo Scientific Pierce BCA (bicinchoninic acid) Protein Assay was used as per manufactures instructions to quantify the protein levels in the cell lysates generated with RIPA buffer prior to Western blot analysis. The microtitre plate methodology was used, briefly the lysates were microfuged; the samples were then prepared in PBS (1:50 and 1:100 dilutions) alongside protein standards provided by the kit. 1:50 ration of A & B solutions were made up,

219 added to the plate and incubated at 37°C for 30 minutes. Then the absorbance was read at 562 nm)

7.19 Transfections

293 T cell or MDCK cells are transfected using either Lipofectamine 2000 (Invitrogen) or Fugene 6 (Roche). The appropriate plasmids were aliquoted into tubes; the fugene 6 transfections were as per the virus rescue method described previously. The lipofectamine 2000 transfections require the dilution of the plasmids into Optimem™ (Gibco) and the dispersal of the transfection reagent into a second bijou of Optimen™ and incubation for 5 mins prior to combination of the two bijous 20 mins prior to addition dropwise to the 70-80% confluent cells that have been incubated for 2 hours in 2 or 3% FCS DMEM for 2 hours.

7.20 PCR amplification

PCR was performed with KOD Taq (Novagen) For KOD, a 25 µl PCR mix was made consisting of 2.5 µl 10X Buffer for KOD DNA Polymerase, 0.4 µM forward and reverse primers, 2.5 l dNTPs 2 mM, 0.2l KOD polymerase (2.5 U/µl), nuclease free water to 25 l.

95° C for 15 seconds

Ta °C 5 seconds/ 1 Kb 30 cycles

72° C for 30 seconds

7.21 Site directed mutagenesis

A QuikChangeXL® Site-Directed Mutagenesis Kit (Stratagene) was used to insert the mutations into the gene segments. Primers (HPLC purified, VHBio) were designed with the altered bases flanked by 10 to 15 nucleotides of non-mutated sequence.

PCR Mix

10x reaction buffer 5 µl

50ng/µl plasmid DNA 1 µl

10µM forward primer 1 µl

10µM reverse primer 1 µl dNTP mix 1 µl

220

QuikSolution 3 µl

H2O 38 µl

PFU Ultra polymerase 1 µl

Cycling conditions:

95˚C for 1 minute

95˚C for 50 seconds

60˚C for 50 minute 16 cycles

68˚C for 1 minutes/kb of plasmid

68˚C for 7 minutes

1 µl of Dpn1 was added to the reaction and incubated at 37˚C for 1 hour, cuts the initial plasmid methylated DNA, leaving the new altered plasmid.

XL10-Gold cells were thawed on ice, 2 µl of β-mercaptethanol was added and incubated for 10 minutes. 2 µl of the treated plasmid mix was added to the cells, which were incubated on ice for an additional 30 minutes. The cells were heat pulsed for 30 seconds at 42˚C, incubated on ice for 2 minutes prior to the addition of 500 µl of preheated NZY+ broth. The cells were placed on a shaking incubator at 225-250 rpm for 1 hour, then spread onto agar plates and incubated overnight.

Once grown up in LB broth, with ampicillin used as a selective agent, the plasmid DNA was extracted with a Qiagen© spin mini-prep kit (as per manufacturer’s instructions) briefly, sterile LB broth with appropriate antibody (amplicilin and kanamycin) was inoculated with a single colony and incubated overnight. The bacterial suspension was centrifuged to remove growth media, then the remaining cell pellet is resuspended with P1 buffer. The cells walls are lysed with a NaOH and SDS solution (P2), a high salt solution (N3) denatures the chromosomal DNA and cell debris, which are separated from the small plasmid DNA by microcentrifugation for 10 mins. The Plasmid DNA is then bound to the Qiagen column, washed with buffers PB and PE, prior to elution in tissue grade water (The elution buffer provided is not used as it can inhibit the reverse genetic transfections). The plasmids generated were sequenced by Lark Technologies™ or the Imperial in-house sequencing service.

7.22 vRNA Extraction

221 vRNA was extracted using the QIAamp viral RNA mini kit (QIAGEN) according to manufacturers instructions. 280 µl of virus stock was lysed using the AVL buffer containing RNAsin (Promega). This was bound to silica membrane, washed and eluted in 30 µl of AVE elution buffer. vRNA was stored at -20°C

7.23 Gel Electrophoresis

DNA was separated on 2% agarose gels diluted with TAE buffer and 1x gel red (Cambridge Bioscience) and run in TAE buffer. Samples were run with commercial DNA size markers at 70- 100V. The DNA bands were visualised using a UV transilluminator. Where required bands were purified using a QIAquick gel extraction kit (QIAGEN), following manufacturer’s instructions. PCR products were eluted in 15 µl nuclease free water and stored at -20ºC.

7.24 Transmission Electron microscopy

Viruses were concentrated through a 30% sucrose cushion. The viruses were placed on a grid to adhere by surface tension, washed to remove ions and placed in 1% urinyl acetate (20sec- 1min) and allowed to dry. A TECNAI G2, MEGAView III microscope was used. Images were taken and analysed with Silverlight software. The gamma wavelength was altered using Photoshop to improve image visability. All TEM images presented in this were taken with the assistance of Mike Hollingshead in the Electron microscopy suite at Imperial College London.

7.25 Sucrose Cushions

Viruses were layered onto 30% sucrose (in PBS), then ultrafuged for 2 hours at 25000 rpm. The Supernatant removed and the viruses resuspened overnight in PBS or virus diluents.

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