Defining and Characterising the anti-HCV Actions of the Interferon-Induced Transmembrane proteins
Sumudu Kumari Narayana B. Science (Biomedical Science) (Hons)
Discipline of Molecular and Cellular Biology School of Biological Sciences The University of Adelaide
A dissertation submitted to The University of Adelaide In candidature for the degree of Doctor of Philosophy in the Faculty of Science October 2015
i Table of Contents
List of Figures and Tables ...... ix
Abstract ...... xiii
Declaration ...... xv
Acknowledgements ...... xvi
Publications Arising from this PhD ...... xvii
Awards Received During PhD ...... xvii
Presentations Arising From PhD ...... xvii
Materials Providers ...... xix
Abbreviations Used ...... xxi
Chapter 1 ...... 1
Introduction ...... 1 1.1 Hepatitis C Virus ...... 1 1.1.1 Epidemiology ...... 1 1.1.2 Transmission ...... 2 1.1.3 Pathogenesis ...... 2 1.1.4 Treatment ...... 3 1.1.5 The HCV genome ...... 5 1.1.6 Classification of genotypes ...... 6 1.1.7 HCV proteins ...... 7 1.1.8 HCV life cycle ...... 11 1.1.9 HCV model systems ...... 13 1.2 Innate Immune Response to HCV ...... 17 1.2.1 Activation of the innate immune response ...... 17 1.2.2 Cellular Recognition of HCV ...... 19 1.2.3 Interferon Transduction Pathway ...... 20
ii 1.2.4 Interferon-stimulated genes ...... 21 1.2.5 HCV Evasion of Innate Immune Response ...... 26 1.3 Interferon-Induced Transmembrane (IFITM) Protein ...... 28 1.3.1 IFITMs: protein structure and cellular distribution ...... 29 1.3.2 Biological functions of the IFITM proteins ...... 32 1.3.3 Antiviral Actions of the IFITM proteins ...... 34 1.3.4 IFITM proteins in hepatocytes and HCV ...... 39 1.4 Hypothesis and Aims ...... 40
Chapter 2 ...... 42
Materials and Methods ...... 42 2.1 General Reagents ...... 42 2.1.1 Transient transfection of plasmid DNA ...... 42 2.1.2 Lentiviral packaging of IFITM protein to generate overexpression cell lines ...... 42 2.2 Tissue Culture Techniques ...... 44 2.2.1 Tissue culture medium ...... 44 2.2.2 Maintenance of cell lines ...... 45 2.2.3 Cryopreservation of cultured cells ...... 45 2.2.4 Resuscitation of frozen cells ...... 46 2.2.5 Trypan blue exclusion ...... 46 2.3 Cultured Cell Lines ...... 46 2.3.1 Huh-7 ...... 46 2.3.2 NNeoC-5B (RG) ...... 46 2.3.3 Huh-7.5 ...... 47 2.3.4 Huh-7+IFITM ...... 47 2.3.5 Huh-7+Empty ...... 47 2.3.6 Huh-7+shIFITM1 ...... 47 2.4 HCVcc Infectious System ...... 48
iii 2.4.1 Generation of HCVcc viral stock ...... 48 2.4.2 General infection protocol for HCVcc ...... 51 2.4.3 HCVpp Assay ...... 51 2.4.4 Extracellular:Intracellular Infectivity Assay ...... 51 2.5 General Molecular Biology Methods ...... 52 2.5.1 Synthetic oligonucleotides ...... 52 2.5.2 Bacterial transformation ...... 53 2.5.3 Mini-preparation (small scale) of plasmid DNA ...... 54 2.5.4 Maxi-preparation (large scale) of plasmid DNA ...... 54 2.5.5 Restriction endonuclease digestion ...... 54 2.5.6 Agarose gel electrophoresis ...... 55 2.5.7 DNA ligation ...... 55 2.5.8 Gel purification ...... 55 2.5.9 DNA sequencing ...... 56 2.5.10 Extraction of total RNA ...... 56 2.5.11 DNAseI treatment of RNA samples ...... 57 2.5.12 Nucleic acid quantification ...... 57 2.5.13 cDNA preparation ...... 57 2.5.14 Polymerase Chain Reaction ...... 58 2.5.15 High Fidelity PCR ...... 58 2.5.16 Real-Time Quantitative PCR ...... 59 2.5.17 Extraction of cellular protein ...... 60 2.5.18 SDS PAGE and protein transfer ...... 60 2.5.19 Western blotting ...... 61 2.5.20 Immunoprecipitation ...... 62 2.5.21 Dual Renilla luciferase assay ...... 62 2.5.22 Site-Directed Mutagenesis ...... 62 2.5.23 Immunofluorescence microscopy ...... 63 2.6 Data Analysis ...... 66
iv Chapter 3 ...... 67
The impact of the IFITM proteins on HCV replication ...... 67 3.1 Introduction ...... 67 3.2 Characterisation of endogenous IFITM proteins in vitro ...... 68 3.3 Regulation of IFITM1 by IFN in hepatocytes ...... 69 3.3.1 IFITM1 is upregulated by IFN-α in Huh-7 cells ...... 69 3.3.2 IFITM1 is upregulated by type I and III IFNs in primary human hepatocytes (PHH) ...... 70 3.4 Knockdown of endogenous IFITM1 reduces the anti-HCV activity of IFN-α ...... 70 3.5 Cloning and characterisation of IFITM1, IFITM2 and IFITM3 ...... 72 3.5.1 Cloning of IFITM1, IFITM2 and IFITM3 into a lentiviral expression vector ...... 72 3.5.2 Transient expression of IFITM proteins decreases HCV replication ...... 73 3.5.3 Producing lentiviral particles encoding IFITM1, IFITM2 and IFITM3 ...... 74 3.5.4 IFITM proteins may have a role at the early stages of HCV infection in vitro ...... 75 3.6 Generation of stable IFITM Huh-7 cell lines ...... 76 3.6.1 Stable expression of IFITM1, IFITM2 and IFITM3 decreases Jc1 replication ...... 77 3.7 Characterising the role of the IFITM proteins on the different stages of the HCV lifecycle ...... 78 3.7.1 IFITM1, IFITM2 and IFITM3 decrease HCV entry into Huh-7 cells ...... 78 3.7.2 The IFITM proteins have no effect on HCV RNA replication ...... 79 3.7.3 The IFITM proteins have no effect on HCV IRES activity ...... 80 3.7.4 The IFITM proteins have no effect on HCV egress ...... 81 3.8 Discussion ...... 82
Chapter 4 ...... 87
v Characterising the anti-HCV effect of IFITM1 at the Molecular Level ...... 87 4.1 Introduction ...... 87 4.2 Localisation of the IFITM proteins in the context of the essential host entry factors for HCV ...... 88 4.2.1 The IFITM proteins do not co-localise with the tight junction protein Occludin (OCLN) ...... 88 4.2.2 The IFITM proteins do not co-localise with the tight junction protein Claudin-1 (CLDN1) at the cell surface ...... 88 4.2.3 The IFITM proteins do not co-localise with the cell surface protein SR-BI 89 4.2.4 IFITM1 co-localises with the HCV entry factor CD81 on the hepatic cell surface ...... 90 4.3 IFITM1 interacts with CD81 on the hepatic cell surface ...... 90 4.4 Mutagenic analysis reveals important regions of IFITM1 for anti-HCV activity ...... 92 4.4.1 Characterisation of a panel of IFITM1 mutants ...... 92 4.4.2 The N-terminal region of IFITM1 is important for its anti-HCV activity .... 93 4.4.3. The C-terminal extension of IFITM1 is important for localisation within the hepatocyte ...... 95 4.5 Discussion ...... 96
Chapter 5 ...... 101
Cellular localisation of IFITM2 and IFITM3 ...... 101 5.1 Introduction ...... 101 5.2 Localisation of IFITM2 and IFITM2 within Huh-7 cells ...... 102 5.2.1 IFITM2 and IFITM3 do not localise to the endoplasmic reticulum (ER) .. 102 5.2.2 IFITM2 and IFITM3 do not localise to the Golgi complex ...... 102 5.2.3 IFITM2 and IFITM3 do not localise to lipid droplets ...... 103 5.2.4 IFITM3 partially co-localises with the early endosome ...... 103 5.2.5 IFITM2 partially co-localises with the late endosome ...... 104
vi 5.2.6 IFITM2 and IFITM3 localise to the lysosome, and IFITM1 partially co- localises with the lysosome ...... 104 5.2.7 The IFITM proteins do not associate with VAP-A in the hepatocyte ...... 105 5.3 Discussion ...... 106
Chapter 6 ...... 111
Post-translational modifications of the IFITM proteins are essential for anti-HCV activity ...... 111 6.1 Introduction ...... 111 6.2 N-terminal tyrosine phosphorylation of IFITM2 and IFITM3 is essential for cellular localisation but not for anti-HCV activity ...... 112 6.2.1 IFITM1, IFITM2 and IFITM3 undergo tyrosine phosphorylation in Huh-7 cells...... 112 6.2.2 Identification of conserved and non-conserved tyrosine residues between the IFITM proteins ...... 113 6.2.3 Y19 and Y20 are responsible for IFITM2 and IFITM3 phosphorylation, while the conserved Y78 is responsible for IFITM1 phosphorylation...... 114 6.2.4 The N-terminal tyrosine residue is required for the endosomal localisation of IFITM2 and IFITM3 ...... 115 6.2.5 IFITM2:Y19A and IFITM3:Y20A enhances anti-HCV activity compared to wildtype ...... 115 6.2.6 IFITM2:Y19A and IFITM3:Y20A co-localise with CD81 on the hepatic cell surface ...... 117 6.3 S-palmitoylation of the IFITM proteins is crucial for anti-HCV activity .. 118 6.3.1 Generation of IFITM mutants targeting S-palmitoylation sites ...... 118 6.3.2 A single conserved cysteine residue in the CIL is important for the anti-HCV properties of the IFITM proteins ...... 119 6.3.3 Mutation of conserved cysteine residues that undergo S-palmitoylation localise the IFITM proteins predominantly to the lysosome ...... 119
vii 6.4 Discussion ...... 120
Chapter 7 ...... 126
Conclusions and Future Directions ...... 126
Appendices ...... 137 Appendix I. General Solutions and Buffers ...... 137 Appendix II. Infectious HCV Constructs...... 140 Appendix III. pGem-T Easy ...... 141 Appendix IV. pLenti6/V5-D-TOPO ...... 142 Appendix V. pLenti6/V5-D-TOPO/IFITM (IFITM1, IFITM2, IFITM3) ...... 143 Appendix VI. PRL-HL ...... 144
References ...... 145
viii List of Figures and Tables
Figure Number On page:
Chapter 1 Figure 1.1 Clinical spectrum of HCV infection 3 Figure 1.2 Progression of HCV-induced liver disease 3 Figure 1.3 HCV Genome and Polyprotein processing 6 Figure 1.4 Global HCV genotype distribution 6 Figure 1.5 Model of HCV entry 11 Figure 1.6 Schematic representation of the HCV lifecycle 12 Figure 1.7 HCV Model Systems 15 Figure 1.8 Cellular recognition pathways of HCV 19 Figure 1.9 IFN-α/β signal transduction pathway 21 Figure 1.10 HCV evasion of the innate immune response 26 Figure 1.11 Schematic representation of IFITM1, IFITM2 and 29 IFITM3 and proposed topological models Table 1.1 Summary of post-translational modifications 30 undergone by IFITM3 Table 1.2 Summary of viruses inhibited by IFITM proteins 34
Chapter 2 Table 2.1 Cell lines and culture conditions used in this study 44 Table 2.2 Primer Sequence 52 Table 2.3 Western Blot Antibody Concentrations 61 Table 2.4 Immunofluorescence Antibody concentrations 64
Chapter 3 Figure 3.1 IFN- α induces the expression of IFITM1 in Huh-7 68 cells Figure 3.2 High level of sequence conservation prevents specific 69 detection of IFITM2 and IFITM3 mRNA in Huh-7 cells Figure 3.3 IFN- α induces the expression of IFITM1 in Huh-7 69 cells Figure 3.4 IFN-α and IFN-λ induces IFITM1 expression in PHH 70 Figure 3.5 shRNA mediated knockdown of endogenous IFITM1 71
ix in Huh-7 cells Figure 3.6 IFITM1 knockdown reduces the anti-HCV activity of 72 IFN-α Figure 3.7 Analysis of IFITM clones following ligation into the 72 lentiviral expression vector Figure 3.8 Transient detection of IFITM1, IFITM2 and IFITM3 73 in Huh-7 cells Figure 3.9 Transient expression of IFITM proteins in Huh-7 cells 73 can decrease HCV replication Figure 3.10 Transient transduction detection of IFITM1, IFITM2 74 and IFITM3 in Huh-7 cells Figure 3.11 The IFITM proteins have an effect on the early stages 75 of HCV infection in vitro Figure 3.12 Characterisation of Huh-7 cell lines stably expressing 76 IFITM1, IFITM2 and IFITM3 Figure 3.13 The IFITM proteins exhibit anti-HCV activity 77 Figure 3.14 The IFITM proteins significantly inhibit HCV entry 78 into Huh-7 cells Figure 3.15 The IFITM proteins have no effect on HCV RNA 79 replication Figure 3.16 The IFITM proteins have no effect on HCV RNA 80 replication Figure 3.17 The IFITM proteins do not modulate HCV IRES 81 activity Figure 3.18 The IFITM proteins have no effect on HCV egress 81
Chapter 4 Figure 4.1 Model of HCV entry 87 Figure 4.2 The IFITM proteins do not co-localise with HCV entry 88 receptor Occludin Figure 4.3 The IFITM proteins do not co-localise with HCV entry 89 receptor Claudin-1 Figure 4.4 The IFITM proteins do not co-localise with HCV entry 89 receptor SR-BI Figure 4.5 The IFITM proteins co-localises with the HCV entry 90 receptor CD81 on the hepatic cell surface Figure 4.6 IFITM1 interacts with the HCV entry receptor CD81 91 on the hepatic cell surface Figure 4.7 Schematic representation of Proximity Ligation Assay 91
x Figure 4.8 IFITM1 interacts with the HCV entry receptor CD81 91 on the hepatic cell surface Figure 4.9 Panel of IFITM1 mutants 92 Figure 4.10 The N-terminal region of IFITM1 is important for its 94 anti-HCV activity Figure 4.11 The N-terminal region of IFITM1 is important for its 95 anti-HCV activity Figure 4.12 The C-terminal region of IFITM1is important for its 95 localisation within the hepatocyte
Chapter 5 Figure 5.1 The IFITM proteins do not localise to the ER within 102 the hepatocyte Figure 5.2 The IFITM proteins do not localise to the Golgi 102 apparatus within the hepatocyte Figure 5.3 The IFITM proteins do not localise to lipid droplets 103 within the hepatocyte Figure 5.4 IFITM3 partially co-localises with the early endosome 103 marker Rab5a Figure 5.5 IFITM3 partially co-localises with the early endosome 103 marker Rab5a Figure 5.6 IFITM2 partially co-localises with the late endosome 104 marker Rab7 Figure 5.7 IFITM2 partially co-localises with the late endosome 104 marker Rab7 Figure 5.8 IFITM1, IFITM2 and IFITM3 co-localise with the 105 lysosomal marker Lamp1 Figure 5.9 IFITM1, IFITM2 and IFITM3 co-localise with the 105 lysosomal marker Lamp1 Figure 5.10 The IFITM proteins do not localise to Vap-A within 105 the hepatocyte Table 1.1 Summary of IFITM1, IFITM2 and IFITM3 107 localisation within Huh-7 cells
Chapter 6 Figure 6.1 Wildtype IFITMs undergo phosphorylation in Huh-7 112 cells Figure 6.2 Identification of conserved and non-conserved tyrosine 113 residues between IFITM1, IFITM2 and IFITM3
xi Figure 6.3 Mutation of a conserved tyrosine residue in the N- 113 terminus of IFITM2 and IFITM3 Figure 6.4 IFITM1 undergoes tyrosine phosphorylation at the 114 conserved Y78 residue Figure 6.5 IFITM2 and IFITM3 N-terminal tyrosine mutants do 114 not undergo phosphorylation in Huh-7 cells Figure 6.6 Tyrosine mutated IFITM2 and IFITM3 have different 115 cellular localisation Figure 6.7 The anti-HCV activity of IFITM2 and IFITM3 is 116 independent of tyrosine residues Y19 and Y20 Figure 6.8 The anti-HCV activity of IFITM2 and IFITM3 is 116 independent of tyrosine residues Y19 and Y20 Figure 6.9 The IFITM2 and IFITM3 N-terminal tyrosine mutants 117 co-localise with CD81on the hepatic cell surface Figure 6.10 Identification of conserved cysteine residues between 118 IFITM1, IFITM2 and IFITM3 Figure 6.11 Mutation of conserved cysteine residues in IFITM1, 118 IFITM2 and IFITM3 Figure 6.12 A single conserved cysteine residue in the CIL is 119 essential for the anti-HCV activity of the IFITM proteins Figure 6.13 Palmitoylation IFITM1 mutants re-localise to the 120 lysosome Figure 6.14 Palmitoylation IFITM2 mutants partially co-localise to 120 the late endosome but primarily re-localise to the lysosome Figure 6.15 Palmitoylation IFITM1 mutants re-localise to the 120 lysosome
Chapter 7 Figure 7.1 Schematic representation of the localisation and 129 potential molecular mechanism of IFITM2 and IFITM3 against HCV
xii Abstract
Hepatitis C virus (HCV) is a significant human pathogen of the liver that in the
majority of infected individuals causes a chronic infection of the liver. This may over
time culminate in the development of severe liver disease such as cirrhosis and hepatocellular carcinoma. Prior to 2012, the only treatment option available for HCV infection was combination therapy with pegylated interferon-α (IFN-α) and ribavirin.
However, the recent development and addition of direct acting antivirals (DAAs) into treatment regimes has significantly improved sustained virological response rates.
While the ultimate aim is for IFN-α free therapy, IFN-α is often required in
combination with the DAAs to reduce the development of viral resistance and due to
cost. It is clear that IFN (either exogenous or endogenous) can induce an antiviral state
in HCV infected cells; however, the exact mechanisms that underpin this action remain
unclear.
The interferon-induced transmembrane (IFITM) family of proteins - IFITM1, IFITM2 and IFITM3 has recently been identified as important host effector molecules of the type I IFN response against a broad range of RNA viruses. During the course of this
PhD study, a number of investigations identified the IFITM proteins to be potent antiviral effectors against HCV; however, the mechanism(s) for this antiviral activity remains contradictory. In this thesis, we demonstrate that IFITM1, IFITM2 and
IFITM3 play an integral role in the IFN response against HCV and act specifically to
inhibit early and late stages of HCV entry to inhibit infection. We reveal that IFITM1
localises to the cell surface in hepatocytes and interacts with the host entry factor CD81
to limit HCV entry. Furthermore, the N-terminus, in particular amino acids 21-28, of
xiii IFITM1 plays an important role in this anti-HCV activity, while the C-terminus is found to be important for localisation to the cell surface.
We also established that in hepatocytes, IFITM2 and IFITM3 localise to the late and
early endosomes respectively, as well as the lysosome, indicating that IFITM2 and
IFITM3 follow the established paradigm of targeting the late entry stages of HCV
infection. Furthermore, we have demonstrated that S-palmitoylation of all three IFITM proteins is essential for both anti-HCV activity and cellular localisation, while the
conserved tyrosine residue in the N-terminus of IFITM2 and IFITM3 plays a
significant role in protein localisation. However, this tyrosine was found to be
dispensable for anti-HCV activity, with mutation of the tyrosine resulting in an
IFITM1-like phenotype with the retention of anti-HCV activity and co-localisation of
IFITM2 and IFITM3 with CD81.
In conclusion, we propose that the IFITM proteins act in a coordinated manner to
restrict HCV infection by targeting the endocytosed HCV virion for lysosomal
degradation and demonstrate that the actions of the IFITM proteins are indeed virus
and cell-type specific. We believe we have significantly added to our understanding of
the interplay between HCV and the host innate immune response and that in the long
term these findings will aid in the generation of novel and targeted anti-HCV
therapeutics for patients chronically infected with HCV.
xiv Declaration
I certify that this work contains no material which has been accepted for the award of
any other degree or diploma in my name in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. In addition, I certify that no part of this work, in the future, will be used in a submission in my name for any other degree or diploma in any other university or other tertiary institution without the prior approval of the University of Adelaide and where applicable, any partner institution responsible for the joint award of this degree.
I give consent to this copy of my thesis when deposited in the University Library, being made available for loan and photocopying subject to the provisions of the Copyright
Act 1968.
I also give permission for the digital version of my thesis to be made available on the web, via the University’s digital research repository, the Library Search and also through web search engines, unless permission has been granted by the University to restrict access for a period of time.
Sumudu Kumari Narayana
October 9th, 2015
xv Acknowledgements
I would like to thank my supervisor Michael Beard for the opportunity to do a PhD in his laboratory and for his continued assistance and mentoring throughout the years.
I offer my sincerest gratitude to Karla Helbig, Nicholas Eyre, Erin McCartney and
Amanda Aloia for their excellent advice, support and technical assistance during my
PhD.
I am most grateful to all members of the Hepatitis C Research Laboratory both past and present. Specifically I would like to acknowledge Edmund Tse, Kate Muller, Kylie Van der Hoek, Guillaume Fiches, Onruedee Khantisitthiporn and Kathleen Davy. I would also like to thank the Department of Molecular and Cellular Biology for the opportunity to undertake a PhD.
I would like to thank my amazing parents for their continued love, support, guidance and encouragement. I would also like to thank my brothers, sister-in-law and nieces,
Tharaka, Mary, Sampath, Lucy and Clara for providing invaluable encouragement and for always making me laugh even when times were hard. Finally I would like to thank
Bella for her unconditional love and company over the years, especially during those long weeks of writing this thesis. I would also like to thank Richard Apps for editing this thesis.
xvi Publications Arising from this PhD
Narayana, S.K., Helbig, K.J., McCartney, E.M, Eyre, N.S., Bull, R.A., Elathla, A., Lloyd, A.R., and Beard, M.R. (2015) The interferon-induced transmembrane proteins IFITM1, IFITM2 and IFITM3 inhibit hepatitis C virus infection. Journal of Biological Chemistry 290(43):25946-59
Awards Received During PhD
2012 School of Molecular and Biomedical Science School Symposium Poster Prize – 2nd Place - $200
2012 Nomination by the Golden Key International Honour Society to attend the 2013 International Scholar Laureate Program (ISLP) Delegation on Medicine
2012 Australian Centre for Hepatitis Virology Travel Award for HCV Research – $2500 National award - one awarded per year for travel to international HCV meeting.
2011 Dawes Top-Up Scholarship, Royal Adelaide Hospital Research Fund Grant Round – funded from 2012 to 2014
Presentations Arising From PhD
INTERNATIONAL
Narayana, S.K., Helbig, K.J., McCartney, E.M. Eyre, N.S., Aloia, A.L. and Beard, M.R. The interferon-induced transmembrane protein (IFITM) family exhibit anti-HCV activity. 20th International Symposium on Hepatitis C Virus and Related Viruses, Melbourne, Australia, 2013. (poster presentation)
Narayana, S.K., Helbig, K.J., Eyre, N.S., McCartney, E.M. and Beard, M.R. The interferon-induced transmembrane protein (IFITM) family exhibit anti-HCV activity. 19th International Symposium on Hepatitis C Virus and Related Viruses, Venice, Italy, 2012. (poster presentation)
K.J. Helbig, J.M. Carr, S.K. Narayana, E.M. McCartney, N.S. Eyre and M.R. Beard. The ISG viperin has novel anti-hepatitis C virus activity through interaction with a
xvii HCV pro-host cell factor. Keystone Symposia 2012 - Innate Immunity, Colorado, USA, 2012. (poster presentation)
McCartney E.M., Helbig, K.J., Eyre, N.S, Narayana, S.K. and Beard, M.R. The role of STAT3 in the life cycle of HCV. 18th International Symposium on Hepatitis C Virus and Related Viruses, Seattle, USA, 2011. (poster presentation)
NATIONAL
Narayana, S.K., Helbig, K.J., McCartney, E.M. Eyre, N.S., Aloia, A.L. and Beard, M.R. The interferon-induced transmembrane protein (IFITM) family exhibit anti-HCV activity. Infection and Immunity Conference, Lorne, Australia, 2014. (poster presentation)
Narayana, S.K., Eyre, N.S., McCartney, E.M., Van der Hoek, K., Helbig, K.J., and Beard, M.R. The interferon-induced transmembrane (IFITM) proteins have anti-HCV activity in vitro. Australian Centre for Hepatitis Virology workshop, Adelaide, Australia, 2012. (oral presentation)
Narayana S.K., Eyre, N.S., Van der Hoek, K., Helbig, K.J., and Beard, M.R. The interferon-induced transmembrane (IFITM) proteins 1 and 3 have anti-HCV activity in vitro. Australian Centre for Hepatitis Virology workshop, Maroochydore, Australia, 2011. (oral presentation)
Beard M.R., Narayana, S.K., Eyre, N.S., Yip, E.Y., Lemon, S.M. and Helbig, K.J. The ISGs viperin and the IFITM family have novel anti-Hepatitis C Virus activity. Infection and Immunity Conference, Lorne, Australia, 2011. (oral presentation)
Narayana S.K., Eyre N.S., Van der Hoek, K., Helbig K.J., and Beard, M.R. Identification of novel interferon stimulated genes (ISGs) that control HCV in vitro. Australian Centre for Hepatitis Virology workshop, Yarra Valley, Australia, 2010. (oral presentation)
xviii Materials Providers
Abcam Cambridge, UK Ambion Texas, USA Amersham Pharmacia Biotech Birminghamshire, UK Amrad Biotech Boronia, VIC, Australia Anogen Ontario, Canada Applied Biosystems Warrington, UK Becton Dickson Labware New Jersey, USA Biomol New Jersey, USA BioRad Laboratories California, USA Cell Signaling Massachusetts, USA Chemicon International Massachusetts, USA Cohu California, USA DAKO California, USA Dynatech Virginia, USA GeneWorks Adelaide, SA, Australia Invitrogen California, USA Merck Darmstadt, Germany Mol Bio Laboratories California, USA Molecular Probes Oregon, USA Nalge Nunc International Illinois, USA Nikkon Sydney, NSW, Australia New England Biolabs Massachusetts, USA Oxis Oregon, USA Olympus New York, USA Panomics Santa Clara, USA Perkin Elmer Massachusetts, USA Promega Wisconsin, USA
xix QIAgen Hilden, Germany Roche Indiana, USA Rockland Pennsylvania, USA Schering-Plough New Jersey, USA Schleicher and Schuell Dassel, Germany Sigma Missouri, USA SPSS Inc Illinois, USA Stratagene California, USA UVP Inc California, USA Vector Laboratories California, USA Vision Systems Mount Waverley, VIC, Australia
xx Abbreviations Used
A adenosine aa amino acids bp base pairs BSA bovine serum albumin BVDV bovine viral diarrhoea virus C cytosine ° C degrees Celsius cDNA complimentary deoxyribosenucleic acid CLDN1 claudin-1 CHC chronic hepatitis C CIL cytoplasmic intracellular loop CMV cytomegalovirus dATP deoxyadenosine-5’-triphosphate dCTP deoxycytosine-5’-tripshosphate DEPC diethyl pyrocarbonate DAA direct acting antiviral dGTP deoxyguanosine-5’-triphosphate
dH2O deionised water DMEM Dulbecco’s Modified Eagle Medium with HEPES DNA deoxyribonucleic acid dNTP deoxyribonucleotide triphosphate dTTP deoxythymidine-5’-triphosphate DENV Dengue virus EBoV Ebola virus EDTA ethylene diamine tetra acetic acid ER endoplasmic reticulum
xxi FCS foetal calf serum FITC fluorescein isothiocyanate FFU focus forming unit g grams G guanosine GAPDH glyceraldehyde-3-phosphate deydrogenase GAS gamma-activated sequence GFP green fluorescent protein HCC hepatocellular carcinoma HBV hepatitis B virus HCV hepatitis C virus hr hour(s) HRP horse radish peroxidase HIV human immunodeficiency virus°°°° HPV human pappilomavirus IAV influenza virus IDU infecting drug use IFITM interferon-induced transmembrane protein
IFN-α interferon alpha IFN-β interferon beta
IFN-γ interferon gamma IFN-λ interferon lambda IRES internal ribosome entry site ISRE interferon stimulated response element JAK janus kinase kb kilobase kDa kilo Dalton L-Agar LB + agar LD lipid droplet
xxii LB Luria Bertani broth LDL low density lipoproteins Luc luciferase µg micrograms µl microlitres µM micromolar mA milliamps mg milligrams ml millilitres mM millimolar MCS Multiple Cloning Site MEM Minimum Essential Medium min minute(s) mRNA messenger RNA MW molecular weight MLV murine leukemia virus ng nanograms nM nanomolar N/A not applicable nt nucleotide NTD N-terminal domain NF-κB nuclear factor-kappa-light chain enhancer OCLN occludin ORF open reading frame PAGE polyacrylamide gel electrophoresis
PBS phosphate buffered saline; 0.15M NaCl, 6M K2HPO4, 2mM
KH2PO4 (pH 7) PAMP pathogen associated membrane patterns
xxiii PCR polymerase chain reaction PHH primary human hepatocyte PRR pathogen recognition receptors PTM post-translational modification pg picograms pmol picomolar RNA ribonucleic acid rpm revolutions per minute RFP red fluorescent protein RdRp RNA-dependent-RNA polymerase RC replication complex RIG-I retinoic acid inducible gene I RT room temperature RT-PCR reverse transcriptase polymerase chain reaction sd standard deviation
SDS sodium dodecyl sulfate
sec second(s)
SEM standard error of mean
ss single stranded
siRNA small interefering RNA
SR-BI scavenger receptor class B member I
STAT signal transducer and activator of transcription
SVR sustained virological response
T T thymidine
TAE 0.04M Tris (pH 8), 0.04M Acetic Acid, 1mM EDTA
TEMED TEMED N,N,N’,N’-tetramethylethyethylenediamine
xxiv Tris 3,3,5,5-tetramethylbenzidine
TLR Toll-like receptor
TYK2 tyrosine kinase 2
U units
UTR untranslated region
V volts
VSV vesicular stomatis virus w/v weight per volume WNV West Nile Virus
xxv Chapter 1
Introduction
1.1 Hepatitis C Virus
1.1.1 Epidemiology
Hepatitis C virus (HCV) is one of the leading causes of liver-disease related morbidity
worldwide. Until the identification of the HCV genome in 1989, it was the leading
cause of ‘non-A, non-B’ hepatitis (Choo et al., 1989). However, since then, great
advances have been made in characterising the molecular biology and the pathogenesis
of HCV. HCV is an enveloped, positive-sense single-stranded RNA virus belonging to
the Hepacivirus genus and the Flaviviridae family (Farci, 2002). It is currently
estimated that there are 150 million people infected with HCV worldwide (Thomas,
2012, Organisation, 2014), while the prevalence in Australia has been estimated at
350,000 individuals (Razali et al., 2009, Razavi et al., 2014). One of the most striking
features of HCV is its tendency towards chronicity, with approximately 80% of
infected individuals developing progressive liver diseases such as liver cirrhosis,
hepatic failure and/or hepatocellular carcinoma (Zhong et al., 2005). With over 8,800 new cases of HCV infection each year in Australia (Razavi et al., 2014), the burden of
HCV-related chronic liver disease is estimated to increase by over 200% in the coming years, resulting in a significant impact on the health system (Davis et al., 2010,
Thomas, 2012, Razavi et al., 2014).
1 1.1.2 Transmission
HCV is a blood borne virus where the most efficient mode of transmission is through
the transfer of blood and blood products. However, the introduction of blood screening
techniques in 1992 virtually eradicated the risk of HCV contraction through blood
transfusion and organ transplantation in the western world. The majority of new
infections in developed countries now occur through injecting drug use (IDU), with sharing of contaminated injecting equipment during intravenous drug injections being the predominant mode of transmission of HCV for the past 40 years (Scott et al., 2015,
Hellard et al., 2015). This mode of transmission accounts for approximately 80% of
HCV transmission cases within Australia. Less common modes of HCV transmission include tattooing, piercing, occupational needle stick injuries, sexual intercourse with infected partners and vertical transmission from mother to baby (Dore et al., 2003,
Mohan et al., 2010, Thomas, 2012). In contrast, un-screened blood transfusions, reuse of syringes and needles, and other unsafe medical procedures account for the significant number of HCV infections in the developing world (Sievert et al., 2011).
1.1.3 Pathogenesis
HCV replication occurs in hepatocytes, although there have been reports of HCV RNA detected in other cell types such as dendritic cells (Pachiadakis et al., 2005), B cells
(Sung et al., 2003), monocytes (Bouffard et al., 1992), lymph node cells (Dietrich et al.,
1997) and digestive tract cells (Tursi et al., 2002). The role of these cells in the HCV
lifecycle remains unknown, and much of the data presented in these studies remains
controversial. HCV is a non-cytopathic virus, and so the liver disease associated with
HCV infection is attributed to the innate and adaptive responses directed towards
clearing HCV infected hepatocytes.
2
Acute HCV infections are generally asymptomatic and therefore difficult to diagnose during the early stages (Gremion and Cerny, 2005). 20% of patients with acute HCV infection are able to clear the virus via the immune response, while the remaining 80% of patients develop a persistent infection, defined as chronic hepatitis C (CHC). Of those individuals who develop CHC, approximately 20% will develop progressive liver disease over a period of 25-30 years [Figure 1.1] (Freeman et al., 2001), culminating in
liver cirrhosis, and in 2% of individuals, in hepatocellular carcinoma (HCC). The
immune response directed against HCV is still considered the main factor in the
development of liver fibrosis and cirrhosis [Figure 1.2]. Cirrhosis is the most advanced
form of liver disease, characterised by extensive scarring that stiffens blood vessels and
distorts the internal structure of the liver impairing its function (Racanelli and
Rehermann, 2003). Advanced HCV-related liver disease is a key factor for liver
transplantation in Australia. The rate of progress of advanced liver disease is influenced
by co-factors such as alcohol consumption and co-infection with other viruses such as
human immunodeficiency virus (HIV) and hepatitis B virus (HBV).
1.1.4 Treatment
Interferon-alpha (IFN-α) monotherapy became available in 1991, and was the first
standard therapy demonstrated to show beneficial effects on patients; however,
response rates were only 10-20% at best. IFN-α is an inducible cytokine capable of inducing an antiviral response, and when combined with the guanine nucleotide, ribavirin (in 1998) response rates improved to 40% (Foster, 2010). Treatment was revolutionised in 2001 upon the addition of a polyethylene glycol group to IFN-α
(pegIFN-α) that increased the half-life of IFN-α and a reduction in the dosage required
3 Infection
Acute Hepatitis 80%
20% Resolution Chronic Hepatitis
20%
2% HCC Cirrhosis
Liver failure
!
Figure 1.1: Clinical spectrum of HCV infection
Figure 1.2: Progression of HCV-induced liver disease
per week (Glue et al., 2000). PegIFN-α in combination with ribavirin induced higher sustained virological response (SVR, defined as undetectable HCV RNA in serum 6 months post-treatment) in patients; however, treatment response was genotype specific, being less effective against genotype 1 infected individuals.
Treatment with pegIFN-α and ribavirin has been the standard of care for a number of years but is only effective in 50% of patients across all genotypes. Duration of treatment is 24-48 weeks depending on genotype and is associated with a number of significant side effects such as flu-like symptoms, severe depression and haemolytic anaemia (Feld and Hoofnagle, 2005, Sharma, 2010). The majority of these side effects are attributed to IFN-α. Additionally, some patients are simply not candidates for treatment such as individuals with a history of mental illness or drug dependency due to issues with compliance and increased side effects, while patients with advanced liver disease, insulin resistance, obesity, significant alcohol consumption and HIV co- infection tend to have lower SVR rates (Thomas, 2012). Thus, a large group of patients remain without treatment, and current research has been directed towards developing targeted antiviral drugs with increased efficacy.
The development of direct acting antiviral (DAAs) agents for HCV, compounds that directly interact with and inhibit HCV proteins has once again transformed the treatment of hepatitis C. DAAs have been developed to target various steps of the
HCV lifecycle, with specific inhibitors targeting the HCV NS3/4A serine protease, the
NS5A protein and the RNA-dependent RNA-polymerase (NS5B) (Aloia et al., 2012).
The first generation of NS3/4A serine protease inhibitors approved included boceprevir and telaprevir and has been used in the treatment of genotype 1 patients with moderate
4 success, although they were often plagued with severe side effects. Current standard of
care for genotype 1 HCV patients is a combination of Peg-IFN-α, ribavirin and a 2nd generation NS3/4A inhibitor simeprevir (Forns et al., 2014). Newly approved, in the
USA and Europe, NS5B polymerase inhibitor sofosbuvir has shown promise against
HCV patients with genotypes 1,2,3 and 4 in combination with ribavirin, and depending
on genotype, pegIFN-α (Keating, 2015). Triple therapy treatments have resulted in an
increase in SVR in these patients, up to 70-80% (Marks and Jacobson, 2012); however,
the treatment is not as promising in patients who have previously failed therapy or have
advanced liver disease. IFN-α remains part of therapy, due to the development of
antiviral resistance towards these compounds when used in monotherapy (Aloia et al.,
2012, Calle Serrano and Manns, 2012). Clinical trials are currently underway for other
compounds, some of which have already been approved in the USA, and suggest that
these drugs are more efficacious, have improved side effects and act across multiple
genotypes (Pol et al., 2012, Jesudian et al., 2013). The next generation of DAAs will
form the standard of care for HCV in the next few years, with the real possibility of a
IFN-free, triple DAA combination that will have minimal side effects and little to no
resistance profile. However, while these new waves of DAA therapies are very
effective they do come at a cost and will only be available to those that can afford it, or
where the cost is subsidized by government or health insurance (Gane, 2012, Kowdley et al., 2014).
1.1.5 The HCV genome
HCV is an enveloped, positive-sense single-stranded RNA virus that encodes a 9.7kb genome that does not enter the nucleus. The open reading frame (ORF) encodes for a single polyprotein precursor approximately 3000 amino acids in length, flanked by
5 highly conserved 5’ and 3’ untranslated regions (UTR) [Figure 1.3]. Host and viral proteases cleave the polyprotein to generate the 10 structural and non-structural HCV proteins (Moradpour et al., 2007).
The 5’ UTR is approximately 341 nucleotides in length and contains an internal ribosomal entry site (IRES) that is essential for the viral RNA to undergo cap- independent translation of the HCV polyprotein. The IRES precedes the initiation codon of the HCV polyprotein, and partially overlaps with an additional sequence in the 5’ UTR that is essential for viral replication. Downstream of the HCV ORF stop codon is the 3’UTR, which consists of 3 main elements that are essential for initiation of viral replication: a variable region, a polyuridine (poly-U/UC) tract of an average 80 nucleotides and a terminal segment designated the X-tail (Niepmann, 2013, Suzuki et al., 2007).
1.1.6 Classification of genotypes
HCV can be classified into 6 main genotypes that differ in nucleotide sequence by 30-
35%. The prevalence of these genotypes varies in geographical distribution [Figure
1.4] and response to IFNα:ribavirin therapy is highly dependent on genotype, as previously discussed in Section 1.14. Genotypes 1 and 3 predominate globally, including Australia (Dore et al., 2003). Genotypes 4-6 tend to be mainly restricted to
Africa, the Middle East and Asia (Bowden and Berzsenyi, 2006). Differences in the nucleotide sequence (between 2—25%) within genotypes can be further classified in to subtypes, designated a, b, c and so on, and these variants arise due to the high mutation rate of the error prone HCV RNA-dependent RNA polymerase (RdRp). The
6
Figure 1.3: HCV Genome and Polyprotein Processing (Moradpour et al., 2007)
Figure 1.4: Global HCV genotype distribution heterogeneous population of related HCV genomes that coexist within an infected
individual is referred to as quasispecies (Moradpour et al., 2007, Pawlotsky, 2003).
1.1.7 HCV proteins
Host and viral proteases cleave the polyprotein precursor, encoded by the HCV ORF,
to give rise to 3 structural proteins (core, E1 and E2), the p7 viroporin and 6 non- structural (NS2, NS3, NS4A, NS4B, NS5A and NS5B) HCV proteins [Figure 1.3]. A brief review of the functions of the HCV proteins is provided below, as the literature surrounding HCV protein function is far beyond the scope of this thesis.
Core: The HCV core protein, located at the N-terminus of the polyprotein, is cleaved
by the endoplasmic reticulum (ER) signal peptidase and has been demonstrated to
localise to the ER, the membranous web (see Section 1.18) and to associate with
cytoplasmic lipid droplets (LD) (McLauchlan et al., 2002, Boulant et al., 2006,
Miyanari et al., 2007). The primary role of the 21kDa core protein appears to be
structural, with involvement in the formation of the viral nucleocapsid via interactions
with NS5A and LDs; however, it is believed to also have a role in viral replication.
Cleavage of the core protein by signal peptide peptidase enables it’s trafficking to LDs, and this cleavage is essential for the assembly of core into new virions at the ER membranes (Ait-Goughoulte et al., 2006). Furthermore, the core protein has been shown to aid HCV pathogenesis by disrupting cellular functions such as inhibiting tumour necrosis factor (TNF)-α mediated apoptosis and NF-κB mediated gene transcription (Dansako et al., 2005). The association of core with LDs has also led to speculation that it might affect lipid metabolism in the cell, leading to steatosis and the development of HCC (Asselah et al., 2006). However, many studies investigating the
7 function of core have been done with exogenous expression and should be interpreted
with caution.
Envelope glycoproteins E1/E2: E1 and E2 are highly glycosylated type 1
transmembrane proteins and form a non-covalent complex that is the major constituent
of the viral particle surface (Goffard and Dubuisson, 2003). The E1/E2 heterodimer
mediates the binding of the HCV virion to the target cell receptors and is thus essential
for the cellular entry of HCV. E1 and E2 have also been shown to play a role in post-
entry fusion with the endosomal membrane as well as in the assembly of new infectious
viral particles (Bartosch et al., 2003, Hsu et al., 2003).
p7: p7 is a small hyrdrophobic protein (63kDa) that is localised to the ER. It has two
transmembrane domains connected by a cytoplasmic loop, with both the N- and C- termini oriented toward the ER lumen (Carrere-Kremer et al., 2002). p7 is often described as a vipoporin; a viral cation channel that allows calcium ions to flow from the ER into the cytoplasm (Griffin et al., 2003, Pavlovic et al., 2003). p7 is not required for RNA replication in a HCV replicon system, but has been shown to be essential for productive infection in vivo in chimpanzees (Sakai et al., 2003). Studies have demonstrated that the ion channel activity of p7 is critical for HCV virus production, with p7 believed to be important for the early stages of virion morphogenesis before assembly (Jones et al., 2007, Steinmann et al., 2007). However, the precise function and role of the p7 ion channel in viral assembly and release remains to be elucidated.
NS2: NS2 is cleaved from NS3 via a cysteine protease that is encoded within the
carboxy terminal of NS2 and the amino terminal of NS3 (Selby et al., 1994). NS2 is
8 localised to the ER and is dispensable for RNA replication, but is required for the
complete HCV replication cycle both in vitro and in vivo. Recent studies suggest that
NS2 plays a central organising role in virus assembly that is independent of its
autoprotease activity. NS2 is thought to act as a mediator between the structural and
non-structural proteins as it is involved in a complex network of interactions with E1,
E2, p7, NS3 and NS5A to form a functional unit capable of driving these proteins
towards the LDs for virus assembly (Thibeault et al., 2001, Lorenz et al., 2006,
Popescu et al., 2011, de la Fuente et al., 2013).
NS3/4A complex: NS3 is a multifunctional protein that contains a serine protease in the
N-terminal and a RNA helicase in the C-terminal regions, respectively (Yao et al.,
1999). The RNA helicase unwinds the RNA duplexes that form during HCV genome replication in an ATP-dependent manner (Tai et al., 1996). The serine protease of NS3
is responsible for the proteolytic cleavage of all the downstream non-structural proteins
[Figure 1.3]. The function of the serine protease is greatly enhanced by its interaction
with NS4A. NS4A acts as a cofactor to stabilize the NS3/4A complex and aids in its
membrane association (Brass et al., 2008, Morikawa et al., 2011). The NS3/4A
complex is located primarily on the membranes of the ER and in replication complexes
but has also been found on mitochondrial membranes (Wolk et al., 2000, Horner et al.,
2011). It plays a role in viral evasion of the innate immune response and persistence of
viral infection by cleaving crucial innate immune signaling adaptor proteins, TIR-
domain-containing adaptor-inducing interferon-β (TRIF) and mitochondrial antiviral
signaling protein (MAVS) (Li et al., 2005b, Meylan et al., 2005). NS3/4A serine
protease has been the target of the first generation of DAAs, namely telaprevir and
9 boceprevir, in which they render the protease activity inactive and this abrogates the
liberation of the HCV non-structural proteins from the polyprotein.
