The N500 Glycan of the Respiratory Syncytial F is Required for Fusion, but Not for Stabilization or Triggering of the Protein

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Heather M. Costello

Graduate Program in Integrated Biomedical Science Program

The Ohio State University

2013

Dissertation Committee:

Mark E. Peeples, Advisor

William C. Ray

Michael J. Oglesbee

Deborah Parris

Copyright by

Heather M. Costello

2013

Abstract

Respiratory syncytial virus (RSV), a paramyxovirus, is the most significant respiratory pathogen in infants and causes 90,000 emergency hospitalizations in the United States and 160,000 deaths worldwide every year. It is also a leading respiratory pathogen in the elderly. RSV provides weak , and the virus infects individuals repeatedly throughout life. The only effective antiviral compound commercially available is a monoclonal , palivizumab

(Synagis®), which is given prophylactically to at-risk infants. Despite intense efforts, no other therapeutic and no vaccines have been approved for use, although several are in development. Most drugs developed against RSV target the fusion (F) glycoprotein. The F protein is responsible for fusing the host cell and viral membranes together to initiate infection. The RSV F protein is unique among other paramyxoviruses in that it does not require its partner attachment

(G) glycoprotein to function as do most other paramyxovirus F . It is the subject of extensive research in hopes of determining its mechanism of action and developing anti-RSV compounds. The mature, cleaved RSV F protein contains N-glycans at asparagine residues 27, 70, and 500. It has previously been reported that none of these N-glycans are required for protein processing or cell surface expression, but that the N500 glycan is required for cell-to-cell fusion

ii in an assay serving as a proxy for virion-cell fusion. In this study, we replicated these results in the full-length protein. We also built N27Q, N70Q, N500Q,

N27/70Q, and N/27/70/500 mutations into a soluble version of the RSV F protein to determine the role, if any, that these N-glycans have in triggering. Following purification, each of the N-glycan mutants migrated near the top of a sucrose velocity sedimentation gradient, similar to the wild type (WT) sF protein, indicating that these proteins were produced and secreted from the cell in the prefusion form. We determined that none of these glycans were responsible for maintaining the prefusion form, and that premature triggering is not the reason for the fusion deficiency of the N500Q mutant. We have previously determined that exposure to low molarity triggers the RSV F protein, resulting in the exposure of its hydrophobic fusion and resultant aggregation. Following dialysis of the WT and mutant sF proteins into a 50 mM Hepes buffer, each migrated to the bottom of a sucrose velocity sedimentation gradient, indicating that the proteins had triggered and aggregated by their hydrophobic fusion . All of the N-glycan sF proteins were triggerable, indicating that the inability of the N500Q mutant to fuse is not the result of an inability to trigger and is likely due instead to an inability to refold properly following triggering. The

N500Q glycan is attached to the HRB domain of the RSV F protein and may guide the HRB as it completes formation of the 6-helix bundle, a characteristic form of the postfusion F protein that brings the virion and cell membranes into close proximity and results in the fusion of these two membranes.

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Dedication

This dissertation is dedicated to the Costello/Krause family with love, especially:

My mother, who has been my best friend and closest confidante for many years;

My brother, whose humor, strength, and intelligence have brought much

amusement and unassuming support;

And especially my father, who is no longer on this earth, but whose faith and

pride in me will resonate forever.

“There are things you do because they feel right & they may make no sense

& they may make no money & it may be the real reason we are here:

to love each other & to eat each other‟s cooking & say it was good.”

-Brian Andreas, StoryPeople

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Acknowledgments

I would like to extend my sincere gratitude to my advisor, Dr. Mark E. Peeples, for his continued support – professional, financial, and emotional - during my time in his laboratory. He has helped me to utilize my strengths, to improve upon my weaknesses, and to ask the important questions. I am particularly grateful for experiencing his ability to connect disparate concepts into new hypotheses. I hope to one day be described as having similar insight.

I also thank the teachers and professors from my past that have introduced me to various fields of biology and entertained my endless questions. I am particularly grateful for Mr. Joseph Kerata‟s entertaining and enlightening 10th grade introduction to biology, Mrs. Jann Ichida for educating my inexperienced, undergraduate self in the basics of bench research and for helping me to write my first “grant”, Dr. Laura Tuhela-Reuning‟s friendship and invaluable academic and professional advice, and for Dr. Jerry Goldstein‟s rigorous curriculum and enthusiasm for all things viral, which led me to choose a laboratory for my doctoral work.

I am grateful for the insights and discussions with all of the members of my dissertation committee: Dr. William Ray, Dr. Deborah Parris, and Dr. Michael

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Oglesbee. They have been an excellent example of what a collaborative and collegial environment should be. My particular gratitude is extended towards

Will, for the gift of many hours of his structural expertise lent towards our cause.

The camaraderie among the members of the Peeples‟ lab has made my time in graduate school joyful (and sometimes crazy). I thank each of them for the lively discussions, problem-solving, and friendship over the years. I would like to express a particular thankfulness to Koi, who has been my best friend and colleague in the laboratory since the day I joined. Together, we have experienced experimental successes and failures, many laughs and dance classes, and grief over the losses of our fathers. I greatly value her friendship and outlook and am extremely grateful to have found a non-genetic sister.

I have trained many students and technicians over my time in the Peeples laboratory and am thankful for the work contributed by each. My gratitude goes especially to the very industrious undergraduate Christopher Weisgarber, the intelligent and curious technician Zachary Risch, and rotating graduate students

Tierra Ware and Eric Pozsgai, who are now laboring in their chosen laboratories.

Thank you also to my colleagues in the Biomedical Sciences Graduate Program and the Center for Vaccines and Immunity, all of whom have played leading roles in my professional development and in research.

I must give a special thank you to Mr. Larry Tarka, who valiantly rescued Chapter

3 from the bowels of my computer late one Saturday night.

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Vita

2001 ...... Eastlake North High School

2005 ...... B.A. Microbiology, Ohio Wesleyan

University

2006 to present ...... Graduate Research Associate,

Biomedical Sciences Graduate

Program, The Ohio State University

Publications

Costello, H.M., Chaiwatpongsakorn, S., Ray, W.C., and M.E. Peeples, Targeting RSV with Vaccines and Small Molecule Drugs. Infect Disord Drug Targets. 2012 Apr;12(2):110-28.