NS4B: NS4B is a conserved hyrdrophobic protein that alters the intracellular
membranes of the ER to initiate the formation of the membranous web in the cytoplasm, which is the site of HCV replication (Egger et al., 2002, Gosert et al., 2003).
NS4B interacts with other HCV non-structural proteins as well as binding viral RNA.
NS4B is a transmembrane protein that traverses the ER membrane and undergoes
oligomerisation. It has been demonstrated to have NTPase activity and shown to have
a role in viral assembly (Einav et al., 2004, Einav et al., 2008, Gouttenoire et al., 2010,
Jones et al., 2009).
NS5A: NS5A is a phosphoprotein with three domains found anchored to the ER and
plays an important role in HCV RNA replication and virus assembly. NS5A can be
found in two phosphorylation states, either basally phosphorylated (56kDa) or
hyperphosphorylated (58kDa) and the phosphorylation status of NS5A appears to effect
the level of HCV replication (Appel et al., 2005, Blight et al., 2000, Appel et al., 2008).
NS5A has been shown to bind HCV RNA (Huang et al., 2005), host factors such as
Rab5a and the vesicle-associated membrane protein-associated protein A (VAP-A)
(Coller et al., 2009, Eyre et al., 2014), as well as other HCV proteins such as core. The
association between NS5A and core has been shown to be crucial for viral particle
assembly at the lipid droplet interface, where NS5A has been demonstrated to be vital
in the transfer of viral RNA to core (Masaki et al., 2008, Jones and McLauchlan, 2010).
While the function of NS5A is not completely understood, it is the target of a number
of new DAAs that have recently been licensed.
10 NS5B: NS5B is an RdRp that is able to initiate de novo RNA synthesis of positive and
negative strand RNA. NS5B is anchored to the ER membrane via its transmembrane
domain (Lohmann et al., 2000, Moradpour et al., 2004, Schmidt-Mende et al., 2001).
Given the importance of this protein and its enzymatic activity, a number of nucleoside and non-nucleoside analgoues have been developed as DAAs.
1.1.8 HCV life cycle
HCV virions are approximately 40-70nm in diameter, and are thought to consist of an icosahedral nucleocapsid composed of core protein oligomers encapsulating the RNA genome. A host-cell derived envelope containing the HCV envelope proteins E1 and
E2 surrounds the nucleocapsid. In circulation, the HCV virions associate with low- and very-low-density lipoproteins (LDLs and VLDLs) to form lipoviralparticles
(Monazahian et al., 2000, Wunschmann et al., 2000).
HCV particles initially attach to the hepatocyte via low-affinity interactions with LDL receptors and glycosaminoglycans (GAGs) (Agnello et al., 1999, Germi et al., 2002), leading to a high-affinity interaction with scavenger receptor class B member 1 (SR-BI)
[Figure 1.5] (Scarselli et al., 2002). Binding to SR-BI has been shown to be critical for
HCV entry, as it exposes the CD81-binding determinants on the HCV E2 glycoprotein thus allowing for the interaction of the viral particle with one of the essential host entry factors, the tetraspanin CD81 (Pileri et al., 1998, Brazzoli et al., 2008, Kapadia et al.,
2007). This interaction results in the lateral movement of the CD81-bound viral particle towards the tight junctions of the hepatocyte, resulting in an interaction with claudin-1 (CLDN1) (Evans et al., 2007, Harris et al., 2010). Recent studies have identified that several transduction pathways are required for this movement along the
11
Figure 1.5: Model of HCV entry (Eyre et al., 2009) cell surface, with epidermal growth factor receptor (EGFR) and ephrin receptor A2
(EphA2) (Lupberger et al., 2011) being essential co-factors for the formation of the
CD81-CLDN1 complex. In addition, the tight junction protein occludin (OCLN) and the cholesterol receptor Niemann-Pick C1-like 1 (NPC1L1) are essential entry factors; however, the precise role of these proteins is currently not well understood (Ploss et al.,
2009, Sainz et al., 2012).
The interaction of HCV bound CD81 with CLDN1 initiates clathrin-mediated endocytosis of the virion (Blanchard et al., 2006), resulting in its traffic to Rab5a- containing early endosomes. Acidification of the endosome results in the release of the viral genome in to the cytoplasm [Figure 1.6]. The fusion process of HCV is not well understood, but it is hypothesised that HCV E1 is the fusion protein that mediates viral- endosome fusion, with a strong interplay with HCV E2. The current model proposes that upon internalization, fusion-primed E1/E2 heterodimers have their confrmation stabilised through insertion into the endosomal membrane, and upon arriving at Rab5a- containing early endosomes, exposure of the viral particle to the acidic environment results in the exposure of the putative E1 fusion peptide and thus allows for viral and endosomal membrane fusion to occur (Douam et al., 2014, Douam et al., 2015). The viral genome is then released into the cytoplasm. HCV proteins are directly translated from the positive-sense RNA, with the binding of the 40S ribosomal subunit to the
HCV IRES. The large HCV polyprotein is subsequently cleaved by both viral and host proteases to produce the structural proteins (core, E1, E2 and p7) and the non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B). Alterations to the ER membranes induced by NS4B result in the formation of a membranous web containing replication complexes (RC), and are the site of HCV replication in the cell (Egger et al.,
12
Figure 1.6: Schematic representation of the HCV lifecycle (a) HCV binding and internalisation (b) Fusion and release (c) IRES-mediated translation and polyprotein processing (d) RNA replication (e) Packaging and assembly (f) Virion maturation and release (Moradpour et al., 2007) 2002, Gosert et al., 2003). The HCV RC also contains non-structural proteins, viral
RNA and a number of other cellular factors such as VAP-A and phosphatidylinositol 4- phosphate (PI-4K) (Gosert et al., 2003, Moradpour et al., 2003). The RdRp NS5B initiates the de novo synthesis of negative-strand RNA, which serves as the template for the synthesis of multiple copies of the HCV genome. A proportion of the newly formed genomes act as templates for translation and replication, while some associate with the core protein to form new virions. The mechanisms of virus assembly and egress have not been completely elucidated; however, assembly of new virions is thought to occur on the cytosolic side of the ER, while complete maturation occurs in the ER lumen. It is thought that the early steps of assembly occur at the site of the LD, where RCs are recruited to the site via an interaction between core and NS5A (Han et al., 2013). This interaction is believed to allow newly replicated RNA to be encapsulated into the new virion, with the core protein aiding the transfer of the RNA from the RC into the virion. The later stages of assembly result in the acquisition of a lipid envelope and the incorporation of the E1 and E2 glycoproteins. The mature virions are then released from the cell via exocytosis in a non-cytolytic manner (Jones and McLauchlan, 2010, Lindenbach and Rice, 2013).
1.1.9 HCV model systems
Study of the HCV lifecycle and pathogenesis has been severely hindered in the past due to the lack of a robust fully permissive cell culture system and a small animal model, with the chimpanzee model being the only permissive animal model. The development of the HCV sub-genomic replicon system in 1999 (Lohmann et al., 1999), the genomic system in 2002 (Ikeda et al., 2002) and the infectious cell culture system in 2005
(Wakita et al., 2005) have revolutionized in vitro HCV research. These in vitro HCV
13 culture systems are utilised throughout this thesis and are discussed in more detail below.
1.1.9.1 Animal models
The chimpanzee model has played an important role in the initial identification and cloning of the HCV genome (Choo et al., 1989), as well as in understanding the host immune response to HCV and in the development of vaccines and drugs. However, the use of this model is limited due to ethical and financial implications, as well as the differences in disease progression between humans and chimpanzees. The generation of the uPA/SCID mouse model, where immunodeficient mice were engrafted with human hepatocytes that enabled active HCV replication within mice (Meuleman et al.,
2005, Mercer et al., 2001), was the first successful small animal model. Despite the use of this model for numerous studies, especially in characterising the innate immune response against HCV (Walters et al., 2006), extensive studies are limited due to the lack of an adaptive immune response and due to high levels of variation in human hepatocyte engraftment (Turrini et al., 2006). Furthermore, the use and development of
uPA/SCID mice is technically challenging and is not available to the majority of
researchers. Similarly, other small animal models have been limited due to the low
levels of HCV viraemia produced (Bukh, 2012, Boonstra et al., 2009). Recent work has
resulted in the generation of a genetically humanized mouse model with a dampened
immune reponse that supports HCV infection (Dorner et al., 2013). This new model
presents different prospects in better understanding HCV infection in vivo and for
testing new drug candidates.
14 1.1.9.2 Cell culture systems
The HCV replicon systems have proven to be crucial for the study of HCV replication,
interaction between host and HCV proteins, and also for the testing of novel antiviral compounds. The genomic (HCV structural and non-structural proteins) and the subgenomic (HCV non-structural proteins) replicons utilise a human hepatoma cell line
(Huh-7), where the genome replicates autonomously under selective pressure without producing infectious viral particles [Figure 1.7-A]. The replicon systems consist of a bicistronic RNA, where a neomycin resistance gene is encoded under the control of the
HCV IRES in the first cistron, while the structural and non-structural proteins are driven by the Encephalomyocarditis virus (EMCV) IRES. RNA, transcribed in vitro via the T7 promoter, is transfected into cells and neomycin resistant clones isolated and characterised to produce replicon cell lines capable of efficient and autonomous HCV
replication with little to no cytopathic effect on the cell (Lohmann et al., 1999,
Pietschmann et al., 2001, Ikeda et al., 2002). The efficient replication of the HCV genome, and the failure to produce infectious viral particles, was found to depend upon mutations that impact on the assembly of virions, referred to as ‘tissue-culture-adapted’ mutations (Lohmann et al., 2001, Pietschmann et al., 2009). Hence the replicon system does not allow for the complete analysis of the HCV lifecycle. The replicon systems have been invaluable in advancing our understanding of the molecular biology of HCV and interactions with the host. These systems have been further enhanced recently by the insertion of reporter genes such as firefly luciferase or fluorescent proteins, thus allowing for high-throughput quantification of HCV replication and for screening assays to determine the efficacy of new inhibitors (Schaller et al., 2007, Boonstra et al.,
2009).
15
Figure 1.7: HCV model systems (Tellinghuisen et al., 2007) 1.1.9.3 Infectious cell culture model
The shortcomings of the replicon systems were overcome in 2005 with the
development of the JFH-1 infectious cell culture system (genotype 2a). The HCV
cDNA was isolated from a Japanese patient with fulminant hepatitis (Kato et al., 2001)
and in vitro transcribed to produce HCV RNA that was subsequently transfected in
Huh-7 cells [Figure 1.7-C]. The clone was able to replicate efficiently and produce
infectious viral particles without the requirement of tissue-culture-adapted mutations.
Infectious supernatants produced from cell culture derived HCV JFH-1 (HCVcc) can
be serially passaged on to naïve Huh-7 cells in vitro, as well as in chimpanzees and
chimeric mice (Wakita et al., 2005, Zhong et al., 2005, Lindenbach et al., 2006). Initial
titres of the clone were low; however, the creation of chimeras of different genotypes,
such as Jc1 (genotype 2a), has produced higher titres (Pietschmann et al., 2006). The
addition of reporter contructs to create luciferase and fluorescent protein tagged viruses
has further enhanced the HCVcc system. Together with the development of the HCV
replicon system, HCVcc system represents the greatest breakthrough in HCV research
and has allowed for the comprehensive study of the full HCV lifecycle.
1.1.9.4 HCV pseudoparticle system
Another important cell culture based model has been the generation of the HCV
pseudoparticle (HCVpp) system that has allowed for the study of the early steps of virus binding and entry into the cell. HCV pseudoparticles utilise highly infectious retroviral or lentiviral cores displaying the HCV glycoproteins E1 and E2, thus mimicking HCV viral entry [Figure 1.7-B]. The expression of a luciferase reporter gene present with the viral core allows for the quantification of entry when infecting
HCV permissive cell lines (Bartosch et al., 2003, Drummer et al., 2003). This system
16 has been instrumental in the discovery of key HCV entry receptors such as glycosaminoglycan, low-density lipoprotein receptor, DC-SIGN (dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin), LC-SIGN (liver-specific intracellular adhesion molecule-3-grabbing non-integrin) and claudin-1, claudin-6 and claudin-9 (Boonstra et al., 2009, Martin and Uprichard, 2013, Kim et al., 2013).
Furthermore, it is extremely useful to study the early steps of HCV binding and entry in the context of the host immune response, such as in recent studies by Raychoudhri et al and Wilkins et al focused on examining the role of ISG56 and IFITM1 in HCV entry
(Raychoudhuri et al., 2011, Wilkins et al., 2013). However, one major shortcoming of the HCVpp assay is that it does not allow for the examination of the late entry stages of
HCV infection such as the fusion and acidification of the endosome resulting in the release of the viral genome into the cytoplasm.
1.2 Innate Immune Response to HCV
1.2.1 Activation of the innate immune response
The host innate immune response is the first line of defense against viral infection and plays an important role in activating the adaptive immune response. This host response is triggered by the cellular recognition of pathogen associated molecular patterns
(PAMPs). PAMPs are signature molecules such as dsRNA, dsDNA, lipopolysaccharide (LPS) and flagellin that are essential for the survival and pathogenesis of pathogens (Mogensen, 2009, Kumar et al., 2011). Evolutionarily conserved specific pathogen recognition receptors (PRRs) recgonise PAMPs triggering an array of anti-microbial reponses through the induction of inflammatory cytokines, chemokines and type I IFN. Several families of PRRs are involved in the innate
17 immune response to microbial pathogens and include; the retinoic acid inducible gene-I
(RIG-I) like receptors (RLRs), Toll-like receptors (TLRs) and the nucleotide-binding
oligomerization domain (NOD) like receptors (NLRs).
Three proteins belong to the RIG-I like receptors, which include RIG-I, melanoma
differentiation associated gene 5 (MDA5) and laboratory of genetics and physiology 1
(LGP2), which sense RNA in the cytosol. RIG-I is an ATP-dependent RNA helicase
that specifically senses short dsRNA with 5’ triphosphorylation, while MDA5 appears
to sense long dsRNA independent of 5’ triphosphorylation (Kato et al., 2008, Pichlmair
et al., 2006). LGP2 binds to both dsRNA and ssRNA, with a higher affinity than RIG-I and MDA5, via its C-terminal domain (Satoh et al., 2010). TLRs are membrane bound receptors found on either the plasma membrane or in endosomes that detect invading pathogens. TLR2 and 4 are found on the cell surface and are important in the recognition of lipopeptides and LPS of bacteria, in turn activating the innate response to bacterial infection (Akira et al., 2006). In contrast, TLRs 3, 7 and 8 are found within endosomes and recognise dsRNA and ssRNA respectively. NLRs are considered key sensors for intraceullar microbes and danger signals, with NOD1 and NOD2 being two well-characterised receptors within this family. These receptors sense bacterial molecules derived from the synthesis and degradation of peptidoglycan (Kanneganti et al., 2007); NOD1 primarily detects gram-negative bacteria through the specific sensing of diaminopimelic acid (Girardin et al., 2003a), while NOD2 is activated by muramyl dipeptide that is found on both gram-positive and –negative bacteria (Girardin et al.,
2003b).
18 1.2.2 Cellular Recognition of HCV
There are 2 main classes of PRRs involved in the host response to HCV, the RLRs and the TLRs (Horner and Gale, 2013, Metz et al., 2013). Activation of these pathways drives the transcription of the type I IFNs (IFNs–α and β) to limit viral replication
[Figure 1.8] while also recruiting adaptive immune cells.
The ability of RIG-I to specifically sense short dsRNA with 5’ triphosphorylation makes it the key sensor of HCV RNA; binding to the exposed 5’ triphosphate and the poly-U/UC tract in the 3’ UTR (Kato et al., 2008, Cui et al., 2008, Saito and Gale,
2008, Saito et al., 2008). Upon binding to HCV RNA, RIG-I undergoes a conformational change resulting in its oligomerisation and translocation from the cytosol to the mitochondrial antiviral signaling (MAVS) protein (also known as IPS-1,
VISA, Cardif). This translocation is facilitated by the interaction of RIG-I with the chaperone protein 14-3-3e and the E3 ubiquitin ligase TRIM25 (Liu et al., 2012a, Jiang et al., 2011, Gack et al., 2007). Interactions between MAVS and RIG-I result in the formation of the MAVS signalosome, which in turn activates the transcription factors interferon regulatory factor 3 (IRF3) and the nuclear factor kappa-light-chain enhancer
(NF-κB). The activation of NF-κB results in the induction of a number of genes, including the cytokines IL-1, IL-6 and TNF-α, and the chemokines IL-8 and RANTES that in turn signal inflammatory responses and the adaptive immune response. Recent studies have also demonstrated MDA5 to play a key role in initiating the IFN response against HCV infection, though the exact HCV PAMP recognised remains unclear (Cao et al., 2015).
19 !"#$
14-3-3" 14-3-3" %&'$
RIG-I RIG-I MAVS TLR TRIM25 TRIM25 Endosome Myd88 TBK1 IKK" TRIF P IRF3
P NF-!B IRF3 P IRF3 IRF3
Pro-inflammatory IFN-# cytokines IFN-$ PRD
Figure 1.8: Cellular recognition pathways of HCV TLR3 and TLR7 recognise dsRNA and ssRNA respectively, though the exact role of
HCV detection is still unknown. Both the receptors reside in the endosome and transmit signals either via TRIF or MyD88 to activate IRF3 and NF-κB (Wang et al.,
2009, Li et al., 2012). Thus the recognition of the HCV PAMP by RIG-I and TLR3
culminates in the phosphorylation, dimerization and translocation of IRF3, as well as
the translocation of NF-κB, to the nucleus resulting in the production of type I IFNs, as well as proinflammatory chemokines and cytokines such as IP-10. The importance of
RIG-I and TLR3 activation in the cellular response to HCV infection is highlighted by the fact that the HCV NS3/4 protease can evade these pathways as discussed in Section
1.2.5.
1.2.3 Interferon Transduction Pathway
IFNs are a family of inducible cytokines more commonly known for their antiviral activity and as mentioned previously IFN- α has been instrumental for HCV therapy for over a decade. However, they are also responsible for the regulation of a diverse number of biological functions such as anti-tumour activity and immune modulation
(Katze et al., 2002, Schroder et al., 2004, Lee et al., 2012, Gonzalez-Navajas et al.,
2012). IFNs can be divided into three types based upon structural and functional properties. Type I IFNs include IFN-α, IFN-β, IFN-δ, IFN-ω, IFN-κ, IFN-τ and IFN-ζ.
Type II IFN consists solely of IFN-γ, while type III IFNs consist of IFN-λ1 (IL-29),
IFN-λ2 (IL-28A) and IFN-λ3 (IL-28B). All IFNs interact with their specific receptors to result in the activation of the JAK-STAT signaling pathway and thus result in the production of hundreds of different effector molecules that exert various biological functions (O'Shea et al., 2015, Villarino et al., 2015).
20 The type I (and III) IFNs act in an autocrine and/or paracrine manner to interact with
their cognate receptors [Figure 1.9]. This specific binding results in the activation of
the receptor associated Janus kinase (JAK) family, specifically JAK1 and TYK1
(Silvennoinen et al., 1993). JAK1 and TYK1 are able to phosphorylate tyrosine
residues on the intracellular domains of interferon-alpha receptor (IFNAR) resulting in
the recruitment of signal transducer and activator of transcription (STAT) 1 and STAT2
via their SH2 domains. STAT1 is then phosphorylated at tyrosine residue 701 and
STAT2 at tyrosine residue 690 via JAK1 and TYK1 respectively. Once
phosphorylated, STAT1 and STAT2 form active heterodimers and associate with
interferon regulatory factor 9 (IRF9) to form the multimeric transcription factor
interferon-stimulated gene factor 3 (ISGF3). ISGF3 translocates into the nucleus,
where STAT1 is further phosphorylated on serine residue 727, enhancing the
transcriptional activation ability of ISGF3 (Gale and Foy, 2005). The end point of this signaling cascade results in the transcription of hundreds of interferon-stimulated genes
(ISGs) via ISGF3 binding to the IFN stimulated response element (ISRE) found in the promoter of the majority of type I IFN genes.
1.2.4 Interferon-stimulated genes
ISGs are the primary effectors of the host response towards a viral infection, with hundreds of ISGs upregulated by IFN (Gale and Foy, 2005, de Veer et al., 2001).
Upregulation of ISGs has been observed in chronically infected HCV patients as well as in acutely and chronically infected chimpanzees (Bigger et al., 2004, Lau et al.,
2008, Helbig et al., 2005, Zhu and Liu, 2003, Urban et al., 2010, Feld et al., 2011).
Due to the lack of any immuno-competent small animal models that are fully
permissive to HCV, the profiling and characterisation of ISGs is largely based on in
21 IFN-" IFN-!
IFN-! IFN-" IFNAR1 IFNAR2
JAK1 TYK2
STAT1
STAT2
STAT1
Y701 P STAT2 P Y690
Anti-viral Immunomodulatory IRF-9 Growth/Differentiation
S727 P ISGF3 STAT1 STAT2 P ISG expression P IRF-9 ISRE
Figure 1.9: IFN-!/" signal transduction pathway vitro models. Importantly, the ISGs found to be upregulated in patients overlap with
those detected in IFN-treated in vitro models.
1.2.4.1 Identification of ISGs targeting HCV
Several discovery-based screens have been performed to identify ISGs involved in controlling HCV in IFN-treated hepatoma cells. Schoggins et al conducted a FACS-
based phenotypic screen in order to determine the anti-HCV profile of 389 ISGs
(Schoggins et al., 2011). ISGs of interest were expressed in a biscistronic lentiviral
vector that also expressed a red fluorescent protein, TagRFP. Lentiviral transduction
was used to express each ISG individually, and these cells were then challenged with a
HCV reporter virus expressing a green fluorescent protein (GFP). Inhibition of viral
replication in ISG-expressing cells was quantified using fluorescence-activated cell
sorting (FACS), where a reduction in the HCV reporter signal indicated inhibition of
viral replication. The strongest antiviral effects were observed for known RNA-sensing
and signaling molecules such as RIG-I, MDA5, IRF2 and IRF7; however, additional
ISGs such as DDIT4, IFI44l, MAP3K14 and OASL were found to inhibit HCV
replication to a lower extent. It is most likely that these ISGs have a more specific
effect against HCV such as slowing down translation. Additionally, none of the ISGs
were able to limit HCV replication to an effect similar to that of IFN treatment, thus
indicating that inhibition of HCV is brought about by the action of multiple ISGs.
Some ISGs can inhibit lentiviral production, and thus this is a major limitation of using
a lentiviral expression system for a large-scale screen; as it failed to identify ISGs
previously characterised to have anti-HCV activity, such as viperin and IFIT1.
22 In contrast to ectopic expression of ISGs, a number of groups have conducted large-
scale RNA interference-based studies to identify specific ISGs. Zhao et al utilised a
whole genome small interfering RNA (siRNA) screen, where genes were knocked
down in Huh-7 subgenomic replicon cells treated with IFN-α and the rescue of HCV
viral replication was assessed (Zhao et al., 2012). This approach identified 93 ISGs, with some of the genes found to be involved in IFN signaling, mRNA processing and translation initiation. Similarly, Metz et al conducted ‘gain-of-function’ siRNA based screens, where the rescue of HCV replication was once again assessed by knocking down candidate gene expression in genomic and/or subgenomic replicon cells treated with either IFN-α or IFN-γ (Metz et al., 2012). The results from this screen were then confirmed using overexpression cell lines stably expressing candidate genes. This screen identified 7 ISGs involved in anti-HCV activity, with several new genes including IFIT3, TRIM14, PLSCR1 and NOS2. Most recently, Li et al conducted a genome-wide siRNA screen using the HCVcc system to identify host proteins involved in the different stages of the HCV life cycle. The study identified 237 host proviral factors and 25 host antiviral factors, and of note for this thesis, identified IFITM1,
IFITM2 and IFITM3 as host antiviral proteins that target HCV entry and IRES- mediated translation respectively (Li et al., 2014). Despite the identification of multiple
ISGs that control HCV replication in large-scale screens, more targeted approaches are
required to ascertain the molecular mechanisms behind the anti-HCV effects of these
ISGs, thus enhancing our knowledge of the HCV-host relationship and to possibly
identify novel targets for antiviral therapy against HCV.
23 1.2.4.2 ISGs with known anti-HCV activity
Only a handful of ISGs capable of controlling HCV have been identified and
characterised for their molecular mechanism against HCV (Metz et al., 2013). These
include, 2’-5’oligoadenylate synthetase (OAS)/ribonuclease L (RNase L), interferon- stimulated gene 20 (ISG20), interferon-induced protein with tetratricopeptide repeats 1
(IFIT1), viperin and cholesterol 25-hyroxylase (CH25H). These will be discussed
below:
2’-5’ OAS/RNase L: RNase L was one of the first ISGs identified to exhibit anti-HCV
activity (Han and Barton, 2002). RNase L is found in a wide range of tissues but
requires stimulation by 2’-5’ OAS to initiate endonuclease activity. 2’-5’ OAS in turn
requires activation by dsRNA, produced during viral RNA replication, allowing it to
bind to RNase L and induce a conformational change that leads to RNase L
homodimerisation and enzymatic function (Floyd-Smith et al., 1981). There are three
different OAS proteins, OAS1, OAS3 and OASL and all three have been shown to
induce RNase L activation in the context of HCV (Kwon et al., 2013). RNase L targets
single stranded regions of RNA, mainly UU or UA dinucleotides, of both viral and
cellular RNA. This non-specific cleavage is the main method by which RNase L
exhibits its antiviral activity; however, in the context of HCV, the cleaved RNA
products are believed to further enhance the IFN response by acting as ligands for RIG-
I and MDA5 (Malathi et al., 2010).
IFIT1: IFIT1 (also known as ISG56) belongs to the IFIT protein family, which includes
IFIT2, IFIT3 and IFIT5 and is characterised by the tetratricopeptide repeat domain folded into a helix-turn-helix structure that mediates protein interaction (D'Andrea and
24 Regan, 2003). Recent studies have identified the IFIT family, especially IFIT1 to be
antiviral against both RNA and DNA viruses including HBV, human papillomavirus
(HPV), HCV and West Nile virus (WNV) (Rathi et al., 2010, Raychoudhuri et al.,
2011, Saikia et al., 2010, Wacher et al., 2007). IFIT1 has been shown to block HCV replication by inhibiting initial HCV polyprotein translation, as IFIT1 is able to bind the ‘e’ subunit of eukaryotic initiation factor 3 (eIF3) to prevent initiation of translation. Recent studies have also demonstrated that IFIT1 recognises viral RNA that have a 5’triphosphate region, such as HCV, in a complex with other IFIT proteins
(Pichlmair et al., 2011). However, the fate of the sequestered RNA remains unknown.
Viperin: Viperin (also known as RSAD2) belongs to the radical S-adenosyl-L-
methionine (SAM) family (Duschene and Broderick, 2010). It is antiviral against a
broad range of viruses, both RNA and DNA, though the exact mechanisms are yet to be
elucidated. Viperin localises to the ER and lipid droplets, both of which are important
sites for HCV replication (Hinson and Cresswell, 2009b, Hinson and Cresswell,
2009a), and has been shown to have a significant effect on HCV replication. Viperin
can significantly limit HCV replication (Jiang et al., 2008, Helbig et al., 2005) and this is thought to be via its ability to interact with NS5A both on the lipid-droplet interface and in RCs (Helbig et al., 2011). Furthermore, viperin is able to interact with VAP-A in RCs and impair its association with NS5A (Helbig et al., 2011, Wang et al., 2012), which is crucial for HCV replication. Recent studies have also demonstrated viperin to have a role in modulating innate immune signaling (Qiu et al., 2009, Saitoh et al.,
2011), suggesting that viperin contributes to the innate immune response on multiple fronts.
25 ISG20: ISG20 belongs to an exonuclease family and is the second IFN-regulated
RNase identified to inhibit RNA virus replication (Degols et al., 2007). ISG20 has
been demonstrated to exert an antiviral effect on a broad range of viruses, including
vesicular stomatis virus (VSV), influenza virus (IAV), human immunodeficiency virus
(HIV) and HCV (Espert et al., 2003, Espert et al., 2005, Zhang et al., 2007, Jiang et al.,
2008). Recent studies have shown that the anti-HCV activity of ISG20 is via its 3’-5’
exonuclease which degrades viral RNA (Zhou et al., 2011, Xu et al., 2013).
CH25H: CH25H is an enzyme that catalyses the formation of 25-hydroxycholesterol
(25HC) from cholesterol. Recent studies have identified CH25H as an ISG against
murine herpesvirus 6 and VSV through the production of 25HC (Liu et al., 2012b), and furthermore CH25H was found to be upregulated in macrophages and dendritic cells in response to TLR activation (Bauman et al., 2009). Anggakusuma et al showed upregulation of CH25H mRNA in HCV-postive liver biopsies and in HCV-infected primary human hepatocytes, and additionally demonstrated CH25H to restrict HCV infection in a genotype-independent manner (Anggakusuma et al., 2015). The enzymatic function activity of CH25H is crucial for its anti-HCV actions, as the by- product 25HC was shown to inhibit the formation of the HCV membranous web, thus restricting HCV infection at the level of RNA replication by preventing the formation of the HCV RC.
1.2.5 HCV Evasion of Innate Immune Response
The ability of HCV to establish a chronic infection in 80% of infections is somewhat
attributed to its ability to suppress and evade the innate immune response [Figure 1.10].
However, it is interesting to note that HAV can also suppress the innate immune
26 IFN#/$ IFNAR1 IFNAR2 CORE CORE JAK1 TYK2 14-3-3! Endosome $" RIG-I !" TRIM25 TRIF STAT1 SOCS3
#" MAVS NS3-4A
STAT2 STAT1 NF-"B E2 NS5A P P P IRF3 %" PKR STAT2
eIF2# P
IFN-# ISG expression IFN-$ PRD ISRE
Figure 1.10: HCV evasion of the innate immune response (1) HCV NS3/4A specifically cleaves MAM-localised MAVS and TRIF preventing downstream signalling, (2) HCV core induces expression of SOCS3 to inhibit the JAK/STAT pathway, (3) HCV directly binds STAT1 preventing phosphorylation, and (4) HCV E2 acts as a competitive inhibitor to prevent PKR interacting with eIF2! response in a similar manner to that of HCV, although HAV is an acute self-limiting
infection (Qu and Lemon, 2010). Clearly the dynamics of the outcome of infection are multifactorial and evasion of the innate response may be one part of a complex process that leads to chronicity. HCV has developed a number of effective strategies to evade the host response, with the main mechanism involving the NS3/4A serine protease.
This multifunctional viral protein is able to attenuate IFN production by targeting both arms of HCV recognition by the hepatocyte. NS3/4A and MAVS are both found associated with multiple intracellular membranes such as the mitochondria, peroxisosomes and mitochondrial-associated membranes (MAMs) (Horner et al., 2011,
Dixit et al., 2010). NS3/4A has been demonstrated to specifically cleave MAM- localised MAVS, thus abrogating its association with intracellular membranes and downstream RIG-I signaling to produce IFN (Baril et al., 2009, Li et al., 2005b, Lin et al., 2006a). NS3/4A is also able to cleave TRIF, the key adaptor protein of the TLR3 signaling pathway. Though the exact mechanism is not known, cleavage by NS3/4A results in a marked decrease in the amount of TRIF, which is suggestive of destabilization and degradation of the protein (Li et al., 2005a, Ferreon et al., 2005).
Cleavage of TRIF disrupts downstream TLR3 signaling. Therefore, one mechanism by which HCV evades the immune response is through NS3/4A’s capability to attenuate the induction of IFN-α/β via the cleavage of key signaling adaptor molecules.
Additionally, HCV is also able to antagonize the IFN signaling pathway, where the
HCV core protein has been shown to induce the expression of suppressor of cytokine signaling (SOCS) proteins, in particular SOCS3. SOCS proteins are known inhibitors of the JAK-STAT pathway and mediate a negative feedback loop on the signaling cascade of IFNAR. It is believed that activation of SOCS3 by HCV core can facilitate
27 the evasion of the actions of IFN (Bode et al., 2003); however, the exact mechanisms remain unknown. HCV core is also able to directly bind to STAT1 (Melen et al., 2004,
Lin et al., 2006b), thus preventing its phosphorylation and downstream activation of anti-HCV ISGs. Other HCV proteins have been implicated in directly interfering with the antiviral functions of specific ISGs, namely protein kinase R (PKR). HCV E2 has been shown to act as a competitive inhibitor for PKR, preventing PKR’s interaction with eIF2α and subsequent kinase activity (Taylor et al., 1999). NS5A has also been shown to inhibit PKR-mediated translational control resulting in increased HCV replication (Appel et al., 2008, Tellinghuisen et al., 2008), though the exact mechanisms remain unclear. Thus, HCV utilises a multitude of different mechanisms to evade the innate immune response in order to establish a chronic infection, and this further highlights the importance of the innate immune response in combating viral infection.
1.3 Interferon-Induced Transmembrane (IFITM) Protein
The IFITM proteins are a family of 14-18kDa proteins that were first discovered over
20 years ago based on their increased expression in screens for IFN-α inducible genes
(Friedman et al., 1984). They have since been identified to belong to a larger family of
proteins, the CD225 protein superfamily, which has over 300 members sharing
homology within the first membrane associated domains and the conserved
intracellular loop (CIL) (Perreira et al., 2013). Humans have been shown to have five
IFITM genes to date – IFITM1 (9-27/Leu-13), IFITM2 (1-8D), IFITM3 (1-8U), IFITM5 and IFITM10, all of which are found clustered around a 26kb region on the short arm of chromosome 11 (Lewin et al., 1991). IFITM5 is not inducible by IFN and is found only in osteoblasts (Moffatt et al., 2008), while little is yet known about the recently
28 discovered IFITM10 (Hickford et al., 2012). The first three IFITM genes are found adjacent to each other, with IFITM5 and IFITM10 found upstream. Phylogenetic studies suggest that IFITM2 and IFITM3 are a result of genetic duplication (Siegrist et al., 2009) explaining the high level of sequence conservation between these genes.
ISRE and gamma-activated sequence (GAS) elements can be found in the promoter regions of IFITM1, IFITM2 and IFITM3, allowing for induction by type I and II IFNs respectively (Lewin et al., 1991). Recent studies have found that IFITM1 can be induced by IFN-λ in certain cell types (Zhou et al., 2007). Conserved homologs of these genes can be found in a diverse range of vertebrates ranging from humans to zebrafish and flounder. IFITM1, IFITM2 and IFITM3 are the only family members to be associated with IFN induction, as well as induction due to viral infection, and are the focus of this thesis.
1.3.1 IFITMs: protein structure and cellular distribution
All three IFITM proteins share high amino acid homology, containing two hydrophobic membrane-associated domains (termed M1 and M2) separated by a CIL but differ at the putative extracellular domains [Figure 1.11]. IFITM2 and IFITM3 contain 20 and
21 amino acid extensions at the N-terminal domain (NTD) respectively, while IFITM1 contains a 13 amino acid extension at the C-terminus [Figure 1.11.i] (Lewin et al.,
1991, Siegrist et al., 2011). The IFITM proteins are ubiquitously expressed; with
IFITM2 and IFITM3 basally expressed in most primary and transformed cells, while in contrast the basal expression of IFITM1 appears to be considerably less. Localisation studies in a number of different cell types (A549, HEK 293 and HeLa cells) have shown IFITM1 to be predominantly found at the plasma membrane with some localisation at early endosomes, while IFITM2 and IFITM3 are found at late
29 i 20 40 60 80 100 120 140 160
IFITM3 IFITM2 IFITM1
NTD M1 CIL M2 CTD ii
Extraluminal
N C C
Intraluminal N C N A B C
Figure 1.11: (i) Schematic representation of human IFITM1, IFITM2 and IFITM3. (ii) Proposed topological models for the human IFITM proteins – A: type III transmembrane protein with extraluminal N- and C-termini, B: intramembranous proteins with intraluminal N- and C-termini and C: type II transmembrane protein with intraluminal N- termini and extraluminal C-termini (also murine IFITM3) endosomes and lysosomes (Wilkins et al., 2013, Feeley et al., 2011, Agnello et al.,
1999, Jia et al., 2012, Huang et al., 2011, Amini-Bavil-Olyaee et al., 2013).
Studies examining the structure and function of the IFITM proteins have primarily focused on IFITM3. Recent studies have shown that IFITM3 undergoes numerous
post-translational modifications (PTM), including tyrosine phosphorylation, S-
palmitoylation, ubiquitination and methylation, which have been summarised below
(Table 1.1).
IFITM3’s NTD: Mutational analysis of the NTD, in particular tyrosine 20 (Y20) that
undergoes phosphorylation, of IFITM3, results in the re-localisation of the protein to the plasma membrane (Jia et al., 2012) from endosomes. This suggests that Y20 is important for the proper trafficking of IFITM3 to the late endosomes, and further work identified Y20 to be part of an endocytic signal motif 20-YEML-23 (consensus
YXX(Ω)). The localisation of IFITM3 within a cell appears to be regulated by the
phosphorylation of Y20 via Fyn kinases. Phosphorylation of Y20 disrupts the YEML
signaling motif, resulting in the sequestration of IFITM3 at the plasma membrane (John
et al., 2013, Jia et al., 2014, Chesarino et al., 2014). Thus, the cellular distribution of
IFITM3 appears to be strongly influenced by its NTD.
S-palmitoylation and ubiquitination: IFITM3 undergoes S-palmitoylation on 3
conserved cysteine residues – C71, C72 and C105, and this PTM is believed to aid the targeting of the protein to the plasma membrane and in some cases to endosomes
(Yount et al., 2010). S-palmitoylation consists of the addition of a palmitoyl group to cytosolic cysteines and will be discussed further in Chapter 6. Mutation of these
30 Table 1.1: Summary of post-translational modifications undergone by IFITM3
Post-translational Position Conservation Functional significance Role in antiviral activity modification Tyrosine phosphorylation Y20 Not present in IFITM1 Deregulates subcellular Interferes with antiviral IFITM2 -Y19 localisation activity - IAV, DENV S-palmitoylation C71, C72, C105 IFITM1 - C50, C51, C84 Targets to plasma Promotes antiviral activity IFITM2 - C70, C71, C104 membrane/endosome - IAV, VSV, DENV Not required for anti-HIV activity Ubiquitination K24, K83, K88, K104 IFITM1 – K3, K62, K67, Improves protein stability Interferes with antiviral K83 and localisation activity - IAV IFITM2 – K23, K, 82, K87, K83 Methylation K88 IFITM1 – K67 Does not alter localisation Interferes with antiviral IFITM2 – K87 or expression levels activity – IAV, VSV
cysteine residues results in a more centralized distribution of IFITM3 within the cell.
S-palmitoylation of IFITM3 is critical for its antiviral action against IAV, VSV and
DENV, but is not important for HIV. Similar results have also been observed for
murine Ifitm1 (Hach et al., 2013). The effects of S-palmitoylation are counteracted by the ubiquitination of IFITM3 at 4 lysine residues – K24, K83, K88 and K104, though ubiquitination at K24 appears to be the most prevalent (Yount et al., 2012, John et al.,
2013). Ubiquitination of IFITM3 occurs independent of S-palmitoylation, but recent studies show that phosphorylation of Y20 results in decreased ubiquitination. It is hypothesised that Y20 phosphorylation might either alter a signaling motif required by ubiquitin ligases or that endocytosis of IFITM3 is required for ubiquitination to occur
(Chesarino et al., 2014). Studies have also identified the E3 ubiquitin ligase NEDD4 to
be responsible for the ubiquitination of IFITM3, and target the protein for degradation
within the lysosomal degradation pathway due to the association between NEDD4 and
lysosomes. Thus, ubiquitination of IFITM3, and its subsequent degradation, promotes
IAV infection (Chesarino et al., 2015).
Methylation and IFITM complexes: IFITM3 undergoes monomethylation at K88,
which is also ubiquitinated, by SET7. Monomethylation of IFITM3 does not alter expression levels or localisation of the protein, but does adversely effect the antiviral actions against IAV and VSV (Shan et al., 2013). Recent studies identified the ability of IFITM3 to interact with itself, as well as with IFITM1 and IFITM2, and found that two phenylalanine residues (F75 and F78) are required for this interaction (John et al.,
2013, Zhao et al., 2014). Though the exact significance of these interactions remains unclear, it is possible that the cellular distribution and function of the IFITM proteins
31 may be influenced by the formation of IFITM homo- and/or heterodimers (John et al.,
2013, Zhao et al., 2014).
Despite the known localisation of the IFITM proteins to membranes, the exact topology of these proteins was only recently elucidated [Figure 1.11.ii]. Initial cell surface immunostaining and flow cytometry studies suggested that the IFITM proteins, in particular IFITM3, were transmembrane proteins with extraluminally located N- and C- termini (Brass et al., 2009, Weidner et al., 2010). However, mass spectrometry analysis identified several PTM sites on IFITM3, which could only occur if the N- and
C-termini were located in the cytosol. Thus, it was suggested that the N- and C-
termini, along with the CIL, are located intraluminally with the hydrophobic domains
interacting with the membrane but not spanning it (Yount et al., 2012, Jia et al., 2012), suggestive of an intramembranous topology. A type II transmembrane topology was described for murine Ifitm3, with an intraluminal N-terminus and CIL but an
extraluminal C-terminus (Bailey et al., 2013). However, a recent study has identified
that the human IFITM proteins also have a type II transmembrane topology, similar to
murine Ifitm3 (Weston et al., 2014). A combination of immunostaining, protease
cleavage assays and biotin-labelling approaches identified that the NTD and CIL of
IFITM1 are located in the cytoplasm and the CTD is extracellular; with IFITM2 and
IFITM3 having the same topology within intracellular membranes.
1.3.2 Biological functions of the IFITM proteins
IFITM1, 2 and 3 have been shown to be involved in a wide range of cellular functions
including anti-proliferation, apoptosis, homotypic cell adhesion and oncogenesis. Anti-
proliferative and apoptotic functions of the IFITM proteins appear to be conserved but
32 the efficiency differs based on cell type as well as the different proteins. IFITM1 has been shown to significantly reduce the anti-proliferative actions of IFN-γ and enhance the expression of the tumour suppression protein p53 in the hepatoma cell line BEL-
7404 and the non-malignant hepatocyte line, Chang liver cells (Yang et al., 2007,
Deblandre et al., 1995). Interactions with p53 can result in partial cell cycle arrest at the G1 phase. In contrast, IFITM2 can result in G1 and subG1 phase arrest independent of p53 activation resulting in an apoptotic state (Joung et al., 2003, Daniel-
Carmi et al., 2009). Similarly, overexpression of IFITM3 has been shown to inhibit the proliferation of IFN-sensitive melanoma cells (Brem et al., 2003). These studies suggest that additional pathways, other than the JAK-STAT pathway, are involved in the cellular actions of the IFITM proteins.
IFITM1 associates with the tetraspanin CD81 on the cell surface of a number of different cell types. Most notably, the IFITM1/CD81 complex interacts with the B- lymphocyte antigen CD19 and complement component receptor 2 signal transduction complex on the surface of B-lymphocytes, where silencing of IFITM1 can result in an anti-proliferative effect on B cells (Takahashi et al., 1990, Evans et al., 1990, Bradbury et al., 1992, Matsumoto et al., 1993, Sato et al., 1997). This complex is also involved in
β1-integrin mediated adhesion to extracellular matrix proteins (Behr and Schriever,
1995). Thus the IFITM proteins contribute to cell adhesion and growth control of lymphocytes and other cell types. The interaction with CD81 has also been shown in mice, with Ifitm1 and Ifitm3 (Smith et al., 2006). Ifitm1 and Ifitm3 have also been identified as early germline markers and have been proposed to have roles in primordial germ cell homing and maturation in mice (Tanaka and Matsui, 2002,
Tanaka et al., 2005).
33 1.3.3 Antiviral Actions of the IFITM proteins
A siRNA screen to identify host proteins that modify the IAV lifecycle first identified
the antiviral nature of the IFITM proteins (Brass et al., 2009). Since then a number of
studies have demonstrated the ability of IFITM1, IFITM2 and IFITM3 to inhibit the replication of a broad range of pathogenic viruses including IAV, DENV, WNV, VSV,
HIV and Ebola virus (EBOV) and during the course of this thesis, HCV [Table 1.2].
The IFITM proteins primarily inhibit enveloped RNA viruses that require low pH- dependent entry into target cells. Recent studies have shown that the IFITM proteins do not inhibit some DNA viruses, in particular human cytomegalovirus (HCMV), HPV and adenoviruses (Warren et al., 2014), as well as some pH-independent RNA viruses such as murine leukemia virus (MLV) and Sendai virus (SeV) (Brass et al., 2009).
While in most instances all three IFITM proteins exhibit antiviral properties against the same viruses, the difference in cellular localisation of these proteins appears to affect restriction efficiency. The majority of the antiviral actions of IFITM3 target viruses that require the late endosomal pathway for entry such as IAV, DENV and Rift Valley fever virus (Brass et al., 2009, Jiang et al., 2010, Feeley et al., 2011, Bailey et al., 2012,
Mudhasani et al., 2013). IFITM3, the most studied of the IFITM proteins, is found predominantly at Rab7-positive late endosomes in many cell types, explaining its specificity to late entry viruses; however, the exact mechanism remains unclear.
IFITM2 is believed to share similar viral specificity as IFITM3, but with weaker effects. In contrast, IFITM1 has been shown to be more proficient than IFITM2 and
IFITM3 at inhibiting viruses that utilise the early endosomal pathway for entry such as
VSV, Jaagsiekte sheep retrovirus (JSRV) and HIV (Weidner et al., 2010, Lu et al.,
2011, Raychoudhuri et al., 2011, Wilkins et al., 2013, Chutiwitoonchai et al., 2013, Li
34 Table 1.2: Summary of viruses inhibited by IFITM proteins
Family Virus Endocytosis pH Requirement IFITM specificity pathway Enveloped Orthomyoxiviridae Influenza A Virus Clathrin-mediated 5.5 IFITM1,2,3 Flaviviridae Dengue Virus Clathrin-mediated 5.5 IFITM1,2,3 West Nile Virus Clathrin-mediated 5.5 IFITM1,2,3 Hepatitis C Virus Clathrin-mediated 6.5 IFITM1 Rhabdoviridae Vesicular Stomatis Virus Clathrin-mediated 6.5 IFITM1,2,3 Rabies Virus Clathrin-mediated <6 IFITM2 and 3 Lagos Bat Virus Clathrin-mediated <6 IFITM2 and 3 Filoviridae Marburg Virus Macropinocytosis 4.5 IFITM1,2,3 Ebola Virus Macropinocytosis 4.5 IFITM1,2,3 Coronaviridae SARS Coronavirus Clathrin-mediated 4.5 IFITM1,2,3 MERS-Coronavirus - <6 IFITM1,2,3 Retroviridae HIV Unknown pH independent Mixed results – IFITM1 Jaagsiekte sheep retrovirus Dynamin-mediated 6.5 IFITM1 best Alphaviridae Semiliki Forest Virus Clathrin-mediated >6 IFITM2 and 3 Bunyaviridae La Crosse Virus Clathrin-mediated 5.5 IFITM1,2,3 Hantaan Virus Clathrin-mediated 5.5 IFITM1,2,3 Andes Virus Clathrin-mediated 5.5 IFITM1,2,3 Rift Valley fever Virus Dynamin-II and 5.5 IFITM2 and 3 Calveolin-I mediated Paramyxovirus Respiratory syncytial virus Macropinocytosis pH independent IFITM1,2,3 Non-Enveloped Reoviridae Reovirus Clathrin-mediated 5.5 IFITM3 et al., 2013a). The cellular localisation of IFITM1 is cell-type dependent, being found on both the plasma membrane and at the early endosome suggesting that the actions of
IFITM1 are also more cell-type dependent. However, this paradigm does not always hold true, with IFITM1 being more proficient at restricting the late entry viruses SARS
CoV, EBOV and Marbug virus (MARV) than IFITM3 (Huang et al., 2011).
Furthermore, the generation of chimeric IFITMs does not translate antiviral properties; for example, the generation of IFITM1 with the NTD of IFITM3 localises similar to wildtype IFITM3 but does not have enhanced anti-IAV activity (Weidner et al., 2010,
John et al., 2013). This indicates that cellular localisation is not the only factor determining the antiviral properties of the IFITM proteins.
The exact mechanism(s) by which the IFITM proteins exert their antiviral actions remain unclear; however, recently two possible indirect and direct mechanisms have emerged. Studies have demonstrated that the IFITM proteins are able to alter the endosomal environment, including expansion, acidification and lipid homeostasis, all of which could contribute to viral inhibition. Of most interest are the changes to lipid homeostasis, as the IFITM proteins have recently been shown to interact with endosome trafficking protein VAP-A. This interaction disrupts the interaction between
VAP-A and the cholesterol regulator oxysterol binding protein (OSBP), resulting in the sequestration of cholesterol within the late endosome. Thus, it is hypothesized that cholesterol accumulation within the late endosome will prevent fusion between intraluminal vesicles and virus-containing compartments resulting in the inhibition of infection (Amini-Bavil-Olyaee et al., 2013). However, this mechanism does not account for the ability of the IFITM proteins to inhibit viruses utilising the early endosomal pathway. The other potential mechanism describes the IFITM proteins
35 blocking viral fusion and preventing the entry of the viral genome into the cytoplasm.
Initial studies suggested that the IFITM proteins, in particular IFITM3, inhibited viral-
host hemifusion (Li et al., 2013a). Hemisfusion is an intermediate stage during
membrane fusion, where the two outer membranes have merged but the inner
membranes remain separate until the fusion pore has formed (Chernomordik and
Kozlov, 2005). However, recent work by Desai et al. suggests that IFITM3 in fact
prevents the release of the viral genome into the cytoplasm by inhibiting viral entry
after hemifusion but prior to fusion pore formation (Desai et al., 2014). It has been
postulated that this method of inhibition occurs through interactions between adjacent
IFITM proteins thus altering membrane (plasma or endosomal) fluidity and curvature,
making the membrane resistant to the viral fusion machinery. In this way, viruses
sensitive to the actions of the IFITM proteins are arrested at the cell surface and
trapped within the endosomal pathway to be degraded by lysosomes or autolysosomes.
This would be an efficient way to inhibit a wide array of viruses.
Other cellular factors also influence the antiviral actions of the IFITM proteins.
Numerous studies have investigated the importance of PTMs of the IFITM proteins on
the antiviral functions. Phosphorylation of IFITM3 at a key tyrosine residue (Y20) has been shown to be detrimental for its antiviral activity, due to the sequestration of
IFITM3 away from the late endosome to the plasma membrane. S-palmitoylation has been shown to aid the antiviral activity of the IFITM proteins against IAV and DENV
(Yount et al., 2010). However, is not required for anti-HIV activity (Chutiwitoonchai et al., 2013). Similarly, methylation and ubiquitination of IFITM3 on specific residues results in decreased antiviral activity against IAV and VSV. IFITM1 expression in hepatocytes is regulated by miR-130A by binding to the 3’UTR, thus regulating the
36 expression of IFITM1 in response to IFN-α (Chowdhury et al., 2012). Thus, there are a multitude of factors that control the function of the IFITM proteins, and more work is required to understand the complex regulation of the IFITM proteins in regard to how these proteins establish an antiviral state against a broad spectrum of viruses.
1.3.3.1 IFITM proteins and in vivo studies
Knockout mice targeting the Ifitm genes were first established by Lange et al to examine the role of murine Ifitm proteins on primordial germ cell development (Lange et al., 2008). However, recently these animal models have been used to examine the role of the murine Ifitm proteins against IAV infection. Studies by Everitt et al and
Bailey et al have demonstrated that Ifitm3 knockout mice are significantly more susceptible to IAV infection compared to wildtype, with more rapid weight loss and respiratory tract lesions observed in the knockout mice upon infection (Everitt et al.,
2012, Bailey et al., 2012). These studies demonstrate a critical role of Ifitm3 in the control of acute IAV infection in vivo. Everitt et al first demonstrated an in vivo role for IFITM3 in humans with the observation that patients hospitalised during the 2009
H1N1 pandemic contained a significant enrichment in the IFITM3 allele, SNP 12252-
C. Further examination revealed that SNP 12252-C generated an IFITM3 variant with a 21a.a truncation at the N-terminus resulting in a decrease in anti-IAV activity (Everitt et al., 2012). Additionally, a recent study has demonstrated that IFITM3 SNP 12252-C is associated with the rapid progression, but not acquisition, of acute HIV-1 infection in
Chinese men having sex with men (MSM) cohort (Zhang et al., 2015). This is the first evidence that a single polymorphism in the IFITM3 gene can be associated with disease outcome.
37 IFITM3 has also been shown to have a role in the murine adaptive immune response.
Wakim et al found a population of lung resident memory CD8+ T cells to maintain
Ifitm3 expression after virus IAV infection, thus rendering these cells less susceptible to reinfection (Wakim et al., 2013). A similar population of memory CD8+ T cells overexpressing Ifitm3 was observed in the brains of mice after VSV infection (Wakim et al., 2012). These studies indicate that the IFITM proteins may have a role in the adaptive immune response in preferentially protecting subsets of immune effector cells.
1.3.3.2 Evasion of the antiviral actions of the IFITM proteins
As mentioned in Section 1.2.4, viruses are able to evade the IFN-α response in multiple different ways. Recent studies have found that several viruses have found ways to evade the antiviral actions of the IFITM proteins. HIV is able to evade the actions of
IFITM1 through mutations to the viral proteins Vpu (at amino acid 34) and Env
(G367E). These mutations allow for increased cell-to-cell transmission of HIV, thus bypassing classical HIV entry; indicating that HIV is able to evolve and overcome the effects of IFITM1 by promoting cell-to-cell transmission (Ding et al., 2014). IAV has also been reported to evade the actions of IFITM3 by targeting the translational regulation of the protein. IFITM3 protein expression is increased in the presence of the eukaryotic translation initiation factor 4B (eIF4B). However, the IAV NS1 protein is able to target eIF4B for lysosomal degradation, preventing translation of IFITM3 and allowing increased IAV replication and evasion of the ISG response (Wang et al.,
2014). The specific targeting of these proteins by viruses perhaps indicates the
importance in the early innate response to viral infection; however, further studies are
required to elucidate the mechanisms used by other viruses to evade the antiviral
actions of the IFITM proteins.
38 1.3.4 IFITM proteins in hepatocytes and HCV
The antiviral properties of the IFITM proteins have been extensively examined in recent years against a number of different RNA viruses and in a number of different cell types. However, despite the ability of the IFITM proteins to significantly limit the
Flaviviruses DENV and WNV, at the commencement of this PhD only a handful of studies have examined the role of the IFITM proteins in the context of HCV infection in hepatocytes.
Several recent overexpression and siRNA knockdown studies have identified IFITM1,
IFITM2 and IFITM3 to be involved in the IFN response against HCV in hepatocytes
(Schoggins et al., 2011, Metz et al., 2012, Li et al., 2014). IFITM3 was first described to have an effect on HCV replication as early as 2003, where exogenous expression of
IFITM3 decreased HCV RNA levels in a HCV subgenomic replicon model system
(Zhu and Liu, 2003), while another group identified IFITM3 to have an effect on HCV
IRES translation (Yao et al., 2011). Similarly, examination of HCV viral RNA and
IFITM3 mRNA expression in the same hepatocyte using single cell laser capture analysis showed that hepatocytes expressing IFITM3 in patient liver samples were mostly HCV negative (Kandathil et al., 2013). During the course of this PhD, IFITM1 was shown to be able to block the entry of HCV into the hepatocyte by interacting with the essential host entry factors CD81 and OCLN at the hepatic tight junction (Wilkins et al., 2013); while another study has reported IFITM1 to have an effect on HCV replication and not entry (Raychoudhuri et al., 2011). While these studies demonstrate that IFITM1 and IFITM3 play a role in controlling HCV infection, they are contradictory in identifying the exact role of these proteins against HCV. Additionally, the role of the related IFITM2 protein on HCV infection remains unclear. Thus, a
39 comprehensive study examining the exact role of each of the IFITM proteins at the different stages of the HCV lifecycle is required.
Moreover, the localisation and regulation of the IFITM proteins within hepatocytes remains limited. Wilkins et al demonstrated IFITM1 to be localised to the plasma membrane of hepatocytes where it interacts with the essential host receptor CD81
(Wilkins et al., 2013). Consequently it would be prudent to identify the specific localisation of IFITM2 and IFITM3 within the hepatocyte and determine whether this localisation contributes to anti-HCV activity. The role of PTM of the IFITM proteins within hepatocytes, and also HCV infection, remains unknown. As mentioned previously in Section 1.3.3, PTMs of the IFITM proteins appear to affect the antiviral actions of these proteins in a virus- and cell-type specific manner, in particular S- palmitoylation. The work of this thesis aims to address these gaps in our knowledge in the relationship between the IFITM proteins and HCV infection, thus expanding our knowledge of the interplay between the host response and HCV infection within hepatocytes.
1.4 Hypothesis and Aims
The overall aim of this thesis is to define and characterise the role of the IFITM proteins on the HCV lifecycle. Initial studies will aim to elucidate whether the IFITM proteins exert anti-HCV activity and define the cellular localisaton of these proteins within the hepatocyte. This will be achieved through the production of liver derived cell lines that specifically overexpress each of the IFITM proteins coupled with in vitro models of HCV replication, namely the HCV replicon and HCVcc (Jc1) models.
40 Subsequent objectives will then focus on characterising the specific roles of the IFITM proteins through mutational analysis.
Hypothesis – The IFITM family of proteins significantly impact HCV replication with each member exerting its effect at a distinct cellular site.
We specifically aim to:
(1) Investigate the antiviral role of IFITM1, IFITM2 and IFITM3 in the context of
the complete HCV life cycle.
(2) Define the cellular localisation and specific antiviral actions of IFITM1 through
mutational analysis.
(3) Define the cellular localisation of IFITM2 and IFITM3 within the hepatocyte.
(4) Investigate the role of key post-translational modifications on the antiviral
actions of the IFITM proteins in the context of HCV infection.
41 Chapter 2
Materials and Methods
2.1 General Reagents
2.1.1 Transient transfection of plasmid DNA
Plasmid DNA was transfected into various cell lines using FuGENE 6 Transfection
Reagent (Promega). Cells were seeded in 12-well cell culture plates 24 hrs prior to
transfection at a density of 7 × 104 cells per well (50-70% confluency at the time of transfection). FuGENE 6 reagent and plasmid DNA were diluted in an appropriate volume of serum free Opti-MEM as per manufacturer’s recommendations, generally at a DNA:FuGENE 6 ratio of 1:1. Following a 20 min incubation at room temperature, the mixture was added drop-wise to cell monolayers; the plates were then gently swirled and returned to normal tissue culture conditions (37ºC, 5% CO2). Assays were performed 24-72 hrs post transfection.
2.1.2 Lentiviral packaging of IFITM protein to generate overexpression cell lines
293T cells were seeded in a 6-well plate at a concentration of 2×105 cells/well to be transfected the following day. The transfection mix were comprised of 2 µg of total plasmid DNA, 5 µl of FuGENE® 6 Transfection Reagent (Roche) and 100 µl of OPTI-
MEM® SFM (Gibco BRL, Invitrogen). The total plasmid DNA was made of equivalent amounts of the candidate ISG lentiviral plasmid DNA and the lentiviral packaging plasmids psPAX2, pMD2.G and pRSV-Rev. The supernatant from the 293T cells was harvested 48 and 72 hrs post transfection, pooled and filtered through a 0.45
µm filter membrane and 1 ml aliquots of the supernatant were stored at -80°C.
42 In order to generate stable cells, Huh-7 cells were seeded at 2 × 105 cells per well in a
6-well cell culture tray prior to transduction with lentiviral particles diluted 1:3. The infectious media was removed from the cells 6 hrs post-transduction, replaced with new media and cells returned to culture for 2-3 days. Transduced cells were selected using 4µg/ml Blasticidin (Invitrogen) and once antibiotic resistance was observed, cells were passaged at low density for 2 weeks and screened for over-expression of the protein of interest. Positive polyclonal cell lines were maintained under normal culture conditions (37°C, 5% CO2) with antibiotic selection continued.
2.1.3 Stable transduction of GIPz shRNA to generate knockdown cell lines shRNA designed to knock down IFITM1 expression were obtained from Open
Biosystems (ThermoScientific) in a pGIPz lentiviral construct encoding a green fluorescent protein. Lentiviral particles expressing IFITM1 shRNA were produced as per 2.1.2, and transduced cells were selected using 3µg/ml Puromycin (Invitrogen).
Once antibiotic resistance was observed, cells were passaged at low density for 2 weeks and screened for knockdown in target protein expression. Scrambled control shRNA were used as a control. In order to screen for knockdown in IFITM1 expression, cells were seeded in 12-well plates at a density of 7 × 104 cells/well and left at 37°C for 24
hours before addition of IFN-α2b (100 IU/ml: Intron A, Schering-Plough). Total RNA
was isolated at 16 hours post treatment for cDNA synthesis and semi-quantitative real-
time PCR.
43 2.2 Tissue Culture Techniques
2.2.1 Tissue culture medium
Cultured mammalian cell lines were maintained in Dulbecco’s Modified Eagle Medium
(DMEM) containing 4.5 g/L D-Glucose, 25 mM HEPES and 2 mM L-glutamine
(Gibco BRL, Invitrogen). Media was supplemented with 10% (w/v) foetal calf serum
(FCS; Trace Biosciences), 12 µg/ml penicillin (CSL), 16 µg/ml gentamycin
(Pharmacia) and other supplements as required (Table 2.1).
Table 2.1 Cell lines and culture conditions used in this study
Cell Line Media Supplements
Huh-7 and Huh-7.5 DMEM 10% FCS, 1% penicillin/gentamycin
NNeoC-5B(RG) DMEM 10% FCS, 1% penicillin/gentamycin, 800 µg/ml G-418
Huh-7+Empty DMEM 10% FCS, 1% penicillin/gentamycin, 4 µg/ml blasticidin
Huh-7+IFITM1 DMEM 10% FCS, 1% penicillin/gentamycin, 4 µg/ml blasticidin
Huh-7+IFITM2 DMEM 10% FCS, 1% penicillin/gentamycin, 4 µg/ml blasticidin
Huh-7+IFITM3 DMEM 10% FCS, 1% penicillin/gentamycin, 4 µg/ml blasticidin
Huh-7+shControl DMEM 10% FCS, 1% penicillin/gentamycin, 3 µg/ml puromycin
Huh-7+shIFITM1 DMEM 10% FCS, 1% penicillin/gentamycin, 3 µg/ml puromycin
Huh-7+IFITM1ΔN6 DMEM 10% FCS, 1% penicillin/gentamycin, 4 µg/ml blasticidin
Huh-7+IFITM1ΔN21 DMEM 10% FCS, 1% penicillin/gentamycin, 4 µg/ml blasticidin
Huh-7+IFITM1ΔC18 DMEM 10% FCS, 1% penicillin/gentamycin, 4 µg/ml blasticidin
Huh-7+IFITM1ΔHNPAP DMEM 10% FCS, 1% penicillin/gentamycin, 4 µg/ml blasticidin
44 Huh7+IFITM2: Y19A DMEM 10% FCS, 1% penicillin/gentamycin, 4 µg/ml blasticidin
Huh7+IFITM3: Y20A DMEM 10% FCS, 1% penicillin/gentamycin, 4 µg/ml blasticidin
2.2.2 Maintenance of cell lines
Cells were maintained in sterile 0.2 µm vented tissue culture flasks (25 cm2, 75 cm2, or
175 cm2), tissue culture dishes (3.5 cm2, 6 cm2 or 10 cm2) or tissue culture trays (6, 12,
24, 48 or 96-well) (Corning Life Sciences). Cells were passaged by removing the culture medium, washing with PBS (see Appendix I) and trypsinising in a small volume of Trypsin-EDT A (see Appendix I) for 3-5 minutes. Trypsinised cells were then resuspended in culture medium and cell counts were performed using Trypan Blue exclusion and a haemocytometer. Cells were grown in a humified incubator (5% CO2) at 37°C and passaged every 2 to 3 days depending on confluency.
2.2.3 Cryopreservation of cultured cells
To preserve cells in liquid nitrogen, cells (80% confluent) were trypsinised, resuspended in culture medium and transferred to sterile 10 ml centrifuge tubes
(Techno-Plas). Cells were then centrifuged at 2,500 rpm for 5 minutes, the culture media removed and resuspended in fresh media and transferred to sterile 1.8 ml
CryoTubeTM vials (Nunc). An equal volume of freezing mixture (50% media, 30%
FCS, 20% DMSO [Sigma]) was added to each cryovial and gently mixed. Cryovials were then transferred to a rate-controlled freezing chamber (Nalgene) containing isopropanol, which was placed at −80°C. A liquid nitrogen storage vessel was used for long term storage of cryopreserved cells.
45 2.2.4 Resuscitation of frozen cells
Cryopreserved cells were thawed in a 37°C water bath and then added to a flask containing culture medium and incubated at 37°C (5% CO2).
2.2.5 Trypan blue exclusion
Cells were counted using Trypan Blue Exclusion. Trypsinised cells were mixed with an equal volume of Trypan Blue (see Appendix I) and counted using a haemocytometer.
The concentration of cells was counted using the equation below :
Cell concentration (cells/ml) = cell count per grid × 2 (dilution factor) × 104
2.3 Cultured Cell Lines
2.3.1 Huh-7
The Huh-7 cell line is a human hepatocellular carcinoma cell line of epithelial origin previously isolated from a 57 year old Japanese male (Nakabayashi et al., 1982).
2.3.2 NNeoC-5B (RG)
The NNeoC-5B (RG) cell line consists of Huh-7 cells harbouring the full HCV genome and expressing the entire HCV polyprotein as previously described (Ikeda et al., 2002).
Under the selective pressure of G418 (800 µg/ml) this cell line autonomously replicates
HCV RNA and produces both the structural and non-structural HCV viral proteins.
However, despite this replication, infectious virions are not produced in this system for reasons that remain unknown. This cell line was a kind gift by Professor Stanley
Lemon (University of Texas Medical Branch, Galveston, Texas, USA). For the remainder of this thesis this cell line will be referred to as HCV genomic replicon cells.
46 2.3.3 Huh-7.5
Huh-7.5 cells are subgenomic replicon cells that were cured of HCV RNA by treatment
with IFN-α. These cells are highly permissive for HCVcc infection and have been
shown to be defective in RIG-I signaling.
2.3.4 Huh-7+IFITM
Huh-7+IFITM cells are Huh-7 cells stably transduced with lentiviral particles
expressing the IFITM1, IFITM2 or IFITM3 gene. Under the selective pressure of
blasticidin, these stable cell lines overexpress each of the proteins and are permissive to
HCVcc infection. For the remainder of the thesis, these cell lines will be referred to as
Huh-7+IFITM1, Huh-7+IFITM2 and Huh-7+IFITM3. Huh-7+IFITM1ΔN6, Huh-
7+IFITM1ΔN21, Huh-7+IFITM1ΔC18, Huh-7+IFITM1ΔHNPAP. Huh-7+IFITM2:
Y19A, and Huh-7+IFITM3:Y20A were generated in the same manner as the wildtype cell lines and maintained under the selective pressure of blasticidin and are permissive to HCVcc infection.
2.3.5 Huh-7+Empty
Huh-7 cells were stably transduced with lentiviral particles expressing an empty vector and maintained under blasticidin selection. For the remainder of the thesis, these cell
lines will be referred to as Huh-7+Empty.
2.3.6 Huh-7+shIFITM1
Huh-7+shIFITM1 cells are Huh-7 cells stably transduced with lentiviral particles
expressing shRNA targeting the IFITM1 gene. Under the selective pressure of
47 puromycin, this stable cell line knocked down expression of IFITM1 and is permissive for HCVcc infection. For the remainder of this thesis, this cell line will be referred to as
Huh-7+shIFITM1.
2.4 HCVcc Infectious System
2.4.1 Generation of HCVcc viral stock
2.4.1.1 Preparation of HCV RNA
To generate a primary viral stock of Jc1 (see Appendix II for infectious HCV constructs), 5 µg of pJc1 was digested with the appropriate restriction enzyme at 37 °C overnight (MluI for pJc1). RNA was then in vitro transcribed using the T7 in vitro transcription kit (NEB), as per manufacturer’s instructions. Samples were subsequently
DNase treated with DNase1 (Ambion) for 15 minutes at 37° C. RNA was extracted as per standard protocol (see 2.5.10). The concentration of RNA was then measured using a spectrophotmeter and RNA integrity checked via agarose gel electrophoresis
(expected bands at ∼ 4.3 and 9.6 kb).
2.4.1.2 HCV RNA transfection
Huh-7.5 cells were cultured in two 175 cm2 flasks until they were near confluence.
Cells were then harvested via trypsinization and washed twice with 10 ml of Opti-
MEM. Cells were then resuspended in Opti-MEM at a concentration of 1 × 107 cells/ml. 0.4 ml of cells were then aliquoted per electroporation cuvette (on ice), to which 10 µg of RNA was added and gently mixed. Cells were then electroporated with a single pulse at 0.27 kV, 100 ohms and 960 µF. The cells were then immediately
48 plated into 175 cm2 flasks (one per electroporation) in complete culture medium
(DMEM + 10% FCS and antibiotics). Cells were then cultured for 2-10 days post-
transfection, as required, via subculture into new culture flasks when cells reached
confluence. Viral containing supernatants were collected at approximately 5 days post
transfection and stored in 50 ml disposable conical tubes.
2.4.1.3 Concentration of HCV viral stocks (PEG precipitation)
The collected viral supernatants were adjusted to 40 ml with complete medium if
necessary and 10 ml of 40% (w/v) polyethylene glycol (PEG) [Sigma] was added to
give a final concentration of 8% (w/v). Tubes were then mixed well by inversion and
incubated at 4 °C overnight. Tubes were then centrifuged at 3900 × g for 30 minutes at
4 °C. Supernatant was then removed and pellets resuspended in a small volume of complete media (1-2ml) and samples of concentrated virus then aliquoted into screw cap microcentrifuge tubes. Samples were then stored at -80 °C and virus titre calculated as per Section 2.1.1.4. Amplification of HCV viral stocks (‘up-scale’) was performed as per Section 2.1.1.5.
2.4.1.4 Titration of infectious HCV
To titrate infectious HCV stocks, Huh-7 cells were seeded into 96-well culture trays at
2 × 104 cells/well. The next day, in 100 µl volumes, serial 10-fold dilutions of virus- containing supernatants were prepared, up to 1 in 10000. The media was then removed from near confluent Huh-7 cells in 96-well trays and replaced with 40 µl of inoculum
(in duplicate for each dilution); the cells were then cultured for 3 hours. Inoculum was then removed and cells washed once with PBS (100 µl/well) and replaced with 100 µl of complete media. The cells were then cultured for a further 3 days. At 3 days post
49 infection the culture supernatant was removed and cell monolayers fixed by the
addition of 100 µl of ice-cold acetone:methanol (1:1) and plates incubated at 4 °C for
15 minutes. The fixation solution was then replaced with 100 µl of PBS and HCV
antigens labelled by the removal of PBS and the addition of anti-HCV antisera (or
purified antibody) diluted appropriately in PBS and containing 1% bovine serum
albumin (BSA) (40 µl/well) and incubated at room temperature for 1 hour. The primary antibody was then removed and cell monolayers washed with PBS (100
µl/well); the appropriate diluted fluorescent-conjugate of secondary antibody was then added to cells (40 µl/well) in PBS containing 1% BSA and incubated at 4 °C for 1 hour. The secondary antibody solution was then removed and cell monolayers washed twice with PBS (100 µl/well). HCV positive cells were then visualized by fluorescence microscopy using a Nikon Eclipse TiE fluorescence inverted microscope and the foci
(distinct clusters) of HCV positive cells in each well (average of duplicates) were counted. To calculate the virus titre the following formula was used:
Titre (focus forming units [ffu/ml]) = number of foci × dilution factor × 25
2.4.1.5 Amplification of HCV viral stocks (‘up-scale’)
The amplification of HCV viral stocks was performed via seeding Huh-7 cells 1.6 ×
106 cells / 75 cm2 flask and cultured overnight. The following day the culture medium
was replaced with 2 × 104 ffu of cell culture-propagated HCV (HCVcc) in 2-3 ml of
complete culture media and the cells returned to culture for 3 hours, after which
complete media was added to a final volume of 10 ml. The cells were then cultured for
a further 3 days. At 3 days post infection the culture supernatant was collected and
cells harvested by trypsinization and subsequently sub-cultured into 175 cm2 flask. The cells were then cultured for a further 2 to 3 days, or until visible cytopathic effect
50 became evident. At this point the virus containing culture supernatants were collected and cleared of cellular debris via centrifugation (3900 × g for 5 minutes). The cleared culture fluid could then be concentrated and titrated.
2.4.2 General infection protocol for HCVcc
Huh-7 cells were seeded at 7 × 104 cells per 12-well culture tray and subsequently infected with Jc1 virus at MOI: 0.03 in a 300 µl volume of complete media for 3 hours.
Cell monolayers were then washed twice with PBS and returned to culture for 24 hr before subsequent harvesting for mRNA quantification experiments (see 2.5.16) or indirect immunofluorescence analysis (see 2.5.24.3).
2.4.3 HCVpp Assay
Huh-7+IFITM stables were electroporated as described in Section 2.4.1.3 and plated into 10cm dishes for 24 hrs. To determine the amount of intracellular infectious virus, the cells were harvested via trypsinisation, resuspended in complete medium, washed twice with 1x PBS and lysed via 4 freeze/thaw cycles at -80°C. Lysates were then clarified by centrifugation at 2300 x g for 5 mins prior to inoculation on to naïve Huh-7 cells. Extracellular medium was collected at the same time. Amounts of intracellular and extracellular infectious virus were determined as in Section 2.4.1.4.
2.4.4 Extracellular:Intracellular Infectivity Assay
Huh-7+IFITM stables were electroporated as mentioned in 2.4.1.2 and plated into
10cm dishes for 24 hrs. To determine the amount of intracellular infectious virus, the cells were harvested via trypsinisation, resuspended in complete medium, washed twice
51 with 1x PBS and lysed via 4 freeze/thaw cycles. Lysates were then clarified by
centrifugation at 2300 x g for 5 mins prior to inoculation on to naïve Huh-7 cells.
Extracellular medium was collected at the same time. Amounts of intracellular and
extracellular infectious virus were determined as mentioned in Section 2.4.1.4.
2.5 General Molecular Biology Methods
2.5.1 Synthetic oligonucleotides
All oligonucleotides listed in Table 2.2 were obtained from GeneWorks at
PCR/sequencing purity. Primers were received in lyphophilised form, diluted in dH2O to 20 µM and stored at -20°C until used. Oligonucleotide concentration was determined using the following formula, assuming an average MW of 330 Daltons per nucleotide:
Oligonucleotide concentration (µM) = Concentration (mg/ml) × 106
Nucleotide length × nucleotide MW
Table 2.2 Primer sequence Gene Sense primer (5’-3’) Anti-sense primer (5’-3’) Application name IFITM1 TCAGGATCCACCATGGACTACAA AGTCTCGAGGCTATGGGCGGCTACT Cloning GGATGACGACGATAAGATGCACA AGTAAC AGGAGGAACATGAGGTGG IFITM2 TCAGGATCCACCATGGACTACAA AGTCTCGAGAATGATGCCTCCTGATC Cloning GGATGACGACGATAAGATGAACC TATCGCTG ACATTGTGCAAACCTTCTCTC IFITM3 TCAGGATCCACCATGGACTACAA AGTCTCGAGCAGTGATGCCTCCTGAT Cloning GGATGACGACGATAAGATGAATC CTATCCATA ACACTGTCCAAACCTTCTTCTC IFITM1 TCAGGATCCACCATGGACTACAA AGTCTCGAGGCTATGGGCGGCTACT Cloning ΔN6 GGATGACGACGATAAGATGGAG AGTAAC GTGGCTGTGCTGGGGGCAC IFITM1 TCAGGATCCACCATGGACTACAA AGTCTCGAGGCTATGGGCGGCTACT Cloning ΔN13 GGATGACGACGATAAGATGCCCC AGTAAC CCAGCACCATCCTTCCA IFITM1 TCAGGATCCACCATGGACTACAA AGTCTCGAGGCTATGGGCGGCTACT Cloning ΔN21 GGATGACGACGATAAGATGTCCA AGTAAC CCGTGATCAACATCCACAGC IFITM1 TCAGGATCCACCATGGACTACAA AGTCTCGAGGCTATGGGCGGCTACT Cloning
52 ΔN28 GGATGACGACGATAAGATGAGCG AGTAAC AGACCTCCGTGCCCGATGT IFITM1 TCAGGATCCACCATGGACTACAA TAGTCTCGAGCTAGTAGACTGTCACA Cloning ΔC13 GGATGACGACGATAAGATGCACA GAGCCGAATA AGGAGGAACATGAGGTGG IFITM1 See Section 4.4.1 See Section 4.4.1 Cloning ΔC18 IFITM1 GCTGGGGGCACCCCACAACCCCG CGGTGGACCTTGGAGGGGCGGGGTT Cloning ΔHNPAP CCCCTCCAAGGTCCACCG GTGGGGTGCCCCCAGC IFITM2 CAACAGCGGCCAGCCTCCCAATG CTCCTCCTTGAGCATTTCGGCATTGG Cloning ΔIFITM2:Y CCGAAATGCTCAAGGAGGAG GAGGCTGGCCGCTGTTG 19A IFITM3 CAGTGGCCAGCCCCCCAATGCTG GCTCCTCCTTGAGCATTTCAGCATTG Cloning ΔIFITM3:Y AAATGCTCAAGGAGGAGC GGGGGCTGGCCACTG 20A IFITM1 TTCAACACCCTCTTCTTGAACTGG CTTCACGGAGTAGGCGAATGCTATA Cloning ΔC5051A GCCGCTCTAGGGTTTATAGCATTC AACCCTAGAGCGGCCCAGTTCAAGA GCCTACTCCGTGAAG AGAGGGTGTTGAA IFITM2 GTCCCTGTTCAACACCCTCTTCAT GTACGCGAATGCTATGAAGCCTAGG Cloning ΔC7071A GAATACGGCCGCCCTAGGCTTCA GCGGCCGTATTCATGAAGAGGGTGT TAGCATTCGCGTAC TGAACAGGGAC IFITM3 GTCCCTGTTCAACACCCTCTTCAT GTAGGCGAATGCTATGAAGCCTAGG Cloning ΔC7172A GAATCCAGCCGCCCTAGGCTTCA GCGGCTGGATTCATGAAGAGGGTGT TAGCATTCGCCTAC TGAACAGGGAC *IFITM1 CAGGCCTATGCCTCCACCGCAAA CCCAGAATCAGGGCCCAGATATTTA Cloning ΔC84A/ AGCCCTAAATATCTGGGCCCTGA GGGCTTTTGCGGTGGAGGCATAGGC IFITM2 TTCTGGG CTG ΔC104A/ *same primer for all 3 mutants – IFITM3 conserved region *same primer for all 3 mutants – conserved ΔC105A region HCV TCTTCACGCAGAAAGCGTCTAG GGTTCCGCAGACCACTATGG RT-PCR RPLPO AGATGCAGCAGATCCGCAT GGATGGCCTTGCGCA RT-PCR IFITM1 CGCCAAGTGCCTGAACATCT CCCGTTTTTCCTGTATTATCTGTA RT-PCR CMV CGCAAATGGGCGGTAGGCGTG ACCGAGGAGAGGGTTAGGGAT Sequencing
2.5.2 Bacterial transformation
Frozen aliquots of the competent E.coli strain DH5α cells (see Appendix I) were
thawed on ice for 5 minutes prior to the addition of 5µg of plasmid DNA. Once the
appropriate volume of plasmid DNA was added the tubes were gently mixed and
incubated on ice for 20 minutes. Cells were then heat shocked at 42 °C for 45 seconds
followed by incubation on ice for a further 2 minutes. 950µl of SOC medium was then
added to the cells and incubated at 37 °C for 60 minutes to allow for the induction of
antibiotic resistance. Cells were then plated on L-Agar plates containing ampicillin
53 (100 µg/ml) and incubated at 37°C overnight. In regard to pGem-T Easy cloning,
IPTG (Sigma-Aldrich) and X-gal (Sigma-Aldrich) were added to the L-Agar plates at
0.5 mM and 80 µg/ml respectively.
2.5.3 Mini-preparation (small scale) of plasmid DNA
A single bacterial colony was inoculated in 10 ml of LB containing 100 µg/ml of ampicillin and incubated at 37°C overnight on a shaking platform. Plasmid DNA was isolated from log phase bacterial cultures using QIAprep Spin Miniprep Kit (Qiagen) as per manufacturer’s instructions. This method involves alkaline cell lysis and silica column purification of plasmid DNA. Samples were stored at -20°C until required.
2.5.4 Maxi-preparation (large scale) of plasmid DNA
LB (3 ml) cultures containing ampicillin (100 µg/ml) were inoculated with a single transformed bacterial colony and incubated for 8 hours at 37°C with shaking. 100 µl of log phase culture was then transferred to LB (200 ml) containing ampicillin (100
µg/ml) and incubated overnight at 37°C with shaking. Plasmid DNA was then isolated using the NucleoBond® Xtra Maxi (Macherey-Nagel) as per the manufacturer’s instructions. Samples were stored at -20°C until required.(Farci, 2002)
2.5.5 Restriction endonuclease digestion
Restriction digests were performed in a 20 µl volume, which contained 1 µg DNA and
10 U of restriction enzyme(s) (New England Biolabs) in the appropriate buffer.
Reactions were incubated at 37°C overnight and digested DNA was then visualized on a 1% agarose gel.
54 2.5.6 Agarose gel electrophoresis
Gel electrophoresis was performed using 1% (w/v) agarose gels. Agarose gels were made by dissolving DNA grade agarose (Progen Biosciences) in 1 × TAE (see
Appendix I). Gels were then cast in a BioRad Wide Mini-Sub® Cell GT tank. DNA samples were mixed with 6 × loading dye (see Appendix I) and subsequently loaded into wells on the agarose gel. The gel was then run at 50-100 V in 1 × TAE. Markers were run simultaneously to estimate product size, 0.5 µg of 1 kb or 100 bp DNA molecular weight markers (New England Biolabs). Following electrophoresis, gels were stained in 3 × GelRedTM Nucleic Acid Gel Stain in DMSO (Biotium Inc) for approximately 15 minutes. DNA was then visualised under ultraviolet light on a
BioRad Universal Hood II gel documentation system using Quantity One® Version 4.6
Basic software (BioRad).
2.5.7 DNA ligation
Ligation reactions were performed in a 20 µl volume containing 0.4 units T4 DNA
Ligase (New England Biolabs), 1 × Ligase Buffer (New England Biolabs), and a 3:1 ratio of amplified insert to destination vector. Reactions were allowed to proceed at
16°C overnight. Successful ligations were confirmed by restriction digest and gel electrophoresis to identify bands of the expected size. Correct ligation mixtures were transformed into competent DH5α cells for colony growth and plasmid purification.
2.5.8 Gel purification
To purify electrophoresed DNA from agarose, DNA bands were excised using a scalpel blade and stored in eppendorf tubes while being visualized under UV light. PCR
55 products and plasmid DNA were then purified using either the MinElute Gel Extraction
kit (Qiagen) or QIAquick Gel Extraction kit (Qiagen) respectively, as per the
manufacturer’s recommendations. Samples were stored at -20°C until required.
2.5.9 DNA sequencing
Sequencing was performed at the Australian Genome Research Facility (AGRF)
utilizing the Purified DNA service. Samples were prepared by mixing 1µg of plasmid
DNA with 9.6pmol of appropriate forward and reverse primer. Data was analysed using
FinchTV version 1.4.0 and compared to known sequences using NCBI BLAST
nucleotide blast search against the human genome:
(http://www.ncbi.nlm.nih.gov/blast/Blast.cgi).
2.5.10 Extraction of total RNA
Total cellular RNA was extracted from cell monolayers adhered to 6- or 12-well dishes
(Corning Life Sciences) using Trizol® Reagent (Invitrogen) according to the
manufacturer’s recommendations. Cell monolayers were lysed directly in Trizol®
Reagent (500 µl per 12-well / 1 ml per 6-well culture plate). Lysates were followed by
the addition of 200 µl chloroform per 1 ml Trizol®. Tubes were then shaken vigorously
for 15 seconds and incubated again at room temperature for 2 minutes. Samples were
then centrifuged at 12,000 × g for 15 minutes at 4°C. Following separation, the top
aqueous layer was transferred to an RNAse-Free 1.5 ml Microfuge tube (Ambion) and
precipitated with isopropanol (0.5 ml per 1 ml Trizol®) for 10 minutes at room temperature. Samples were again centrifuged for 15 minutes at 12,000 × g at 4°C.
Supernatant was aspirated and the pellet vortexed with 1 ml 70% (v/v) ethanol and
56 centrifuged at 7,500 × g for 5 minutes at 4°C. After removal of the ethanol wash, the
resultant RNA pellet was air dried and resuspended in 10 µl RNAse-free dH2O and stored at -80 °C.
2.5.11 DNAseI treatment of RNA samples
RNA samples were treated with DNAseI to remove any contaminating DNA. To each
10 µl sample 2 units RNase-free DNaseI (Ambion) and 1 × DNAseI buffer (Ambion) were added and tubes incubated at 37°C for 15 minutes. Following incubation, 1 µl
DNAse Inactivation Reagent (Ambion, provided with the RNAqueous-4PCR RNA
Extraction kit) was added to each sample and incubated at room temperature for 2 minutes, after which samples were centrifuged at 10,000 × g for 1 minute. Supernatant was transferred to a fresh tube and stored at -80°C until further use.
2.5.12 Nucleic acid quantification
All DNA and RNA preparations were diluted in dH20 and quantitated using the
TM SmartSpec 3000 UV spectrophotometer (BioRad Laboratories). A260nm readings were
taken and sample concentrations calculated by multiplying the absorbance reading by
the appropriate dilution factor and 50 (DNA) or 40 (RNA) as an A260nm reading of 1 is equivalent to 50mg/ml DNA and 40mg/ml RNA.
[Nucleic Acid] = A260nm x dilution factor x (50 or 40) µg/ml
2.5.13 cDNA preparation
First strand synthesis was carried out with 1 µg total RNA combined with 1 µg random
hexamer primer (GeneWorks) and diluted to a total volume of 14 µl with dH2O. Tubes
57 were incubated at 70°C for 5 minutes, then on ice for a further 5 minutes. 1 × M-MLV
RT reaction buffer (Promega), 0.5 µmol dNTP mix (0.5 µmol each dATP, dCTP,
dGTP, dTTP; Promega), 40 units rRNAsin® RNase Inhibitor (Promega), 200 units M-
MLV RT, RNase H(-) Point Mutant (Promega) and 3.25 µl dH2O were added to each
tube, samples mixed gently and incubated at room temperature for 10 minutes, then
42°C for 50 minutes. Samples were then diluted to a final volume of 100 µl with dH2O and stored at -20°C.
2.5.14 Polymerase Chain Reaction
PCR reactions were performed using AmpliTaq Gold™ (Applied Biosystems) DNA
polymerase. Reactions were carried out in a 50 µl volume containing 10 ng of DNA
template, 2.5 µl each of forward and reverse primers (at 20 µM) (Table 2.2), 1 µl of
dNTP mix (at 0.2 mM with 10 mM each of dATP, dCTP, dGTP and dTTP), 5 µl of 10×
™ PCR Buffer, 4 µl of magnesium chloride (MgCl2) (at 25 mM), 1 U of AmpliTaq Gold and dH2O. PCR reaction conditions were comprised of denaturation at 95°C for 4 min,
followed by 30 cycles of 95°C for 30 sec, 57°C for 30 sec and 72°C for 3 min and 20
sec, with a final extension at 72°C for 7 min. All reactions were carried out in a
MyCycler Thermocycler (BioRad) and stored at 4°C. Results were analysed via
agarose gel electrophoresis.