Goldstein, G., Robinson, A.R., Vorobej, C.L., Aypar, U., and H.M. Costello, Inhibition of Replication of T2 Bacteriophage in E. Coli by 6-diazo-5-oxo-L- norleucine (DON). Research & Reviews: J Microb. & Virol. 2012; 2(1).

Fields of Study

Major Field: Integrated Biomedical Science Program

Minor Field: Molecular Virology and Gene Therapy

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Table of Contents

Abstract ...... ii

Dedication ...... iv

Acknowledgments ...... v

Vita ...... vii

List of Tables ...... xi

List of Figures ...... xii

Chapter 1: Respiratory Syncytial Virus...... 1

Introduction ...... 1

RSV Taxonomy and Classification ...... 3

RSV Biology ...... 4

Life Cycle ...... 5

RSV Proteins ...... 9

Advances in RSV Antiviral Drug Development ...... 25

Antibodies targeting RSV...... 25

Small Molecules with Antiviral Activity Against RSV ...... 27

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Advances in RSV Vaccine Development ...... 39

Live, Attenuated and Vector-Based Viruses as Vaccine Candidates

...... 41

Subunit Vaccines ...... 43

Recent Promising Developments Towards an RSV Vaccine ...... 47

Conclusions ...... 54

Chapter 2: The N500 Glycan of the Respiratory Syncytial Virus F (Fusion)

Protein is Required for Fusion, but Not for Stabilization or Triggering of the

Protein ...... 58

Introduction ...... 58

Materials and Methods ...... 62

Results...... 67

Discussion ...... 76

Chapter 3: Anti-RSV Compounds R170591 and TMC353121 Function by

Stabilizing the Prefusion Form of the F Protein and Inhibiting Triggering .. 80

Introduction ...... 80

Materials and Methods ...... 83

Results...... 87

Discussion ...... 94

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Chapter 4: Relevance and Future Directions ...... 98

Role of N-glycans on the RSV F protein ...... 98

R170591 and TMC353121 resistant mutations of the RSV F protein ...... 100

Disulfide bond mutations to stabilize the prefusion form ...... 103

Determining the structure of the native RSV F prefusion protein ...... 106

Bibliography...... 109

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List of Tables

Table 1.1 Mutations Selected by RSV Antivirals……………………………..28

Table 4.1 Mutations Selected by Y198-binding RSV Antivirals…………. 101

Table 4.2 Introduced Disulfide Bond Constructs Attempted……………….104

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List of Figures

Figure 1.1 Negative-sense of respiratory syncytial virus shown with gene organization and proteins resulting from open reading frames ...... 7

Figure 1.2 Cartoon representing the components and organization of the RSV virion...... 10

Figure 1.3 Cartoon illustrating the life cycle of RSV ...... 13

Figure 1.4 Cartoon of the 298 amino long RSV G protein ...... 16

Figure 1.5 Positions of N-glycans of RSV F protein shown as arrows and residue positions ...... 17

Figure 1.6 Cleavage of the RSV F protein ...... 18

Figure 1.7 Cartoon of dynamic regions of the F protein that refold to initiate fusion ...... 20

Figure 1.8 Monomer structure of the prefusion (A) RSV F protein (PDB structure

4JHW), and the crystal structure of the postfusion RSV F protein (B) (PDB structure 3RRR) ...... 22

Figure 1.9 Soluble version of the RSV F protein (sF) ...... 24

Figure 1.10 Chemical structures of three Y198-binding drugs, TMC353121,

R170591, and VP-14637 ...... 31

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Figure 2.1 Syncytia formation of HEK 293T cells transiently transfected with WT

RSV F protein and single-glycosylation mutants...... 69

Figure 2.2 Cell surface expression and fusion of N-glycosylation mutants...... 70

Figure 2.3 A soluble version of the RSV F protein (sF) was generated by replacing the transmembrane (TM) and cytoplasmic domains with FLAG and 6-

His tags...... 71

Figure 2.4 WT and mutant sF protein transfected Freestyle 293 cell lysates and cell supernatants as examined by Western blotting for the F protein...... 72

Figure 2.5 Velocity sedimentation gradients of WT and mutant sF proteins ...... 75

Figure 2.6 Cartoon representing the 6-HB of the RSV F protein in its postfusion conformation ...... 77

Figure 3.1 RSV neutralization by anti-RSV compounds...... 87

Figure 3.2 Cell-to-cell fusion in the presence of R170591 and TMC353121 ..... 89

Figure 3.3 Velocity sedimentation gradients of sF protein following dialysis into buffers of various molarities ...... 91

Figure 3.4 Velocity sedimentation gradients of sF protein following incubation with anti-RSV compounds at 37°C for 2 h ...... 93

Figure 3.5 sF protein and liposome co-flotation following incubation with and in the presence of various anti-RSV compounds ...... 93

Figure 3.6 Velocity sedimentation gradients of sF protein following incubation with TMC353121 for 2 h at 37°C and dialysis against a 50 mM Hepes buffer containing 30 μM TMC353121 ...... 94

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Figure 4.1 Trimer model of the RSV F protein...... 103

Figure 4.2. Cell surface expression of two potentially disulfide bonded full-length

RSV F proteins, modeled after two published disulfide bond partners ...... 105

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Chapter 1: Respiratory Syncytial Virus

Introduction

Respiratory syncytial virus (RSV) was isolated in 1956 from a displaying symptoms of respiratory infection (206). The virus was eventually identified as the most significant cause of pediatric respiratory . RSV infections result in 90,000 pediatric emergency hospitalizations in the U.S. and

160,000 deaths worldwide each year, mostly in the developing world where access to supportive healthcare is limited (67, 102, 111, 148). Adults and infants suffering maladies such as immune deficiencies (147, 220, 236), congenital heart defects (183, 289), chronic lung ailments such as cystic fibrosis (2), malnutrition

(3), or premature birth (113) are at particularly high risk for developing severe

RSV disease (285) in the lower respiratory tract, bronchiolitis and pneumonia.

RSV infects individuals repeatedly throughout life (115) and causes significant disease in the elderly, second only to virus in non-epidemic years (84,

284, 295). Because of these risks, RSV has been the target of intensive research and vaccine and antiviral drug development efforts (145).