2.5.15 High Fidelity PCR
High fidelity PCR reactions were performed using Platinum® Taq (Invitrogen), a high
fidelity DNA polymerase. Reactions were carried out in a 50 µl volume containing 10
ng of DNA template, 1.25 µl each of forward and reverse primers (at 20 µM) (Table
58 2.2), 1 µl of dNTP mix (at 0.2 mM with 10 mM each of dATP, dCTP, dGTP and dTTP), 10 µl of 5x iProof HF buffer, 1 U of Platinum® Taq High Fidelity DNA
Polymerase and dH2O. PCR reaction conditions were comprised of denaturation at
94°C for 1 min, followed by 30 cycles of 94°C for 20 sec, 55°C for 20 sec and 68°C for
2 min and cooling at 4°C. All reactions were carried out in a MyCycler Thermocycler
(BioRad) and where applicable were stored at 4°C. Results were analysed via agarose
gel electrophoresis.
2.5.16 Real-Time Quantitative PCR
Real-time quantitative PCR (qRT-PCR) was performed to determine relative levels of
® HCV RNA using the comparative CT method of SYBR Green PCR Master Mix
(Applied Biosystems). 5 µl cDNA was combined with 10 µl SYBR® Green PCR
Master Mix and 300 nM of each forward and reverse primers (Table 2.2) in duplicate.
All cDNA samples were combined with 10 µl SYBR® Green PCR Master Mix and 300
nM each forward and reverse primers for the housekeeping gene ribosomal protein
large PO (RPLPO; Table 2.2) to normalize input cDNA levels. Reaction conditions
were controlled by an ABI PRISM 7000 Sequence Detection System (Applied
Biosystems) and comprised denaturation at 95°C for 10 minutes followed by 40 cycles
of 95°C for 15 seconds and 60°C for 1 minute. A final dissociation step of 60°C for 10
minutes followed, to facilitate melt curve analysis. Data was analysed using ABI Prism
7000 SDS Software and the t hreshold was set at 0.2 for all experiments.
59 2.5.17 Extraction of cellular protein
Total protein was extracted from cell monolayers using RIPA buffer (see Appendix I).
Culture media was removed and cells washed in PBS. 1 µl of proteinase inhibitor
cocktail (Sigma) was added per 100 µl of RIPA buffer. 100 µl of this mixture was then
added to each well. The plates were then incubated on ice for 20 minutes with gentle
agitation every 5 minutes. The wells were then scraped and the lysates collected and
added to eppendorfs that were then centrifuged at 14,000 rpm for 10 minutes at 4 °C.
The protein containing supernatant was then removed and stored at -20 °C.
2.5.18 SDS PAGE and protein transfer
SDS PAGE was performed as previously described (Laemmli, 1970). A 12%
separating gel (see Appendix I) was cast in a BioRad Mini-PROTEAN® Tetra Cell gel tank and layered with dH2O to prevent oxidation and enhance gel polymerisation. Once set, a 5% stacking gel (see Appendix I) was layered onto the separating gel and a comb inserted to form wells. Protein samples were prepared for electrophoresis by boiling for
5 minutes with 1 × SDS PAGE sample buffer (see Appendix I). 50 µg samples were then loaded onto the gel alongside 7.5 µg Precision Plus Protein® Standards -
Kaleidoscope (BioRad) and run at 100 volts for 1-2 hours in SDS-PAGE running buffer (see Appendix I). Gels were equilibrated in cold western transfer buffer (see
Appendix I) for 5 minutes and proteins transferred to a Hybond ECL membrane
(Amersham Pharmacia Biotech) in western transfer buffer for 1 hour at 100 volts in a
BioRad Mini Trans Blot Electrophoretic Transfer Cell.
60 2.5.19 Western blotting
Following transfer, membranes were blocked with 5% skim milk (Diploma) in 0.1%
PBS-T (see Appendix I) for 1 hour with gentle agitation. Membranes were rinsed twice
in 0.1% PBS-T and incubated in the appropriate concentration of primary antibody
diluted in 0.1% PBS-T (Table 2.3) overnight at 4°C (see Table 2.3 for antibody
concentrations). Membranes were rinsed twice in 0.1% PBS-T and incubated once for
15 minutes and three times for 5 minutes each in fresh changes of 0.1% PBS-T to
remove any unbound primary antibody. Membranes were then incubated in the
appropriate horseradish peroxidase-conjugated secondary antibody (Table 2.3) for 1
hour with gentle agitation and washed as described above. Protein detection was
carried out using either the ECL Plus Western blotting detection reagent kit (Amersham
Pharmacia Biotech) or the Supersignal® West Femto Maximum Sensitivity Substrate detection kit (ThermoScientific) with chemiluminescent detection as per manufacturer’s instructions. Protein bands were visualised by exposure to Kodak
BioMax film for 30 seconds to 20 minutes depending on signal intensity.
Table 2.3 Western Blot Antibody concentrations 1 °Antibody Concentration Company FLAG 1:1000 O/N 4°C Sigma Aldrich IFITM1 1:1000 O/N 4°C Proteintech Phosphotyrosine 1:1000 O/N 4°C Millipore
β-actin 1:10,000 O/N 4°C Sigma Aldrich
2 °Antibody Anti-Mouse IgG ( HRP-linked) 1:10,000 1 hr @ room temp Cell Signaling Anti-Rabbit IgG, ( HRP-linked) 1:15,000 1 hr @ room temp Cell Signaling
61 2.5.20 Immunoprecipitation
Near-confluent cells in 6-well culture plates were washed with PBS before lysis in 0.5
ml of NP-40 lysis buffer (Appendix I) containing protease (Sigma) and phosphatase
inhibitors (Calbiochem) on ice for 20 min. Lysates were pre-cleared by incubation with
20µl of protein A/G PLUS agarose (Santa Cruz Biotechnology) for 1 hr. Samples were
then centrifuged at 13000×g for 5 min at 4°C, and supernatants were collected and
incubated with 1µg of anti-FLAG mAb overnight. 30µl of protein A/G PLUS agarose
was then added to each sample prior to incubation for 1 hr. Beads were then pelleted by
centrifugation at 13000×g for 5 min at 4°C and washed 5 times with 500µl of NP-40
lysis buffer before resuspension in 40µl of 2 x SDS-PAGE sample buffer and Western
blot analysis (see 2.5.19).
2.5.21 Dual Renilla luciferase assay
The relative luciferase activity of the IRES promoter element was measured using the
Luciferase Assay System (Promega). Overexpression cells were seeded at a density of
7 x 104 cells/well and transfected with pRL-HL, where the integrated Renilla luciferase acted as a transfection control. Transfections were performed using FuGENE 6 transfection reagent (Roche Applied Science) as mentioned previously. After 24 hours cell lysates were collected in 80 µl of Passive Lysis Buffer by a single freeze-thaw cycle. 20 µl of cell lysate was then added to an optical tray and luciferase out-put measured on a Glow Max machine (Promega).
2.5.22 Site-Directed Mutagenesis
Primers were designed using the QuikChange Primer design tool
(http://www.genomics.agilent.com/primerDesignProgram.jsp?&_requestid=447214)
62 provided by Agilent Technologies. Mutagenesis was conducted using the QuikChange®
II XL Site-Directed Mutagenesis Kit (Stratagene) as per manufacturer’s instructions.
Successful mutants were screened via sequencing (see 2.5.9) and samples were stored at -20°C.
2.5.23 Immunofluorescence microscopy
2.5.24.1 Acetone/Methanol Fixation
Cells were cultured on 24-well cell culture trays prior to fixation. Cell monolayers were then washed with 1× PBS, before fixation with ice cold acetone/methanol mixture
(1:1) for 15 mins at 4ºC. The fixative was removed and cells were then washed with
1× PBS. Cells were then blocked by incubation for 30 mins with 5% BSA (Sigma-
Aldrich) in PBS at room temperature, prior to immunofluorescence labelling (see
2.5.24.3).
2.5.24.2 4% Paraformaldehyde Fixation
Cells were cultured on 24-well cell culture trays prior to fixation. Cell monolayers were then washed with 1× PBS, before fixation with ice cold 4% paraformaldehyde
(w/v) for 20 mins at room temperature. The fixative was removed and cells were then washed with 1× PBS. Cells were then permeabilised by incubation for 10 mins with
0.1% Triton-X 100 (Sigma-Aldrich) in PBS at room temperature and blocked for 30 mins with 5% BSA in PBS at room temperature, prior to immunofluorescence labeling
(see 2.5.24.3).
63 2.5.24.3 Immunofluorescence labelling
Huh-7 cells were seeded at 4 × 104 cells per well in 24-well cell culture trays
containing sterile, 0.2% (w/v) gelatin-coated coverslips and fixed with either
acetone/methanol (see 2.5.24.1) or 4% paraformaldehyde (see 2.5.24.2). Cells on
coverslips were labelled by incubation with 200 µl of primary antibody (Table 2.4)
diluted in PBS containing 1% BSA (w/v) for 1 hr at room temperature. After washing
twice with PBS, bound primary antibody was detected by incubation for 1 hr in the
dark at 4ºC with an appropriately diluted secondary antibody fluorescent conjugate
(Table 2.4) in PBS containing 1% BSA. Following labelling, samples were washed
twice with 1× PBS and stained with the nuclear stain DAPI (Sigma-Aldrich) for 10 min
in the dark. Cells were washed twice with 1×PBS, before addition of 0.5 ml of FACS
fixative solution (Appendix 1). After incubation n (at least 30 min) at 4ºC, fixative
solution was aspirated and replaced with PBS. Coverslips were mounted on glass
slides with ProLong® Gold antifade reagent (Invitrogen). Unfixed and fixed cell
monolayers were visualised using a Nikon Eclipse TiE fluorescence inverted
microscope and images were captured using NIS Elements software.
Table 2.4 Immunofluorescence Antibody concentrations 1 °Antibody Concentration Company FLAG 1:200 1 hr @ room temp Sigma-Aldrich Occludin 1:200 1 hr @ room temp Invitrogen Claudin 1:200 1 hr @ room temp Invitrogen SR-BI 1:200 1 hr @ room temp Novus Biologicals CD81 1:200 1 hr @ room temp BD Pharmingen™ HCV patient antisera 1:50 1 hr @ room temp Pooled patient serum (genotype 2) Rab5a 1:100 O/N 4°C Cell Signaling Rab7 1:100 O/N 4°C Cell Signaling Lamp1 1:100 O/N 4°C Cell Signaling
64 2 °Antibody Anti-Mouse Alexa 488 1:200 1 hr @ room temp Invitrogen Anti-Rabbit Alexa 488 1:200 1 hr @ room temp Invitrogen Anti-Mouse Alexa 555 1:200 1 hr @ room temp Invitrogen Anti-Rabbit Alexa 555 1:200 1 hr @ room temp Invitrogen Anti-Human Alexa 488 1:200 1 hr @ room temp Invitrogen Anti-Human Alexa 555 1:200 1 hr @ room temp Invitrogen Anti-Mouse Cy5 1:200 1 hr @ room temp Invitrogen
2.5.24.4 Fluorescence Energy Resonance Transfer (FRET) Analysis
Cells were cultured on 0.2% (w/v) gelatin-coated coverslips in 24 well culture plates
prior to fixation with 4% paraformaldehyde and immunofluorescence labelling (see
2.5.24.3). Acceptor photobleaching was carried out using Alexa 555 (Invitrogen) and
Cy5 (Invitrogen) conjugated secondary antibodies. Images of the acceptor and donor flurophores were acquired using a Zeiss Axioplan2 upright microscope, using a
63PlanApo objective (Carl Zeiss AG, Oberkochen, Germany). Acceptor photobleaching was performed at maximum light intensity for 30-180 seconds, followed by reimaging of the donor and acceptor fluorophores (an automated process
ensuring identical imaging conditions). The FRET signal (increase in signal post-
bleach) was determined by the subtraction of the pre- from post-bleach donor image
using ImageJ software. Cell surfaces positive for both proteins being examined were
selected, and the average intensity in that region was compared on the aligned pre- and post-bleach images (the plug-in StackReg was utilised to control for lateral image
displacement, and the FRET signal was displayed using the ‘‘fire’’ lookup table). At least 10 different cells positive for both proteins measured were measured in two independent experiments to ensure reproducibility. Negative slides were prepared by omitting the primary antibody for the acceptor molecule.
65 2.5.25.5 Duolink® In situ Proximity Ligation Assay
Cells were cultured on 0.2% (w/v) gelatin-coated coverslips in 24 well culture plates
prior to fixation with 4% paraformaldehyde. Proximity ligation assay (PLA) was conducted using the Duolink® In situ kit (Olink® Biosciences) as per manufacturer’s
instructions. Positive interactions visualised using a Nikon Eclipse TiE fluorescence
inverted microscope and images were captured using NIS Elements software.
2.6 Data Analysis
All data was statistically analysed by way of unpaired student t-tests using GraphPad
Prism 5.
66 Chapter 3
The impact of the IFITM proteins on HCV replication
3.1 Introduction
The IFITM family of proteins (IFITM1, IFITM2 and IFITM3) are induced by
interferon (IFN) and are expressed in a number of different cell types and associated
with numerous cellular functions including immune cell signaling, cell adhesion and
oncogenesis. Recent studies have identified IFITM1, IFITM2 and IFITM3 to be
capable of inhibiting a broad range of RNA viruses, including the Flaviviruses DENV
and WNV (Brass et al., 2009, Jiang et al., 2010), although the exact molecular
mechanism(s) that underpin this remain unknown. Microarray analysis of HCV infected patient liver tissue in our laboratory (unpublished data) has also identified the
IFITM proteins, in particular IFITM1, to be significantly upregulated. Given this
observation and previous findings in the literature, we sought to investigate the role of
the IFITM proteins on the HCV lifecycle, as it is conceivable that this family of
proteins may contribute to the antiviral activity of IFN-α against HCV. We therefore
sought to investigate the role of the IFITM family on HCV infection. However, during the course of this PhD thesis, several studies identified IFITM1 and IFITM3 to have an anti-HCV effect (Raychoudhuri et al., 2011, Yao et al., 2011, Wilkins et al., 2013,
Kandathil et al., 2013), while a number of siRNA screens also identified all of the
IFITM proteins to play a role in HCV infection (Metz et al., 2013). Although these studies have demonstrated anti-HCV activity for IFITM1 and IFITM3 in vitro, they appear contradictory in terms of defining the exact stage of the HCV lifecycle targeted by these proteins. In addition, no work to date has been conducted in examining the
67 role of the highly analogous IFITM2 protein in the context of HCV infection. Hence,
the aim of this chapter was to comprehensively investigate the role of IFITM1, IFITM2 and IFITM3 in the context of the HCV lifecycle in hepatocytes.
3.2 Characterisation of endogenous IFITM proteins in vitro
The IFITM family of proteins is known to be IFN inducible; however, their regulation
by IFNs in hepatocytes remains unexplored. In order to investigate the dynamics of
mRNA expression for each of the IFITM proteins, attempts were made to design
specific primers for real-time PCR analysis (Table 2.2). Specific primers were designed
for IFITM1; however, this proved difficult for IFITM2 and IFITM3 due to the high
level of sequence conservation between the two proteins. To determine whether
IFITM1 mRNA expression could be specifically detected, Huh-7 cells were treated
with 50 IU/ml IFN-α over a timecourse, for up to 72 hrs. At each time point total RNA
was extracted using Trizol® and RT-PCR then performed to generate cDNA, which was subsequently used in real-time PCR to measure IFITM1 mRNA levels, with results normalized to the house-keeping gene RPLPO. IFITM1 was found to be specifically upregulated in Huh-7 cells following IFN-α, with a 280-fold increase in mRNA expression observed at peak expression [Figure 3.1]. Unforutnately, primers specific for IFITM2 and IFITM3 could not be designed preventing the analysis of the mRNA profile for these proteins in Huh-7 cells. This problem was also encountered with commercial antibodies that claimed to specifically detect either IFITM2 or IFITM3 via
Western blot analysis. Numerous antibodies were tested that claimed to be specific for
IFITM1, IFITM2 and IFITM3 during the course of this PhD on Huh-7 cells specifically expressing each of the IFITM proteins (described in Section 3.6). Antibodies targeting
68 400 mRNA
200
20
10
0 Relative fold change IFITM1 change fold Relative 0 20 40 60 80 Time with IFNα (hrs)
Figure 3.1: IFN-α induces the expression of IFITM1 in Huh-7 cells. Huh-7 cells were treated with IFN-α (50 IU/ml) for up to 72hrs and total RNA recovered. IFITM1 mRNA levels were quantitated by real-time PCR and normalised to control protein RPLPO. IFITM1 mRNA expression peaked at 16hrs post IFN-α treatment, whilst non-specific upregulation was observed for IFITM2/3. IFITM1 were found to specifically detect the IFITM1 protein at sizes corresponding to
the literature; however, none of the antibodies were able to bind specifically to either
IFITM2 or IFITM3 [Figure 3.2]. This resulted in all endogenous protein expression
studies being limited to IFITM1 only.
3.3 Regulation of IFITM1 by IFN in hepatocytes
3.3.1 IFITM1 is upregulated by IFN-α in Huh-7 cells
To further characterise IFITM1 expression in Huh-7 cells upon stimulation with IFN-α
at the mRNA and protein level, we treated cells with 50 IU/ml IFN-α over a
timecourse, for up to 72 hrs. At each time point total RNA was extracted using Trizol® and RT-PCR then performed to generate cDNA, which was subsequently used in real- time PCR to measure IFITM1 mRNA levels, with results normalized to the house- keeping gene RPLPO. Results demonstrated that IFITM1 mRNA expression was induced within 4 hrs of IFN-α treatment, with expression peaking at 16 hrs post- treatment [Figure 3.3.i] resulting in an approximately 280-fold increase from baseline expression. These results were corroborated by a concurrent experiment, where total protein was extracted at each timepoint post-IFN-α treatment and a Western blot specific for IFITM1 performed. IFITM1 proteins levels can first be detected within 4 hrs of IFNα treatment, with expression peaking and plateauing from 16 hrs onwards
[Figure 3.3.ii]. Immunofluorescence analysis of Huh-7 cells treated with IFN-α for 24 hrs and probed with an IFITM1-specific antibody demonstrated that within the hepatocyte, IFITM1 localises to the cell periphery with some cytoplasmic localisation
[Figure 3.3.iii]. Taken together, these results demonstrate that IFITM1 mRNA and
69 Empty IFITM1 IFITM2 IFITM3
15 kDa anti-IFITM1
18 kDa 17 kDa 15 kDa anti-IFITM2
18 kDa 17 kDa 15 kDa anti-IFITM3
anti-!-actin 42 kDa
Figure 3.2: High level of sequence conservation prevents specific detection of IFITM2 and IFITM3 protein in Huh-7 cells. To determine if commercial IFITM1, IFITM2 and IFITM3 anitbodies were specific for the respective proteins, total protein was extracted from Huh-7+IFITM cell lines for Western blot analysis. Extracted protein was immunoblotted with antibodies specific for IFITM1, IFITM2, IFITM3 and "-actin. Results demonstrate that the IFITM1 antibody detected IFITM1 specifically, with sizes corresponding to the literature. Antibodies for IFITM2 and IFITM3 detected non- specific bands as well as all the IFITM proteins. i 400 mRNA
300
200
100
0
Relative fold change IFITM1 change fold Relative 0 20 40 60 80 Time with IFN! (hrs)
ii !"#$%&"'(%)*+,%-(./0% IFITM1 0 4 8 16 24 48 72
15 kDa anti-IFITM1
42 kDa anti-!-actin
iii
Figure 3.3: IFN-! induces the expression of IFITM1 in Huh-7 cells. Huh-7 cells were treated with IFN-! (50 IU/ml) for up to 72hrs and total RNA (i) and cellular lysate (ii) recovered. (i) IFITM1 mRNA levels were quantitated by real-time PCR and normalised to control protein RPLPO. IFITM1 mRNA expression peaked at 16hrs post IFN-! treatment which corresponded to protein expression (ii), also peaking at 16hrs post treatment. (iii) IFITM1 localises to the cell periphery, with some cytoplasmic localisation upon treatment with IFN-! for 24hrs (60x magnification). protein expression can be significantly induced in vitro by stimulating Huh-7 cells with
IFN-α for up to 16 hrs.
3.3.2 IFITM1 is upregulated by type I and III IFNs in primary human hepatocytes
(PHH)
Huh-7 cells are an immortal cell line of hepatocellular carcinoma origin and in order to
confirm IFITM1 mRNA expression in a primary cell line, PHHs were utilised. PHHs
are isolated from single donors, and thus allow for the in vitro study of primary liver
cells and were purchased from Lonza. Cells were treated with either 50 IU/ml IFNα or
25ng/ml IFN-λ for 24 hrs prior to total RNA extraction using Trizol® and RT-PCR
performed to generate cDNA, which was subsequently used in real-time PCR to
measure IFITM1 mRNA levels, with results normalized to RPLPO levels. As can be
seen in Figure 3.4, IFITM1 mRNA was significantly upregulated 50- and 90-fold
following stimulation with either IFN-α [Figure 3.4.i] or IFN-λ [Figure 3.4.ii]
respectively. Collectively, these results demonstrate that IFITM1 is upregulated by both type I and III IFNs in PHHs. Interestingly IFN-λ has recently been shown to be expressed by hepatocytes and is an important IFN in the control of HCV (Sheahan et al., 2014).
3.4 Knockdown of endogenous IFITM1 reduces the anti-HCV activity
of IFN-α
IFN (type I, II and III) is a potent inhibitor of HCV replication; however, the full complement of IFN-induced genes responsible for this inhibition is not known. In order to investigate if IFITM1 is partially responsible for this anti-HCV effect, we created a
70 i 80 mRNA
60 ****
40
20 ****P <0.0001 0 Control IFNα Relative fold change in IFITM1 in change fold Relative
ii
100 *** mRNA 80
60
40
20 ***P =0.0007 0
Relative fold change IFITM1 change fold Relative Control IFN λ
Figure 3.4: IFN-α and IFN-λ induces IFITM1 expression in PHH. PHH were treated with either (i) 50 IU/ml IFN-α or (ii) 25ng IFN-λ for 24hrs. Total RNA was extracted and real-time PCR used to quantitate IFITM1 mRNA levels. RPLPO was used as a control and IFITM1 mRNA levels were expressed as a fold change relative to no treatment. These results demonstrated that type I and type III IFNs are able to signifcantly induce the expression of IFITM1 in PHH (data represented as a mean ± SEM, n=3). number of Huh-7 cell lines stably expressing shRNA targeting IFITM1 mRNA.
Briefly, these stable cell lines were generated by transducing Huh-7 cells with lentiviral
particles expressing shIFITM1, followed by antibiotic selection with puromycin.
Resistant cells were isolated (method described in Section 2.1.3) to generate cells with
a stable knock down of IFITM1 expression. A non-specific targeting shRNA was used
as a control, referred to as Huh-7+shControl in this thesis. To validate this shRNA
approach, the Huh-7+shIFITM1 cells were stimulated with 100 IU/ml of IFN-α for 24
hrs and real-time PCR performed to measure IFITM1 mRNA levels. IFITM1 mRNA
levels were expressed as a fold change relative to the control shRNA. While all the
IFITM1 shRNAs tested were able to knockdown levels of IFITM1 mRNA produced following IFN-α stimulation, cells expressing IFITM1 shRNA #4 were able to produce the least IFITM1, with an 80% reduction in IFITM1 mRNA compared to control
[Figure 3.5.i]. To ascertain if the Huh-7+shIFITM1 cells had a successful knockdown for IFITM1 protein, total protein was harvested and Western blots specific for IFITM1 were performed. Significant decreases in detectable IFITM1 protein in all the Huh-
7+shIFITM1 cell lines were seen, with no detectable levels of IFITM1 protein in the cells expressing IFITM1 shRNA #4 [Figure 3.5.ii]. For the remainder of this thesis, cell lines expressing IFITM1 shRNA #4 were used and will be referred to as Huh-
7+shIFITM1. Taken together, these results demonstrate that we successfully generated
Huh-7 cells that stably knocked down IFITM1 expression at both the mRNA and protein level.
To determine whether endogenous IFITM1 had a role in the anti-HCV activity of IFN-
α, the Huh-7+shIFITM1 cells were either pretreated with 50 IU/ml IFN-α for 24 hrs before HCV Jc1 infection or treated with IFN-α for 24 hrs after Jc1 infection. Real-
71 i 1.5
1.0 mRNA
0.5 *** ** IFITM1 **P = 0.0051 **** ***P = 0.0002 Relative fold change in change fold Relative ****P <0.0001 0.0 Control #1 #2 #3 #4
ii Control #1 #2 #3 #4 15 kDa anti-IFITM1
42 kDa anti-!-actin
Figure 3.5: shRNA mediated knockdown of endogenous IFITM1 in Huh-7 cells. To determine the level of knockdown obtained by a panel of shRNAs targeting IFITM1, Huh-7+shIFITM1 cell lines were treated with 100 IU/ml of IFN!. (i) Total RNA was extracted and real-time PCR used to quantitate IFITM1 mRNA levels. RPLPO was used as a control and IFITM1 mRNA levels were expressed as a fold change relative to no treatment (data represented as a mean ± SEM, n=3). (ii) Total protein was extracted and immunoblotted with antibodies specific for IFITM1 and "-actin. These results show that shRNA #4 knocked down IFITM1 mRNA and protein expression most significantly. time PCR was utilised to measure HCV RNA levels, with HCV RNA levels expressed as a fold change relative to no IFN-α treatment. No differences in HCV replication levels were observed between the control and IFITM1 knockdown cell lines upon post-
IFN-α treatment; however, Huh-7 cells in which IFITM1 has been knocked down, the efficiency of the antiviral effects of IFN-α is slightly but significantly attenuated
[Figure 3.6]. These results suggest that IFITM1 plays an important, but not exclusive, role in the antiviral effects of IFN-α against the early stages of HCV infection in vitro,
an observation that will be further explored in this thesis.
3.5 Cloning and characterisation of IFITM1, IFITM2 and IFITM3
3.5.1 Cloning of IFITM1, IFITM2 and IFITM3 into a lentiviral expression vector
IFITM1 and IFITM3 have previously been shown to limit HCV in vitro (Wilkins et al.,
2013, Yao et al., 2011), However, the ability of IFITM2 to limit HCV has not been examined. To examine the potential anti-HCV properties of all three IFITM proteins, each of the cDNAs encoding the IFITM proteins were cloned into the lentiviral expression vector pLenti6/V5-D-TOPO, encoding a N-terminal FLAG tag to facilitate detection. Briefly, full length IFITM1, IFITM2 and IFITM3 were amplified from cDNA generated from Huh-7 cells stimulated with 1000 IU/ml IFN-α for 16 hrs.
Purified amplicons were cloned into the pLenti6/V5-D-TOPO vector using the BamHI and XhoI restriction sites [Figure 3.7], with double digest of candidate clones yielding 2 fragments corresponding to the 6.9 kb pLenti6/V5-D-TOPO vector and the respective insert with 402 bp for IFITM1 (Lane 2), 423 bp for IFITM2 (Lane 6) and 426 bp for
IFITM3 (Lane 10). A pLenti6/V5-D-TOPO vector re-ligated with itself was used as a
72 1.5
Control shRNA 1.0 IFITM1 shRNA
0.5
0.10 *
in HCV replication HCV in 0.05 Relative fold change *P = 0.035 0.00 -IFN pre-IFN post-IFN
Figure 3.6: IFITM1 knockdown reduces the anti-HCV activity of IFN-α. To investigate whether the knockdown of IFITM1 effected anti-HCV activity of IFN-α, the Huh-7+shIFITM1 cell line and its control cell line were either pretreated for 24 hrs before Jc1 infection (MOI: 0.03) or post-treated 24 hrs after Jc1 infection for 16 hours with 50 IU/mL of IFN-α. Total RNA was extracted and real-time PCR used to quantitate HCV RNA levels. RPLPO was used as a control and HCV RNA levels were expressed as a fold change relative to no treatment. These results demonstrated that IFITM1 plays an important but not exclusive role against the early stages of HCV infection in vitro (data represented as a mean ± SEM, n=3). !
! IFITM1 IFITM2 IFITM3 ! M1 1 2 3 4 5 6 7 8 9 10 11 12 M2
8.0kb 6.0kb
IFITM1, 500bp IFITM2, 400bp IFITM3
!
Figure 3.7: Analysis of IFITM clones following ligation into the lentiviral expression vector. The mini-preparation plasmids from the ligated products were digested with BamHI and XhoI restriction enzymes. Lane M1 and M2: 1kb and 10bp DNA markers respectively. Lanes 1/5/9: Uncut IFITM1, IFITM2 and IFITM3 (pLenti6/V5-D-TOPO) plasmid (6.9kb) clones respectively. Lanes 2/6/10: Double digest of IFITM1 (402bp), IFITM2 (423bp) and IFITM3 (426bp) (pLenti6/V5-D- TOPO) plasmid clones with BamHI and XhoI respectively. Lanes 3/7/11: Single digest of IFITM1, IFITM2 and IFITM3 (pLenti6/V5-D-TOPO) plasmid clones with BamHI respectively. Lanes 4/8/12: Single digest of IFITM1, IFITM2 and IFITM3 (pLenti6/V5- D-TOPO) plamsid clones with XhoI respectively. negative control and will be referred to as the empty vector control for the remainder of this thesis.
To confirm IFITM protein expression under the control of the CMV promoter of the pLenti6/V5-D-TOPO vector, Huh-7 cells were transfected with each of the IFITM clones and the empty vector control for 24 hrs and then total protein harvested and
Western blots performed using an antibody directed against the FLAG-tag, in combination with the detection of the loading control protein β-actin. Western blot analysis demonstrated the detection of the 15kDa IFITM1 protein, the 18kDa IFITM2 protein and the 17kDa IFITM3 protein, with protein sizes corresponding to that in the literature [Figure 3.8.i]. Immunofluorescence analysis on transfected cells, probed with an anti-FLAG antibody, demonstrated successful expression of IFITM1, IFITM2 and
IFITM3 [Figure 3.8.ii]. We noted no impact on cellular growth with exogenous expression of the IFITM proteins.
3.5.2 Transient expression of IFITM proteins decreases HCV replication
We next sought to examine the effect of transiently expressing IFITM1, IFITM2 and
IFITM3 on HCV replication. Huh-7 cells were transfected with plasmids expressing each of the IFITM proteins or the empty vector control for 24 hrs followed by Jc1 infection (MOI: 0.03) for 24 hrs. We consistently achieved a transfection efficiency of
approximately 35-40% and thus only a proportion of cells expressed both IFITM
protein and harboured HCV replication. Real-time PCR was utilised to examine HCV
RNA levels, with levels expressed as a fold change relative to the empty vector control.
Figure 3.9 depicts that each of the IFITM proteins are able to decrease HCV replication
73
i
IFITM1 IFITM2 IFITM3 Empty
FLAG-IFITM218 kDa anti-FLAG FLAG-IFITM317 kDa FLAG-IFITM115 kDa
anti- -actin !-actin42 kDa !
ii
Empty IFITM1
IFITM2 IFITM3
Figure 3.8: Transient detection of IFITM1, IFITM2 and IFITM3 in Huh-7 cells. To determine the expression of the IFITM proteins in a transient assay, Huh-7 cells were transfected with each of the IFITM proteins or an empty vector control for 24hrs prior and examined via Western blot and immunofluorescence analysis. (i) Total protein was extracted and immunoblotted with antibodies specific for FLAG and "-actin. Results indicated successful expression of IFITM1 (15kDa), IFITM2 (18kDa) and IFITM3 (17kDa), with sizes corresponding to the literature. (ii) Immunofluorescence analysis of transfected cells with FLAG antibody demonstrated expression of IFITM proteins within Huh-7 cells in vitro (60x magnification). Transfection Infect with Jc1 RNA extraction
24hrs 24hrs
1.5
1.0 * * *
0.5 in HCV replication HCV in Relative fold change change fold Relative *P <0.05 0.0 Empty IFITM1 IFITM2 IFITM3
Figure 3.9: Transient expression of IFITM proteins in Huh-7 cells can decrease HCV replication. To determine whether the IFITM proteins are able to limit HCV replication, Huh-7 cells were transiently transfected with the IFITM proteins and an empty vector control for 24hrs prior to Jc1 infection (MOI: 0.03) for 24hrs. Total RNA was extracted and real-time PCR used to quantitate HCV RNA levels. RPLPO was used as a control and HCV RNA levels were expressed as a fold change relative to empty vector. These results show that transient expression of IFITM proteins can decrease HCV replication in vitro (data represented as a mean ± SEM, n=3). by approximately 35% in a transient expression system and is most likely under-
represented for the reasons outlined above. However, this data indicates for the first
time that all three IFITM proteins are able to limit HCV replication in vitro.
3.5.3 Producing lentiviral particles encoding IFITM1, IFITM2 and IFITM3
Lentiviral vectors contain several advantageous characteristics that aid gene transfer,
such as (i) transduced genes integrate into the chromosome of the target cell allowing for stable, long-term expression, (ii) the integrated vectors do not encode viral proteins thus avoiding virus-specific immune responses directed towards the transduced cells and (iii) the vectors allow for the insertion and subsequent transfer of sequences up to
10 kb in size (Naldini et al., 1996, Zufferey et al., 1997). In addition, high transduction efficiency obtained in cultured cells makes this system useful for high-level transient expression of a gene of interest in in vitro studies. The expression vector used in this thesis, pLenti6/V5-D-TOPO is a lentiviral expression vector and can be packaged into lentiviral particles expressing a protein of interest, as described in Section 2.1.2.
Briefly, 293T cells were co-transfected with pLenti6/V5-D-TOPO harbouring IFITM cDNA and the packaging plasmids (Section 2.1.2) and pooled supernatant 48 and 72 hrs post-transfection was applied on to naïve Huh-7 cells. Cells were transduced with supernatant containing lentiviral particles encoding FLAG-tagged IFITM1, IFITM2 and IFITM3 for 72 hrs to obtain optimal protein expression and total protein was harvested and Western blots performed using an antibody directed against the FLAG- tag, in combination with the detection of the loading control protein β-actin [Figure
3.10.i]. Western blots demonstrated the detection of the 15kDa IFITM1 protein, the
18kDa IFITM2 protein and the 17kDa IFITM3 protein as observed in Figure 3.8.i.
Immunofluorescence analysis of transduced cells, probed with an anti-FLAG antibody,
74 i Empty IFITM1 IFITM2 IFITM3 18 kDa 17 kDa anti-FLAG 15 kDa
42 kDa anti-! actin
ii
Empty IFITM1
IFITM2 IFITM3
Figure 3.10: Transient transduction detection of IFITM1, IFITM2 and IFITM3 in Huh-7 cells. To determine the expression of the IFITM proteins in a transient assay, Huh-7 cells were trasnduced with lentivital particles expressing each of the IFITM proteins and an empty vector control for 72hrs prior and examined via Western blot and immunofluorescence analysis. (i) Total protein was extracted and immunoblotted with antibodies specific for FLAG and "-actin. Results indicated successful expression of IFITM1 (15kDa), IFITM2 (18kDa) and IFITM3 (17kDa), with sizes corresponding to the literature. (ii) Immunofluorescence analysis of transduced cells with FLAG antibody demonstrated expression of IFITM proteins within Huh-7 cells in vitro (60x magnification). demonstrated a high level of transduction (approximately 60%), as well as further confirming the expression of IFITM1, IFITM2 and IFITM3 derived from lentiviral particles [Figure 3.10.ii]. This method of IFITM protein expression is significantly greater than transient expression using a transfection reagent.
3.5.4 IFITM proteins may have a role at the early stages of HCV infection in vitro
To determine whether expression of IFITM proteins by transient expression of lentiviral transduction in Huh-7 cells would yield similar anti-HCV activity as discussed previously, we transduced Huh-7 cells with lentiviral particles encoding
IFITM1, IFITM2 and IFITM3, as well as an empty vector control for 72 hrs prior to
Jc1 infection (MOI: 0.03) for 24 hrs. Real-time PCR was utilised to measure HCV
RNA levels, with levels expressed as a fold change relative to the empty vector control.
Results demonstrate that the expression of IFITM1 and IFITM2 can limit HCV replication by approximately 40%, while IFITM3 limits replication by 20% [Figure
3.11.i]. This data corresponds with the results obtained in Figure 3.9. The converse of this experiment was also conducted, where Huh-7 cells were first infected with Jc1
(MOI: 0.03) before being transduced with IFITM expressing lentiviral particles for 72 hrs. Real-time PCR was conducted as described above and interestingly the data showed that the IFITM proteins had no significant effect on the HCV RNA levels of an established HCV infection [Figure 3.11.ii]. In these experiments, variations in the degree of impact of IFITM protein expression on HCV RNA levels may also be influenced by the transduction efficiency of each of the lentiviral preparations, as the proportion of cells transduced with the respective IFITM lentiviruses may vary. These results are indicative that IFITM1, IFITM2 and IFITM3 are inhibiting early stages of
75 i Pre-transduction 1.5
1.0 * ** ** 0.5 in HCV replication HCV in
Relative fold change change fold Relative *P < 0.005 **P = 0.03 0.0 Empty IFITM1 IFITM2 IFITM3
ii Post-transduction 1.5
1.0
0.5 in HCV replication HCV in Relative fold change change fold Relative
0.0 Empty IFITM1 IFITM2 IFITM3
Figure 3.11: The IFITM proteins have an effect on the early stages of HCV infection invitro. To determine whether the IFITM proteins act early or late in HCV infection in vitro, Huh-7 cells were either transduced with lentiviral particles expressing the IFITM proteins for 72hrs prior to Jc1 infection (MOI : 0.03) (i) or transduced 24hrs after Jc1 infection (MOI: 0.03) for 72hrs (ii). Total RNA was extracted and real-time PCR used to quantitate HCV RNA levels. RPLPO was used as a control and HCV RNA levels were expressed as a fold change relative to empty vector. These results show that the IFITM proteins were able to significantly reduce HCV infection when expressed in Huh-7 cells prior to infection but not after an infection was already established, suggesting that the IFITM proteins effect the early stages of HCV infection in vitro (data represented as a mean ± SEM, n=3). HCV infection in vitro and not at the level of RNA replication and further studies are
required to confirm this observation.
3.6 Generation of stable IFITM Huh-7 cell lines
To overcome the limitation of low level of IFITM expression within Huh-7 cells using
transient based approaches and the unavailability of specific antibodies and real-time
PCR primers for IFITM2 and IFITM3, Huh-7 cells stably expressing each of the IFITM
proteins were generated. Stable expression of ISGs of interest in a target cell line,
either overexpression or inducible, have previously been used successfully to
investigate the antiviral nature of ISGs (Jiang et al., 2008, Jiang et al., 2010, Zhou et al., 2011, Weidner et al., 2010). Briefly, these stable cell lines were generated by transducing Huh-7 cells with lentiviral particles expressing IFITM1, IFITM2 and
IFITM3, followed by antibiotic selection with blasticidin to select for stable integration of the IFITM expressing plasmid into the cellular DNA. Resistant cells were isolated
(method described in Section 2.1.2) to generate polyclonal overexpression cell lines. In order to confirm specific expression in the polyclonal cell lines, total protein was harvested from all the cell lines and Western blots performed using an antibody directed against the FLAG-tag, in combination with the detection of the loading control protein β-actin. Western blot analysis demonstrated the detection of the 15kDa
IFITM1 protein, the 18kDa IFITM2 protein and the 17kDa IFITM3 protein [Figure
3.12.i]. There was no detection of any IFITM protein in the empty vector control cell line. Immunofluorescence analysis of the cell lines confirmed increased but variable
expression of the proteins within the cells [Figure 3.12.ii]. The images also highlight
the localisation of each of the IFITM proteins within the Huh-7 cells; IFITM1 primarily
at the cell periphery, corresponding with the endogenous IFITM1 localisation observed
76 i Empty IFITM1 IFITM2 IFITM3 18 kDa 17 kDa anti-FLAG 15 kDa
42 kDa anti-! actin
ii Empty IFITM1
IFITM2 IFITM3
Figure 3.12: Characterisation of Huh-7 cell lines stably expressing IFITM1, IFITM2 and IFITM3. To detect the expression of the IFITM proteins in blasticidin resistant Huh-7+IFITM cell lines, cells from each cell line were either fixed for immunofluorescence analysis or total protein was extracted for Western blot analysis. (i) Extracted protein was immunoblotted with antibodies specific for FLAG and "-actin. Results indicated that each cell line expressed unique proteins, with sizes corresponding to the literature. (ii) Immunofluorescence analysis of each cell line with FLAG antibody identified that approximately 80% of the cells expressed the IFITM proteins. IFITM1 was detected on the cell periphery, whilst strong cytoplasmic expression was detected for IFITM2 and IFITM3. The empty vector control cell line expressed no detectable IFITM proteins (60x magnification). in Figure 3.3.iii, while IFITM2 and IFITM3 were found predominantly in the
cytoplasm at intracellular compartments. Collectively, these results show that we have successfully generated polyclonal cell lines that stably overexpress each of the IFITM proteins. A more detailed description of the localisation of IFITM1, IFITM2 and
IFITM3, and how this relates to HCV replication, is discussed in Chapters 4 and 5.
3.6.1 Stable expression of IFITM1, IFITM2 and IFITM3 decreases Jc1 replication
Initially we sought to examine the effects of IFITM1, IFITM2 and IFITM3 stable expression on the complete HCV life cycle through the use of the infectious HCV Jc1 system. To investigate this, the Huh-7+IFITM stable cells were infected with Jc1
(MOI: 0.03) for 3 hrs and HCV RNA quantified up to 72 hrs thereafter. Real-time PCR to measure HCV RNA levels was performed, with levels expressed as a fold change relative to the empty vector control. Results clearly demonstrate that stable expression of IFITM1, IFITM2 and IFITM3 significantly decreases HCV RNA levels by 77%,
61% and 57% respectively within 24 hrs of infection [Figure 3.13.i], and is consistent with our transient expression data. Similar results were obtained upon extending HCV infection to 48 and 72 hrs; with HCV RNA levels significantly decreasing by 69%,
66% and 65% at 48 hrs; and by 82%, 69% and 78% at 72 hrs for IFITM1, IFITM2 and
IFITM3 respectively. Concurrently, Huh-7+IFITM cells were infected with Jc1 for 72 hrs and immunofluorescence performed using pooled anti-HCV serum and anti-FLAG antibodies to detect HCV antigen and the IFITM proteins respectively. Figure 3.13.ii depicts a “viral exclusion” phenotype, where cells stably expressing the IFITM proteins do not appear to be infected with HCV, as there is no HCV antigen staining, while
HCV infection is present in neighbouring cells lacking IFITM expression. The polyclonal nature of our IFITM stable cell lines means that the majority of cells express
77 i 1.5 Empty IFITM1 1.0 IFITM2 IFITM3
** 0.5 * ** ** ** * in HCV replication HCV in ** * *P <0.05 Relative fold change change fold Relative ** **P <0.05 0.0 ** 24hr 48hr 72hr
ii !"#$%&'() !"#$*+,)-./0123) 4/05/6) 7%7849) ) ) 7%784:) ) ) 7%784;)
Figure 3.13: The IFITM proteins exhibit anti-HCV activity. To determine whether the IFITM proteins were antiviral against HCV, the Huh-7+IFITM and Huh-7+Empty vector cell lines were infected with Jc1 (MOI: 0.03) and examined via real-time PCR and immunofluorescence analysis. (i) Total RNAwas extracted from cells 24-72 hrs post- infection and total HCV RNA levels quantitated by real-time PCR. RNA levels were normalised to the control protein RPLPO and were expressed as a fold change relative to the empty vector control. Results demonstrate that stable expression of IFITM1, IFITM2 and IFITM3 was able to significantly decrease HCV RNA levels as early as 24hrs post- infection (data represented as a mean ± SEM, n=3). (ii) Huh-7+IFITM cell lines were probed with FLAG and HCV serum antibodies 72hrs post Jc1 infection. Results showed that HCV infection was only present in cells that did not express any of the IFITM proteins (60x magnification). the IFITM proteins to varying levels. It is therefore interesting to note that in those cells
infected with HCV, the corresponding IFITM expression is absent or relatively low
compared to expression in those cells not replicating HCV. Collectively these results demonstrate that stable IFITM1, IFITM2 and IFITM3 expression significantly affects the permissiveness of Huh-7 cells for Jc1 infection.
3.7 Characterising the role of the IFITM proteins on the different
stages of the HCV lifecycle
3.7.1 IFITM1, IFITM2 and IFITM3 decrease HCV entry into Huh-7 cells
As our data above was suggestive of a role for the IFITM proteins during the early
stages of HCV infection, the HCVpp system was utilised to examine the role of these
proteins on HCV entry into the hepatocyte. The HCVpp system has been extensively used to assay HCV entry, and has also been instrumental in the identification of key
HCV entry receptors such as CLDN-1 (Bartosch et al., 2003). Briefly, Huh-7+IFITM and Huh-7+Empty cells were transduced for up to 72 hrs with pseudoparticles
expressing HCV E1/E2, MLV or VSV glycoproteins, as well as a non-functional
envelope as a negative control. VSVpp and MLVpp were utilised as positive and
negative controls respectively, as previous studies have demonstrated IFITM1 and
IFITM3 to limit VSVpp entry while having no effect on MLVpp entry (Brass et al.,
2009). Total cellular lysate was then harvested and a luciferase assay performed.
Results were demonstrated as a fold change relative to empty vector. Stable expression
of IFITM1, IFITM2 and IFITM3 in Huh-7 cells resulted in significant decreases (55%
and 30% respectively) in entry of pseusoparticles expressing the HCV envelope
compared to control [Figure 3.14]. Results for the MLV and VSV pseudoparticles
78 Empty 108 IFITM1 IFITM2 IFITM3 107
*** **** * * *** 106 RLU
105
*P < 0.02 ***P < 0.003 ****P < 0.0001 104 HCV VSV MLV
250000
200000 * *
SD 150000
± ***
100000 RLUs RLUs 50000
0 Empty IFITM1 IFITM2 IFITM3
Figure 3.14: The IFITM proteins significantly inhibit HCV entry into Huh-7 cells. To investigate whether the IFITM proteins had an effect at the level of HCVentry, Huh-7+IFITM and Huh-7+Empty vector cell lines were transduced with pseudoparticles containing envelopes of HCV, MLV, VSV and a non-functional (NFV) envelope as a negative control for 24 hrs. Viral entry was determined by luciferase activity in Relative Light Units (RLU) 72hrs post-infection. The results indicate that IFITM, IFITM2 and IFITM3 are able to inhibit HCV entry in vitro. mirrored the observations previously noted in the literature. Thus, these results demonstrate that IFITM1, IFITM2 and IFITM3 are acting at the level of HCV entry in vitro.
3.7.2 The IFITM proteins have no effect on HCV RNA replication
Our previous results demonstrate that the IFITM proteins, when expressed post- establishment of HCV infection, had no effect on virus production and suggest that they do not act at the level of HCV RNA replication. To formally investigate this in more detail we used the HCV replicon system. The HCV genomic replicon system allows for the autonomous replication of the HCV proteins without the production of infectious viral particles and thus provides the best model for studying HCV RNA replication in the absence of HCV enry and egress (Ikeda et al., 2002). To determine whether the IFITM proteins had an adverse effect on HCV RNA replication, HCV genomic replicon cells were transduced with lentiviral particles expressing IFITM1,
IFITM2 and IFITM3 for a 72 hr period, to achieve maximum protein expression. Real- time PCR was utilised to measure HCV RNA levels, with levels expressed as a fold change relative to the empty vector control. Results from this experiment showed that none of the IFITM proteins had a significant effect on HCV RNA replication [Figure
3.15.i]. In fact, immunofluorescence analysis of replicon cells transduced with IFITM proteins and probed using pooled anti-HCV serum and anti-FLAG antibodies respectively, revealed IFITM1, IFITM2 and IFITM3 expression in cells replicating
HCV, which is in contrast to the ‘viral exclusion’ phenotype observed in Figure 3.13.ii.
[Figure 3.15.ii]. This confirms that the IFITM proteins do not have a direct effect on
HCV RNA replication.
79 i 2.0
1.5
1.0
0.5 in HCV replication HCV in Relative fold change change fold Relative
0.0 Empty IFITM1 IFITM2 IFITM3
ii !"#$%&'() !"#$*+,)-./0123) 4/05/6) 7%784;) )) ) 7%784: )) ) 7%7849
Figure 3.15: The IFITM proteins have no effect on HCV RNA replication. To investigate whether the IFITM proteins had an effect at the level of HCV RNA replication, genomic replicon cells were transduced with each of the IFITM proteins and an empty vector control. (i) Total RNA was extracted and real-time PCR used to quantitate HCV RNA levels. RPLPO was used as a control and HCV RNA levels were expressed as a fold change relative to the empty vector. The results showed that the IFITM proteins had no significant effect on HCV RNA replication (data represented as a mean ± SEM, n=3). (ii) Immunofluorescence analysis of the transduced genomic cells using FLAG and HCV serum antibodies showed expression of the IFITM proteins in cells replicating HCV, indicating that the proteins had no effect on HCV RNA replication (60x magnification). In a complementary experimental approach, HCV replicon cell lines stably expressing
the IFITM proteins were generated as described in Section 3.4. Briefly, these stable
cell lines were generated by transducing HCV genomic replicon cells with lentiviral particles expressing IFITM1, IFITM2 and IFITM3, followed by antibiotic selection with blasticidin. Resistant cells were isolated (method described in Section 2.1.2) to generate polyclonal overexpression cell lines. Firstly, we sought to determine that stable expression of the IFITM proteins did not alter HCV RNA replication in the HCV replicon cells. Immunofluorescence analysis of these cells probed with pooled anti-
HCV serum and anti-FLAG antibodies demonstrated no loss of HCV RNA replication as the replicon cells expressing the IFITM proteins had the same level of HCV antigen present as the control cells [Figure 3.16.i]; while co-expression of the IFITM proteins in cells replicating HCV was also observed as in Figure 3.15.ii. To quantitatively assess the level of HCV RNA replication in these cells, we harvested total RNA from
HCV replicon cells stably expressing IFITM1, IFITM2 and IFITM3. Real-time PCR was used to measure HCV RNA levels, with levels expressed as a fold change relative to the empty vector control. Figure 3.16.ii demonstrates that the stable expression of the IFITM proteins in the HCV replicon does not significantly reduce HCV RNA replication. Collectively, this data indicates that the ability of IFITM1, IFITM2 and
IFITM3 to limit HCV infection is not at the level of HCV RNA replication.
3.7.3 The IFITM proteins have no effect on HCV IRES activity
THE HCV IRES precedes the initiation AUG codon of the HCV polyprotein and is
essential for the binding of the host 40S ribosomal unit to initiate translation. In order
to investigate whether the IFITM proteins were able to alter HCV translation, HCV
IRES activity was examined in the presence of IFITM1, IFITM2 and IFITM3. Briefly,
80 i Empty IFITM1
IFITM2 IFITM3
ii 2.0
1.5
1.0
0.5 in HCV replication HCV in Relative fold change change fold Relative
0.0 Empty IFITM1 IFITM2 IFITM3
Figure 3.16: The IFITM proteins have no effect on HCV RNA replication. To further confirm that the IFITM proteins had no effect at the level of HCV RNA replication, genomic replicon cells stably expressing each of the IFITM proteins and an empty vector control were examined via immunofluorescence analysis and (i) Immunofluorescence analysis of genomic replicon cells stably expressing the IFITM proteins using FLAG and HCV serum antibodies showed co-expression of IFITM proteins in cells replicating HCV, indicating that the proteins had no effect on HCV RNA replication (60x magnification). (ii) Total RNA was extracted and real-time PCR used to quantitate HCV RNA levels. RPLPO was used as a control and HCV RNA levels were expressed as a fold change relative to the empty vector. The results confirmed that the IFITM proteins had no significant effect on HCV RNA replication (data represented as a mean ± SEM, n=3). a construct containing the HCV IRES linked to the luciferase gene (pRL-HL) was
transfected into Huh-7+IFITM cells for 48 hrs (Ikeda et al., 2002). 24 hrs post- transfection, empty vector control cells were treated with 100ng/ml of IL-29 for 24 hrs as a positive control based on previous studies demonstrating that IL-29 treatment limits HCV IRES activity (Kanda et al., 2012). Total lysate was then harvested and a luciferase assay performed, with Renilla luciferase used as a transfection control.
Results were expressed as a fold change relative to the untreated empty vector control.
Figure 3.17 shows that while IL-29 treatment reduced HCV IRES activity by approximately 40%, the expression of the IFITM proteins had no measurable effect on luciferase output, suggesting that the proteins do not modulate HCV IRES activity in vitro.
3.7.4 The IFITM proteins have no effect on HCV egress
Finally we sought to examine the role of IFITM1, IFITM2 and IFITM3 on HCV egress using an extracellular:intracellular infectivity assay to assess the number of infectious particles in the supernatant in relation to that within the cell (see Section 2.4.3). The rationale was that if the IFITM proteins have an impact on egress then the
extracellular:intracellular ratio will be altered due to retention of HCV virions within
the cell. Briefly, Huh-7+IFITM cells, as well as the empty vector cells, were
electroporated with Jc1 RNA and after 24 hrs supernatants and cells were collected for
analysis of infectious virions using a focus-forming assay (FFA). 72 hrs post-infection
cells were subjected to immunofluorescence analysis and probed with pooled anti-HCV
serum antibodies. FFU/ml were determined for each condition, and expressed as a ratio of extracellular FFU to intracellular FFU. Figure 3.18 demonstrates that the expression of IFITM1, IFITM2 and IFITM3 have no significant effect on the release of
81 2.0
1.5
1.0
** 0.5 (NormalisedRenilla) to **P < 0.005
Fold Change in IRES-Luc Output 0.0 - +IL-29 IFITM1 IFITM2 IFITM3 Vector
Figure 3.17: The IFITM proteins do not modulate HCV IRES activity. To investigate if the IFITM proteins were modulating HCV IRES activity, the Huh-7+IFITM cell lines were transiently transfected with pRL-HL for 48 hrs. 24 hrs post-transfection vector control cells were treated with 100ng/ml IL-29 for 24hrs. Total cellular lysates were extracted and a luciferase assay performed to measure HCV IRES luciferase activity. Renilla luciferase was used as a transfection control, and results were expressed as a fold change relative to the empty vector control cell line. These results indicated that the IFITM proteins do not modulate IRES luciferase output (data represented as a mean ± SEM, n=3). Freeze/thaw and apply S/N to naïve Electroporate Huh-7 cells Huh-7+IFITM cells Immunofluorescence analysis of FFA 24hrs 72hrs
20
15
10 FFU/ml
5
0
Ratio of extracellular/intracelullar extracellular/intracelullar of Ratio Emtpy IFITM1 IFITM2 IFITM3
Figure 3.18: The IFITM proteins have no effect on HCV egress. To investigate if the IFITM proteins had an effect at the level of HCV egress, the Huh-7+IFITM cell lines were electroporated with Jc1 RNA, left for 24 hrs, and extracellular and intracellular supernatant was applied on to naïve Huh-7 cells for 3 hrs. Huh-7+Empty vector cell lines were used as a control. At 72 hrs post-infection, cells were stained for HCV antigen using pooled anti-HCV serum and focus forming units (FFU/ml) calculated. FFU/ml is expressed as a ratio of extracellular FFU to intracellular FFU. These results indicated that the IFITM proteins have no effect on HCV egress (data represented as a mean ± SEM, n=3). infectious viral particles, indicating that the proteins have no effect on HCV egress in vitro.
3.8 Discussion
The IFITM proteins are a family of IFN-inducible proteins that have recently been
identified to play a role in the antiviral response of IFN. Initial studies identified the
antiviral nature of the IFITM proteins through a siRNA screen against IAV, with viral like particles (VLPs) from different viruses used to characterise the antiviral properties
of the IFITM proteins (Brass et al., 2009). Interestingly, an initial experiment
demonstrated that HCV infection was not inhibted by IFITM3 as co-expression of the
HCV core protein and IFITM3 was observed in the same cells (Brass et al., 2009).
Numerous independent studies in the last few years have demonstrated the ability of
IFITM1, IFITM2 and IFITM3 to limit the replication of a broad range of RNA viruses,
including the Flaviviruses DENV and WNV (Brass et al., 2009, Jiang et al., 2010).
These proteins appear to limit late stages of viral entry; however, the exact molecular
mechanisms are yet to be elucidated. At the beginning of this PhD, the antiviral nature
of the IFITM proteins had not been investigated in the context of HCV infection, and based on the ability of these proteins to limit infection of related Flaviviruses, it seemed plausible that these proteins may play a role in the anti-HCV response of IFN.
The aim of this chapter was to clarify the role of IFITM1, IFITM2 and IFITM3 using a combination of the HCV infectious model system and replicon system to establish at the molecular level, a role for the IFITM proteins in the HCV life cycle.
82 Firstly, we established that IFITM1 is upregulated in the presence of IFN-α in Huh-7 cells in vitro [Figure 3.3], as well as in PHHs in the presence of IFN-α and IFN-λ
[Figure 3.4]. Ideally these experiments would also have been conducted for IFITM2
and IFITM3; however, the design of real-time PCR primers specific for each of these
proteins was problematic due to the high level of conservation in protein sequence
[Figure 3.1]. This problem also translated to the acquisition of specific antibodies for
IFITM2 and IFITM3, where all commercially bought antibodies detected both IFITM2 and IFITM3, and in some cases IFITM1, thus preventing any reliable investigations
[Figure 3.2]. Thus, initial studies into IFN-α-induced upregulation of the IFITM proteins were limited to IFITM1 in Huh-7 cells. However, since all three protein promoters contain absolute conservation of both ISRE and GAS elements, it is conceivable that IFITM2 and IFITM3 would be upregulated in Huh-7 cells in a similar manner to IFITM1 in vitro. As the study of endogenous IFITM proteins would not be possible in Huh-7 cells (for the reasons outlined above), we cloned each of the IFITM proteins with an N-terminal FLAG tag to facilitate detection. These restrictions were not limited only to our study, as most of the published work regarding the antiviral nature of the IFITM proteins use HA- or FLAG-tagged IFITM proteins (Brass et al.,
2009, Weidner et al., 2010, Jia et al., 2012, John et al., 2013, Amini-Bavil-Olyaee et al., 2013). We have demonstrated that the tagged IFITM constructs can be successfully expressed in our cell lines and display cellular localisation consistent with the literature
[Figure 3.8 & 3.10], where IFITM1 was found to primarily localise to the plasma membrane and IFITM2 and IFITM3 found in punctate intracellular compartments within the cytoplasm (Huang et al., 2011, Wilkins et al., 2013).
83 The FLAG-tagged IFITM1, IFITM2 and IFITM3 constructs were used in both transient expression assays and in a stable expression model. Both transient [Figure 3.9 & 3.11] and stable [Figure 3.13] expression of the IFITM proteins significantly decreased HCV replication following infection with HCV Jc1. These results were further corroborated using immunofluorescence analysis, where a ‘viral exclusion’ phenotype was observed in Huh-7 cells overexpressing each of the IFITM proteins [Figure 3.13.ii].
Interestingly, in the context of an established HCV infection, transient expression of the
IFITM proteins had no significant impact on HCV replication [Figure 3.11.ii] and suggests that the IFITM family may block the HCV lifecycle at an early stage with no impact on HCV replication. Collectively, these results demonstrate that IFITM1,
IFITM2 and IFITM3 are able to significantly decrease HCV replication in vitro.
During the course of this PhD these findings have been confirmed by a number of studies that have identified IFITM1 and IFITM3 to have anti-HCV effects (Wilkins et al., 2013, Raychoudhuri et al., 2011, Yao et al., 2011, Metz et al., 2012); however, our observations regarding the anti-HCV nature of IFITM2 are novel.
The fact that the expression of the IFITM proteins reduced HCV replication in the
HCVcc (Jc1) system to such a large degree, while conversely having no effect on cells with an established HCV infection, could be indicative of IFITM1, IFITM2 and
IFITM3 playing a role on HCV entry. As such, this possibility was investigated using the HCVpp assay and Figure 3.14 confirms the findings by Wilkins et al (a study focused on IFITM1 only) and demonstrates that IFITM1 expression significantly decreases HCV entry into Huh-7 cells in vitro. Similar reductions in HCVpp entry were observed in cells expressing IFITM2 and IFITM3, demonstrating that the anti-HCV activity observed for these proteins is at the level of HCV entry. These results link in
84 with the general observation that the IFITM proteins act on the late entry stages of
RNA viruses, though the exact mechanisms are still unknown.
We went on to investigate the role of the IFITM proteins on the other stages of the
HCV lifecycle. Transient and stable expression of the IFITM proteins in HCV
genomic replicon cells demonstrated that IFITM1, IFITM2 and IFITM3 had no
significant effect on HCV RNA replication [Figure 3.15 & 3.16]. Interestingly, the
‘viral exclusion’ phenotype observed in immunofluorescence analysis using the HCVcc
system was no longer evident upon expression of the IFITM proteins in the genomic
replicon cells, with cells harbouring IFITM expression and HCV replication frequently
noted. Furthermore, the IFITM proteins were also demonstrated to have no effect on
HCV IRES promoter activity [Figure 3.17]. Our findings that the IFITM proteins have
no effect on HCV RNA replication and translation are in contrast to recent publications
by Yao et al and Li et al that showed IFITM2 and IFITM3 play important roles limiting
IRES-mediated translation and HCV RNA replication respectively. Yao et al utilised a
Huh-7.5 cell line constitutively expressing a fusion protein of STAT1 and the mouse estrogen receptor that can form a homodimer upon 4-hydroxytamoxifen (4-HT) treatment allowing downstream ISG expression to examine effects on HCV IRES- mediated translation (Yao et al., 2011). While a significant decrease of IRES-mediated translation may be observed in these cell lines, IFITM3 may not be the only ISG upregulated in this model system that may contribute to the observed effect on IRES- mediated translation, while more specific studies were carried out in HeLa cells and not hepatocytes. Thus any discrepancy in results is likely due to the different model systems used. Similarly, Li et al identified IFITM2 and IFITM3 as potential host
antiviral factors in a siRNA screen targeting different stages of the HCV lifecycle (Li et
85 al., 2014). Though we cannot repeat this observed effect, we propose that the IFITM proteins may in part limit HCV RNA replication, but the primary effect occurs at the level of HCV entry as in other RNA viruses. Finally we demonstrated in Figure 3.18 that IFITM1, IFITM2 and IFITM3 had no noteworthy effect at the level of HCV egress from Huh-7 cells using an extracellular:intraceullar infectivity assay.
In summary, collectively these results conclusively establish the ability of the IFITM
proteins to significantly decrease HCV infection in vitro, particularly at the level of
HCV entry. The next logical step would be to examine the localisation of these
proteins within the hepatocyte, especially in regard to the essential HCV host entry
receptors, to determine the exact molecular mechanisms of the IFITM anti-HCV
activity and this will be further discussed in Chapter 4.
86 Chapter 4
Characterising the anti-HCV effect of IFITM1 at the Molecular Level
4.1 Introduction
HCV enters the hepatocyte through sequential interactions with a number of host receptors, with the four essential receptors identified as SR-BI, CD81, CLDN1 and
OCLN [Figure 4.1]. Briefly, the HCV virion first interacts with SR-BI, allowing the virion to further associate with CD81 on the hepatic cell surface. This interaction results in the movement of the bound HCV virion to the hepatic tight junction where further interactions with CLDN1 and OCLN allow for clathrin-mediated endocytosis of the HCV virion into the hepatocyte (Evans et al., 2007, Harris et al., 2010, Blanchard et al., 2006). IFITM1 has previously been demonstrated to interact with CD81 on the surface of a number of different cells, most notably on the surface of B cells to reduce
B cell receptor engagement needed for cell stimulation (Levy et al., 1998). In the previous chapter of this thesis, we demonstrated that the IFITM proteins are able to limit HCV entry into the hepatocyte in vitro, and given the previous findings in the literature we surmised that the IFITM proteins, in particular IFITM1, may be interacting with the essential host entry receptors to limit HCV entry. In order to elucidate the molecular mechanisms at play in the ability of IFITM proteins to limit
HCV entry, we (1) examined the potential interactions between the IFITM proteins and essential HCV entry receptors and (2) performed mutational analysis of IFITM1 to determine essential regions involved in its anti-HCV action.
87 Figure 4.1: Model of HCV entry (Eyre et al., 2009) 4.2 Localisation of the IFITM proteins in the context of the essential
host entry factors for HCV
4.2.1 The IFITM proteins do not co-localise with the tight junction protein
Occludin (OCLN)
The first HCV receptor we examined was the tight junction protein OCLN. As OCLN is a protein that localises to tight junctions the use of polarized hepatocytes would have been ideal. Huh-7 cells do not polarize under cell culture conditions. However, as all
HCV receptors are required for HCV entry into Huh-7 cells and as we were only interested in ascertaining any potential associations between the IFITM proteins and the HCV entry receptors, the Huh-7+IFITM cells were used. Figure 4.2 shows immunofluorescence analysis of Huh-7+IFITM cells probed using anti-OCLN and anti-
FLAG antibodies to detect endogenous OCLN and the IFITM proteins respectively.
As expected, OCLN can be seen at the cell surface exclusively. Despite both cytoplasmic and cell surface IFITM1 localisation, no co-localisation between these two molecules was observed, which is in contrast to the study by Wilkins et al where an interaction between IFITM1 and OCLN was demonstrated (Wilkins et al., 2013).
Similar results are noted for IFITM2 and IFITM3, which is not surprising considering the intracellular nature of these proteins.
4.2.2 The IFITM proteins do not co-localise with the tight junction protein
Claudin-1 (CLDN1) at the cell surface
Next we examined IFITM localisation in the context of the tight junction protein
CLDN1, which forms an essential CD81/CLDN1 complex that initiates clathrin- mediated endocytosis (Evans et al., 2007, Harris et al., 2010). Immunofluorescence
88 !"#$% &'()*+",% &'()-../012'% 345641%
$*$738%
$*$739%
$*$73:%
;<=>?%
Figure 4.2: The IFITM proteins do not co-localise with HCV entry receptor Occludin. To investigate whether the IFITM proteins co-localised with the HCV entry receptor Occludin, the Huh-7+IFITM overexpression cell lines were utilisied and then immunofluorescence performed for !-FLAG (red) and !-Occludin (green). No co- localisation with Occludin was observed for any of the IFITM proteins (60x magnification). analysis was performed on Huh-7+IFITM cells using anti-CLDN1 and anti-FLAG
antibodies to detect endogenous CLDN1 and the IFITM proteins respectively [Figure
4.3]. Despite the cell surface localisation of CLDN1, no co-localisation between
IFITM1 and IFITM3 with CLDN1 was observed. Interestingly, a more cytoplasmic
localisation of CLDN1 was observed in cells stably expressing IFITM2. Furthermore,
there appears to be some co-localisation between CLDN1 and IFITM2 in the cytoplasm
that may suggest an ability for IFITM2 to sequester CLDN1 away from the cell
surface. Additional experiments are required to ascertain the nature of this potential
association and will be further discussed in Section 4.5.
4.2.3 The IFITM proteins do not co-localise with the cell surface protein SR-BI
The first host receptor to interact with the HCV virion is SR-BI and was the next entry
receptor investigated in the context of the IFITM proteins. It has been well documented in previous studies that SR-BI has both a cell surface and a cytoplasmic-
Golgi localisation in Huh-7 cells, with fixed and permeabilised cells showing the
cytoplasmic localisation (Eyre et al., 2010). Immunofluorescence analysis was
performed on Huh-7+IFITM cells using anti-SR-BI and anti-FLAG antibodies to detect endogenous SR-BI and the IFITM proteins respectively. Figure 4.4 depicts the cytoplasmic localisation of wildtype SR-BI in Huh-7 cells, while also illustrating that none of the IFITM proteins co-localise with SR-BI in the cytoplasm. In order to reveal the cell surface localisation of endogenous SR-BI, an antibody that targets the extracellular loops of SR-BI is required, with labeling prior to fixation. Thus these results demonstrate that IFITM1, IFITM2 and IFITM3 do not associate with SR-BI in
Huh-7 cells in vitro.
89 !"#$% &'()*+",% &'()-.&/01')2% 345640%
$*$732%
$*$738%
$*$739%
:;<=>%
Figure 4.3: The IFITM proteins do not co-localise with HCV entry receptor Claudin-1. To investigate whether the IFITM proteins co-localised with the HCV entry receptor Claudin-1, the Huh-7+IFITM overexpression cell lines were utilisied and then immunofluorescence performed for !-FLAG (red) and !-Claudin-1 (green). No co-localisation with Claudin-1 was observed for IFITM1 and IFITM3, but some association observed between IFITM2 and Claudin-1 in the cytoplasm (60x magnification). DAPI anti-FLAG anti-SR-BI Merged
IFITM1
IFITM2
IFITM3
Empty vector
Figure 4.4: The IFITM proteins do not co-localise with HCV entry receptor SR- BI. To investigate whether the IFITM proteins co-localised with the HCV entry receptor SR-BI, the Huh-7+IFITM overexpression cell lines were utilisied and then immunofluorescence performed for !-FLAG (green) and !-SR-BI (red). No co- localisation with SR-BI was observed for any of the IFITM proteins (60x magnification). 4.2.4 IFITM1 co-localises with the HCV entry factor CD81 on the hepatic cell
surface
As mentioned previously, the tetraspanin CD81 is known to interact with IFITM1 on
the surface of B cells and since CD81 is one of the key host receptors for HCV entry into the hepatocyte we hypothesised this association would also be present in Huh-7
cells. Figure 4.5 shows immunofluorescence analysis of Huh-7+IFITM cells probed
using anti-CD81 and anti-FLAG antibodies to detect endogenous CD81 and the IFITM
proteins respectively. Confocal analysis identified extensive co-localisation between
the cell surface IFITM1 and CD81, with no co-localisation observed between CD81
and the predominantly intracellular IFITM2 and IFITM3. These results confirm that
IFITM1 and CD81 co-localise on the hepatic cell surface in vitro and support the
possibility that an association between these two proteins may be the mechanism by
which IFITM1 disrupts the HCV entry process as observed in Figure 3.14.
4.3 IFITM1 interacts with CD81 on the hepatic cell surface
Co-localisation between two proteins does not always confer an interaction between the
proteins, as the limit of resolution for immunofluorescence is approximately 200nm. To
extend our confocal analysis and to determine whether IFITM1 and CD81 could
physically interact on the cell surface, FRET and proximity ligation assays (PLA) were
utilised. Both these techniques rely on using specific antibodies to detect interacting proteins within 10nm of each other, but differ in the methods used. Briefly, for FRET
analysis, Huh-7+IFITM1 and control cells were probed with anti-FLAG and anti-CD81
antibodies to detect IFITM1 and endogenous CD81 respectively, and then FRET
analysis was performed using the acceptor-photobleaching technique as described in
Section 2.5.24.4. FRET analysis relies on the non-radiative transfer of energy from an
90 anti-FLAG anti-CD81 Merge
IFITM1
IFITM2
IFITM3
Figure 4.5: IFITM1 co-localises with the HCV entry receptor CD81 on the hepatic cell surface. To investigate whether the IFITM proteins co-localised with the HCV entry receptor CD81, the Huh-7+IFITM overexpression cell lines were utilisied and then immunofluorescence performed for !-FLAG (green) and !-CD81 (red). No co- localisation with CD81 was observed for IFITM2 and IFITM3, however strong co- localisation was observed between CD81 and IFITM1 on the cell surface using confocal microscopy (60x magnification). excited donor molecule to a suitable acceptor molecule in close proximity (<10nm).
Significant positive FRET was observed on the cell surface between IFITM1 and CD81
[Figure 4.6], indicating an interaction occurs at the cell surface between these two proteins. A control of no primary antibody for the acceptor was utilised to confirm specificity of interaction. These results were corroborated by a concurrent experiment where Huh-7+IFITM1 cells were probed with anti-FLAG and anti-CD81 antibodies as described above and then PLA analysis was performed as described in Section 2.5.25.5.
This technique is distinctive from FRET analysis in that this assay relies on the interaction of DNA strands attached to secondary antibodies and the resultant formation of an oligonucleotide circle [Figure 4.7]. The DNA is amplified by rolling circle amplification and is recognised by labelled complementary probes, resulting in the visualisation of bright foci when viewed under fluorescence microscopy
(Fredriksson et al., 2002). The presence of bright red foci in Figure 4.8.ii denotes close proximity interactions between IFITM1 and CD81. No red foci were present in the control Huh-7+Empty cell line [Figure 4.8.i]. Figures 4.8.iii and 4.8.iv demonstrate that the fluorescent red foci are only present when both primary antibodies are bound to their target proteins as incubation with one primary antibody or a generic IgG antibody results in no fluorescence. This confirms that the PLA secondary antibodies are not able to bind non-specifically and result in false positive interactions. Collectively, these results strongly suggest that IFITM1 is able to interact with CD81 on the Huh-7 cell surface. The localisation of IFITM2 and IFITM3 within Huh-7 cells, and in the context of HCV infection, will be explored further in Chapter 5.
91 ***
Positive n= 25 negative n=10 Difference of intensity fire-map displaying areas of significant FRET (FLAG anti-rabbit and CD81 anti-mouse primary antibodies) Control = no primary antibody for acceptor *** P < 0.0001
Figure 4.6: IFITM1 interacts with the HCV entry receptor CD81 on the hepatic cell surface. To investigate whether the IFITM1 interacts with the essential HCV entry receptor CD81, the Huh-7+IFITM1 overexpression cell line was utilisied and FRET analysis performed using the acceptor-photobleaching technique. Strong interaction between IFITM1 and CD81 was observed. No primary antibody for the acceptor was used as a control. Figure 4.7: Schematic representation of Proximity Ligation Assay (Abnova) i Huh-7+Empty: FLAG/CD81 ii Huh-7+IFITM1:FLAG/CD81
iii IgG control iv anti-CD81 only
Figure 4.8: IFITM1 interacts with the HCV entry receptor CD81 on the hepatic cell surface. To further confirm the interaction between IFITM1 and CD81, the Huh-7+IFITM1 cell line was utilised and a DuoLink In situ Proximity Ligation Assay was conducted using anti-FLAG and anti-CD81 antibodies. (i) Huh-7+Empty cells were used as a control. (ii) The red dot staining identifies sites of positive interaction between IFITM1 and CD81 on the cell surface. Non-specific binding controls were also used with a isotype control (iii) and incubation with only one primary antibody (iv). Strong interaction between IFITM1 and CD81 was observed (60x magnification). 4.4 Mutagenic analysis reveals important regions of IFITM1 for anti-
HCV activity
4.4.1 Characterisation of a panel of IFITM1 mutants
Little is known about the functional aspects of the IFITM proteins, in particular
IFITM1. As previously described, IFITM1 has a distinct amino acid sequence
compared to IFITM2 and IFITM3, where it has a C-terminal extension (13 a.a) and a truncated N-terminal region (21 a.a) but almost identical intramembranous regions
[Figure 4.9.i]. Additionally, within Huh-7 cells, IFITM1 has a distinct cellular localisation [Figure 3.12] compared to IFITM2 and IFITM3 and thus we hypothesised that these differences in the extraluminal regions influenced the potent anti-HCV activity of IFITM1. Mutagenesis studies by Lu et al identified that in the context of
HIV replication, the first 21 amino acids of the N-terminal region of IFITM1 are not
required for its anti-HIV activity and that the C-terminal region was equally
dispensable (Lu et al., 2011). However, these studies are in the context of one cell line
and one viral infection, and in order to further understand the anti-HCV mechanisms of
IFITM1, a panel of truncation mutants targeting both the N- and C-termini were created
[Table 2.2 and Figure 4.9.i]. Previous studies have shown that deleting more than 29
amino acids from the N-terminus of IFITM1 resulted in a complete loss of protein
expression (Lu et al., 2011), and so we created a panel of mutants with 6 (ΔN6), 13
(ΔN13), 21 (ΔN21) and 28 (ΔN28) amino acid deletions from the N-terminus. Next we
deleted the 13 amino acid C-terminal extension of IFITM1 (ΔC13), thus making it
similar to IFITM2 and IFITM3. During the cloning process, either through
mismatched primer binding or error in polymerase activity, 18 amino acids of the
IFITM1 C-terminus were deleted and we continued to use this C-terminal truncation
mutant in this work (ΔC18). Besides the 21 amino acid N-terminal extension, the N-
92 i 20 40 60 80 100 120 140 160
IFITM3 HNPAP TM I TM2 IFITM2 HNPAP TM I TM2 IFITM1 PSTIL TM I TM2 IFITM1 ! C-13 a.a TM I TM2 IFITM1 ! C-18 a.a TM I TM2 IFITM1 ! N-6 a.a TM I TM2 IFITM1 ! N-13 a.a TM I TM2 IFITM1 ! N-21 a.a TM I TM2 IFITM1 ! N-28 a.a TM I TM2 IFITM1 ! PSTIL HNPAP TM I TM2
ii IFITM1 !N6 !N13 !N21 !N28 !C13 !C18 !PSTIL 15 kDa anti-FLAG
42 kDa anti-!-actin
Fig 4.9: Panel of IFITM1 mutants. (i) Schematic representation of a panel of truncation mutants targeting both the N- and C-termini of IFITM1. Site-directed mutagenesis was also conducted on the N-terminus of IFITM1, changing the PSTIL region to the HNPAP region conserved in IFITM2 and IFITM3. (ii) To detect the expression of the IFITM1 mutants in a transient assay, total protein was extracted 24 hrs post-transfection into Huh-7 cells. Extracted protein was immunoblotted with antibodies specific for FLAG and "-actin. Results showed changes in protein size corresponding to the truncations. terminal region of IFITM2 and IFITM3 is highly analogous to IFITM1 with the exception of 5 amino acids [Figure 4.9.i]. The PSTIL region found between amino acids 15-19 in IFITM1 is different to the conserved HNPAP region found in IFITM2 and IFITM3. In order to examine this conserved sequence of IFITM2 and IFITM3 in
comparison to IFITM1, the PSTIL region of IFITM1 was mutated to HNPAP, thus
resembling the N-terminus of IFITM2 and IFITM3. All of these mutants were cloned
into the pLenti6/V5-D-TOPO vector with N-terminal FLAG tags as described in
Section 3.3.1.
To confirm expression of our mutant IFITM1 proteins under the control of the CMV promoter of the pLenti6/V5-D-TOPO vector, Huh-7 cells were transfected with each of the IFITM1 mutant clones and wildtype IFITM1 for 24 hrs and then total protein
harvested and Western blots performed using an antibody directed against the FLAG-
tag, in combination with the detection of the loading control protein β-actin. Western
blots demonstrated the detection of the 15kDa wildtype IFITM1 protein and the
IFITM1 mutants with a decrease in protein size corresponding to the deletions observed
[Figure 4.9.ii].
4.4.2 The N-terminal region of IFITM1 is important for its anti-HCV activity
To determine the anti-HCV nature of our IFITM1 mutants, Huh-7 cells were
transfected with each of the IFITM proteins and the empty vector control for 24 hrs
followed by HCV Jc1 infection (MOI: 0.03) for 24 hrs. Real-time PCR to measure
HCV RNA levels was performed, and levels were expressed as a fold change relative
to the empty vector control. Due to the transient nature of the experiment
(approximately 35-40% transfection efficiency), only a 30% knockdown in HCV
93 infection was observed in cells expressing wildtype IFITM1, consistent with the results observed in Figure 3.9. Deleting the 6 (ΔN6) and 13 (ΔN13) amino acids from the N- terminus and 13 (ΔC13) and 18 (C18) amino acids from the C-terminus resulted in no change to the anti-HCV activity of IFITM1 [Figure 4.10]. Interestingly, in contrast to the HIV studies, the deletion of 21 (ΔN21) and 28 (ΔN28) amino acids from the N- terminus abrogated the anti-HCV activity of IFITM1. Furthermore, the alteration of the PSTIL region to HNPAP also resulted in a loss of anti-HCV activity, indicating that the N-terminal region may be crucial for the anti-HCV activity of IFITM1.
In order to reduce the variations within an experiment due to differences in transfection efficiency between proteins, as well as to improve protein expression in the difficult to transfect Huh-7 cells, Huh-7 cells stably expressing each of the IFITM mutants were generated. Briefly, these stable cell lines were generated by transducing Huh-7 cells with lentiviral particles expressing the IFITM1 mutants followed by antibiotic selection with blasticidin. Resistant cells were isolated (method described in Section 2.1.2) to generate polyclonal overexpression cell lines. Protein expression was confirmed by immunofluorescence analysis for the successfully generated Huh-7+IFITM1 ΔN6,
ΔN21, ΔC18 and ΔPSTIL cell lines. Unfortunately, despite numerous attempts, polyclonal stable cells could not be created for IFITM1 ΔN13, ΔN28 and ΔC13 mutants as these mutants could not be packaged and expressed in the form of lentiviral particles. The possible reasons for this will be discussed further in Section 4.5.
To corroborate the observations of Figure 4.10, Huh-7+IFITM1, IFITM1 mutants and empty vector control cells were infected with HCV Jc1 (MOI: 0.03) for 24 hrs. Real- time PCR was utilised to measure HCV RNA levels, with levels expressed as a fold 94 1.5
1.0 * * * * *
0.5 in HCV replication HCV in Relative fold change change fold Relative *P < 0.04 0.0 Empty IFITM1 ΔN6 ΔN13 ΔN21 ΔN28 ΔC13 ΔC18 ΔPSTIL Anti-HCV activity + + + - - + + -
Fig 4.10: The N-terminal region of IFITM1 is important for its anti-HCV activity. To determine the antiviral activity of the IFITM1 mutants, Huh-7 cells were transfected with the IFITM1 mutants 24 hrs prior to infection with Jc1 (MOI: 0.03). Total RNA was harvested 24 hrs post-infection and and total HCV RNA levels quantitated by real- time PCR. RNA levels were normalised to the control protein RPLPO and were expressed as a fold change relative to wildtype IFITM1. Results indicate that the that the N-terminal region plays an important role in the anti-HCV activity of IFITM1 in vitro (data represented as a mean ± SEM, n=3). change relative to the empty vector control. Figure 4.11 confirms the observation that
the N-terminal region (ΔN21) is important for the anti-HCV activity of IFITM1.
However, in contrast to the transient data, the stable overexpression of the PSTIL
mutant retained anti-HCV activity. Collectively, this data indicates for the first time
that the N-terminal region of IFITM1 is important to limit HCV infection in vitro.
4.4.3. The C-terminal extension of IFITM1 is important for localisation within the
hepatocyte
To ascertain whether the loss of anti-HCV function of various IFITM1 mutants was
due to an alteration in cellular localisation, we performed immunofluorescence analysis
on our library of IFITM1 mutants. Immunofluorescence of transiently expressed
wildtype and IFITM1 mutants was conducted as only a handful of IFITM1 mutants
were stably expressed in Huh-7 cells. Sequential deletions or alterations to the IFITM1
N-terminus did not significantly alter localisation, consistent with the anti-HCV
activity observed in Figures 4.10 and 4.11, with the mutants found primarily at the cell periphery with increasing cytoplasmic localisation [Figure 4.12]. Deletion of 28 a.a from the N-terminus resulted in a complete cytoplasmic relocalisation of IFITM1, which was not unexpected as a previous study showed that the deletion of 29 a.a resulted in the complete loss of IFITM1 expression (Lu et al., 2011). Interestingly, the deletion of the C-terminal 13 a.a and 18 a.a that makes IFITM1 more like IFITM2 and
IFITM3 resulted in the redistribution of IFITM1 to a more cytoplasmic localisation.
These results indicate that the C-terminal extension of IFITM1 is essential for the localisation of the protein to the hepatic cell surface; however, also imply that the
cellular localisation of IFITM1 may not be important for anti-HCV activity as these
deletions retained antiviral activity.
95 1.5
1.0 *
* * 0.5 ** in HCV replication HCV in
Relative fold change change fold Relative *P < 0.04 **P = 0.004 0.0 Empty IFITM1 N6 N21 C18 PSTIL Anti-HCV activity + + - + +
Fig 4.11: The N-terminal region of IFITM1 is important for its anti-HCV activity. To determine the antiviral activity of the IFITM1 mutants, Huh-7+IFITM and Huh-7+ IFITM1 mutants were infected with Jc1 (MOI: 0.03). Total RNA was harvested 24 hrs post-infection and and total HCV RNA levels quantitated by real-time PCR. RNA levels were normalised to the control protein RPLPO and were expressed as a fold change relative to wildtype IFITM1. Results indicate that the that the N-terminal region may play an important role in the anti-HCV activity of IFITM1 in vitro (data represented as a mean ± SEM, n=3). !"!#$%& !"!#$%&'()& !"!#$%&'(%*&
!"!#$%&'(+%& !"!#$%&'(+,& !"!#$%&'-.#!/&
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Fig 4.12: The C-terminal region of IFITM1 is important for its localisation within the hepatocyte. To investigate the localisation of the IFITM1 mutants in relation to wildtype IFITM1, the Huh-7+IFITM1 overexpression cell lines were utilisied and then immunofluorescence performed for !-FLAG (red). The N-terminal IFITM1 mutants mostly retain wildtype localisation until #N28, while the C-terminal mutant had a more disperesed cytoplasmic localisation, suggesting that the C-terminal region of IFITM1 is important for localisation within the hepatocyte (60x magnification). 4.5 Discussion
This chapter builds on the results generated in Chapter 3, where we identified that
IFITM1, IFITM2 and IFITM3 were limiting HCV infection at the level of HCV entry.
As outlined previously, HCV entry into the hepatocyte requires sequential interactions with four key host receptors SR-BI, CD81, CLDN-1 and OCLN [Figure 4.1]. The exact molecular mechanism(s) behind the anti-HCV activity of the IFITM proteins are unclear; however, results from this chapter provide evidence that IFITM1 limits HCV infection through an interaction with CD81 and that 21-28 a.a within the NTD of
IFITM1 may be critical for this activity.
Using the Huh-7+IFITM overexpression cells generated in Chapter 3 and specific antibodies targeting endogenous HCV receptor proteins, we showed through immunofluorescence analysis that IFITM1 is able to co-localise with CD81 on the
Huh-7 cell surface [Figure 4.5]. No co-localisation was observed between IFITM2 and
IFITM3 and any of the HCV entry receptors. This was not entirely unexpected as these two proteins are found predominantly at intracellular compartments within the cytoplasm and not at the cell surface. Further work is required to identify the cellular localisation of IFITM2 and IFITM3 and will be further discussed in Chapter 5.
Similarly, no co-localisation was observed between IFITM1 and the other essential
HCV entry receptors. Interestingly, some co-localisation was observed between
IFITM2 and intracellular CLDN-1 in the cytoplasm [Figure 4.3]. Studies have previously demonstrated that CLDN-1 recycles at intracellular vesicles in kidney, colon and lung epithelial cells and that this recycling action requires endosomal sorting complex required for transport (ESCRT) machinery (Dukes et al., 2011). Furthermore,
Farquhar et al have shown that CD81 and CLDN-1 co-endocytose and fuse with Rab5-
96 expressing endosomes during HCV internalisation (Farquhar et al., 2012). Thus, determining the intracellular localisation of IFITM2 (Chapter 5) and further analysis to identify any interactions between these proteins is required to determine whether
IFITM2 has a role in either altering CLDN-1 traffick within Huh-7 cells or sequestering the receptor away from the cell suface and thus aiding HCV entry inhibition.
Our immunofluorescence analysis of co-localisation between IFITM1 and CD81 is suggestive of an interaction and hence other methods to confirm interaction were required. We went on to identify an interaction between IFITM1 and CD81 using
FRET and PLA [Figure 4.6 & 4.8]. It is clear that at least for the resolutions of these techniques that IFITM1 and CD81 interact at the cell surface. These results are supported by the work of Wilkins et al that was published during the course of this PhD
(Wilkins et al., 2013). This study further went on to identify an interaction between
IFITM1 and OCLN at hepatic tight junctions via co-immunoprecipation analysis, which is in contrast to our observations in Figure 4.2. The cells used in this model system are un-polarised and thus we cannot confirm the observed interaction between
IFITM1 and OCLN at tight junctions. Wilkins et al hypothesise that the anti-HCV activity of IFITM1 is through the disruption of the assembly of HCV co-receptor complexes, thus interfering with the HCV entry process. This hypothesis is plausible as there are numerous studies highlighting the importance of receptor movement for the
HCV entry process, such as the association of hras with CD81, following activation of
EGFR, which is required for the lateral diffusion of CD81 to form the CD81/CLDN-1 complex essential for HCV entry (Zona et al., 2013). Though we can neither confirm nor contradict this hypothesis, further work is required to identify the exact role of
97 IFITM1 in the HCV entry process. As mentioned above, recent studies have identified
EGFR, and potentially protein kinase A, to play an important role in receptor movement and in facilitating the internalization of the CD81/CLDN-1 complex, while multiple signaling pathways have also been shown to regulate this association
(Lupberger et al., 2011, Farquhar et al., 2008, Liu et al., 2012c, Kim et al., 2013). In addition, NPC1L1 and transferrin receptor 1 have also been identified to be important for HCV entry (Sainz et al., 2012, Martin and Uprichard, 2013). Thus given time, the tight junction localisation of IFITM1 would need to be confirmed using a polarised hepatic cell line such as HepG2, while also examining IFITM1 in the context of the other HCV entry co-receptors and signaling pathways.