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Although there are no effective antivirals against RSV, many at-risk infants in developed countries are treated prophylactically with palivizumab (Synagis®), a humanized monoclonal antibody (mAb) developed by MedImmune, LLC, and sold through Abbott Laboratories. Repeated treatment with this mAb (one injection per month for the 5 winter months of the infant‟s first year) results in a

55% reduction in hospital admissions (1). Palivizumab is currently the only option widely used to prevent severe RSV disease in at-risk infants, but because it is costly and requires monthly administration by medical staff during RSV season, it is not a practical treatment for at-risk infants in many countries (271).

The small molecule drug, ribavirin, is the only antiviral drug approved for treatment of RSV disease, but its use has largely been discontinued due to significant doubts concerning its efficacy in vivo (114, 291).

No vaccine is currently available to protect against RSV disease. Vaccine development has been approached with caution due to the failure of the initial, formalin-inactivated RSV vaccine in the 1960‟s (93, 148, 150). Although that vaccine stimulated the production of against the F protein in recipients, the antibodies did not inhibit fusion between the virus and host cell and were not protective (209). Vaccinees remained susceptible to RSV infections, displaying an increased frequency of lower respiratory tract disease that resulted in a high hospitalization rate and two infant deaths. Pathology analysis of these two infants found a high level of inflammation in the lungs, including infiltrating eosinophils (150). Eosinophilia is characteristic of a TH2 2 response, suggesting that the vaccine had primed recipients toward a TH2 immune response, leading to enhanced inflammation and disease pathology

(169). Experiments in mice were consistent with this idea (137-139, 299).

Another factor possibly linked to the enhanced disease is that anti-RSV antibodies produced by the recipients following vaccination formed immune complexes during subsequent natural infection (237). The results of this vaccine trial have strongly shaped current RSV vaccine development, such that the only vaccines under consideration for infants and children are live, attenuated vaccines (107).

There is a great need for an effective vaccine and for antiviral drugs against

RSV, and considerable effort has focused on these objectives, but so far the field is still without an approved product. Most vaccines and drugs under development target the major glycoproteins of RSV: the F and G glycoproteins.

These glycoproteins are responsible for attaching the virion to a host cell and fusing its membrane with the membrane of the cell.

RSV Taxonomy and Classification

RSV is a member of Pneumovirinae, one of the two subfamilies of the

Paramyxoviridae family within the order Monomegavirales. There are two genera of the Pneumovirinae subfamily, Pneumovirus, which includes RSV

(human, bovine, and ovine) and mouse pneumovirus, and Metapneumovirus, which includes metapneumovirus (human and avian).

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The other subfamily is the Paramyxovirinae. There are five genera of this subfamily, Respirovirus, which contains human parainfluenza viruses type 1 and type 3, Rubulavirus, which contains and parainfluenzavirus 5 viruses, Avulavirus, which contains Newcastle disease virus,

Morbillivirus, which contains measles virus, and Henipavirus, which contains

Hendra and Nipah viruses.

The subfamilies Paramyxovirinae and Pneumovirinae differ in a number of ways.

The Pneumovirinae have narrower nucleocapsids and very different attachment proteins than the Paramyxovirinae (67, 166). Other distinguishing differences are the number of encoded proteins, the types of accessory proteins, the sizes of homologous proteins, the gene order, and the sequence relatedness (67, 166).

RSV Biology

RSV contains a 15 kb single-stranded, negative-sense RNA genome with 10 genes that encode 11 proteins (Fig 1.1). The RNA genome (and antigenome) is covered by the nucleoprotein (N), forming a helical nucleocapsid upon which the large polymerase (L) protein, phosphoprotein (P) and the M2-1 protein sit. All four of these proteins are required for mRNA transcription and so are present in the virion (67). The virus contains a lipid bilayer envelope derived from the host cell plasma membrane and which is decorated with the three glycoproteins of

RSV (Fig. 1.2).

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Life Cycle

Attachment and Entry

RSV is spread via aerosolized droplets from infected persons to the respiratory tracts of uninfected persons. At the outset, RSV infects the upper respiratory tract, causing -like symptoms. However, infection can move quite readily to the lower respiratory tract, particularly during the initial infection, causing more severe disease. RSV attaches to the host cell receptor via its G attachment glycoprotein. The host cell receptor has been reported to be glycosaminoglycans such as heparan sulfate in immortalized cells (89, 116, 117,

160), but as heparan sulfate is not present on the luminal surface of the human airway (322), the receptor used for attachment in vivo is almost certainly a different receptor. RSV infects only ciliated cells on the apical surface, suggesting that the in vivo receptor for RSV is present only on ciliated cells and only on the apical surface (323).

As is required of all enveloped viruses, RSV must next fuse with a host using its fusion (F) glycoprotein to initiate infection (Fig. 1.3). The current understanding regarding RSV entry is that fusion occurs at the cell surface based on the ability of the F protein to refold and cause fusion at neutral pH. However, there have been several compelling studies recently that point to a possible role of endosomal-mediated or pinocytotic entry by RSV (156, 161,

254). This theory is particularly attractive because it would explain how the virus

5 is able to avoid the meshwork scaffolding the plasma membrane, which it would otherwise encounter following cell surface fusion.

Transcription, , and Genome Replication

Regardless of the route of cell entry, the viral nucleocapsid is delivered to the of the cell as a result of membrane fusion (Fig. 1.3). The cell‟s nucleus does not appear to be involved in any step of RSV replication, as the virus can reproduce in enucleated cells and in the presence of actinomycin D

(91, 224). After the virion enters the cytoplasm, transcription using the negative sense genome begins (Fig. 1.3). The transcription of RSV genes begins at the 3‟ . The polymerase protein (L) stops at the end of each gene at the “gene end” signal. The mRNA transcript is polyadenylated by a stuttering mechanism of the polymerase, repeatedly copying a poly U tract at the end of each gene, after which the transcript is released (162, 164). The polymerase travels along the intergenic region to the “gene start” of the next gene, where it reinitiates transcription (Fig. 1.1). The RSV polymerase has a propensity to disassociate from the DNA in the intergenic regions resulting in the RSV genes being transcribed in a decreasing gradient: the 3‟ most gene (NS1) producing the most transcripts and the 5‟ most gene (L polymerase) the fewest transcripts (66, 159,

163, 164). The viral polymerase also caps each transcript at its 5‟ end and methylates the cap (180).