Having firmly established an interaction between IFITM1 and CD81, we then focused on identifying the important regions of IFITM1 involved in this interaction and anti-
HCV activity. Despite IFITM1 imposing antiviral actions against a number of viruses, there is limited work examining the important regions of IFITM1 that facilitate this activity. Lu et al identified that deleting 29 amino acids from the IFITM1 N-terminus resulted in loss of protein expression, while also demonstrating that the N- and C- termini of IFITM1 were not important for anti-HIV activity (Lu et al., 2011). Based on this study we generated a panel of IFITM1 deletion mutants targeting 6, 13, 21 and 28 amino acids of the N-terminus and 13 and 18 amino acids of the C-terminus. Further examination of the conserved protein sequence between the three IFITM proteins identified a 5 amino acid difference in the N-terminus between IFITM1 (PSTIL) and
IFITM2/3 (HNPAP) [Figure 4.9.i]. This region in IFITM1 was mutated to resemble
IFITM2 and IFITM3. Expression of each of the FLAG-tagged mutants was confirmed transiently via Western blot [Figure 4.9.ii]; however, problems were encountered when
98 attempting to package the mutants into lentiviral particles to generate polyclonal stable
overexpression cell lines. As mentioned in Section 4.4.2 the mutants that could not be packaged (ΔN13, ΔN28 & ΔC13) were sublconed into a new lentiviral backbone where the control protein was successfully packaged and expressed while the mutants were not. Numerous attempts were made to resolve the issue with no success, and due to time constraints these mutants were only utilised in a transient manner.
The IFITM1 N- and C-terminal mutants were examined for anti-HCV activity using transient and stable overexpression systems. Both transient [Figure 4.10] and stable
[Figure 4.11] expression of the IFITM1 mutants revealed the N-terminus, excluding the
first 13 a.a, to be important for anti-HCV activity. Interestingly, no significant change in localisation was observed for the N-terminal mutants, though increasing intracellular localisation was observed for the ΔN21 and ΔN28 mutants [Figure 4.12]. The loss of anti-HCV activity but no change to localisation lead us to hypothesise that the N-
terminus of IFITM1 is the domain that interacts with CD81 in the cytoplasm. This is
plausible, as the termini of CD81 are intracellular and, based on the latest topology
studies (Weston et al., 2014), so is the N-terminus of IFITM1 [Figure 1.11]. Further
work would need to be done to confirm this hypothesis, initially by determining any
loss of IFITM1 and CD81 interaction amongst the N-termini mutants using FRET
analysis. Localisation studies between panels of CD81 mutants along with the IFITM1
mutants would also aid in identifying which region of CD81 interacts with IFITM1.
The C-terminal extension found on IFITM1 does not appear to play a role in the anti-
HCV activity of IFITM1; however, it does appear to be important for the localisation of
the protein at the cell periphery [Figure 4.10 & 4.11]. In fact, the deletion of the 13 99 amino acid extension, along with the ΔC18 mutant, resulted in a more cytoplasmic
localisation of IFITM1. This localisation change was not unexpected, as previous
studies have identified the C-terminus of the IFITM proteins to provide protein stability
(Weidner et al., 2010). Thus we hypothesise that the C-terminal extension on IFITM1
may be responsible for targeting the protein to the plasma membrane. Retention of anti-HCV activity despite a significant change in localisation of the C-terminus of
IFITM1, however, was unexpected. IFITM2 and IFITM3 have intracellular localisation and exhibit significant anti-HCV activity so it is plausible that the deletion of the C- terminal extension of IFITM1 resulted in a change of phenotype resembling IFITM2 and IFITM3. This hypothesis would need to be investigated further by examining the localisation of the IFITM1 C-terminal mutants in conjunction with IFITM2 and
IFITM3, using different tags to facilitate detection in the same cell. Similarly, the localisation of the IFITM1 mutants could be examined in context of the localisation of
IFITM2 and IFITM3 within the hepatocyte, which will be discussed further in Chapter
5.
Taken together, our results and previously published findings indicate that IFITM1 limits HCV entry through interactions with CD81 at the hepatic cell surface. The N- terminal region is crucial for this antiviral activity and we hypothesise that this region interacts with CD81, while the C-terminal region is important for ensuring the localisation of IFITM1 to the hepatic cell surface. New insights into the cellular functions of an antiviral protein, particularly understanding the expression and function of these proteins, allows for the improvement of current antiviral therapies.
100 Chapter 5
Cellular localisation of IFITM2 and IFITM3
5.1 Introduction
The localisation of a protein within the cell provides an indication to the role of that protein in cellular function. The results in Chapter 4 identified IFITM1 to localise to the surface of Huh-7 cells and interact with the HCV entry receptor CD81, and this provides a rational basis for its antiviral activity. However, the localisation of IFITM2 and IFITM3 within the hepatocyte remains unclear, but we do know from our studies in
Chaper 3 that in contrast to IFITM1, IFITM2 and IFITM3 predominantly localise within vesicles in the cytoplasm. Several studies have recently shown IFITM2 and
IFITM3 to partially co-localise at the late endosome and lysosome in a variety of different cell types (Feeley et al., 2011, Huang et al., 2011, Amini-Bavil-Olyaee et al.,
2013, John et al., 2013), providing an explanation for the hypothesis that these proteins act by inhibting the late entry stages of viral infection. A number of cellular organelles have been shown to be required for HCV RNA replication and viral assembly and egress, including the endoplasmic reticulum, Golgi complex, lipid droplets and the early and late endosomes (Egger et al., 2002, Dubuisson and Cosset, 2014, Miyanari et al., 2007, Blanchard et al., 2006, Manna et al., 2010). To this end we examined the localisation of the IFITM proteins, particularly IFITM2 and IFITM3, against a panel of organelle markers. Hence, the aim of this chapter was to define the cellular localisation of IFITM2 and IFITM3 to provide an insight into the anti-HCV mechanisms of these proteins.
101 5.2 Localisation of IFITM2 and IFITM2 within Huh-7 cells
5.2.1 IFITM2 and IFITM3 do not localise to the endoplasmic reticulum (ER)
We first sought to determine whether the IFITM proteins localised to the ER within
Huh-7 cells, as the ER is the site of HCV replication complex formation (Egger et al.,
2002). Figure 5.1 shows immunofluorescence analysis of Huh-7+IFITM cells transfected with a synthetic ER marker protein, a red fluorescent protein (RFP) with the
ER retention signal KDEL (ER-RFP) (Kadereit et al., 2008) and probed with anti-
FLAG antibodies to detect the IFITM proteins. The cytoplasmic network of tubular membranes characteristic of ER distribution can be observed. However, we could not detect co-localisation with IFITM2 and IFITM3 that were found at more punctate intracellular compartments. Furthermore, in the presence of IFITM1, IFITM2 and
IFITM3 we noted no alteration or disruption of ER integrity. These results suggest that
IFITM2 and IFITM3 do not localise with the ER in Huh-7 cells in vitro.
5.2.2 IFITM2 and IFITM3 do not localise to the Golgi complex
Next we examined the localisation of IFITM2 and IFITM3 in the context of the Golgi complex, an essential site of HCV assembly (Dubuisson and Cosset, 2014). Huh-
7+IFITM cells were transfected with a synthetic Golgi-specific marker protein, galactocysl-transferase-RFP (Golgi-RFP) (Kadereit et al., 2008) and probed with anti-
FLAG antibodies to detect the IFITM proteins [Figure 5.2]. Despite the punctate cytoplasmic localisation of the Golgi complex, IFITM2 and IFITM3 were found not to localise to the Golgi complex within Huh-7 cells. However, this was not unexpected, as IFITM2 and IFITM3 did not have any discernable effect on HCV egress (Section
3.7.4).
102 !"#$%& !-.-(/& *01203&
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Figure 5.1: The IFITM proteins do not localise to the endoplasmic reticulum (ER) within the hepatocyte. To investigate whether the IFITM proteins localised to the ER, the Huh-7+IFITM overexpression cell lines were transfected with a ER-RFP expression plasmid and then immunofluorescence performed for !-FLAG (green) (60x magnification). No co-localisation with the ER was observed for any of the IFITM proteins. !"#$%& -./0123(4& *56057&
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Figure 5.2: The IFITM proteins do not localise to the Golgi apparatus within the hepatocyte. To investigate whether the IFITM proteins localised to the Golgi apparatus, the Huh-7+IFITM overexpression cell lines were transfected with a Golgi-RFP expression plasmid and then immunofluorescence performed for !-FLAG (green) (60x magnification). No co-localisation with the Golgi was observed for any of the IFITM proteins. 5.2.3 IFITM2 and IFITM3 do not localise to lipid droplets
Lipid droplets (LD) have recently been shown to be an integral part of the HCV lifecycle, with a close association between LD and the ER membrane believed to be essential for HCV RNA replication and assembly (Miyanari et al., 2007). Thus the
localisation of the IFITM proteins in the context of LDs was determined through
immunofluorescense analysis. Huh-7+IFITM cells were probed with anti-FLAG
antibodies and the fluorescent LD stain (neutral lipid stain) BODIPY to detect IFITM
proteins and endogenous LDs respectively. Figure 5.3 depicts that despite the
cytoplasmic localisation of both LD and IFITM2 and IFITM3, these proteins do not
localise to LD within Huh-7 cells.
5.2.4 IFITM3 partially co-localises with the early endosome
Following binding of HCV to the heptocyte cell surface it enters the hepatocyte
through clathrin-mediated endocytosis resulting in the traffic of the virion along actin
stress fibres to Rab5a-containing early endosomes (Dubuisson and Cosset, 2014).
Fusion and acidification of the endosome results in the release of the HCV genome into
the cytoplasm where it is directly translated (Dubuisson and Cosset, 2014, Blanchard et
al., 2006). Huh-7+IFITM cells were transfected with an mCherry-Rab5a expression
plasmid (Eyre et al., 2014) for 24 hrs and immunofluorescence analysis was performed
using anti-mCherry antibodies to detect transfected Rab5a, and anti-FLAG antibodies
to detect the exogenously expressed IFITM proteins [Figure 5.4]. Despite the similar
intracellular localisation pattern observed for both IFITM2 and IFITM3, only IFITM3
was found to partially co-localise at Rab5a-containing early endosomes [Figure 5.5].
This data indicates that IFITM2 localises to a different organelle in the hepatocyte
compared to IFITM3.
103 ()*+,& -./!01& $34536&
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!"!#$'&
!"!#$%&
Figure 5.3: The IFITM proteins do not localise to lipid droplets within the hepatocyte. To investigate whether the IFITM proteins localised to the lipid droplets, the Huh-7+IFITM overexpression cell lines were utilised and then immunofluorescence performed for !-FLAG (red) and BODIPY (green) (60x magnification). No co- localisation with the lipid droplets was observed for any of the IFITM proteins. !"#$%& -./0.&1"23455%& *45647&
'(')*8&
'(')*+& '(')*+&
'(')*,&
Figure 5.4: IFITM3 partially co-localises with the early endosome marker Rab5a. To investigate whether the IFITM proteins co-localised with early endosomes, the Huh-7+IFITM overexpression cell lines were transfected with a mCherry-Rab5a expression plasmid and then immunofluorescence performed for !-FLAG (green) and !-mCherry (red) (60x magnification). Partial co-localisation between IFITM3 and Rab5a was observed. !"!#$%& '()*(&+,-./001& $/02/&
IFITM3 Rab5a - mCherry Merge
Figure 5.5: IFITM3 partially co-localises with the early endosome marker Rab5a. To investigate whether the IFITM proteins co-localised with early endosomes, Huh-7+IFITM2 and Huh-7+IFITM3 cells were transfected with mCherry-Rab5a expression plasmid and stained with anti-FLAG (green) and anti-mCherry antibodies (red). Partial co-localisation was observed between Rab5a and IFITM3, and not IFITM2 (60x magnification, 20 µm scale bar). 5.2.5 IFITM2 partially co-localises with the late endosome
A number of different studies have identified IFITM2 and IFITM3 to partially co- localise to the late endosome in a few different cell types (Feeley et al., 2011, Huang et al., 2011). Rab7, a member of the Rab protein family is associated with late endosomes and is commonly used as a marker for the late endosome. It is also important in the
HCV lifecycle with evidence that Rab7 along with Rab5a may facilitate the formation of NS4B altered membrane foci, which are important for the setting up of the HCV replication complex (Manna et al., 2010). Immunofluorescence analysis was performed on Huh-7+IFITM cells using using anti-Rab7 antibodies to detect endogenous Rab7, and anti-FLAG antibodies to detect the exogenously expressed IFITM proteins [Figure
5.6]. As seen previously, IFITM2 exhibited a punctate intracellular staining pattern and in some instances co-localised with Rab7 at distinct foci within the cell [Figure
5.7]. Interestingly, no such co-localisation was observed between IFITM3 and Rab7.
This demonstrates that despite the high amino acid similarity between IFITM2 and
IFITM3, these proteins localise to distinct organelles within the hepatocyte.
5.2.6 IFITM2 and IFITM3 localise to the lysosome, and IFITM1 partially co- localises with the lysosome
Having previously determined that IFITM2 and IFITM3 partially localise to late and early endosomes respectively, there remains the question as to the localisation of the remaining pool of these proteins. We therefore next examined the localisation of the
IFITM proteins in the context of the lysosome, based on previous studies demonstrating IFITM2 and IFITM3 can localise to the lysosome (Huang et al., 2011).
Immunofluorescence analysis was performed on Huh-7+IFITM cells using anti-
LAMP1 to detect endogenous LAMP1 (lysosomal protein marker), and anti-FLAG
104 !"#$%& 2345& *./0.1& 2345&
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'(')*-&
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Figure 5.6: IFITM2 partially co-localises with the late endosome marker Rab7. To investigate whether the IFITM proteins co-localised with late endosomes, the Huh-7+IFITM overexpression cell lines were utilisied and then immunofluorescence performed for !-FLAG (green) and !-Rab7 (red) (60x magnification). Partial co- localisation between IFITM2 and Rab7 was observed. IFITM2 Rab7 Merge
!"!#$%& '()*& $+,-+&
Figure 5.7: IFITM2 partially co-localises with the late endosome marker Rab7. To investigate whether the IFITM proteins co-localised with late endosomes, Huh-7+IFITM2 and Huh-7+IFITM3 cells were stained with anti-FLAG (green) and anti-Rab7 (red) antibodies. Partial co-localisation between IFITM2 and Rab7 was observed (60x magnification, 20 µm scale bar). antibodies to detect the exogenously expressed IFITM proteins [Figure 5.8]. Figure 5.9 identified both IFITM2 and IFITM3 to localise to the lysosome, but interestingly we observed that intracellular IFITM1 partially co-localised with the lysosome as well. As the lysosome is the site of protein degradation, it is interesting to note that all three
IFITM proteins partially localise to the lysosome, indicating a possible role in targeting the HCV virion for degradation.
5.2.7 The IFITM proteins do not associate with VAP-A in the hepatocyte
Having established the cellular localisation of IFITM1, IFITM2 and IFITM3 to specific cellular compartments, we next investigated the interaction of the IFITM proteins with
VAP-A, a known host pro-viral factor important for HCV replication, with the rationale being that any interaction may disrupt this proviral function (Tu et al., 1999). A recent study identified that the IFITM proteins were able to interact with VAP-A, an ER- associated protein known for modulating cholesterol homeostasis, resulting in the disruption of VAP-A interacting with oxysterol-binding protein (OSBP). Thus it was hypothesised that the IFITM3-VAP-A interaction disrupted cholesterol homeostasis within late endosomes and so might be the mechanism by which IFITM3 limits IAV
(and other viral) infection (Amini-Bavil-Olyaee et al., 2013). Based on this evidence,
we examined the localisation of the IFITM proteins in the context of VAP-A. Huh-
7+IFITM cells were transfected with a VAP-A expression plasmid tagged with
mCherry (Eyre et al., 2014), and probed with anti-FLAG antibodies to detect the
IFITM proteins. The localisation of the IFITM proteins was consistent with previous
observations, while VAP-A revealed a cytoplasmic pattern of expression, as previously described [Figure 5.10]. In no instance was co-localisation between the IFITM proteins
105 !"#$%& -.*/+& *12314&
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Figure 5.8: IFITM1, IFITM2 and IFITM3 co-localise with the lysosomal marker Lamp1. To investigate whether the IFITM proteins co-localised with lysosomes, the Huh-7+IFITM overexpression cell lines were utilisied and then immunofluorescence performed for !-FLAG (green) and !-Lamp1 (red) (60x magnification). Co-localisation of IFITM1, IFITM2 and IFITM3 with Lamp1 was observed. IFITM1 Lamp1 Merge
IFITM2 Lamp1 Merge
IFITM3 Lamp1 Merge
Figure 5.9: IFITM1, IFITM2 and IFITM3 co-localise with the lysosomal marker Lamp1. To investigate whether the IFITM proteins co-localised with lysosomes, Huh-7+IFITM cells were stained with anti-FLAG (green) and anti-Lamp1 (red) antibodies. Co-localisation of IFITM1, IFITM2 and IFITM3 with Lamp1 was observed (60x magnification, 20 µm scale bar). !"#$%& -.#/0/"12344%& *34536&
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Figure 5.10: The IFITM proteins do not localise to Vap-A within the hepatocyte. To investigate whether the IFITM proteins localised to the Vap-A, the Huh-7+IFITM overexpression cell lines were transfected with a Vap-A-mCherry plasmid and then immunofluorescence performed for !-FLAG (green) (60x magnification). No co- localisation with Vap-A was observed for any of the IFITM proteins. and VAP-A detected. This suggests that in Huh-7 cells the anti-HCV actions of the
IFITM proteins are not dependent on an association with VAP-A.
5.3 Discussion
During the course of this PhD, the cellular localisation of IFITM2 and IFITM3, particularly IFITM3, has been examined in a number of different cell lines (A549,
HEK293, HeLa) in relation to their antiviral activity against other viruses. These studies have identified IFITM2 and IFITM3 to partially co-localise with late endosomes and lysosomes within the majority of cells. However, some differences in cellular localisation of the IFITM proteins have been observed in different cell types.
Thus, in this chapter we sought to ascertain the cellular localisation of IFITM2 and
IFITM3 in Huh-7 cells to gain insight into the potential molecular mechanism of these proteins against HCV. Results from this chapter provide strong evidence that IFITM2 and IFTM3 localise to both endosomes and lysosomes, and consequently we propose that the anti-HCV actions of these two proteins follow the recently described paradigm of IFITM2 and IFITM3 targeting newly entered virions for lysosomal degradation.
Firstly, we identified a panel of organelles to examine the localisation of IFITM2 and
IFITM3 in Huh-7 cells. These included the previously described organelles such as the late endosome and lysosome, as well as organelles described to be crucial for the establishment of a productive HCV infection such as the ER, Golgi complex and lipid droplets. Using the Huh-7+IFITM cells and either specific antibodies or fluorescently tagged proteins, we demonstrated that IFITM2 and IFITM3 partially co-localise to the late and early endosome respectively, in addition to localising to the lysosome [Figures
5.5, 5.7 & 5.9]. IFITM1 was included in these immunofluorescence analyses as some
106 cytoplasmic localisation of this protein was observed despite being predominantly at
the cell surface. Interestingly, we observed that intracellular IFITM1 partially co-
localises with the lysosome [Figure 5.9]. Though these observations were not solely unexpected, it is interesting to note the difference in localisation of the IFITM proteins between cell types and, considering the similarity of these proteins, especially IFITM2 and IFITM3 [Table 5.1]. Previous studies have not noted a change in IFITM protein expression upon viral infection, therefore it would be important to conduct further experiments to determine whether IFITM2 and IFITM3 localisation is altered upon infection with HCV.
The common localisation of IFITM2 and IFITM3 at endosomes and lysosomes, as well
as the broad antiviral properties of these proteins, has led many to hypothesise that a
generalised mechanism targeting viral entry exists (Bailey et al., 2014). One potential
mechanism is that the IFITM proteins block specific sites of viral-cell fusion, thus
restricting viral entry and/or viral release of the genome into the cytoplasm. However,
this location-dependent theory does not hold true for viruses such as Machupo virus
(MACV) and Lassa virus (LASV), where entry is not inhibited by IFITM-enriched
endosomes. Another potential mechanism, with more supporting evidence, is the
possibility that the IFITM proteins alter membrane properties to prevent the formation
of fusion pores (Desai et al., 2014). The ability of the IFITM proteins to alter
membrane fluidity and curvature to prevent viral hemifusion (the first stage of fusion)
provides an explanation for this hypothesis (Li et al., 2013a). Furthermore, IFITM3-
mediated cholesterol enrichment of endolysosomal membranes has also been shown by
several independent studies (Amini-Bavil-Olyaee et al., 2013, Lin et al., 2013).
107 Table 5.1: Summary of IFITM1, IFITM2 and IFITM3 localisation within Huh-7 cells
Cellular Localisation Cell Surface Early Late Lysosome Endosome Endosome IFITM1 ✓ ✗ ✗ ✓ IFITM2 ✗ ✗ ✓ ✓ IFITM3 ✗ ✓ ✗ ✓ ! As mentioned previously, HCV entry relies on the fusion of the HCV virion to the early endosomal membrane resulting in the release of the HCV genome into the cytoplasm
(Dubuisson and Cosset, 2014). Based on the localisation of IFITM2 and IFITM3 within
Huh-7 cells, we hypothesise that IFITM2 and IFITM3 limit HCV infection by preventing viral-endosomal fusion thereby ‘trapping’ the endocytosed virion within the endocytic pathway and target it for lysosomal degradation. It is plausible that the early endosomal membrane is altered through IFITM-mediated loss of membrane fluidity or
IFITM3-mediated cholesterol enrichment thus preventing fusion and HCV RNA release. Amini-Bavil-Olyaee et al provided evidence that IFITM3-mediated cholesterol enrichment of endosomal membranes is through an interaction with VAP-A, a known modulator of cholesterol homeostasis (Amini-Bavil-Olyaee et al., 2013). Our results, however, indicate that the IFITM proteins do not associate with VAP-A in hepatocytes, and so any alterations to membrane function would not be via VAP-A. Furthermore,
Lin et al proposed that IFITM-induced alterations to endosomal membrane function promoted fusion with opposing membranes expressing IFITM proteins (Lin et al.,
2013). Endosomal-lysosomal degradation is a sequential process requiring endosomal maturation starting at the early endosome, moving to the late endosome and culminating at the fusion of the late endosome with the lysosome, resulting in the degradation of trapped particles (Schulze et al., 2009). Thus, we postulate that HCV is trapped in the early endosome after clathrin-mediated endocytosis, due to IFITM3- mediated alterations at the early endosomal membrane preventing fusion, and subsequently targeting the virion for degradation via the late endosome and lysosome through interactions between the different IFITM proteins found at these organelles.
Further work is required to confirm this hypothesis, such as determining whether the
IFITM proteins restrict the release of HCV RNA to the cytoplasm, whether pore
108 formation in the endosome is restricted in the presence of the IFITM proteins, and also
using live-imaging to determine whether the virus is indeed targeted for lysosomal
degradation.
Another potential mechanism by which IFITM2 and IFITM3 may restrict HCV infection is by preventing the formation of a functional HCV replication complex (RC).
The HCV RC is the site of RNA replication and requires NS4B induced alteration of the ER membrane. Pertinent to this thesis, studies have shown that the recruitment of
Rab5a and Rab7 is required for the formation of the HCV RC (Manna et al., 2010).
Thus it is possible that IFITM2 and IFITM3 are able to either interact with Rab5a and
Rab7 specifically, or alter the endosomal membranes the Rab proteins are associated with to prevent the recruitment of these proteins to the replication complex. This hypothesis could rationalize the discrepancy in the IFITM2 and IFITM3 anti-HCV activity observed upon infection with Jc1 [Figure 3.13] that is not accounted for in the
HCVpp analysis [Figure 3.14]. However, our replicon data [Figures 3.15 & 3.16] indicate that the IFITM proteins have no effect on RNA replication, which would also indicate no effect on the formation of the replication complex. Nevertheless it is possible that IFITM2 and IFITM3 may only have an effect on newly formed replication complexes with previously formed RCs being resistant, and would provide an explanation for the prolonged anti-HCV activity observed in Chapter 3. Further work would be required to confirm this hypothesis, where IFITM and Rab protein expression would be examined via microscopy in the presence of HCVcc infection for longer periods of time. Localisation of the Rab proteins to HCV NS4B in cells not expressing the IFITM proteins would be used as a control, given NS4B remodels the ER membrane to result in the formation of the replication complex (Hugle et al., 2001).
109 Collectively, the results in this chapter demonstrate IFITM2 and IFITM3 to partially localise to the late and early endosomes respectively, with all three IFITM proteins localising to the lysosome. Thus, these results suggest that IFITM2 and IFITM3 act on
HCV virions that have bypassed restriction by IFITM1 at the cell surface, and in a coordinated manner limit HCV infection by altering the site of virus-cell fusion.
Further questions remain to define the mechanism of action of these proteins, in particular the site of this potential disruption and whether any cofactors are involved.
The results from this chapter support the theory that IFITM2 and IFITM3 have a generalised mechanism of action allowing for a broad range of antiviral activity against low-pH dependent virus entry, including HCV.
110 Chapter 6
Post-translational modifications of the IFITM proteins are essential for anti-HCV activity
6.1 Introduction
Post-translational modifications (PTMs) of the IFITM proteins have been identified to
play a crucial role in the localisation and function of these proteins. Studies have
examined the PTM status of the IFITM proteins and identified that these proteins undergo tyrosine phosphorylation (Jia et al., 2012, Jia et al., 2014), ubiquitination
(Yount et al., 2012, Chesarino et al., 2014), S-palmitoylation (Yount et al., 2010) and
methylation (Shan et al., 2013). A single tyrosine residue in the NTD of IFITM3 has
been shown to be essential for antiviral activity against IAV, DENV and VSV, while S- palmitoylation of all IFITM proteins was found to be important against IAV and
DENV but not HIV (John et al., 2013, Jia et al., 2012, Chutiwitoonchai et al., 2013). In
the previous chapters of this thesis we have demonstrated the importance of the IFITM
proteins against HCV infection; however, no work has been performed to date investigating the role of IFITM PTMs in the context of HCV infection. Therefore, the aim of this chapter is to investigate whether the IFITM proteins undergo tyrosine phosphorylation and S-palmitoylation in hepatocytes in vitro and to determine whether these PTMS are important for the cellular localisation and anti-HCV properties of these proteins.
111 6.2 N-terminal tyrosine phosphorylation of IFITM2 and IFITM3 is essential for cellular localisation but not for anti-HCV activity
6.2.1 IFITM1, IFITM2 and IFITM3 undergo tyrosine phosphorylation in Huh-7 cells.
Initial studies that identified the importance of N-terminal tyrosine phosphorylation of
IFITM3 were carried out in HEK-293T cells, and indicated that IFITM1 and IFITM2 did not undergo tyrosine phosphorylation (Jia et al., 2012). To determine whether this was analogous in Huh-7 cells, we investigated the tyrosine phosphorylation status of
FLAG-tagged IFITM1, IFITM2 and IFITM3 via immunoprecipitation analysis as described in Section 2.5.20. Briefly, total protein was harvested from Huh-7+IFITM cells in the presence of a phosphatase inhibitor cocktail and immunoprecipitated with anti-FLAG antibodies overnight. The precipitated samples were probed by Western blot with antibodies specifically targeting phosphorylated tyrosine (pY), in combination with anti-FLAG and anti-β-actin antibodies to detect the IFITM proteins and the loading control protein β-actin respectively. Western blots specific for pY show the
detection of bands at 15kDa, 18kDa and 17kDa corresponding to the size of IFITM1,
IFITM2 and IFITM3 respectively, as previously observed in Chapter 3 and in the
literature [Figure 6.1]. These results demonstrate that all three IFITM proteins undergo
tyrosine phosphorylation in Huh-7 cells in vitro, in contrast to the results in the
literature, in which as outlined above, only IFITM3 is phosphorylated (Jia et al., 2012).
112 Figure 6.1: Wildtype IFITMs undergo phosphorylation in Huh-7 cells. To determine whether the IFITM proteins undergo phosphorylation, the Huh-7+IFITM overexpression cells were utlitised and total protein extracted in the presence of phosphatase inhibitors for co-immunoprecipitation and Western blot analysis. Extracted protein was immunoprecipitated with α-FLAG antibodies, followed by blotting for α- phospho-tyrosine and α-FLAG. Cell lysates were also immunoblotted with antibodies specific for FLAG and β-actin. Results indicate that all the IFITM proteins undergo phosphorylation in Huh-7 cells. 6.2.2 Identification of conserved and non-conserved tyrosine residues between the
IFITM proteins
In order to investigate potential phosphorylation sites within IFITM1, IFITM2 and
IFITM3, we utilised the NetPhos 2.0 Server provided by the Center for Biological
Sequence Analysis (Technical University of Denmark). Three potential phosphorylation sites were identified for IFITM1, and two each for IFITM2 and
IFITM3, with one conserved tyrosine residue found between all three IFITM proteins in the conserved intracellular loop (CIL) [Figure 6.2]. IFITM1 contains two other potential phosphorylation sites in its 13 a.a C-terminal extension at sites Y112 and
Y125, while the previously identified N-terminal tyrosine residue in IFITM3 (Y20) is conserved in the N-terminus of IFITM2 (Y19) but not IFITM1 by virtue of its N- terminal truncation. In order to determine whether the conserved or non-conserved tyrosine residues are responsible for IFITM tyrosine phosphorylation, we conducted mutational analysis of the non-conserved tyrosine residues found at the termini of the proteins. The ΔC13 and ΔC18 IFITM1 mutants generated in Chapter 4 sequentially target the Y112 and Y125 tyrosine residues respectively and so were utilised for the next set of experiments. To target the non-conserved tyrosine residues of IFITM2 and
IFITM3, we generated tyrosine to alanine mutants at Y19A and Y20A respectively
[Figure 6.3.i]. To confirm IFITM2:Y19A and IFITM3:Y20A protein expression from the pLenti6/V5-D-TOPO vector, Huh-7 cells were transfected with wildtype or tyrosine mutant IFITM2 and IFITM3 for 24 hrs and then total protein harvested and Western blots performed using an antibody directed against the FLAG-tag, in combination with the detection of the loading control protein β-actin. Western blots demonstrated the detection of the 18kDa IFITM2 protein and the 17kDa IFITM3 protein, with successful expression of IFITM2:Y19A and IFITM3:Y20A observed at a slightly higher
113 IFITM3 MNHTVQTFFSPVNSGQPPNYEMLKEEHEVAVLGAPHNPAPPTSTVIHIRSETSVPDHVVW 60 IFITM2 MNHIVQT-FSPVNSGQPPNYEMLKEEQEVAMLGGPHNPAPPTSTVIHIRSETSVPDHVVW 59 IFITM1 ------MHKEEHEVAVLGPPPSTILPRSTVINIHSETSVPDHVVW 39
IFITM3 SLFNTLFMNPCCLGFIAFAYSVKSRDRKMVGDVTGAQAYASTAKCLNIWALILGILMTIL 120 IFITM2 SLFNTLFMNTCCLGFIAFAYSVKSRDRKMVGDVTGAQAYASTAKCLNIWALILGIFMTIL 119 IFITM1 SLFNTLFLNWCCLGFIAFAYSVKSRDRKMVGDVTGAQAYASTAKCLNIWALILGILMTIG 99
IFITM3 LIVIPVL------IFQAYG-- 133 IFITM2 LVIIPVL------VVQAQR-- 132 IFITM1 FILLLVFGSVTVYHIMLQIIQEKRGY 125
Conserved and non-conserved tyrosine residues IFITM1: Y78, Y112, Y125 IFITM2: Y19, Y98 IFITM3: Y20, Y99
Fig 6.2: Identification of conserved and non-conserved tyrosine residues bewteen IFITM1, IFITM2 and IFITM3. i 20 40 60 80 100 120 140 160
IFITM3 Y TM I TM2 IFITM2 Y TM I TM2 IFITM3 !Y20A A TM I TM2 IFITM2 !Y19A A TM I TM2
ii IFITM2 Y19A IFITM3 Y20A
18 kDa anti-FLAG 17 kDa
42 kDa anti-!-actin
Fig 6.3: Mutation of a conserved tyrosine residue in the N-terminus of IFITM2 and IFITM3. (i) Schematic representation of tyrosine to alanine site-directed mutagenesis conducted on the conserved tyrosine residues Y19 and Y20 found in IFITM2 and IFITM3, respectively. (ii) To detect the expression of the IFITM2 and IFITM3 tyrosine mutants in a transient assay, total protein was extracted 24 hrs post- transfection into Huh-7 cells. Extracted protein was immunoblotted with antibodies specific for FLAG and "-actin. Results showed slight changes in protein size. molecular weight compared to wildtype, consistent with sizes observed in the literature
[Figure 6.3.ii]. The slower mobility of the mutants could be due to either changes in protein confirmation or in the ability to bind to the SDS due to the mutations inserted
(Jia et al., 2012).
6.2.3 Y19 and Y20 are responsible for IFITM2 and IFITM3 phosphorylation, while the conserved Y78 is responsible for IFITM1 phosphorylation.
To determine whether the conserved or non-conserved tyrosine residues are responsible for the phosphorylation status of the IFITM proteins, we investigated the ability of the wildtype and IFITM mutants (M1:ΔC13, M1:ΔC18, IFITM2:Y19A & IFITM3:Y20A) to undergo phosphorylation via immunoprecipitation as described in Section 2.5.20.
Briefly, Huh-7 cells were transfected with each of the wildtype and mutant IFITM proteins for 24 hrs and total protein was harvested in the presence of a phosphatase inhibitor cocktail and immunoprecipitated with anti-FLAG antibodies overnight. The precipitated samples were probed by Western blot with antibodies specifically targeting pY, in combination with anti-FLAG and anti-β-actin antibodies to detect the IFITM proteins and the loading control protein β-actin respectively. A complete loss in phosphorylation was observed for IFITM2:Y19A and IFITM3:Y20A compared to wildtype IFITM2 and IFITM3 [Figure 6.5]. Conversely, both M1:ΔC13 and M1:ΔC18 retained phosphorylation ability similar to wildtype [Figure 6.4]. Collectively these results indicate that the CIL Y78 residue is responsible for the phosphorylation of
IFITM1, while IFITM2 and IFITM3 tyrosine phosphorylation is dependent on the expression of the conserved N-terminal tyrosine residue (Y19 or Y20).
114 Figure 6.4: IFITM1 undergoes tyrosine phosphorylation at the conserved Y78 residue. To determine whether the mutants targeting the non-conserved tyrosine residues of IFIMT1 are responsible for tyrosine phosphorylation, the Huh-7 cells were transiently transfected with the respective plasmid DNA for 48 hrs and total protein extracted in the presence of phosphatase inhibitors for co-immunoprecipitation and Western blot analysis. Extracted protein was immunoprecipitated with α-FLAG antibodies, followed by blotting for α-phospho-tyrosine and α-FLAG. Cell lysates were also immunoblotted with antibodies specific for FLAG and β-actin. These results demonstrate that the conserved Y78 residue is responsible for IFITM1 phosphorylation in Huh-7 cells. IFITM2 IFITM3 Y19A Y20A 18 kDa anti-Tyr-P 17 kDa
IFITM2 Y19A IFITM3 Y20A 18 kDa 17 kDa anti-FLAG IP: ! -FLAG
Figure 6.5: IFITM2 and IFITM3 N-terminal tyrosine mutants do not undergo phosphorylation in Huh-7 cells. To determine whether the mutants targeting the non- conserved tyrosine residues in IFITM2 and IFITM3 undergo phosphorylation, the Huh-7 cells were transiently transfected with the respective plasmid DNA for 48 hrs and total protein extracted in the presence of phosphatase inhibitors for co- immunoprecipitation and Western blot analysis. Extracted protein was immunoprecipitated with !-FLAG antibodies, followed by blotting for !-phospho- tyrosine and !-FLAG. Cell lysates were also immunoblotted with antibodies specific for FLAG and "-actin. These results demonstrate Y19 and Y20 are responsible for the phosphorylation of IFITM2 and IFITM3 in Huh-7cells, respectively. 6.2.4 The N-terminal tyrosine residue is required for the endosomal localisation of
IFITM2 and IFITM3
Since the localisation and anti-HCV properties of ΔC13 and ΔC18 have already been examined in Chapter 4, where we demonstrated a role for the C-terminal region of
IFITM1 in cellular localisation but not anti-HCV activity, we focused on the tyrosine to alanine mutants IFITM2:Y19A and IFITM3:Y20A. We extended our observations to
the cellular localisation of IFITM2:Y19A and IFITM3:Y20A compared to wildtype, as
mutation of Y20 has previously been reported to change IFITM3 localisation from the
endosome to the plasma membrane (Jia et al., 2012, Jia et al., 2014).
Immunofluorescence analysis confirmed this redistribution, with the localisation of
IFITM3:Y20A within Huh-7 cells resembling that of IFITM1 on the cell surface
[Figure 6.6]. IFITM2:Y19A also displayed a change in localisation though not as
striking as that of IFITM3:Y20A, where the majority of protein localisation was
redistributed to the plasma membrane with some perinuclear localisation still present.
This suggests that phosphorylation is a key PTM that targets these proteins to the
endosomes.
6.2.5 IFITM2:Y19A and IFITM3:Y20A enhances anti-HCV activity compared to
wildtype
Previous studies have demonstrated that the tyrosine to alanine mutation of Y20 in
IFITM3 abrogated the antiviral properties of the protein against IAV, DENV and VSV
(Jia et al., 2012, John et al., 2013), and hypothesised that tyrosine phosphorylation was
required for the endocytosis of IFITM3 to the early endosome where it can exert
antiviral activity. However, more recent studies have contradicited this hypothesis by
suggesting Y20 to be part of an endocytic signal motif 20-YEML-23 (consensus
115 !"!#$'& !"!#$'&(&)*+,& !"!#$'&
!"!#$%& !"!#$%&(&)'-,&
Fig 6.6: Tyrosine mutated IFITM2 and IFITM3 have different cellular localisation. To investigate whether the tyrosine to alanine mutations in IFITM2 and IFITM3 effects cellular localisation, Huh-7 cells were transfected with the tyrosine mutants 24 hrs prior to immunofluorescence performed for !-FLAG (red). Both mutants changed from the wildtype endosomal-localisation to a more plasma membrnane localisation (60x magnification). YXX(Ω)), where the localisation of IFITM3 within a cell is regulated by the
phosphorylation of Y20 via Fyn kinases (Jia et al., 2014, Chesarino et al., 2014).
Despite opposing hypotheses on the role of tyrosine phosphorylation on IFITM3, the
Y20 residue appears to be essential for the antiviral actions of IFITM3. In order to determine whether this phenotype was true for HCV, Huh-7 cells were transfected with
IFITM2, IFITM3, the tyrosine mutants and the empty vector control for 24 hrs followed by Jc1 infection (MOI: 0.03) for 24 hrs. Real-time PCR was performed to measure HCV RNA levels and levels were expressed as a fold change relative to the empty vector control. In contrast to the results in the literature, we observed
IFITM2:Y19A and IFITM3:Y20A in a transient expression assay to retain anti-HCV
activity at levels similar to wildtype; however, these results were not significant [Figure
6.7].
In order to reduce the variations within experiments, as described in Section 4.4.2,
Huh-7 cells stably expressing each of the tyrosine mutants IFITM2:Y19A and
IFITM3:Y20A were generated. Briefly, these stable cell lines were generated, as we
have done previously, by transducing Huh-7 cells with lentiviral particles expressing
the tyrosine mutants followed by antibiotic selection with blasticidin. Resistant cells
were isolated (method described in Section 2.1.2) to generate polyclonal
overexpression cell lines. Protein expression was confirmed by Western blot and
immunofluorescence analysis (results not shown). To corroborate the observations of
Figure 6.7, Huh-7 cells stably expressing IFITM2, IFITM3, the tyrosine mutants and
empty vector control cells were infected with Jc1 (MOI: 0.03) for 24 hrs. Real-time
PCR was performed to measure HCV RNA levels, with HCV RNA levels expressed as a fold change relative to the empty vector control. Figure 6.8 confirms the retention of
116 2.0
1.5
1.0
0.5 in HCV replication HCV in Relative fold change change fold Relative
0.0 Empty IFITM2 Y19A IFITM3 Y20A
Fig 6.7: The anti-HCV activity of IFITM2 and IFITM3 is independent of tyrosine residues Y19 and Y20. To determine the antiviral activity of the tyrosine mutants, Huh-7 cells were transfected with the tyrosine mutants 24 hrs prior to infection with Jc1 (MOI: 0.03). Total RNA was harvested 24 hrs post-infection and and total HCV RNA levels quantitated by real-time PCR. RNA levels were normalised to the control protein RPLPO and were expressed as a fold change relative to empty vector control. Results indicated that the anti-HCV activity of IFITM2 and IFITM3 is independent of the conserved N-terminal tyrosine residue (data represented as a mean ± SEM, n=3). 1.0
0.8 ** 0.6 **
0.4 ** **
in HCV replication HCV in 0.2 Relative fold change change fold Relative **P < 0.004 0.0 Empty IFITM2 Y19A IFITM3 Y20A
Anti-HCV activity + + + +
Fig 6.8: The anti-HCV activity of IFITM2 and IFITM3 is independent of tyrosine residues Y19 and Y20. To determine the antiviral activity of the tyrosine mutants, Huh-7+IFITM and Huh-7+IFITM tyrosine mutant cells were infected with Jc1 (MOI: 0.03). Total RNA was harvested 24 hrs post-infection and and total HCV RNA levels quantitated by real-time PCR. RNA levels were normalised to the control protein RPLPO and were expressed as a fold change relative to empty vector control. Results indicated that the anti-HCV activity of IFITM2 and IFITM3 is independent of the conserved N-terminal tyrosine residue (data represented as a mean ± SEM, n=3). anti-HCV activity, and in fact shows that IFITM2:Y19A and IFITM3:Y20A have enhanced anti-HCV properties compared to wildtype. Interestingly, the enhanced anti-
HCV activity of both IFITM2:Y19A and IFITM3:Y20A resemble the drop in HCV
activity observed in Figure 3.13 in the presence of IFITM1. Together these results
demonstrate that phosphorylation at Y19 and Y20 are not essential for the anti-HCV
activity of IFITM2 and IFITM3 respectively.
6.2.6 IFITM2:Y19A and IFITM3:Y20A co-localise with CD81 on the hepatic cell surface
The results in this chapter have demonstrated IFITM2:Y19A and IFITM3:Y20A to have enhanced anti-HCV activity (compared to wildtype IFITM2 and IFITM3), as well as re-distribute to the cell surface from an endosomal/lysosomal localisation. As these changes mirror the actions of IFITM1, we investigated the localisation of
IFITM2:Y19A and IFITM3:Y20A in the context of CD81 to ascertain a possible mechanism. Figure 6.9 shows immunofluorescence analysis of Huh-7 cells expressing wildtype IFITMs, IFITM2:Y19A and IFITM3:Y20A, that were probed using anti-
CD81 and anti-FLAG antibodies to detect endogenous CD81 and the mutated IFITM proteins respectively. Significant co-localisation was observed between the tyrosine mutants IFITM2:Y19A and IFITM3:Y20A and CD81, thus mirroring the localisation of IFITM1. These results signify for the first time that the enhanced anti-HCV properties of IFITM2:Y19A and IFITM3:Y20A may be through an association with
CD81 at the cell surface, while also indicating that a single mutation in IFITM2 and
IFITM3 could revert the phenotype of these proteins to resemble IFITM1.
117 !"!#$.& '()*+,-.& $/01/2& '()*+,-.& $/01/2&
!"!#$3& '()*+,-.& $/01/2&
!"!#$%& '()*+,-.& $/01/2&
*+*,-1/0(43) !"#$%&'() -56758)
*+*,-./0123) !"#$%&'() -56758)
Fig 6.9: The IFITM2 and IFITM3 N-terminal tyrosine mutants co-localise with CD81 on the hepatic cell surface. To investigate whether the IFITM2:Y19A and IFITM3:Y20A mutants co-localised with the HCV entry receptor CD81, the Huh-7+IFITM (wildtype and mutant) expression cell lines were immunoprobed for !- FLAG (red) and !-CD81 (green). Strong co-localisation was observed between CD81 and both the IFITM tyrosine mutants (60x magnification). 6.3 S-palmitoylation of the IFITM proteins is crucial for anti-HCV activity
6.3.1 Generation of IFITM mutants targeting S-palmitoylation sites
Yount et al (2010) identified that the IFITM proteins undergo S-palmitoylation at three specific cysteine residues, two consecutive cysteine residues found in the first membrane-associated domain (M1) (IFITM1:C50/51, IFITM2:C70/71,
IFITM3:C71/72), while the other cysteine residue is in the CIL (IFITM1:C84,
IFITM2:C104, IFITM3:C105) [Figures 6.10 and 6.11.i] (Yount et al., 2010).
Additional studies during the course of this PhD have identified S-palmitoylation of the
IFITM proteins, particularly the C72 residue of IFITM3 to be vital to maintain antiviral activity against IAV and DENV (Yount et al., 2012, John et al., 2013). However, a recent study has also noted that S-palmitoylation is dispensable for the antiviral properties of the IFITM proteins against HIV (Chutiwitoonchai et al., 2013). This raises questions as to the role of S-palmitoylation in the antiviral actions of the IFITM proteins and suggests possible selectivity of antiviral effects. In order to examine whether S-palmitoylation of the IFITM proteins was important for anti-HCV activity, double (IFITM1:C50/C51A, IFITM2: C70/C71A, IFITM3:C71/C71A) and single
(IFITM1:C84A, IFITM2:C104A, IFITM3:C105A) cysteine to alanine mutants were generated for each of the IFITM proteins [Figure 6.11.i]. These mutations were based on previous studies in the literature (Chutiwitoonchai et al., 2013, John et al., 2013), with the basis that mutation of the cysteine residues would block S-palmitoylation of the IFITM proteins. Protein expression was confirmed by immunofluorescence analysis of Huh-7 cells transfected with each of the palmitoylation IFITM mutants. Figure
6.11.ii demonstrates that mutation of the cysteine residues within the M1 and CIL domains results in a redistribution of IFITM1 from the cell surface to punctate
118 IFITM3 MNHTVQTFFSPVNSGQPPNYEMLKEEHEVAVLGAPHNPAPPTSTVIHIRSETSVPDHVVW 60 IFITM2 MNHIVQT-FSPVNSGQPPNYEMLKEEQEVAMLGGPHNPAPPTSTVIHIRSETSVPDHVVW 59 IFITM1 ------MHKEEHEVAVLGPPPSTILPRSTVINIHSETSVPDHVVW 39
IFITM3 SLFNTLFMNPCCLGFIAFAYSVKSRDRKMVGDVTGAQAYASTAKCLNIWALILGILMTIL 120 IFITM2 SLFNTLFMNTCCLGFIAFAYSVKSRDRKMVGDVTGAQAYASTAKCLNIWALILGIFMTIL 119 IFITM1 SLFNTLFLNWCCLGFIAFAYSVKSRDRKMVGDVTGAQAYASTAKCLNIWALILGILMTIG 99
IFITM3 LIVIPVL------IFQAYG-- 133 IFITM2 LVIIPVL------VVQAQR-- 132 IFITM1 FILLLVFGSVTVYHIMLQIIQEKRGY 125
Conserved cysteine residues IFITM1: C50/51, C84 IFITM2: C70/71, C104 IFITM3: C71/72, C105
Fig 6.10: Identification of conserved cysteine residues bewteen IFITM1, IFITM2 and IFITM3. Each of the IFITM proteins have two conserved cysteine residues in the first membrane-associated domain (M1) and one conserved cysteine in the conserved intracellular loop (CIL) (Yount et al, 2010, Nat Chem Biol)
i 20 40 60 80 100 120 140 160
IFITM3 TM I CC C TM2 IFITM2 TM I CC C TM2 IFITM1 TM I CC C TM2 IFITM3 !C7172A TM I AA TM2 IFITM2 !C7071A TM I AA TM2 IFITM1 !C5051A TM I AA TM2 IFITM3 !C105A TM I A TM2 IFITM2 !C104A TM I A TM2 IFITM1 !C84A TM I A TM2
ii IFITM1 IFITM1:C5051A IFITM1:C84A
IFITM2 IFITM2:C7071A IFITM2:C104A
IFITM3 IFITM3:C7172A IFITM3:C105A
Fig 6.11: Mutation of a conserved cysteine residues in IFITM1, IFITM2 and IFITM3. (i) Schematic representation of cysteine to alanine site-directed mutagenesis conducted on the conserved cysteine residues found in IFITM1, IFITM2 and IFITM3. (ii) Immunofluorescence analysis of transfected cells with anti-FLAG antibody demonstrated expression of the IFITM cysteine mutants within Huh-7 cells in vitro (60x magnification). compartments within the cytoplasm. Cysteine mutants for IFITM2 and IFITM3 are
retained within the cytoplasm, but appear to have a more clustered perinuclear
localisation. Clearly S-palmitoylation has a significant effect on the cellular localisation
of the IFITM proteins and indicates a possible role in the anti-HCV actions of these
proteins.
6.3.2 A single conserved cysteine residue in the CIL is important for the anti-HCV
properties of the IFITM proteins
Next we sought to determine whether the loss of S-palmitoylation capabilities of the
IFITM proteins altered anti-HCV activity through a transient assay. Huh-7 cells were
transfected with wildtype IFITM proteins, the palmitoylation mutants and the empty
vector control for 24 hrs followed by Jc1 infection (MOI: 0.03) for 24 hrs. Real-time
PCR was performed to measure HCV RNA levels, with levels expressed as a fold change relative to the empty vector control. The conserved cysteine residue in the CIL
(IFITM1:C84, IFITM2:C104, IFITM3:C105) were found to be essential for anti-HCV activity, as mutation of this cysteine in each IFITM proteins abrogated anti-HCV activity [Figure 6.12]. IFITM3, however, also requires the two cysteine residues found in the M1 domain (C71/C72) for its anti-HCV activity.
6.3.3 Mutation of conserved cysteine residues that undergo S-palmitoylation localise the IFITM proteins predominantly to the lysosome
Preliminary immunofluorescence analysis of the IFITM palmitoylation mutants indicated a change in cellular localisation from wildtype, and based on the loss of anti-
HCV activity of the CIL mutants in Figure 6.12, we sought to determine whether this loss in anti-HCV activity was due to change in cellular localisation.
119 2.0
1.5
1.0 ** ** ** ***
in HCV replication HCV in 0.5 Relative fold change change fold Relative **** **P < 0.01 ***P = 0.003 0.0 ****P < 0.0001
Empty IFITM1 IFITM2 IFITM3 M1:C84A M2:C104A M3:C105A M1:C5/051A M2:C70/71A M3:C71/72A Anti-HCV activity + + - + + - + - -
Fig 6.12: A single conserved cysteine residue in the CIL is essential for the anti- HCV acitivity of the IFITM proteins. To determine the antiviral activity of the palmitoylation mutants, Huh-7 cells were transfected with the palmitoylation mutants 24 hrs prior to infection with Jc1 (MOI: 0.03). Total RNA was harvested 24 hrs post- infection and and total HCV RNA levels quantitated by real-time PCR. RNA levels were normalised to the control protein RPLPO and were expressed as a fold change relative to empty vector control. Results indicate that the conserved cysteine residue in the CIL (M1:C84A, M2:C104A & M3:C105A) is important for anti-HCV activity, as well as C71/72 for IFITM3 (data represented as a mean ± SEM, n=3). Immunofluorescence analysis of transiently expressed wildtype and IFITM1 mutants
was conducted in relation to subcellular markers previously identified in this thesis – (i)
IFITM1 mutants against CD81 and Lamp1, (ii) IFITM2 mutants against Rab7 and
Lamp1 and (iii) IFITM3 mutants against Rab5a and Lamp1. Mutation of the conserved
cysteine residues in IFITM1, and concomitant S-palmitoylation, completely abrogated
its interaction with CD81, with both mutants observed to re-localise from the cell
surface to the lysosome [Figure 6.13]. A similar lysosomal redistribution was observed
for the IFITM3 palmitoylation mutants, where IFITM3:C71/72A and IFITM3:C105A
were no longer observed to co-localise with the early endosome marker Rab5a and now
exclusively localised to Lamp1 positive lysosomes [Figure 6.15]. Interestingly,
IFITM2:C70/71A was found to retain partial co-localisation at the late endosome,
while the CIL IFITM2 mutant (C104A) was found to re-localise to the lysosome
[Figure 6.14]. The re-localisation of the CIL cysteine IFITM mutants, and
IFITM3:C71/72A, in conjuction with the abrogation of anti-HCV activity indicate that
these mutants may be targeted for lysosomal degradation. Similarly, the retention of
partial late endosome and lysosome localisation for the IFITM2:C70/71A and
IFITM1:C50/51A mutants respectively, could account for the retention of anti-HCV
properties for these mutants in Figure 6.12.
6.4 Discussion
The role that PTMs play in the localisation and antiviral activity of the IFITM proteins has been demonstrated by a number of studies during the latter stages of this PhD.
Ubiquitination (at positions K2, K83, K88 & K104) and monomethylation (at position
K88) of IFITM3 decreases its antiviral activity against IAV and VSV (Shan et al.,
2013, Yount et al., 2012). Conversely, phosphorylation at a single tyrosine residue in
120 i -.-/*$& !"#"$%& !(,%& !'($& !'($& !'($&
ii -.-/*$& !"#"$%& !(,%& )%*+$& )%*+$& )%*+$&
Fig 6.13: Palmitoylation IFITM1 mutants re-localise to the lysosome. To investigate whether the cysteine to alanine mutations in IFITM1 effected cellular localisation, Huh-7 cells were transfected with wildtype and palmitoylation mutants 24 hrs prior to immunofluorescence performed for (i) !-FLAG (red) and anti-CD81 (green) or (i) anti-FLAG (green) and anti-Lamp1 (red). IFITM1 palmitoylation mutants were found to predominantly localise at the lysosome (60x magnification). i ./.0+1& !"#"$%& !$#-%& '()"& '()"& '()"&
ii ./.0+1& !"#"$%& !$#-%& *%+,$& *%+,$& *%+,$&
Fig 6.14: Palmitoylation IFITM2 mutants partially co-localise to the late endosome but primarly re-localise to the lysosome . To investigate whether the cysteine to alanine mutations in IFITM2 effected cellular localisation, Huh-7 cells were transfected with wildtype and palmitoylation mutants 24 hrs prior to immunofluorescence performed for (i) !-FLAG (green) and anti-Rab7 (red) or (i) anti- FLAG (green) and anti-Lamp1 (red). IFITM2:C7071A mutants partially co-localises to the late endosome were found to predominantly localise at the lysosome (60x magnification). i !"!#$%& +,*,-(& +*81(& ./01/23+45667& ./01/23+45667& ./01/23+45667&
ii !"!#$%& +,*,-(& +*81(& '($)*& '($)*& '($)*&
Fig 6.15: Palmitoylation IFITM3 mutants re-localise to the lysosome. To investigate whether the cysteine to alanine mutations in IFITM2 effected cellular localisation, Huh-7 cells were transfected with wildtype and palmitoylation mutants (and mCherry-Rab5a expression plasmid) 24 hrs prior to immunofluorescence performed for (i) !-FLAG (green) and anti-mCherry (red) or (i) anti-FLAG (green) and anti-Lamp1 (red). IFITM3 palmitoylation mutants were found to predominantly localise at the lysosome (60x magnification). the NTD (Y20) and S-palmitoylation of conserved cysteine residues (C71, C72 &
C105), specifically C72, is essential for the antiviral activity of IFITM3 against IAV,
VSV and DENV. We wanted to extend these observations to all three IFITM proteins
in the context of HCV infection, and due to time constraints, focused on tyrosine
phosphorylation and S-palmitoylation. The aim of this chapter is to investigate the role
of PTMs on the anti-HCV properties of the IFITM proteins, and our results
demonstrate that S-palmitoylation is essential for the cellular localisation and anti-HCV
actions of all the IFITM proteins, while IFITM2 and IFITM3 require tyrosine phosphorylation at the NTD.
Firstly we focused on tyrosine phosphorylation of the IFITM proteins. We demonstrated that IFITM1, IFITM2 and IFITM3 undergo tyrosine phosphorylation in vitro in Huh-7 cells [Figure 6.1]. Closer examination of the protein sequences identified a conserved tyrosine residue in the CIL as a potential phosphorylation target for all 3 proteins, while specific non-conserved targets were identified for IFITM1 in the C-terminal extension region (Y112 and Y125) and in the NTD of IFITM2 and
IFITM3 (Y19 and Y20 respectively). Examination of the phosphorylation ability of mutants targeting the non-conserved residues of each of the IFITM proteins identified that IFITM1 undergoes tyrosine phosphorylation at Y78 in the CIL, while Y19 and
Y20 are responsible for the phosphorylation status of IFITM2 and IFITM3 respectively
[Figures 6.4 & 6.5].
The next set of experiments focused on the IFITM2 and IFITM3 tyrosine mutants
IFITM2:Y19A and IFITM3:Y20A, as previous studies have shown Y20 to be important for the localisation and antiviral activity of IFITM3 (Jia et al., 2012, John et
121 al., 2013). We confirmed the importance of this conserved tyrosine residue for cellular localisation, as expression of both IFITM2:Y19A and IFITM3:Y20A resulted in a redistribution of the proteins to the cell surface compared to the endosomal wildtype localisation [Figure 6.6]. Consequently, the localisation of the IFITM2:Y19A and
IFITM3:Y20A resembled that of wildtype IFITM1. Transient and stable expression systems were utilised to determine the anti-HCV properties of the IFITM2:Y19A and
IFITM3:Y20A mutants. Both transient [Figure 6.7] and stable [Figure 6.8] expression
of the tyrosine mutants revealed no change in anti-HCV activity; in fact, stable
expression of the mutants resulted in enhanced anti-HCV activity resembling the
decrease observed in the presence of IFITM1. In order to determine whether the loss of
a single tyrosine residue reverted IFITM2 and IFITM3 to biologically resemble
IFITM1, we examined the localisation of IFITM2:Y19A and IFITM3:Y20A in the
context of CD81. Significant co-localisation was observed between the IFITM2 and
IFITM3 tyrosine mutants and CD81 [Figure 6.9] thus mirroring the actions of IFITM1.
Hence, for the first time we demonstrate that a single tyrosine residue conserved in two out of the three IFITM proteins while determining the cellular localisation of the proteins does not necessarily regulate antiviral activity against HCV. Further work is required to confirm an interaction between CD81 and IFITM2:Y19A and
IFITM3:Y20A, as well as enhanced restriction at the level of HCV entry compared to wildtype via a HCVpp assay. However, our results suggest that an association with
CD81 is the mechanism by which the tyrosine mutants retain anti-HCV activity. It is not surprising that this observation has not been previously noted, as the antiviral activity of IFITM1 at the plasma membrane appears to be unique against HCV. Most studies have noted the plasma membrane localisation of IFITM1 but attribute the antiviral activity to intracellular IFITM1. Thus, the retention of antiviral activity of the
122 IFITM2 and IFITM3 tyrosine mutants against HCV but not other viruses is not
unexpected, as IFITM restriction of viral infection at the site of entry through the
disruption of virus-host receptor interactions is unique to HCV infection. Based on our results, we hypothesise that while the IFITM proteins probably act broadly at the site of viral-cell fusion, the exact mechanisms of antiviral activity of these proteins is both virus and cell-type specific.
We also generated cysteine to alanine mutants targeting three conserved cysteine residues known to undergo S-palmitoylation (Yount et al., 2010), in each of the IFITM proteins to determine whether this PTM was important for anti-HCV activity. Our data indicates for the first time that the conserved cysteine residue found in the CIL is important for the anti-HCV activity of all three IFITM proteins, while C71 and C72 found within M1 of IFITM3 are also important for anti-HCV activity [Figure 6.12]. We also demonstrate for the first time that mutation of the cysteine residues within the CIL results in each of the IFITM proteins re-localising to the lysosome, while mutation of the cysteine residue within the M1 region results in IFITM1 and IFITM3 localising predominantly to the lysosome but IFITM2 to retain late endosome localisation
[Figures 6.13, 6.14 & 6.15]. The loss of wildtype localisation of the CIL mutants validates the complete abrogation of anti-HCV activity observed for these mutants as shown in Figure 6.12 and suggests possible degradation of these mutants at the lysosome. However, further work is required to confirm this hypothesis. These results indicate the importance of this cysteine residue and hence S-palmitoylation for the anti-
HCV activity of these proteins. The addition of palmitoyl groups is associated with
protein stability, tethering proteins to membrane cytosolic surfaces and protein
association with lipid rafts, thus explaining the importance of S-palmitoylation for the
123 antiviral activity of the IFITM proteins (Blaskovic et al., 2013). It is interesting, however, to note, that the C50/51A and C70/71A mutations in IFITM1 and IFITM2 respectively, retain partial wildtype localisation and this provides an explanation for the anti-HCV activity observed for these mutants. Nevertheless, only the partial retention of subcellular localistation for these mutants indicates a possiblity that these proteins are also able to associate with either the virus or a new host protein to limit HCV infection, highlighting the versatility of these proteins to retain anti-HCV activity.
Further work is required to confirm that endogenous IFITM proteins undergo S-
palmitoylation via a biotin switch assay and identify the enzyme that causes S-
palmitoylation of the IFITM proteins in hepatocytes. In the instance of IFITM1, while
we propose that the primary mechanism of anti-HCV acitivity is through the disruption
of HCV entry, it is possible that this may not be the sole mechanism based on the
results observed for the C50/51A mutant. However, it is also of importance to note that
despite localising predominantly at the cell surface, CD81 cycles to and from the cell
surface resulting in a pool of intracellular protein (Farquhar et al., 2012). Thus it is
probable that the C50/51A mutant for IFITM1 either associates with this pool of
intracellular CD81, or there is another minor mechanism at play resulting in HCV infection being targeted by multiple fronts. Additionally, HCV core and NS4B proteins
are associated with increased expression and activity of fatty acid synthetase (FASN)
within hepatocytes (Nasheri et al., 2013). FASN is selectively required for the
expression of CLDN-1 and thus plays a role in regulating viral entry and production
(Yang et al., 2008, Nasheri et al., 2013). FASN is also associated with the process of
palmitoylation of a number of different proteins in different cell types. Thus, it is not
inconceivable that the increase in FASN in HCV infected cells could contribute to the
S-palmitoylation of IFN-induced IFITM proteins in infected hepatocytes thus
124 contributing to the anti-HCV activity of the IFITM proteins. In support of this hypothesis, work conducted in our laboratory by a colleague has shown a 2-fold increase in IFITM1 expression in the presence of lipid in Huh-7 cells (Tse et al., 2015).
So, it would be prudent to investigate the S-palmitoylation status of the IFITM proteins in the presence and absence of HCV infection.
In conclusion, the results in this chapter demonstrate that tyrosine phosphorylation of
IFITM2 and IFITM3, as well as S-palmitoylation of all three proteins, is essential for the anti-HCV activity of the IFITM proteins. Our results also provide an insight into the regulation of IFITM expression and localisation within the hepatocyte, and thus confirm the possibility that the IFITM proteins act in a sequential manner to limit HCV infection into the hepatocyte that is both virus and cell-type specific.
125 Chapter 7
Conclusions and Future Directions
HCV has emerged as a significant human pathogen and is one of the most common causes of clinically significant liver disease in many countries (Razali et al., 2009).
One of the most striking features of HCV infection is that approximately 70-80% of infected individuals will develop a chronic infection and in some cases progressive liver disease, characterised by fibrosis, cirrhosis and in some cases HCC (Zhong et al.,
2005). No preventative vaccine is available and prior to 2012, treatment was limited to combination therapy with pegylated IFN-α and the antiviral agent, ribavirin (Feld and
Hoofnagle, 2005). However, the addition of the newly approved DAAs into treatment regimes has significantly improved SVR rates to the point where greater than 95% of infected individuals can be cured. While an IFN free treatment regime is the ultimate goal, at this point IFN-α therapy is still required in combination with DAAs to reduce the development of viral resistance (Aloia et al., 2012, Calle Serrano and Manns, 2012) and to enhance the host innate immune response against a viral infection. However, the molecular mechanisms that underpin the antiviral actions of IFN, not only for HCV, are not fully understood.
In general, viral infection of a target cell results in the activation of a number of viral recognition pathways triggered by viral proteins and/or replication intermediates, resulting in the induction of IFN and ultimately ISGs (Sen, 2001, Smith et al., 2005,
Gale and Foy, 2005). While the number of ISGs with direct antiviral activity is expanding, there is a paucity of information regarding the complete spectrum of ISGs responsible for the restriction of HCV infection (and many other viruses).
126 Understanding ISG expression during viral infection is not only important for
understanding the host-viral relationship but also has the ability to uncover novel
antiviral strategies. As such the main focus of this thesis is to investigate the molecular
mechanism(s) that underpin the antiviral actions of one family of ISGs, the IFITM
proteins.
Through transcriptome analysis of HCV infected liver biopsy samples and in vitro
stimulation of Huh-7 cells with IFN-α (unpublished data), our laboratory has identified
the IFITM proteins (IFITM1, IFITM2 and IFITM3) to be significantly upregulated in
expression. The antiviral properties of the IFITM proteins were first identified in 2009
against IAV, DENV and WNV through siRNA screens and VLP assays (Brass et al.,
2009). These studies were preliminary and exploratory in nature; however, they
suggested that the IFITMs have a role at the level of viral entry although the molecular
mechanism(s) were not established. Since then numerous studies have demonstrated
the IFITM proteins to have antiviral activities against a broad range of RNA viruses
including HIV, VSV and Ebola virus (Lu et al., 2011, Weidner et al., 2010, Huang et
al., 2011, Wrensch et al., 2015). The extensive nature of the IFITM protein’s antiviral
actions, in conjunction with increased expression of these proteins in HCV infected
patients, led us to hypothesise that the IFITM proteins would be able to limit HCV
infection. However, during the course of this PhD, a number of studies have reported
IFITM1 and IFITM3 to limit HCV infection in vitro (Raychoudhuri et al., 2011,
Wilkins et al., 2013, Yao et al., 2011), while several siRNA screens identified the
IFITM proteins to be important against HCV (Metz et al., 2012, Li et al., 2014). The results of these studies confirm our hypothesis that the IFITM family of proteins can limit HCV infection in vitro, however, they are contradictory in defining the target of
127 this antiviral activity. For example, in 2011 Raychoudhri et al reported that IFITM1 specifically targeted HCV RNA replication in order to limit HCV infection in vitro
(Raychoudhuri et al., 2011). In contrast, Wilkins et al demonstrated that IFITM1 targeted HCV entry into the hepatocyte through disruptions in co-receptor interactions
(Wilkins et al., 2013). Clearly these studies are contradictory and thus, the aim of this
PhD study was to comprehensively examine the anti-HCV activity of IFITM1, IFITM2 and IFITM3 at every stage of the HCV lifecycle. We confirm and extend the studies in the literature by corroborating the anti-HCV actions of IFITM1 and IFITM3, while also demonstrating for the first time the anti-HCV properties of IFITM2 and establishing that the IFITM proteins target both the early and late entry stages of HCV infection.
Our studies have shown that the IFITM proteins have no discernable effect on the post- entry stages of HCV infection such as RNA replication, translation or viral egress, which is in contrast to the original studies by Brass et al and Raychoudhri et al (Brass et al., 2009, Raychoudhuri et al., 2011). However, the IFITM proteins were able to limit HCV entry (Chapter 3). Due to the high level of amino acid conservation between the IFITM proteins, it would be assumed that the subcellular localisation of these proteins would be similar. However, this was not the case, with the localisation of each of the IFITM proteins found to be unique in Huh-7 cells. IFITM1 was found to primarily localise to the cell surface (Chapter 4), IFITM2 to the late endosome and
IFITM3 to the early endosome, while all three IFITM proteins were found to localise to lysosomes to some extent (Chapter 5). In-depth studies into the localisation of IFITM1 revealed an interaction with the essential HCV receptor CD81 at the cell surface
(Chapter 4) indicating a possible mechanism where IFITM1 could inhibit HCV entry.
These results corroborated the recent study by Wilkins et al, which revealed that
128 IFITM1 limits HCV infection by disrupting HCV co-receptor assembly (Wilkins et al.,
2013). Unique to this study, we extended investigations into the molecular mechanism of IFITM1 to reveal that the N-terminus, in particular amino acids 21-28, play an important role in the anti-HCV activity of IFITM1, while the C-terminus was found to be important for protein localisation. Time constraints precluded us from defining a precise role for the IFITM1 NTD in HCV entry inhibition. Clearly future work is required to examine the hypothesis that the NTD of IFITM1 is responsible for the interaction with CD81 resulting in the inhibiton of HCV entry. It will also be important in future studies to utilise polarised HepG2 cells, as well as liver tissue from mice treated with or without IFN, to definitively ascertain the cellular localisation, co- receptor interactions and role of IFITM1 in inhibiting HCV entry.
The ability of IFITM2 and IFITM3 to restrict HCV entry, together with the localisation of these proteins at both the early and late endosomes and lysosomes indicated that
IFITM2 and IFITM3 followed the established paradigm of targeting the late entry stages of HCV infection. The IFITM proteins have been demonstrated to limit the late entry stages of a number of RNA viruses (Brass et al., 2009, Jiang et al., 2010, Huang et al., 2011, Feeley et al., 2011, Bailey et al., 2012, Mudhasani et al., 2013); however, the exact mechanisms of actions remain unclear. While the anti-HCV actions of
IFITM1 are unique for HCV infection (interaction with CD81), we postulate that in the case of IFITM2 and IFITM3, the HCV virion is trapped within the endocytic pathway after clathrin-mediated endocytosis and subsequently targeted for lysosomal degradation via IFITM protein interactions at these organelles [Figure 7.1]. The ability of the IFITM proteins, in particular IFITM3, to alter membrane fluidity and membrane curvature to prevent viral hemifusion, as well as evidence that IFITM-induced
129 .'/&
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'0123456#78951289& -'+,& 869:;<2:=5=&
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()*+,)& +<=:=:78H0128& ()*.& 869:=:78&I& !"!#$-& !"!#$'& 2345)2)678&&
D@=5:6&
%D%EF#%D%EF& !"!#$%& +128&>69:=:78& 562841;G:6=& !"!#$-& !"!#$'& /)01%& +<=:=:78&
Figure 7.1: Schematic representation of the localisation and potential moleuclar mechnism of IFITM2 and IFITM3 against HCV. IFITM2 and IFITM3 may exert anti-HCV activity by altering the site of vius-cell fusion and targeting the virion for degrdation via the endo-lysosomal pathway. alterations to endosomal membrane fusion promotes fusion with opposing membranes
expressing IFITM proteins provided a basis for this hypothesis (Li et al., 2013a, Li et
al., 2013b, Desai et al., 2014). However, further questions remain to define the exact mechanism of action of these proteins. The accepted hypothesis that the IFITM proteins, particularly IFITM2 and IFITM3, prevent virus-cell fusion by altering the site of fusion is based largely on circumstantial evidence. Therefore further work is required to focus on and identify the site of this potential disruption, whether it is in the lumina or the membrane, and also to determine whether any cofactors are involved in this process (Bailey et al., 2014). Insights into the cellular functions of the IFITM
proteins could lead to strategies to specifically induce IFITM protein expression or
emulate the function of these proteins through small molecule mimics to improve
existing anti-HCV therapies (and possible other antivirals) and to achieve increased
SVR rates by also potentially bypassing IFN treatment.
While the focus on ascertaining the molecular functions of the IFITM proteins against
HCV is important for the development of novel therapeutics, it is prudent to note that
the IFITM proteins may have more than one function within the hepatocyte upon
upregulation by IFN. Recent studies have identified the well-characterised ISG viperin
to have a role in modulating immune signaling in addition to its antiviral actions
(Saitoh et al., 2011). As IFITM3 is found to localise to the early endosome in
hepatocytes (Chapter 5), the site where TLR3 is able to recognise HCV RNA as a
pathogen (Wang et al., 2009), it is conceivable that IFITM3, like viperin, may have a
role in augmenting TLR3 signaling and contributing to the host innate immune
response in multiple ways. In fact it is plausible that IFITM1 and IFITM2 may aid this
modulation either in a combined or individual manner. Further studies are required to
130 determine whether the IFITM proteins may have a role in augmenting TLR3 signaling
or other signaling pathways, but this possible role could provide an explanation for the
prolonged anti-HCV activity of the IFITM proteins (Chapter 3). As the TLR3 pathway
is one of the first lines of defense against HCV, it is conceivable that a complex
regulation and interplay of co-factors exists during HCV infection that remains to be discovered. It is also plausible that the IFITM proteins may have another role involving exosomes based on recent studies in the literature. Exosome formation occurs upon the fusion of early and late endosomes to result in the formation of multivesicular endosomes that then fuse with the plasma membrane to release exosomes to the extracellular environment (Denzer et al., 2000). A recent study identified the transmission of an antiviral state from DENV infected cells to uninfected cells via exosomes containing IFITM3 (Zhu et al., 2014). Our results demonstrate that in hepatocytes, IFITM2 and IFITM3 are found at the late and early endosomes respectively, and thus it is plausible that the IFITM proteins may be packaged into exosomes. Furthermore, studies have demonstrated HCV infected hepatocytes to release exosomes, though most of these studies have focused on the presence of HCV
RNA within these exosomes (Bukong et al., 2014). Therefore, further studies would be required to determine whether any of the IFITM proteins are released into the extracellular environment via exosomes and transmit antiviral effects from HCV infected to uninfected cells. These findings could also lead to further understanding the
‘bystander effect’ observed in HCV infection, as it is not the virus but the innate and adaptive immune responses to infection that result in liver damage. The identification of IFITM protein-containing exosomes also provides a potentially novel strategy for antiviral therapy, with the use of exosomes as vehicles for drug delivery. These questions can only be answered using a small animal model capable of HCV infection
131 such as the recently established uPA-SCID mouse model with humanized liver or the new genetically humanized mouse model that supports HCV infection (Meuleman et al., 2005, Walters et al., 2006, Dorner et al., 2013). Such models would allow for the investigation of the feasibility and efficacy of using specifically engineered exosomes containing antiviral agents for treating HCV infection.
One important aspect of determining the cellular roles of the IFITM proteins includes understanding the regulation of protein expression in different cell types. Several studies have demonstrated the IFITM proteins, in particular IFITM3, to undergo a number of post-translational modifications (PTMs) including tyrosine phosphorylation, methylation, S-palmitoylation and ubiquitination (Yount et al., 2010, Yount et al.,
2012, Jia et al., 2012, John et al., 2013, Shan et al., 2013). Furthermore, some of these
PTMs have been demonstrated to influence the antiviral actions of the IFITM proteins.
With this in mind, we investigated the importance of phosphorylation and S- palmitoylation on the antiviral action of the IFITM proteins (Chapter 6). A single tyrosine residue present in the NTD of IFITM2 and IFITM3, but not IFITM1, was found to undergo phosphorylation and to our surpise was not important in the anti-
HCV activity of these proteins. Of particular interest, mutation of this specific tyrosine residue resulted in the retention of anti-HCV activity for both IFITM2 and IFITM3, and furthermore resulted in an IFITM1-like phenotype, with the proteins relocalising to the cell surface. While previous studies have described this change in localisation and attributed the conserved tyrosine residue to be part of an endocytic motif (Jia et al.,
2014, Chesarino et al., 2014), the retention of anti-HCV activity is unique. Closer examination revealed the IFITM2 and IFITM3 tyrosine mutants to co-localise with
CD81 on the cell surface; a mechanism of action unique against HCV and further work
132 is required to define a possible interaction. However, we would assume that given that
IFITM1 interacts with CD81 (as determined by FRET and PLA), this would also be
true for unphosphorylated IFITM2 and IFITM3. Interestingly, the antiviral activity of
IFITM2 and IFITM3 was enhanced upon re-localisation to the cell surface, possibly
suggesting that cell surface expression of the IFITM proteins is crucial and the primary
mode in limiting HCV entry.
The anitivial activity of the IFITM proteins against IAV and DENV was found to be
dependent on S-palmitoylation of specific cysteine residues, but not important for anti-
HIV activity (Chutiwitoonchai et al., 2013, Yount et al., 2010). Mutational analysis
targeting cysteine residues previously demonstrated to undergo S-palmitoylation in all
three IFITM proteins revealed a single conserved cysteine residue in the conserved
intracellular loop (CIL) to be crucial for the anti-HCV properties of these proteins,
while the M1 cysteine residue in IFITM3 was also found to be crucial. Significant
redistribution from wildtype localisation was observed for all the IFITM CIL mutants,
and the IFITM3 M1 domain mutant, validating the abrogation of anti-HCV activity
observed. Interestingly, we observed retention of anti-HCV activity, and partial
retention of wildtype localisation, in the mutants targeting the cysteine residues in the
M1 domain of IFITM1 and IFITM2 but not IFITM3. We hypothesise that this may be due to a novel association with either a viral or host factor and further work is required to investigate this hypothesis. For the first time we demonstrate a unique feature of the
IFITM proteins against HCV infection, where a single mutation in IFITM1, IFITM2 and IFITM3 can still maintain significant anti-HCV activity despite significantly altered localisation. This suggests that the exact mechanisms of antiviral activity for the IFITM proteins are both virus and cell-type specific.
133 Although the understanding of protein regulation plays a crucial role in revealing the
mechanism of actions of antiviral proteins, regulation and mutational analysis at the
genetic level is equally important in predicting both disease and treatment outcome. In
2009, treatment procedures for HCV were transformed with the identification that single nucleotide polymorphisms (SNP) found upstream of the IL28B gene had strong correlations to treatment outcome, where the SNP rs12979860 is associated with positive treatment response and rs8099917 being one of the biggest predictors of non- response (Ge et al., 2009, Halfon et al., 2011). Polymorphisms in IL28B have since become the most important pre-treatment predictor of IFN treatment response, better than standard indicators such as ethnicity, baseline viral load and BMI for patients infected with genotype 1, though these factors still remain important. Similar polymorphism predictors have been identified for the IFITM proteins, with recent studies by Everitt et al identified a SNP in the IFITM3 allele, SNP 12252-C, in patient’s hospitialised during the 2009 H1N1 (IAV) pandemic. Individuals homozygous for rs12252-C were found to have more severe H1N1 infections compared to those that did not contain this variation (Everitt et al., 2012, Zhang et al., 2013).
More recently, SNP 12252-C in IFITM3 has been associated with rapid disease progression in acute HIV-1 infected MSM cohorts in China (Zhang et al., 2015).
Additionally, polymorphisms in IFITM1 and IFITM3 have also been associated with susceptibility to ulcerative colitis in Korean populations (Seo et al., 2010, Mo et al.,
2013), while significantly upregulated IFITM protein expression in colorectal tumours have been identified as a new molecular marker for diagnostic purposes (Andreu et al.,
2006). This further highlights that the IFITM proteins have roles beyond antiviral activity. Given these observations and the importance of IL28B polymorphisms in predicting HCV treatment outcome, examining IFITM gene expression for mutations or
134 SNPs within or upstream of the gene to determine whether potential changes correlated with patient response to IFN treatment would be reasonable. Genetic analysis would need to be conducted in patients infected with different HCV genotypes as well as in patients with chronic infection, and response to treatment monitored to determine any correlation between IFITM genetic variation and treatment outcome. As IFITM polymorphisms have been observed in both viral infection and cancer situations, it is plausible that polymorphisms correlating to HCV infection, and HCC, outcomes occur.
It would be interesting to determine whether any possible polymorphisms are specific for a HCV genotype and the rate of occurrence of these genetic variations in different ethnic groups. This could provide another mechanism to improve pre-treatment predictors for individuals infected with all genotypes of HCV.
In summary, we have demonstrated a new family of ISGs, the IFITM proteins -
IFITM1, IFITM2 and IFITM3 are able to significantly restrict HCV infection in vitro via distinct mechanisms. Our data indicates that the IFITM proteins may be acting in a sequential and combined manner to limit HCV entry by directly targeting HCV-host receptor interactions as well as the process of HCV genome release into the cytoplasm.
Based on our studies, we envisage that the IFITM proteins act to limit HCV entry at various stages of entry and these are summarised in Figure 7.1. In the HCV infected liver, an increase in IFN expression can drive the expression of the IFITM proteins in both infected and uninfected bystander hepatocytes. IFITM1 at the cell surface interacts with CD81 to limit HCV/CD81 interactions and hence entry. However, if this first line of defence is breached, IFITM3 can act at the level of Rab5a-positive early endosomes and even possibly at the late endosome stage, through interactions with
IFITM2, at Rab7-positive late endosomes. These late stages would inhibit HCV-
135 endosome fusion and ultimately target HCV viral particles to the lysosome for degradation. Furthermore we established the importance of PTMs, particularly S- palmitoylation and tyrosine phosphorylation of IFITM1, IFITM2 and IFITM3 in the anti-HCV activities of these proteins. Similarly we have determined the role of the N-
and C-terminal domains of the IFITM proteins in localisation within the hepatocyte and
the importance of this localisation against HCV infection. All of the studies conducted
in this thesis are in vitro, and while they provide valuable insight into a new family of
ISGs controlling HCV, the use of a small animal model, such as the genetically
humanized mouse model that supports HCV infection (Dorner et al., 2013), is urgently
required to further advance our understanding of the interplay between the host innate
immune response and HCV and to aid in the generation of novel and targeted anti-HCV
therapeutics.
136 Appendices
Appendix I. General Solutions and Buffers
The following solutions were obtained from the Central Services Unit, School of
Molecular and Biomedical Science, University of Adelaide.
FCS SDS
L-Agar + ampicillin plates Ampicillin
Luria Broth TAE
PBS Tris
GTS SOC media
Saline EDTA
All solutions were obtained at working concentrations or diluted in dH2O accordingly.
Competent Cells
The genotype of the α-Select Chemically Competent Cells used in this was:
- + deoR endA1 recA1 relA1 gyrA96 hsdR17(rk mk ) supE44 thi-1 Δ(lacZYA-argFV169) Φ80δlacZΔM15 F- γ-
137
Solution Components
RIPA Buffer 1% NP-40 5% sodium deoxycholate 0.1% SDS in PBS
Cell Lysis Buffer 1M Tris [pH 7.5] 0.5M EDTA 4M NaCl 0.5% NP-40
10X TAE Loading Buffer 60% sucrose 1% Sarkosyl 1X TAE 0.1% bromophenol blue 0.1% xylene cyanol
12% Separating Gel 12% acrylamide (Sigma) 0.4M Tris (pH 8.8) 0.1% SDS 0.1% ammonium persulfate (Sigma) 0.025% TEMED (Sigma)
5% Stacking Gel 5% acrylamide (Sigma) 0.13M Tris (pH 6.8) 0.1% SDS 0.1% ammonium persulfate (Sigma) 0.1% TEMED (Sigma)
SDS PAGE Running Buffer 2.9% Trisma Base 14.14% glycine 1% SDS
138 2X Laemmli Buffer 4% SDS 16% glycerol 0.02% bromophenol blue
10% β-mercaptoethanol 0.1M Tris (pH 6.8)
SDS PAGE Transfer Buffer 0.3% Tris Base 1.44% glycine 20% methanol
Acetone:Methanol 50% acetone 50% methanol
0.2% (w/v) gelatin 0.2g gelatin 100ml 1xPBS
Gel-Red Solution 30µl Gel-Red 2.5mL 4M NaCl
100mL dH2O 1% Agarose 1g Agarose 100ml 1xTAE buffer
139
Appendix II. Infectious HCV Constructs. Full length JFH1 construct (GT2a) and J6CF (GT2a). The chimeric Jc1 construct consisting of the structural proteins and NS2 of the J6CF clone with the non-structural proteins of JFH1.
140
Appendix III. pGem-T Easy
141
\
Appendix IV. pLenti6/V5-D-TOPO
142 !"!#$%&'!"!#$(&'!"!#$) !"#$ !"#
!"#$%" !
Appendix V. pLenti6/V5-D-TOPO/IFITM (IFITM1, IFITM2, IFITM3)
143 Appendix VI. PRL-HL (Gift from Professor Stanley Lemon)
144
Paper Accepted by Journal of Biological Chemistry - Published online 9th September 2015 (Papers in press) crossmark
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 43, pp. 25946–25959, October 23, 2015 © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
The Interferon-induced Transmembrane Proteins, IFITM1, IFITM2, and IFITM3 Inhibit Hepatitis C Virus Entry* Received for publication, April 15, 2015, and in revised form, September 6, 2015 Published, JBC Papers in Press, September 9, 2015, DOI 10.1074/jbc.M115.657346 Sumudu K. Narayana‡§¶, Karla J. Helbig‡§¶, Erin M. McCartney‡§¶, Nicholas S. Eyre‡§¶, Rowena A. Bullʈ, Auda Eltahlaʈ, Andrew R. Lloydʈ, and Michael R. Beard‡§¶1 From the ‡School of Biological Sciences, and the §Research Centre for Infectious Diseases, University of Adelaide, Adelaide, South Australia 5005, Australia, the ¶Centre for Cancer Biology, SA Pathology, Adelaide, South Australia, 5000, Australia, and the ʈInflammation and Infection Research Centre, School of Medical Sciences, The University of New South Wales, Sydney, NSW 2052, Australia
Background: Interferon-induced transmembrane (IFITM) proteins limit a broad range of RNA viruses. Downloaded from Results: Tyrosine phosphorylation of IFITM2 and IFITM3, and S-palmitoylation of the IFITM proteins, are crucial for anti- hepatitis C virus (HCV) activity. Conclusion: IFITM2 and IFITM3 are able to limit HCV infection by targeting the late entry stages of the virus. Significance: IFITM proteins inhibit HCV at early and late stages of entry. http://www.jbc.org/ The interferon-induced transmembrane (IFITM) family of imately 80% of infected individuals develop a chronic life long proteins have recently been identified as important host effector infection with a proportion progressing to significant liver dis- molecules of the type I interferon response against viruses. ease such as fibrosis, cirrhosis, and in some cases hepatocellular IFITM1 has been identified as a potent antiviral effector against carcinoma (3). The newly approved direct acting antivirals have hepatitis C virus (HCV), whereas the related family members improved sustained virological response rates (upwards of at UNIVERSITY OF ADELAIDE (CAUL) on December 14, 2015 IFITM2 and IFITM3 have been described to have antiviral 95%), however, interferon-␣ (IFN-␣) therapy is still required in effects against a broad range of RNA viruses. Here, we demon- combination to reduce the development of viral resistance (4). strate that IFITM2 and IFITM3 play an integral role in the inter- IFN, whether endogenous or exogenous, induces the expres- feron response against HCV and act at the level of late entry sion of hundreds of interferon-stimulated genes (ISGs), the pri- stages of HCV infection. We have established that in hepato- mary host effectors to mediate an antiviral state against viral cytes, IFITM2 and IFITM3 localize to the late and early endo- infection. Only a handful of ISGs have been characterized in somes, respectively, as well as the lysosome. Furthermore, we limiting HCV replication, and although the spectrum of ISGs have demonstrated that S-palmitoylation of all three IFITM capable of controlling HCV is emerging many more anti-HCV proteins is essential for anti-HCV activity, whereas the con- ISGs remain to be discovered and characterized. served tyrosine residue in the N-terminal domain of IFITM2 Five interferon-induced transmembrane (IFITM) proteins and IFITM3 plays a significant role in protein localization. How- have been identified in humans to date: IFITM1, IFITM2, ever, this tyrosine was found to be dispensable for anti-HCV IFITM3, IFITM5, and IFITM10. IFITM1, IFITM2, and IFITM3 activity, with mutation of the tyrosine resulting in an IFITM1- are inducible by both type I and II IFN (5). IFITM5 is not IFN- like phenotype with the retention of anti-HCV activity and co- inducible and little is known about the function of IFITM10 (6, localization of IFITM2 and IFITM3 with CD81. In conclusion, 7). IFITM1, IFITM2, and IFITM3 have recently been identified we propose that the IFITM proteins act in a coordinated manner as antiviral mediators conferring resistance against a broad to restrict HCV infection by targeting the endocytosed HCV range of viruses including influenza A virus (8, 9), West Nile virion for lysosomal degradation and demonstrate that the virus (WNV) (8, 10), Dengue virus (11, 12), vesicular stomatis actions of the IFITM proteins are indeed virus and cell-type virus (VSV) (13), human immunodeficiency virus (HIV) (14), specific. SARS coronavirus and Marbug virus (15). The IFITM proteins are the first known ISGs to target the late entry step of viral entry by preventing viral-cell fusion, although exact mecha- Hepatitis C virus (HCV)2 is a major public health problem, nisms still remain unclear. They primarily inhibit RNA viruses with over 185 million people infected worldwide (1, 2). Approx- that require low pH-dependent entry into target cells (16, 17). The localization of IFITM3 to the late endosome and lysosome explains the unique antiviral actions of these ISGs, whereas * This work was supported in part by National Health & Medical Research IFITM1 has been found to localize to the plasma membrane Council of Australia Grant 1053206. The authors declare that they have no conflicts of interest with the contents of this article. and the early endosome. IFITM1 has recently been demon- 1 Senior Research Fellow (ID 626906). To whom correspondence should be strated to significantly restrict HCV entry into hepatocytes by addressed: Dept. of Molecular and Cellular Biology, School of Biological disrupting the sequential interactions between the virus and the Sciences, The University of Adelaide, Australia. E-mail: michael.beard@ adelaide.edu.au. 2 The abbreviations used are: HCV, hepatitis C virus; IFITM, interferon-induced transmembrane protein; ISG, interferon stimulated gene; VSV, vesicular assay; FFU, focus-forming units; m.o.i., multiplicity of infection; CIL, con- stomatis virus; CLDN1, Claudin-1; OCLN, occludin; PLA, proximity ligation served intracellular loop; IRES, internal ribosome entry site.