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Figure 1.1. Negative-sense genome of respiratory syncytial virus shown with gene organization and proteins resulting from open reading frames. Each gene has a gene start (GS) and gene end (GE), used to signal the polymerase complex when to begin and terminate transcription. The 3‟ end of the genome contains a leader sequence and the 5‟ end of the genome contains a trailer sequence, both used in genome and antigenome replication.

RSV codes for 10 subgenomic mRNAs (Fig. 1.1). One of these mRNAs is translated into two proteins, M2-1 and M2-2, from different open reading frames

(18, 61). All other viral proteins are produced from their own unique mRNA. The glycoproteins, G, F, and SH, are translated by ribosomes which attach to the endoplasmic reticulum (ER). During translation, the majority of each protein is translocated into the lumen while remaining anchored in the ER membrane. In the ER, the F, G, and a portion of the SH proteins are N-glycosylated. The proteins are transferred to the cis compartment of the Golgi apparatus. As the proteins are transported through to the trans Golgi compartment, the N- glycosylations of the proteins are clipped back and decorated with additional, different sugars. In addition, the G protein is O-glycosylated, and the F protein is cleaved into its active form by a furin-like . Vesicles containing the F, G, and SH proteins are pinched off from the trans Golgi and are transported intracellularly to the surface of the cell where they fusion with the plasma

7 membrane, resulting in the luminal facing proteins being exposed on the outside of the cell (Fig. 1.3).

The NS1, NS2, N, P, M, M2-1, M2-2, and L proteins are all translated by ribosomes and released into the cytoplasm. Some of these proteins are required for transcription and replication of the viral genome. The L and P proteins make up the complete, active polymerase complex. M2-1 is an anti-termination protein that is required for mRNA transcription by the viral polymerase but not for genome replication (122). The N protein is the major protein of the RSV nucleocapsid and remains bound to the viral genomic RNA during both replication and transcription (Fig. 1.3). The M2-2 protein acts as a negative regulator of transcription (5, 18, 46). As it accumulates in the cytoplasm, it signals a shift from viral transcription to genome replication.

The leader sequence at the 3‟ end of the genome serves as a promoter for the viral polymerase complex to copy the genome template into the antigenomic

RNA, For this synthesis, the polymerase disregards all transcription signals and does not cap or polyadenylate the nascent RNA strand in order to successfully synthesize a complete antigenome, a positive-sense copy of the viral genome.

Like the genome, the antigenome is also encapsidated with the N protein. The trailer sequence at the 3‟ end of the antigenome is the promoter used by the viral polymerase to initiate production of the genomic RNA. Many more genomic nucleocapsids are present in infected cells than antigenomic nucleocapsids.

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Virion Assembly and Egress

As the glycoproteins are transported to lipid rafts on the apical surface of the cell, the matrix protein (M) binds to one or both of the cytoplasmic regions of the G and F proteins and to the nucleocapsid (associated with all of its polymerase proteins) (31, 90, 247). The M protein inhibits virus transcription prior to budding

(100). The M protein is thought to also orchestrate the assembly of the virion, and the inclusion of viral glycoproteins in the lipid rafts is believed to be the initiation point of assembly (99, 127, 282).

After the components of the virus are assembled, the virus buds directly out of the lipid rafts on the cell‟s plasma membrane (30, 32) (Fig. 1.3). It is unclear by which mechanism the virus buds and pinches off, although it has been shown that the apical recycling endosome pathway regulates the directional budding of nascent virions (29) and that the budding of RSV virions is ESCRT-independent

(290). Cellular actin and cellular microtubules have also been shown to be involved in virion assembly and egress (29, 37, 142).

RSV Proteins

The Nucleocapsid/Polymerase Proteins

The N, P, L, and M2-1 proteins are packaged into the virion to implement immediate transcription of viral genes into mRNA upon infection (60, 97) (Fig.

1.2). The N protein binds to and coats the viral genome and antigenome and is

9 the major protein of the nucleocapsid. The N protein assembles on the RNA to form a helical nucleocapsid, with the RNA protected from nuclease digestion. It is smaller than the N protein of other Paramyxovirus family members (391 amino as compared to its counterparts‟ 489 – 553 amino acids). While the N protein binding to RNA is thought to be the switch from mRNA transcription to replication in other paramyxoviruses, it has been clearly shown that this is not the case for RSV (15, 292). The N-terminus of the protein is involved in RNA binding and self-assembly, while the C-terminus seems to be involved in giving flexibility to the RNA-N protein complex and in interacting with the P protein (15, 88, 95,

210, 266).

The RSV P protein, like the N protein, is smaller than P proteins of other

Paramyxovirus family members, at 241 amino acids

(as compared to 391-602 Figure 1.2. Cartoon representing the components amino acids). The P protein is and organization of the RSV virion. The three glycoproteins, SH, F, and G decorate the lipid envelope of the virion. The M protein forms a self-associated a multimer component of the layer beneath the lipid bilayer. Inside the virion is its negative-sense RNA genome coated with N proteins. polymerase complex and Proteins necessary for immediate transcription following infection, the L, P, and M2-1 proteins, are also packaged associates with and maintains inside the virion. solubility of N proteins not yet incorporated into the nucleocapsid (95, 266). The P protein is highly 10 phosphorylated, which is important for stability and activity of the polymerase complex (80).

The RSV L protein is the main component of the polymerase complex. It is by far the largest RSV protein, at 2,165 amino acids. As the most promoter-distal gene in the RSV genome, it is present in low levels in infected cells. The L protein is an that is able to transcribe, cap, methylate the cap and polyadenylate each of the 10 mRNAs. It also replicates the complete genome and antigenome

RNAs (235). Each L protein has six domains, each with a distinct function. For example, domain II is responsible for RNA-binding, domain III functions in polymerization, and domain VI caps the mRNAs (180, 235, 269).

The M2-1 Protein

The RSV M2 gene has two open reading frames (ORFs). The first ORF is translated into the M2-1 protein, a 194 amino acid protein that contains a zinc- finger motif and functions as a transcription processivity factor (58-60, 121). The

M2-1 protein maintains polymerase association to the RNA genome during mRNA transcription (87, 121). There are two electrophoretically distinct species of the M2-1 protein due to differential phosphorylation (121). The role of this phosphorylation is unknown.

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The M2-2 Protein

The second ORF of the M2 mRNA codes for the 90 amino acid M2-2 protein.

This ORF is accessed by ribosomal termination and reinitiation of translation

(61). Cells infected with M2-2 deleted RSV have higher amounts of mRNA and lower amounts of genome and antigenome than cells infected with WT RSV (18), leading to the belief that the M2-2 protein functions as a negative regulator of

RSV transcription. As the amount of the M2-2 protein rises in the infected cell, the polymerase complex switches from transcription to mostly genome replication

(18). Its mechanism of action, however, is unknown.

The Non-structural NS1 and NS2 Proteins

The NS1 (139 amino acids) and the NS2 (124 amino acids) proteins are the most abundant viral mRNAs in an infected cell. They are not absolutely essential for viral growth (66, 81, 281, 283, 304) but deletion of these genes reduces virus yield significantly (281, 283). They inhibit, mainly through the actions of NS1, the induction of the IFN response by interfering with the phosphorylation and nuclear localization of IFN regulatory factor (IRF3) (27, 267). IRF3 is a transcription factor that induces the expression of IFN-α and IFN-β.

These proteins also inhibit IFN-α, β, γ, and λ signaling, largely through the actions of NS2. RSV targets STAT2, a signaling molecule in the JAK/STAT pathway, for degradation (243). Without STAT2, amplification of IFN expression and the expression of other antiviral genes and genes involved in innate and 12 adaptive immunity are not activated. NS1 and NS2 also delay apoptosis of the host cell (23).

NS1 may also have a role in RNA synthesis, as overexpression of the protein in a minigenome system inhibits both transcription and genome replication (14).

Deleting NS1 and/or NS2 from the virus results in attenuation of virus production even in Vero, a cell line that does not produce IFN, indicating that the NS proteins may also serve a role other than interferon inhibition (132, 281, 283).

Figure 1.3. Cartoon illustrating the life cycle of RSV. Main steps are highlighted in number order: attachment, fusion, transcription, translation, genome replication, assembly, and egress of progeny virion. 13

The Matrix Protein

The 256 amino acid matrix (M) protein underlies the lipid bilayer envelope of the virus in a self-associated layer (188) (Fig. 1.2). The M protein is the regulator that orchestrates assembly of the nascent virion and is thought to initiate budding from the cell surface (100, 188, 227). The M protein associates with one or both of the cytoplasmic tails of the G and/or F glycoproteins and with the N protein which associates the nucleocapsid with the integral membrane viral glycoproteins. The M protein association with the nucleocapsid also inhibits RNA synthesis prior to assembly (100).

The Glycoproteins

SH protein

The SH protein is a small (64 amino acid) type II membrane protein (Fig. 1.2) that is nonessential for viral replication (63, 221). It is present intracellularly in at least four different forms: SH0, SHg, SHp, and SHt. SH0 is nonglycosylated and full- length, SHg is full-length and contains one N-linked glycan, SHp is full-length and has a polylactosaminoglycan attached to the sole N-linked glycan, and SHt is nonglycosylated and is shorter as it initiated at the second start codon in the

ORF. SH0 and SHp are the most abundant forms of the SH protein present on the virion (8, 221).

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RSV with the SH gene deleted is still fully infectious in vitro and infects and replicates similarly to WT RSV in mouse lower respiratory tract and chimpanzee upper respiratory tract (35, 304). It was only moderately attenuated in mouse upper respiratory tract and chimpanzee lower respiratory tract (35, 304). It appears to be dispensable for viral growth in vitro and somewhat involved in replication in vivo. While the function of the SH protein remains largely unknown, it has been shown to attenuate apoptosis in the infected cell (92). Additionally, it increases membrane permeability when expressed in bacteria, and modeling of the SH protein indicates that it may form a pentamer that may function as a membrane channel for cations (63, 94, 228).

G protein

The G protein is the main viral attachment protein (303), although the F protein also displays some attachment function (279). The G protein has no sequence similarity to the attachment glycoproteins of viruses in the other subfamily,

Paramyxovirinae, although like these others, it is a type II integral membrane protein (303) anchored in the membrane by its N-terminus (Fig. 1.2, 1.4). It facilitates attachment of the virus to the surface of cultured immortalized cells by binding to glycosaminoglycans, primarily heparan sulfate (89, 116, 117, 160,

176). The G protein also facilitates RSV infection of primary well differentiated human airway epithelial (HAE) cultures. RSV infects primarily the ciliated cells in these cultures, entering from the apical surface (323). But, since heparan sulfate

15 is not detectable on their apical surface (322), the RSV receptor on airway cells is likely to be a different molecule. The G protein is also produced in a secreted form that may act as a decoy to sequester anti-G antibodies and to modulate lymphocyte migration, enabling more efficient virus infection and spread (36).

The cell-associated G protein is 90 kDa in molecular mass, 60% of which is carbohydrate. Most of this carbohydrate is O-linked to the protein, similar to the mucins (303) (Fig. 1.4). The molecular mass of the G protein varies somewhat depending on the cell line that produces it, as a result of differences in glycosylation, cleavage or other modifications (96, 165). Its cleavage in Vero cells by an unknown protease removes the C-terminus and inactivates its attachment function (165). G protein produced in well differentiated HAE cultures is much larger (165).

Figure 1.4. Cartoon of the 298 amino acid long RSV G protein. The N-terminal location of its cytoplasmic domain (CT) and transmembrane domain (TM), two mucin-like regions with their N- linked (squares) and O-linked (small ovals) glycans, and central cysteine noose which contains the 4 conserved cysteines, the central conserved domain (CCD), and the CX3C domain, followed by the heparin-binding domain (HBD) are shown. The position of the mAb 131-2G binding site and the peptide included in the experimental G2Na vaccine are indicated. The position of the second Met that is used to initiate translation of a truncated form of the G protein is also indicated. This form is proteolytically cleaved to remove its truncated TM domain and released from the cell as soluble G protein.

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The structure of the G protein has not yet been solved. Its unglycosylated central region contains 4 cysteines that form disulfide bonds in a cysteine noose pattern

(105) (Fig. 1.4). NMR analyses of this region have identified loop structures

(106, 170) that are similar to a domain in the tumor necrosis factor receptor

(TNFR) (170). The 4 cysteines of the G protein are in the same position as the 4 cysteines in this TNFR domain (170).