25946 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290•NUMBER 43•OCTOBER 23, 2015 IFITM Proteins Control HCV Entry essential host co-receptors, in particular CD81 (18). Ray- nated Huh-7ϩIFITM1, Huh-7ϩIFITM2, Huh-7ϩIFITM3, and choudhri et al. (19, 20) have also reported the anti-HCV actions Huh-7ϩVector. Lentiviral particles were prepared by co-trans- of IFITM1, whereas the expression of IFITM1 has been shown fecting 293T cells with equivalent amounts of the packaging to be regulated by HCV through the up-regulation of mir-130a. vectors psPAX2 (Addgene 12260), pRSV-Rev (Addgene 12253), Several genomic screens have identified IFITM2 and IFITM3 as and pMD2.G (Addgene 12259), along with the IFITM and possible anti-HCV effectors at the level of HCV RNA replica- empty plasmid DNAs. Supernatant containing virus was col- tion and translation, however, more comprehensive studies are lected 48 and 72 h post-transfection, filtered (0.45 m), and required to characterize the relative anti-HCV activity and the applied to Huh-7 cells at a 1:5 ratio with normal culture mode of action (21). medium. Polyclonal cell populations were selected with 3 HCV entry into the hepatocyte is coordinated through g/ml of blasticidin. sequential interactions with the essential co-receptors: SR-BI, Virus Generation and Infection—Cell culture-propagated CD81, Claudin-1 (CLDN1), and Occludin (OCLN). The entry HCV particles (HCVcc, Jc1) were generated as described else- pathway is not completely understood, although it is postulated where (28) and used experimentally at m.o.i. 0.03. Pseudovi- that HCV binds LDL receptors and heparin sulfate glycosami- ruses encoding luciferase were generated by co-transfection of Downloaded from noglycans leading to high-affinity interactions with SR-BI and 293T cells with the packaging plasmid pNL43-LucRE and the CD81 on the hepatic surface (22). The interaction of HCV VSV-G envelope expression plasmid pMD2.G (Addgene plas- bound CD81 with the tight junction molecule CLDN1 initiates mid 12259) for generation of VSVpp, the expression plasmid clathrin-mediated endocytosis of the HCV virion resulting in pE1E2-GT1b (212) for generation of HCVpp, the expression its traffic along actin stress fibers to Rab5a-containing early plasmid pMLV for generation of MLVpp, or empty plasmid http://www.jbc.org/ endosomes (22, 23). Fusion and acidification of the endosome pcDNA3 for generation of Env-pp. Supernatants were har- results in the release of the viral genome to the cytoplasm where vested at 48 h post-transfection and filtered (0.45 m). Virus- it is directly translated. Because HCV entry into the hepatocyte containing cell culture supernatants were incubated with target is low pH-dependent and utilizes the endocytic pathway, it is cells, seeded at 8 ϫ 103 cells/cm2 the day before (unless other- plausible that IFITM2 and IFITM3 may contribute to the anti- wise specified), overnight before washing with PBS, and
HCV response of IFN in a mechanism similar to that observed returned to culture. Pseudoparticle infections were performed at UNIVERSITY OF ADELAIDE (CAUL) on December 14, 2015 for other viruses. in the presence of 10 g/ml of Polybrene. At 72 h post-infection To this end, we investigated the role of IFITM2 and IFITM3 luciferase activity was measured using a Luciferase Assay Sys- in the context of the full HCV life cycle, including entry, trans- tem (Promega) and a GloMax 96 microplate luminometer (Pro- lation, replication, and egress. We present evidence that mega). Specific HCVpp, MLVpp, and VSVpp infectivity levels IFITM2 and IFITM3 display anti-HCV activity that may com- were determined by subtraction of the luciferase signals asso- plement the anti-HCV activity of IFITM1 (18) by inhibiting the ciated with the use of non-enveloped pseudoparticles (Env-pp). late stages of HCV entry, possibly in a coordinated manner by Huh-7ϩIFITM stables were electroporated as described (3) trapping the virion in the endosomal pathway and targeting it and plated into 10-cm dishes for 24 h. To determine the amount for degradation at the lysosome. Furthermore, we demonstrate of intracellular infectious virus, the cells were harvested via that post-translational modifications, in particular S-palmitoy- trypsinization, resuspended in complete medium, washed lation and tyrosine phosphorylation to contribute to the cellu- twice with 1ϫ PBS, and lysed via 4 freeze/thaw cycles at Ϫ80 °C. lar localization and the anti-HCV activities of the IFITM Lysates were then clarified by centrifugation at 2300 ϫ g for 5 proteins. min prior to inoculation on to naive Huh-7 cells. Extracellular medium was collected at the same time. Amounts of intracel- Experimental Procedures lular and extracellular infectious virus were determined by Plasmid DNA and Transfections—Human IFITM genes were focus forming assay (3). PCR-amplified from cDNA synthesized from Huh-7 cells stim- Immunoblotting—Western blotting was performed as de- ulated with IFN-␣ (1000 units/ml) for 16 h using primers and scribed elsewhere (29) and used the following antibodies: cloned, in-frame, into BamHI and XhoI sites of pLenti6/V5-D- mouse anti-FLAG (Sigma) diluted at 1/1000 and mouse anti- TOPO (Invitrogen). A FLAG tag was attached to the N termi- phosphotyrosine (Millipore) diluted at 1/1000. Mouse anti-hu- nus of each IFITM. Transfection of all plasmids was performed man -actin (Sigma) was used to control loading of protein at using FuGENE6 (Roche Applied Science) according to the 1:10,000. Appropriate secondary antibodies labeled with horse- manufacturer’s recommendations. Mutant versions of each radish peroxidase (Cell Signaling) were used, and bound pro- protein were constructed into pLenti6/V5-D-TOPO utilizing a tein was detected by chemiluminescence using SuperSignal QuikChange II XL Site-directed Mutagenesis system (Strat- West Femto (Pierce). agene, La Jolla, CA). Plasmids pLenti6-mCherry-Rab5a, pRC- Immunoprecipitation—Immunoprecipitation of FLAG-tagged CMV-Viperin, and pRL-HL have been previously described proteins was carried out as described (29), with samples har- (24–26). vested in the presence of phosphatase inhibitor (Calbiochem). Establishment of Cell Lines and Culture Conditions—The Immunofluorescence Microscopy—Cells were grown on 0.2% human hepatoma cell lines Huh-7 and the HCV genomic rep- gelatin-coated coverslips overnight, transfected where applica- licon line NNeoC-5B(RG) were maintained as previously ble, and fixed the following day using acetone/methanol (1:1) described (27). Huh-7 cells stably expressing the IFITM pro- for 5 min on ice for standard fluorescence microscopy or with teins and an empty vector control were generated and desig- 4% parafomaldehyde for 10 min on ice followed by a 10-min
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25948 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290•NUMBER 43•OCTOBER 23, 2015 IFITM Proteins Control HCV Entry
incubation in 0.1% Triton X-100 in PBS for confocal micros- genes contain ISRE and GAS elements (5). Thus we hypothe- copy, prior to incubation with primary antibodies at room sized that based on the high similarity in protein sequence and temperature for 1 h. Cells were washed in PBS and incubated the recent findings in the literature, IFITM2 and IFITM3 may with secondary antibodies for1hat4°Cbefore being also have a significant antiviral activity against HCV. Initially mounted with ProLong Gold reagent (Invitrogen). Images we investigated the ability of Huh-7 cells and primary human were acquired with a Bio-Rad Radiance 2100 Confocal or a hepatocytes to express IFITM1 in the presence of IFN-␣/. As Nikon TiE inverted microscope. Mouse monoclonal anti- expected IFN-␣ and - induced expression of IFITM1 mRNA FLAG and rabbit polyclonal anti-FLAG were obtained from and protein in a concentration- and time-dependent manner Sigma. Rabbit monoclonal antibodies against Rab5a, Rab7, and (data not shown). Due to the high level of protein conservation Lamp1 were obtained from Cell Signaling. Mouse monoclonal between IFITM2 and IFITM3, we were unable to design spe- anti-CD81 was obtained from BD Pharmingen, rabbit poly- cific RT-PCR primers or obtain specific antibodies for either clonal anti-mCherry was obtained from BioVision, and human protein and thus we were unable to extend these observations anti-HCV serum was generated as previously described (3). to endogenous IFITM2 or IFITM3. To determine the role of HCV RNA Quantitation—Extraction of total cellular RNA, endogenous IFITM1 following IFN-␣ stimulation, we gener- Downloaded from first-strand cDNA synthesis, and real-time RT-PCR was per- ated a polyclonal Huh-7 cell line stably expressing shRNA spe- formed as described elsewhere (26). cifically targeting IFITM1 mRNA (Fig. 1B, i). These cells were Fluorescence Energy Resonance Transfer (FRET) Analysis— either pretreated with 50 IU/ml of IFN-␣ for 24 h followed by FRET by acceptor photobleaching was carried out essentially as HCV Jc1 infection or infected with Jc1 prior to stimulation with described previously (30). IFN-␣ (Fig. 1B, ii). Interestingly the anti-HCV activity of IFN-␣ http://www.jbc.org/ Proximity Ligation Assay (PLA)—Cells were cultured on 0.2% is attenuated in IFITM1 shRNA cells compared with controls (w/v) gelatin-coated coverslips in 24-well culture plates prior to only in the pre-IFN-␣ treatment setting. These results suggest fixation with 4% paraformaldehyde. Proximity ligation assay that IFITM1 plays an important, but not exclusive, role in the (PLA) was conducted using the Duolink In situ kit (Olink antiviral effects of IFN-␣ against the early stages of HCV infec- Biosciences) as per the manufacturer’s instructions. Positive tion in vitro. interactions visualized using a Nikon Eclipse TiE fluorescence at UNIVERSITY OF ADELAIDE (CAUL) on December 14, 2015 To characterize the relative anti-HCV roles of IFITM1, inverted microscope and images were captured using NIS Ele- IFITM2, and IFITM3 and their cellular localization, we gener- ments software. ated polyclonal Huh-7 cells stably expressing each of the IFITM Statistics—Results are expressed as mean Ϯ S.E. Student’s t proteins (Huh-7ϩIFITM) with a N-terminal FLAG tag to facil- test was used for statistical analysis. p Ͻ 0.05 was considered to be significant. All statistical analysis was performed using Prism itate detection. Protein expression in these stable cell lines was 6 (GraphPad Software). confirmed by immunofluorescence and immunoblot analysis (Fig. 1C). In Huh-7 cells, IFITM1 localized predominantly to Results the cell surface with some intracellular localization, whereas IFITM1, IFITM2, and IFITM3 Limit HCV Infection in Vitro— IFITM2 and IFITM3 localized to specific intracellular compart- Several functional genomics screens have identified IFITM1, ments within the cytoplasm. HCV infection (Jc1) of the stable ϩ IFITM2, and IFITM3 as potential anti-HCV effector molecules Huh-7 IFITM cell lines and analysis of HCV RNA 24 h there- (21, 31, 32), with a recent study by Wilkins et al. (18) character- after demonstrated that IFITM1, IFITM2, and IFITM3 were izing the anti-HCV nature of IFITM1. Examination of the able to significantly decrease HCV RNA levels by 77, 61, and IFITM family of proteins (Fig. 1A) revealed that all three IFITM 57%, respectively (Fig. 1D), compared with vector control. Sim- proteins share high amino acid homology, containing two ilar results were obtained upon extending HCV infection to 48 hydrophobic membrane-associated domains (M1 and M2) sep- and 72 h. Interestingly, Huh-7ϩIFITM cells infected with Jc1 arated by a conserved intracellular loop (CIL) but differing at for 72 h depicted a “viral exclusion” phenotype, where cells their N- and C-terminal domains. In contrast to IFITM1, expressing the IFITM proteins do not appear to be infected IFITM2 and IFITM3 contain 20 and 21 amino acid extensions with HCV, whereas neighboring cells lacking IFITM expression at the N-terminal domain, respectively, whereas IFITM1 con- were infected (Fig. 1E). These results corroborate the known tains a 13-amino acid extension at the C terminus (5, 33). It has anti-HCV activity of IFITM1, and for the first time demonstrate also been shown that the promoter regions of all three IFITM the anti-HCV nature of IFITM2 and IFITM3.
FIGURE 1. IFITM2 and IFITM3 inhibit HCV infection. A, schematic representation of human IFITM1, IFITM2, and IFITM3. IFITM1 differs from IFITM2 and IFITM3 with a 21-amino acid truncation at the N terminus and a 13-amino acid extension at the C terminus. B, i, Huh7ϩshControl and Huh-7ϩshIFITM1 cells were stimulated with 100 IU/ml of IFN-␣ for 16 h. Total RNA was harvested for RT-quantitative PCR for IFITM1 mRNA levels (data represented as a mean Ϯ S.E. with a significance of ****, p Ͻ 0.0001 calculated using a Student’s t test). ii, Huh7ϩshControl and Huh-7ϩshIFITM1 cells were either pretreated for 24 h before Jc1 infection (m.o.i. ϭ 0.03) or post-treated 24 h after Jc1 infection for 16 h with 50 IU/ml of IFN-␣. Total RNA was harvested for RT-quantitative PCR for HCV RNA levels (data represented as a mean Ϯ S.E. with a significance of *, p ϭ 0.035, calculated using a Student’s t test). C, Huh-7ϩIFITM and Huh-7ϩvector control cells were either stained with a mouse monolconal anti-FLAG antibody, followed by an Alexa 555-conjugated anti-mouse IgG (i) or cellular lysate harvested to detect specific IFITM protein expression (ii). D, Huh-7ϩIFITM and Huh-7ϩvector control cells were infected with HCV Jc1 (m.o.i. 0.03). Total RNA was harvested at the indicated time points for RT-quantitative PCR for HCV RNA levels (data represented as a mean Ϯ S.E. with a significance of *, p Ͻ 0.05; **, p Ͻ 0.005 calculated using a Student’s t test). E, Huh-7ϩIFITM and Huh-7ϩvector control cells were infected with HCV Jc1 (m.o.i. 0.03) and immunofluorescence analysis conducted 72 h later using mouse monoclonal anti-FLAG and human anti-HCV serum antibodies, followed by an Alexa 555-conjugated anti-mouse IgG and an Alexa 488-conjugated anti-human IgG, respectively.
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FIGURE 3. IFITM1, IFITM2, and IFITM3 have no effect on HCV RNA replication, translation, or egress. A, i, HCV genomic replicon cells were transduced with each of the IFITM proteins, viperin, and the vector control for 48 h and total RNA harvested for RT-quantitative PCR for HCV RNA levels (data represented as a mean Ϯ S.E.). ii, HCV genomic replicon cells were transduced with IFITM proteins and the vector control and immunofluorescence analysis conducted 48 h later using mouse monoclonal anti-FLAG and human anti-HCV serum antibodies, followed by an Alexa 555-conjugated anti-mouse IgG and an Alexa 488- conjugated anti-human IgG respectively. B, Huh-7ϩIFITM and Huh-7ϩvector control cells were transiently transfected with IRES-luc for 48 h. 24 h post-transfection vector control cells were treated with 100 ng/ml of IL-29 for 24 h. Luciferase activity determined 48 h post-transfection (data represented as a mean Ϯ S.E.). C, Huh-7ϩIFITM and Huh-7ϩvector control cells were electroporated with Jc1 and extracellular and intracellular supernatant was applied to naive Huh-7 cells 24 h post-electroporation. At 72 h post-infection focus-forming units were enumerated using human anti-HCV serum antigens. i, extracellular FFU and intracellular FFU titers expressed as FFU/ml. ii, FFU/ml is expressed as a ratio of extracellular FFU to intracellular FFU (data represented as a mean Ϯ S.E.).
IFITM Proteins Inhibit HCV Infection at an Early Stage of ticles (HCVpp) to enter Huh-7ϩIFITM cells in comparison to Infection—Recent studies have identified IFITM1 to be able to vector control cells (Fig. 2A). As expected, we observed a signif- limit HCV infection at the level of HCV entry through an inter- icant reduction in the entry of HCVpp into cells expressing action with the essential entry co-receptor CD81 (18). To deter- IFITM1 compared with control. Similar reductions in HCVpp mine whether this mechanism was also true for IFITM2 and entry were observed in cells expressing IFITM2 and IFITM3, IFITM3, we compared the ability of HCV (E1/E2) psuedopar- demonstrating that the anti-HCV activity observed for these
FIGURE 2. IFITM1, IFITM2, and IFITM3 limit HCV entry into Huh-7 cells. A, Huh-7ϩIFITM and Huh-7ϩvector control cells were transduced with pseudoparticles containing envelopes of HCV, MLV, and VSV. Viral entry was determined by luciferase activity 72 h post-transduction (data represented as a mean Ϯ S.E. with a significance of *, p Ͻ 0.02; ***, p Ͻ 0.003; ****, p Ͻ 0.0001 calculated using a Student’s t test). B, Huh-7ϩIFITM cells were stained with rabbit polyclonal anti-FLAG and mouse anti-CD81 antibodies, followed by an Alexa 488-conjugated anti-rabbit IgG and an Alexa 555-conju- gated anti-mouse IgG. C, Huh-7ϩIFITM1 cells were stained with a rabbit polyclonal anti-FLAG and mouse anti-CD81, followed by an Alexa 555- conjugated anti-rabbit IgG and a CY5 goat anti-mouse IgG, respectively. Cells were analyzed on a Zeiss Axioplan microscope using FRET (Carl Zeizz, Oberkochen, Germany). DIF, difference in fluorescence.
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FIGURE 4. IFITM2 and IFITM3 partially co-localize with early and late endosomes, whereas all three IFITM proteins co-localize with lysosomes. A, Huh-7ϩIFITM2 and Huh-7ϩIFITM3 cells were transfected with mCherry-Rab5a expression plasmid. 24 h later cells were stained with mouse monoclonal anti-FLAG and rabbit polyclonal anti-mCherry antibodies, followed by an Alexa 488-conjugated anti-mouse IgG and an Alexa 555-conjugated anti-rabbit IgG (20 m scale bar). B, Huh-7ϩIFITM2 and Huh-7ϩIFITM3 cells were stained with mouse monoclonal anti-FLAG and rabbit monoclonal anti-Rab7 antibodies, followed by Alexa 488-conjugated anti-mouse IgG and Alexa 555-conjugated anti-rabbit IgG (20 m scale bar). C, Huh-7ϩIFITM cells were stained with mouse monoclonal anti-FLAG and rabbit monoclonal anti-Lamp1 antibodies, followed by an Alexa 488-conjugated anti-mouse IgG and an Alexa 555-conjugated anti-rabbit IgG (20 m scale bar). proteins is also at the level of HCV entry. Based on these results 2C) and PLA (data not shown). In contrast, IFITM2 localized and even though IFITM2 and IFITM3 show a predominantly solely to the cytoplasm and hence did not co-localize with CD81, intracellular localization, we next examined the localization of however, IFITM3, whereas predominantly cytoplasmic, did par- IFITM2 and IFITM3 in relationship to all HCV entry factors tially co-localize with CD81 in some instances (Fig. 2B). None of (CD81, SR-BI, CLDN1, and OCLN). We confirmed the co-local- the other HCV entry receptors tested (SR-BI, CLDN1, and OCLN) ization (Fig. 2B) of IFITM1 with CD81 and extended this to show a were found to interact with any of the IFITM proteins as deter- physical interaction between IFITM1 and CD81 via FRET (Fig. mined by FRET and PLA (data not shown).
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FIGURE 5. Conserved N-terminal tyrosine residue is important for IFITM2 and IFITM3 cellular localization but not anti-HCV activity. A, schematic representation of IFITM2 and IFITM3 and the tyrosine mutant derivatives. B, Huh-7ϩIFITM2, Huh-7ϩIFITM3, Huh-7ϩIFITM2:Y19A, and Huh-7ϩIFITM3:Y20A cells treated with a phosphatase inhibitor mixture before cellular lysates were harvested. IFITM proteins were precipitates with anti-FLAG antibodies and then tested in Western blotting with anti-tyrosine phosphorylation antibodies. C, IFITM2, IFITM3, and tyrosine mutant overexpression cells were stained with mouse monoclonal anti-FLAG, followed by an Alexa 555-conjugated anti-mouse IgG. D, i, Huh-7ϩIFITM2, Huh-7ϩIFITM3, Huh-7ϩIFITM2:Y19A, Huh-7ϩIFITM3:Y20A, and Huh-7ϩvector control cells were infected with HCV Jc1 (m.o.i. 0.03). Total RNA was harvested at the indicated time points for RT-quantitative PCR for HCV RNA levels (data are represented as mean Ϯ S.E. with a significance of p ϭ 0.004 calculated using a Student’s t test); ii, Huh-7ϩIFITM2:Y19A and Huh-7ϩIFITM3: Y20A were infected with HCV Jc1 (m.o.i. 0.03) and immunofluorescence analysis was conducted 72 h later using mouse monoclonal anti-FLAG and human anti-HCV serum antibodies, followed by an Alexa 555-conjugated anti-mouse IgG and an Alexa 488-conjugated anti-human IgG, respectively. IP, immunoprecipitation.
We next investigated the role of the IFITM proteins on the proteins and cells harboring HCV replication suggesting that rest of the HCV life cycle. Transient expression of each of the the IFITM proteins do not impact upon HCV RNA replication. IFITM proteins did not significantly effect HCV RNA replica- Furthermore, the IFITM proteins had no effect on HCV IRES tion in genomic HCV replicon cell lines (Fig. 3A, i). This is in promoter activity, upon transfection of the Huh-7ϩIFITM cells contrast to the anti-HCV activity of the well characterized ISG with a construct containing the HCV IRES driving a luciferase viperin that served as a positive control (30, 34). Interestingly, reporter gene (Fig. 3B). Finally we examined HCV egress using the viral exclusion phenotype observed using the HCVcc sys- an extracellular:intracellular infectivity assay, where Huh- tem was no longer evident upon expression of the IFITM pro- 7ϩIFITM and vector control cells were electroporated with teins in these cells (Fig. 3A, ii), with co-expression of IFITM HCV Jc1 RNA to bypass entry and after 24 h the extracellular
OCTOBER 23, 2015•VOLUME 290•NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 25953 IFITM Proteins Control HCV Entry and intracellular supernatant was applied onto naive Huh-7 cells prior to measurement of HCV infectivity by a focus-form- ing assay. IFITM1, IFITM2, and IFITM3 did not limit HCV egress following analysis of both raw HCV focus-forming units (FFU) (Fig. 3C, i) and the ratio of extracellular FFU:intracellular FFU (Fig. 3C, ii). Collectively this data demonstrates that all three IFITM proteins block de novo HCV infection with no detectable effect on other stages of the HCV life cycle, indicat- ing that IFITM1, IFITM2, and IFITM3 primarily act on the early stages of HCV infection. IFITM2 and IFITM3 Co-localize with Early and Late Endo- somes and Lysosomes in Hepatocytes—The cellular localization of IFITM2 and IFITM3, particularly IFITM3, has been exam- ined in a number of different cell lines (A549, HEK293, and Downloaded from HeLa) in relationship to their antiviral activity against other viruses (9, 15, 16, 35). These studies have identified that IFITM2 and IFITM3 partially co-localize with late endosomes and lyso- somes within the majority of cells. We sought to investigate this localization within Huh-7 cells, and probed the Huh-7ϩIFITM http://www.jbc.org/ cells with specific antibodies targeting early endosomes (Rab5a), late endosomes (Rab7), and lysosomes (Lamp1). Co- localization immunofluorescence analysis found that in Huh-7 cells, IFITM2 and IFITM3 partially co-localized with late and early endosomes, respectively (Fig. 4, A and B). Interestingly, both IFITM2 and IFITM3 were found to co-localize with lyso- at UNIVERSITY OF ADELAIDE (CAUL) on December 14, 2015 somes, whereas partial co-localization between intracellular IFITM1 and lysosomes was also observed in some cells (Fig. 4C). Although these observations were not solely unexpected, it is interesting to note the difference in localization of the IFITM proteins between cell types. In the context of this study, the endosomal localization of IFITM2 and IFITM3 is most note- worthy, as HCV entry requires Rab5a-positive endosomes, FIGURE 6. IFITM2:Y19A and IFITM3:Y20A mutants co-localize with CD81 whereas the formation of the HCV replication complex re- on the cell surface. Huh-7ϩIFITM cells and Huh-7ϩIFITM2:Y19A and Huh- quires both Rab5a and Rab7 (36). 7ϩIFITM3:Y20A cells were stained with rabbit polyclonal anti-FLAG and The Conserved N-terminal Tyrosine Residue Common to mouse anti-CD81 antibodies, followed by an Alexa 555-conjugated anti-rab- bit IgG and an Alexa 488-conjugated anti-mouse IgG. IFITM2 and IFITM3 Crucial for Cellular Localization but Does Not Affect Anti-HCV Activity—Tyrosine residue 20 (Tyr-20) in the N-terminal domain of IFITM3 has recently been identified ylated tyrosine (Tyr(P)). A complete loss in phosphorylation to be important for both cellular localization and antiviral activ- was observed for Y19A and Y20A compared with wild-type ity, particularly against influenza A virus, Dengue virus, and IFITM2 and IFITM3, respectively (Fig. 5B). We extended our VSV (16, 37). Furthermore, recent studies have demonstrated observations to the cellular localization of IFITM2:Y19A and Tyr-20 to be part of an endocytic signal (YEML) targeting IFITM3:Y20A compared with wild-type, as mutation of Tyr-20 IFITM3 to the late endosome (38, 39). We examined the pro- has previously been reported to change IFITM3 localization tein sequences between the three IFITM proteins and discov- from the endosome to the plasma membrane and abrogate anti- ered that this N-terminal tyrosine residue is conserved in viral activity. Immunofluorescence analysis confirmed this IFITM2 (Tyr-19) but is not present in IFITM1 due to the 21- redistribution, with the localization of Y20A within Huh-7 cells amino acid N-terminal truncation (Fig. 5A). Based on this obser- resembling that of wild-type IFITM1 on the cell surface (Fig. vation, we decided to investigate the importance of this con- 5C). Y19A also displayed a change in localization although not served N-terminal tyrosine residue on the anti-HCV activity of as striking as that of Y20A, where the majority of protein local- IFITM2 and IFITM3. We generated tyrosine to alanine mu- ization was redistributed to the plasma membrane with some tants for IFITM2 and IFITM3, Y19A and Y20A, respectively, perinuclear localization still present (Fig. 5C). We next sought and created polyclonal constitutively expressing cell lines as to determine whether this change in localization would alter previously described. First we sought to confirm the loss in the anti-HCV activity of IFITM2 and IFITM3. HCV infection of phosphorylation upon mutating the tyrosine residue, where cells stably expressing either wild-type or tyrosine mutant IFITM2, IFITM3, and the tyrosine mutant cells were treated IFITM2 and IFITM3 for 24 h revealed no loss in anti-HCV with a phosphatase inhibitor mixture prior to harvesting for activity in cells expressing the tyrosine mutants compared with immunoprecipitation. The precipitated samples were probed vector control (Fig. 5D, i). The viral exclusion phenotype was by immunoblot with antibodies specifically targeting phosphor- retained in cells expressing the tyrosine mutants, where Jc1
25954 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290•NUMBER 43•OCTOBER 23, 2015 IFITM Proteins Control HCV Entry Downloaded from http://www.jbc.org/ at UNIVERSITY OF ADELAIDE (CAUL) on December 14, 2015 FIGURE 7. IFITM1, IFITM2, and IFITM3 require palmitoylation for anti-HCV activity. A, schematic representation of IFITM1, IFITM2, and IFITM3 and the palmitoylation mutant derivatives. B, Huh-7 cells were transfected with wild-type IFITM proteins, the palmitoylation mutants, and vector control (empty) for 24 h prior to infection with HCV Jc1 (m.o.i. 0.003). Total RNA was harvested 24 h post-infection for RT-quantitative PCR for HCV RNA levels (data represented as a mean Ϯ S.E. with a significance of **, p Ͻ 0.001; ***, p ϭ 0.003; ****, p Ͻ 0.0001 calculated using a Student’s t test). infection was only observed in neighboring cells lacking residue of IFITM3 to be vital for antiviral activity against influ- IFITM2:Y19A and IFITM3:Y20A expression (Fig. 5D, ii). Fur- enza A virus and Dengue virus (16, 41, 42). Interestingly, it has thermore, it can be noted that the Y19A and Y20A mutants been noted that S-palmitoylation of the IFITM proteins is not exhibited enhanced anti-HCV properties compared with wild- required for their anti-HIV activity (43). To examine whether type. These results were unexpected based on previous studies S-palmitoylation of the IFITM proteins was important for anti- where the mutation of the tyrosine resulted in loss of antiviral HCV activity, double (M1, C50A/C51A; M2, C70A/C71A; M3, activity for IFITM3. Interestingly, the enhanced anti-HCV ac- C71A/C72A) and single (M1, C84A; M2, C104A; M3, C105A) tivity of both Y19A and Y20A resemble the level of anti-HCV cysteine to alanine mutants were generated for each of the activity observed in Fig. 1C for IFITM1. To investigate whether IFITM proteins (Fig. 7A). Transient expression of wild-type the loss of a single tyrosine residue reverted IFITM2 and and palmitoylation mutants prior to infection with HCV (Jc1) IFITM3 to an IFITM1-like phenotype, we examined the local- for 24 h revealed the single cysteine residue in the CIL of each ization of Y19A and Y20A in the context of CD81. Significant IFITM proteins to be important for anti-HCV activity, as a co-localization was observed between the IFITM2 and IFITM3 complete loss of anti-HCV activity was observed for the CIL tyrosine mutants and CD81 (Fig. 6), mirroring the localization mutants compared with vector control (Fig. 7B). IFITM3, how- of IFITM1. These results demonstrate for the first time that the ever, also requires the two cysteine residues found in the first conserved N-terminal tyrosine residue in IFITM2 and IFITM3 membrane-associated domain (M1) for its anti-HCV activity. is important for cellular localization but not for the anti-HCV Previous studies have reported conflicting roles of S-palmitoy- properties of these proteins. lation on IFITM localization within the cell. Yount et al. (41) Palmitoylation of the IFITM Proteins Is Important for Anti- demonstrated that S-palmitoylation played a role in the clus- HCV Activity—S-palmitoylation is a post-translational modifi- tering of IFITM3 near the ER, whereas John et al. (16) showed cation resulting in the addition of a palmitoyl group to cytosolic the converse, where the removal of the cysteine residues cysteine residues and in many instances is essential for protein resulted in significant clustering within the cell. To determine stability, localization, and association with lipid rafts (40). whether a similar change in localization occurred in Huh-7 Yount et al. (41) identified for the first time that the IFITM cells, the cellular localization of the palmitoylation mutants was proteins undergo S-palmitoylation at three specific cysteine examined in relationship to previously identified subcellular residues: two consecutive cysteine residues found in the first markers (Fig. 8). We demonstrate for the first time that muta- membrane-associated domain (M1), whereas the other cys- tion of the cysteine residue within the CIL resulted in each of teine residue is in the CIL. Additional studies have identified the IFITM proteins predominantly localizing to the lysosome. S-palmitoylation of the IFITM proteins, particularly the Cys-72 This redistribution was also observed for M1 palmitoylation
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IFITM2, respectively, retain partial wild-type localization and this provides an explanation for the anti-HCV activity observed for these mutants. Nevertheless, the partial retention of subcel- lular localization for these mutants indicates a possibility that these proteins are also able to associate with either the virus or a new host protein to limit HCV infection, highlighting the versatility of these proteins to retain anti-HCV activity.
Discussion The antiviral actions of the type I interferon response is key in the control of viral infection and the foundation behind interferon therapy for chronic hepatitis C virus infection that is still used in conjunction with the new wave of direct acting antivirals. IFN induces the expression of hundreds of antiviral Downloaded from ISGs; however, the subset of ISGs essential to mediate the IFN response against HCV and many other viruses is yet to be elucidated. Previous studies using genome-wide siRNA screens have identified the importance of IFITM1, IFITM2, and IFITM3 http://www.jbc.org/ in the antiviral response against HCV, whereas recent stud- ies have identified a specific role for IFITM1 against HCV entry (18). Here we confirm and extend these studies further by first corroborating the ability of IFITM1 to interact with CD81 to limit HCV entry, whereas also establishing IFITM2 and
IFITM3 as anti-HCV ISGs that target the late-entry stages of at UNIVERSITY OF ADELAIDE (CAUL) on December 14, 2015 viral infection. IFITM2 and IFITM3 have no discernable effect on the post-entry stages of HCV infection such as RNA repli- cation, translation, or viral egress but were able to limit HCV entry. Although the observed restriction of HCV entry was not as significant as that observed for IFITM1 (using a HCVpp assay), the localization of IFITM2 and IFITM3 at both early and late endosomes as well as lysosomes indicates that these pro- teins are following the established paradigm of acting at the late entry stages of HCV entry. Our data suggests that the IFITM FIGURE 8. Cysteine mutations targeting the CIL region of the IFITM pro- proteins may be acting in a sequential and combined manner to teins re-localize to the lysosome. i, Huh-7 cells transiently transfected for 24 h with either wild-type IFITM1 or the respective IFITM1 palmitoylation limit HCV entry by directly targeting HCV-host receptor inter- mutants were stained with mouse monoclonal anti-FLAG and either mouse actions as well as the processes of uncoating and release of the anti-CD81 or rabbit monoclonal anti-Lamp1 antibodies, followed by an Alexa 488-conjugated anti-mouse IgG and an Alexa 555-conjugated anti-rabbit HCV genome into the cytoplasm (Fig. 9). IgG. ii, Huh-7 cells transiently transfected or 24 h with either wild-type IFITM2 Endosomal-lysosomal degradation is a sequential process or the respective IFITM2 palmitoylation mutants were stained with mouse requiring endosomal maturation starting at the early endo- monoclonal anti-FLAG and either rabbit monoclonal anti-Rab7 or rabbit monoclonal anti-Lamp1 antibodies, followed by an Alexa 488-conjugated some, moving to the late endosome and culminating in the anti-mouse IgG and an Alexa 555-conjugated anti-rabbit IgG. iii, Huh-7 cells fusion of the late endosome with the lysosome, resulting in the transiently transfected for 24 h with either wild-type IFITM1 or the respective degradation of trapped particles (44). We hypothesize that IFITM1 palmitoylation mutants, as well as a mCherry-Rab5a expression plas- mid, were stained with mouse monoclonal anti-FLAG and either rabbit poly- although IFITM1 limits HCV infection by disrupting HCV co- clonal anti-mCherry or rabbit monoclonal anti-Lamp1 antibodies, followed receptor assembly as demonstrated by Wilkins et al. (18), by an Alexa 488-conjugated anti-mouse IgG and an Alexa 555-conjugated anti-rabbit IgG. IFITM2 and IFITM3 limit HCV infection by preventing viral- endosomal fusion thereby “trapping” the endocytosed virion mutants C50A/C51A and C71A/C72A in IFITM1 and IFITM3, within the endocytic pathway targeting it for lysosomal degra- respectively. Interestingly, the C70A/C71A mutant of IFITM2 dation. The ability of the IFITM proteins to alter membrane retained partial co-localization at the late endosome and lyso- fluidity and curvature to prevent viral hemifusion provides an some similar to wild-type. The loss of localization of the CIL explanation for this hypothesis (45). Furthermore, IFITM3-me- mutants, and IFITM3, C71A/C72A, validates the complete diated enrichment of endolysosomal membranes has also been abrogation of anti-HCV activity observed for these mutants in shown by several independent studies, and whereas it is possi- Fig. 7B and suggest possible degradation of these mutants at the ble that IFITM3-induced cholesterol accumulation contributes lysosome. This indicates the importance of this cysteine residue to changes in endosomal membrane function within hepato- and hence conceivably S-palmitoylation for the anti-HCV cytes, it is not through an interaction with VAPA, as was shown activity of these proteins. It is interesting, however, to note, that by Amini-Bavil-Olyaee et al. (35) (data not shown). Li et al. (46) the C50A/C51A and C70A/C71A mutations in IFITM1 and proposed that IFITM-induced alterations to endosomal mem-
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.FIGURE 9. Model of the role of IFITM1, ؊2, and -3 on the HCV life cycle. IFITM1 interacts with CD81 on the cells surface to limit HCV entry into the hepatocyte HCV virions that bypass this inhibition enter the cell via clathrin-mediated endocytosis and enter Rab5a- and IFITM3-positive early endosomes. Alterations to endosomal membrane function traps the HCV virion within the early endosome, which is targeted for endosomal-lysosmal degradation via interactions between IFITM proteins, found in opposing endosomal and lysosomal membranes.
brane function promoted fusion with opposing membranes this tyrosine residue in the IFITM2 and IFITM3 N-terminal expressing IFITM proteins. Thus, we postulate that HCV is domain for the localization of these proteins at endosomal trapped in the early endosome after clathrin-mediated endocy- compartments, but unexpectedly found that the N-terminal tosis due to IFITM3-mediated alterations at the early endo- domain tyrosine mutants enhanced anti-HCV activity com- somal membrane preventing fusion and subsequently targeting pared with wild-type. The IFITM2 and IFITM3 tyrosine the virion for degradation via the late endosome and lysosome mutants co-localize with CD81 on the hepatic cell surface thus through interactions between the different IFITM proteins mirroring the localization and potentially the anti-HCV activity found at these organelles. of IFITM1. For the first time we demonstrate a unique feature Post-translational modifications of the IFITM proteins have of the IFITM proteins against HCV infection, where a single been identified to play a crucial role in the localization and mutation in IFITM1, IFITM2, and IFITM3 can still maintain function of these proteins. Our data indicates for the first time significant anti-HCV activity despite significantly altered local- that the conserved cysteine residue found in the CIL is impor- ization. This suggests that whereas the IFITM proteins proba- tant for cellular localization and anti-HCV activity of all three bly act broadly at the site of viral-cell fusion, the exact mecha- IFITM proteins. In addition, Cys-71 and Cys-72 found within nisms of antiviral activity of these proteins is both virus and the first membrane-associated domain of IFITM3 are also cell-type specific. important for anti-HCV activity. S-palmitoylation of Cys-72 has been shown to be essential for the ability of IFITM3 to In this study we demonstrate that the IFITM proteins may restrict influenza A virus and Dengue virus (16, 41). The addi- act sequentially to limit HCV infection, and support the theory tion of palmitoyl groups is associated with protein stability, that the IFITM proteins, in particular IFITM2 and IFITM3 localization, and the association of proteins with lipid rafts, thus exhibit antiviral activity by altering virus-cell fusion. Further explaining the importance of S-palmitoylation for the antiviral questions remain to define the mechanism of action of these activity of the IFITM proteins. Interestingly, we also noted that proteins, in particular the site of this potential disruption and mutation of the M1 cysteine residues in IFITM1 and IFITM2 whether any cofactors are involved. Insights into the cellular retained anti-HCV activity despite partial retention of wild- functions of the IFITM proteins could lead to strategies to spe- type localization, perhaps suggesting a novel association with cifically induce IFITM protein expression or mimic the func- either a viral or host factor. We also confirmed the importance tion of these proteins to complement not only existing anti- of the highly conserved YXXF motif and the phosphorylation of HCV therapies but to also target other viral infections.
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Kellam, P., and Brass, A. L. (2013) The CD225 domain of IFITM3 is re- Author Contributions—M. R. B. and S. K. N. designed the study and quired for both IFITM protein association and inhibition of influenza A wrote the paper. S. K. N. and E. M. M. produced all IFITM con- virus and Dengue virus replication. J. Virol. 87, 7837–7852 structs and performed all antiviral and immunofluorescence assays. 17. Bailey, C. C., Zhong, G., Huang, I. C., and Farzan, M. (2014) IFITM-family N. S. E. produced confocal images and provided Rab5a-mCherry proteins: the cell’s first line of antiviral defense. Annu. Rev. Virol. 1, constructs. R. A. W. and A. E. performed HCVpp experiments. 261–283 K. J. H. and A. R. L. provided technical advice and critical evaluation 18. Wilkins, C., Woodward, J., Lau, D. T., Barnes, A., Joyce, M., McFarlane, N., of the manuscript. All authors analyzed the results and approved the McKeating, J. A., Tyrrell, D. L., and Gale, M., Jr. (2013) IFITM1 is a tight final version of the manuscript. junction protein that inhibits hepatitis C virus entry. Hepatology 57, 461–469 19. Raychoudhuri, A., Shrivastava, S., Steele, R., Kim, H., Ray, R., and Ray, R. B. (2011) ISG56 and IFITM1 proteins inhibit hepatitis C virus replication. References J. Virol. 85, 12881–12889 1. Thomas, D. L. (2013) Global control of hepatitis C: where challenge meets 20. Bhanja Chowdhury, J., Shrivastava, S., Steele, R., Di Bisceglie, A. M., Ray, opportunity. Nat. Med. 19, 850–858 R., and Ray, R. B. (2012) Hepatitis C virus infection modulates expression 2. McCartney, E. M., Helbig, K. J., Narayana, S. K., Eyre, N. S., Aloia, A. L., of interferon stimulatory gene IFITM1 by upregulating miR-130A. J. Virol. and Beard, M. R. (2013) Signal transducer and activator of transcription 3 86, 10221–10225 Downloaded from is a proviral host factor for hepatitis C virus. Hepatology 58, 1558–1568 21. Li, Q., Zhang, Y. Y., Chiu, S., Hu, Z., Lan, K. H., Cha, H., Sodroski, C., 3. Zhong, J., Gastaminza, P., Cheng, G., Kapadia, S., Kato, T., Burton, D. R., Zhang, F., Hsu, C. S., Thomas, E., and Liang, T. J. (2014) Integrative func- Wieland, S. F., Uprichard, S. L., Wakita, T., and Chisari, F. V. (2005) Ro- tional genomics of hepatitis C virus infection identifies host dependencies bust hepatitis C virus infection in vitro. Proc. Natl. Acad. Sci. U.S.A. 102, in complete viral replication cycle. PLoS Pathog. 10, e1004163 9294–9299 22. Dubuisson, J., and Cosset, F. L. (2014) Virology and cell biology of the 4. Aloia, A. L., Locarnini, S., and Beard, M. R. (2012) Antiviral resistance and hepatitis C virus life cycle: an update. J. Hepatol. 61, S3-S13 http://www.jbc.org/ direct-acting antiviral agents for HCV. Antivir. Ther. 17, 1147–1162 23. Blanchard, E., Belouzard, S., Goueslain, L., Wakita, T., Dubuisson, J., 5. Lewin, A. R., Reid, L. E., McMahon, M., Stark, G. R., and Kerr, I. M. (1991) Wychowski, C., and Rouillé, Y. (2006) Hepatitis C virus entry depends on Molecular analysis of a human interferon-inducible gene family. Eur. clathrin-mediated endocytosis. J. Virol. 80, 6964–6972 J. Biochem. 199, 417–423 24. Eyre, N. S., Fiches, G. N., Aloia, A. L., Helbig, K. J., McCartney, E. M., 6. Moffatt, P., Gaumond, M. H., Salois, P., Sellin, K., Bessette, M. C., Godin, McErlean, C. S., Li, K., Aggarwal, A., Turville, S. G., and Beard, M. R.
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OCTOBER 23, 2015•VOLUME 290•NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 25959 Microbiology: The Interferon-induced Transmembrane Proteins, IFITM1, IFITM2, and IFITM3 Inhibit Hepatitis C Virus Entry
Sumudu K. Narayana, Karla J. Helbig, Erin Downloaded from M. McCartney, Nicholas S. Eyre, Rowena A. Bull, Auda Eltahla, Andrew R. Lloyd and Michael R. Beard J. Biol. Chem. 2015, 290:25946-25959. doi: 10.1074/jbc.M115.657346 originally published online September 9, 2015 http://www.jbc.org/
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