F protein

The F protein trimer is responsible for fusing the with the host cell plasma Figure 1.5. Positions of N-glycans of RSV F protein shown as membrane (16, arrows and residue positions. Solid arrows indicate glycans found in all strains. The grey arrow indicates the strain-specific glycan. 167, 252). The

Pneumovirinae F protein is unique among paramyxoviruses in that it is the only glycoprotein necessary for cell-cell fusion (16) as well as for infection of cultured, immortalized cells (16, 144, 279). However, infection is not as efficient or productive as it is

17 with a virus containing the G protein, probably because the G protein enhances attachment to the target cell.

Figure 1.6. Cleavage of the RSV F protein. As the F0 precursor travels through the trans-Golgi, it is cleaved by a furin-like protease to yield the mature F1+F2 protein, releasing a 27 amino acid peptide (pep27).

The RSV F protein is a type I glycoprotein whose precursor F0 monomer is modified by the addition of either 5 or 6 N-linked glycans and assembled into a trimer in the endoplasmic reticulum. N-linked glycans are incorporated at residues N27, N70, N116, N126, and N500 in all RSV strains (41, 109). An additional N-linked glycan at residue N120 is present on the F protein of the Long strain (Fig. 1.5). The N500 glycan has been shown to be essential for efficient

18 syncytial formation, though the reason for this has been difficult to determine using the full-length F protein (328).

During passage through the Golgi, these glycans mature and the protein is cleaved by a furin-like enzyme in two places, releasing a 27 amino acid peptide

(pep27) with, depending on the strain, two or three attached glycans (104, 327)

(Fig. 1.6). The second cleavage site is unique to the RSV F protein (104, 327).

The resulting F protein is composed of the N-terminal F2 protein linked by two disulfide bonds to the transmembrane F1 protein. All paramyxovirus F proteins have a disulfide bond at the top of the prefusion F protein that links the F1 and F2 subunits. However, the second disulfide bond, located at the bottom of the head, is unique to and characteristic of members of the Pneumovirinae subfamily.

Once cleaved, the RSV F protein is fully active. The highly hydrophobic fusion peptide at the cleavage-created N-terminus of the F1 protein resides in a pore in the side of the F protein trimer (197). The F protein is expressed on the cell surface and from that position is able to cause cell-cell fusion, or to be incorporated into virions, where it causes virion-cell fusion, an essential step that initiates infection.

The paramyxovirus F protein trimer on the cell or virion surface is in a

“metastable” prefusion form, like a compressed spring, waiting to be triggered

(182, 251). Following a triggering event, it refolds into the postfusion form with its signature 6-helix bundle (6-HB) that brings the viral and cellular membranes 19 together enabling them to fuse (Fig. 1.7). For structural analysis, the F protein has been produced in a soluble form that lacks its transmembrane and cytoplasmic domains, thereby avoiding aggregation that would be caused by the hydrophobic transmembrane domain.

Figure 1.7. Cartoon of dynamic regions of the F protein that refold to initiate fusion. The three N-terminal α-helices in pre-HRA are connected by two non-helical peptides in the prefusion form. Upon triggering these non-helical connecting peptides refold into α-helices, completing the long HRA α-helix (red) and in the process thrust the F1 N-terminal fusion peptide into the target cell membrane. HRA trimerizes, the molecule folds in half, and the HRB α-helices (blue) insert into the grooves on the surface of the HRA trimer forming the stable 6-helix bundle (6-HB). As a result, the virion and cell membranes are brought together and initiate membrane fusion. The “head” region of the F protein does not rearrange during the triggering and refolding events and, therefore, is not represented in this cartoon.

The crystal structures of both the prefusion and postfusion RSV F protein were recently solved (197, 198, 273) (Fig. 1.8). The structure of the postfusion RSV F protein was solved first, by two separate groups, by deleting nine or ten of the eighteen N-terminal residues of the protein‟s fusion peptide (198, 273) (Fig. 20

1.8B). This mutant protein is produced in the postfusion form, complete with the

6-HB. Its structure is similar to the previously solved human parainfluenza virus

3 (hPIV3) postfusion structure (317). A soluble version of the prefusion RSV F protein structure has only very recently been solved after adding a fibritin trimerization domain to the C-terminus and complexing it with a prefusion F protein-specific antibody (197) (Fig. 1.8A).

Upon triggering, four short α-helices and the loop regions connecting them

(BLUE, in the upper left region of Fig. 1.8A) consolidate into one very long α- helix (BLUE, Fig 1.8B), thrusting the hydrophobic fusion peptide at its N-terminus

(GRAY, on the left side of the molecule, Fig. 1.8A) into the target cell membrane, thereby linking the two membranes. The resulting “pre-hairpin intermediate,” shown in the conceptual cartoon in Fig. 1.7, folds back on itself to form the 6- helix bundle, bringing the two membranes together in the postfusion form and initiating fusion. During the transition from the prefusion to the postfusion conformation, the central “Head” region of the F protein (Fig. 1.8A) remains relatively constant. It is not included in the cartoon in Fig. 1.7 for simplicity.

The highlighted amino acids on the prefusion RSV F protein structure (Fig. 1.8A) indicate drug-resistant mutations in the F protein of viruses. The same drug- resistant mutations are displayed on the postfusion RSV F protein structure Fig.

1.8B). The drugs were discovered through high-throughput viral neutralization assays using large libraries of compounds. Drug resistant mutants were grown

21

Figure 1.8. Monomer structure of the prefusion (A) RSV F protein (PDB structure 4JHW), and the crystal structure of the postfusion RSV F protein (B) (PDB structure 3RRR) (197, 198). We have maintained the HRB domain (red α-helix) in approximately the same position in both models, as a fixed reference point. Following triggering, the pre-HRA domain (BLUE in A) becomes one long α-helix (BLUE in B), inserts its N-terminal fusion peptide (GRAY in A) into the target membrane and folds back (B), bringing the virion and cell membranes together. The fusion peptide was not resolved in the postfusion structure. During this fold-back, the “Head” domain maintains its position relative to HRA, but rotates approximately 180 degrees, bottom to top, relative to HRB. This rotation causes the peptide that connects HRB to the Head to wind off the Head. Cysteines, all of which are involved in disulfide bonds appear as yellow balls. Drug- resistant mutations appear as red balls. Residues 140 and 144 are not present in the postfusion structure, because this region of the fusion peptide (the 9 N-terminal residues) were deleted from the nucleotide sequence to prevent aggregation of the postfusion form. Residue Y198 is presented as blue spheres. Image modified from RSV F protein monomer structures published in (197, 198).

22

in the presence of each drug and isolated after numerous passages and will be described below.

The mechanism by which these drugs disrupt F protein function is difficult to determine, but there are several obvious possibilities. A drug could induce premature triggering, prevent triggering, disrupt movements involved in refolding, or prevent formation of the final postfusion 6-helix bundle conformation. Which of these events are disrupted by each drug is not known. We have recently produced a „soluble‟ RSV F protein that lacks its transmembrane and cytoplasmic domains and is in the fully cleaved, prefusion form (Fig. 1.9). We have also identified conditions that cause it to trigger, refolding into its postfusion form (43).

This soluble F protein will allow us to ask questions about the mechanism of action of antiviral drugs targeting the F protein.

The F and G glycoproteins are highly immunogenic and most, if not all, RSV- neutralizing antibodies are raised against these two proteins (146, 261). The most potent neutralizing antibodies in high-titered convalescent serum react with the RSV F protein and recognize the prefusion conformation (185). Monoclonal antibodies (mAbs) that specifically recognize the prefusion RSV F protein are also much more potent at neutralizing RSV than mAbs against the region of F that does not change during triggering and therefore are present on both the prefusion and postfusion F proteins.

23

Most antiviral

compounds that have

been discovered target

the F protein, even

though these

compounds were

identified by screening

chemical libraries for

their ability to block

Figure 1.9. Soluble version of the RSV F protein (sF). sF was RSV infection, generated by replacing the transmembrane (TM) and cytoplasmic domains with FLAG and 6-His tags. highlighting the

importance of the F protein in infection and as a target for antivirals. The essential role of the F protein in initiating RSV infection, its availability on the outer surface of the viral envelope, its crucial function in membrane fusion, and its dramatic conformational changes that cause membrane fusion, make the F protein the most potent target for RSV antiviral drug development.

Alternative F protein cleavage hypothesis

A recent report presented evidence that all of the F1 protein is cleaved at what appears to be an additional position during entry into the target cell. This cleavage appears to also be caused by a furin-like protease, following a

24 macropinocytotic entry, resulting in the removal of 2 kDa from F1. The authors proposed that this cleavage induces the protein to trigger, and that the 2 kDa fragment removed is pep27 (161). However, the authors did not take into account that pep27 has two attached N-glycans and is actually closer to 8 kDa.

The same group published another recent report examining the structure of the whole virion by electron cryotomography and found that most of the F protein present on the virus is in the postfusion form (178). This observation is not consistent with cleavage in the target cell of all of the F protein as the cause of triggering. The location of their observed cleavage of F1 during virion entry remains unclear, as does its importance.

Advances in RSV Antiviral Drug Development

Antibodies targeting RSV

Palivizumab and Motavizumab. The humanized mAb palivizumab (Synagis®,

MedImmune) targets the fusion (F) glycoprotein (136, 238), inhibiting fusion but not attachment (128). Motavizumab (Numax®, MedImmune), a second generation of palivizumab with much greater binding and neutralizing activity was subsequently developed by affinity maturation in vitro (101). The crystal structure of motavizumab complexed with its peptide binding site from the F protein (position indicated in Fig. 1.8) had suggested that although this neutralizing mAb would be able to bind to both the prefusion and postfusion 25 monomers (196), in the native trimer its binding site would be partially hidden by the neighboring monomer. This prediction was based on the crystal structure of the prefusion parainfluenza virus 5 (PIV5) F protein. However, the two crystal structures of the postfusion RSV F protein (198, 273) revealed that the palivizumab/motavizumab binding site is available for antibody binding in the postfusion structure. Swanson, et al. (273) further suggested that this antibody- binding site is likely to be available in the prefusion F protein since the structure and orientation of this domain does not change relative to its neighboring region when the PIV3 postfusion F protein and the PIV5 prefusion F protein are compared. Questions over the comparative potency of motavizumab relative to palivizumab and concerns over injection site reactions led the FDA to recommend that motavizumab not be licensed for prophylactic use (229).

Motavizumab has recently been tested in a clinical trial for its efficacy in the treatment of severe RSV disease (56), but the results have not yet been published.

RI-001. This high titer, human immunoglobulin against RSV was developed by

ADMA Biologics, Inc. The immunoglobulin was isolated from otherwise healthy human adults following RSV infection. It was effective in treating three immunocompromised patients with lower respiratory tract RSV infection (83). A phase II trial for this immunoglobulin preparation has been completed, but the results have not yet been published (54).

26 mAb 131-2G. This non-neutralizing mAb against the G protein of RSV inhibits the G protein from binding to cells expressing the CX3C chemokine receptor 1

(CX3CR1) and, therefore, appears to bind near the CX3C chemokine motif in the central region of the G protein (Fig. 1.4) (9-11, 203). Purified G protein induces leukocyte chemotaxis in vitro, and mAb131-2G inhibits this activity (124, 286).

Administration of mAb 131-2G to mice one day before RSV inoculation reduces pulmonary inflammation (9-11, 203) suggesting that it may decrease inflammation and pathogenicity if used prophylactically (123). In addition to reducing inflammation, mAb 131-2G also facilitates viral clearance in mice (123,

203, 242), so it is possible that the enhanced viral clearance was responsible for the observed reduction in inflammation. Regardless of the mechanism, such a mAb might be a useful complement to palivizumab for protecting at-risk infants

(242).

Small Molecules with Antiviral Activity Against RSV

Compounds that Bind to Y198 in the F Protein

Three of the most studied RSV small-molecule antivirals have been found to select similar drug-resistant mutants (Table 1.1) indicating that they target the same region in the RSV F protein. We will discuss each briefly, followed by a discussion of their possible mode of inhibition.

27

Table 1.1. Mutations Selected by RSV Antivirals

Drug Company/Developer Resistant Mutants Ref. MAb palivizumab Medimmune/ N268I (325) AstraZeneca K272Q,M,N,T,E, (326) S275F,L (328) F Drugs Fusion HRA (392-394) (398 -400) (486-489) (Y198 binders) Peptide BMS-433771 Bristol-Myers F140I D392G D489Y (51) Squibb V144A K394R VP-14637 ViroPharma; T400A D486E (78) (MDT-637) MicroDose F488I, V, Y, L (207) Therapeutx (+N517I) D489Y TMC353121 Janssen Research S398L D486N (12) (Improved upon Foundation; S398L E487D (249) JNJ2408062/R Tibotec/Johnson & (+K394R) D489Y (207) 170591) Johnson K399I (77) P13 Lundin, et al. N197T T400I (181) C15 Lundin, et al. D489G (181) Other F Drugs RFI-641 Wyeth G446E (207) (CL-309623) and CL-387626 BTA-9981 Biota; MedImmune/ None reported AstraZeneca Peptides Trimeris None reported T-67 (HRA) T-118 (HRB) G Drug MBX-300 Microbiotix L97P, F101L, I107T, I114T (153) F163P, F165L, F168S, F170S, I189T L215P F265L, L274P N Drug RSV604 Arrow; Novartis N105D, K107N, I129L, L139I (44) siRNA ALN-RSV01 Alnylam None reported ALN-RSV02 Alnylam None reported

BMS-433771. Bristol-Myers Squibb identified the fusion inhibitor BMS-233675 by screening a chemical library for compounds neutralizing RSV infection, and modified it to generate the orally-active and bioavailable azabenzimidazole molecule BMS-433771 (48, 50, 51). BMS-433771 interacts with the F protein and does not inhibit attachment (184). It was effective in inhibiting RSV infection of BALB/c mice when a single dose was administered prior to RSV inoculation, 28 but not when administered 1 h after exposure (48), suggesting that it might be useful prophylactically but not therapeutically. Despite these promising results,

BMS-433771 was not developed further for clinical testing due to realignment of company priorities (199).

VP-14637. ViroPharma identified a lead compound, VP-14637, from a panel of low molecular weight compounds in a HEp-2 cell-based high-throughput virus neutralization screen. It is a triphenolic compound with potent activity that targets the RSV F protein (77, 78, 195, 207). The initial clinical trials of VP-14637 were discontinued and VP-14637 was sold to MicroDose Therapeutx, who is continuing to develop VP-14637 with Gilead (204) VP-14637 has since been reformulated as a dry inhalational powder and renamed MDT-637. It is currently in Phase I trials.

TMC353121. In a screen of 130,000 compounds for the ability to block RSV infection, Johnson & Johnson/Tibotec discovered R170591 (JNJ-2408068) (12,

25, 26). Although extremely potent when tested in both tissue culture and the lungs of cotton rats, JNJ-2408068 was found to have long tissue retention, making it unsatisfactory for further development as an antiviral compound (25,

314). Lead optimization guided by molecular modeling resulted in a morpholinopropyl derivative, TMC353121, which retained the potency of its predecessor, but lacked its long tissue retention (25, 26). TMC353121 inhibits both virus-cell and cell-cell fusion in vitro, and maintains 50% effectiveness when

29 added to cells as late as 15 h post-inoculation (249). In recent studies with

BALB/c mice, TMC353121 was effective in reducing viral load and pathology in the lungs even when administered as late as 2 days post-inoculation (222).

Pharmacokinetic and pharmacodynamic studies of TMC353121 in cotton rats infected with RSV found the effective dose to be more than 2,000 fold higher than that required in cultured cells (0.07 ng/ml), partly because of activity lost through binding to serum proteins (26, 248). Johnson & Johnson/Tibotec continues to pursue TMC353121.

Mechanism of action for the Y198-binding group of compounds. All three of these compounds selected viral mutants in the aa 486-489 region of the F protein and therefore are likely to bind to the F protein similarly. However, a photoreactive, radiolabeled analog of BMS-433771 did not identify 486-489 as the attachment site of the compound, cross-linking instead to Y198 in the F protein (49). In a crystal structure of the RSV 6-helix bundle, TMC353121 was found attached to Y198 (249). Both experiments clearly identify Y198 as a critical part of the binding site for these compounds.

In the prefusion F protein structure, Y198 is found in the pre-HRA domain, the site of dramatic protein refolding during triggering (Fig. 1.8A). Because of the dynamics of this region, we hypothesize that it could be involved in triggering the

F protein. Interestingly, none of the RSV mutants selected by these drugs were in

Y198, suggesting that this residue plays an essential role in the function of the F

30 protein (Table 1.1). We have recently confirmed the importance of Y198 by showing that the Y198A mutation results in an F protein incapable of fusion even though it reaches the plasma membrane (Chaiwatpongsakorn, S., Ray, W.,

Costello, H.M., and M.E. Peeples, manuscript in preparation).

In the prefusion form, the aromatic side group of Y198 faces the aromatic side group of residue F223. The structure of each of these Y198-binding compounds has at least two aromatic rings (Fig. 1.10). It is possible that the drug aromatic rings bind to and stack on the aromatic rings of each Y198 and F223, locking the pre-HRA in place and preventing refolding.

The 486-489 mutation site shared by drug resistant

mutants selected with all three of these compounds

is in the HRB α-helix, at a position that would be very

close to Y198 in the postfusion 6-helix bundle (Fig.

1.8B). These changes may have enlarged the

hydrophobic space between HRA and HRB in this

region enabling the compounds to bind without

disrupting 6-HB formation, thereby allowing Figure 1.10. Chemical structures of three Y198- membrane fusion to occur (49, 50). In this scenario, binding drugs, TMC353121, R170591, and VP-14637. these compounds would likely bind to Y198 during

refolding, probably following the extension and trimerization of HRA but preceding or concomitant with intercalation of the HRB

31 helices that complete 6-helix bundle formation (184). In the co-crystallized 6- helix bundle, TMC353121 bound to both the HRA and HRB peptides within a hydrophobic pocket (249). VP-14637 and BMS-433771 have also been suggested to bind to this hydrophobic pocket (49, 77, 78, 249). Interestingly,

TMC353121 did not prevent 6-helix bundle formation, but it distorted its conformation. However, this distortion is limited to the membrane distal end of the 6-helix bundle (249). It is unclear how a local distortion some distance from the membranes could prevent fusion without preventing 6-helix bundle formation.

Additionally, it is unclear how mutations in four consecutive residues (486-489) in an α-helical structure could all result in the accommodation of a small molecule inhibitor. Because of its α-helical structure, two of these residues in HRB would be facing the HRA helix, at most.

Following selection with TMC353121 or VP-14637, viral mutants in two additional regions of the “head” (aa 392-394 and aa 398-400) were found (Fig. 1.8). BMS-

433771, in addition, selected mutations in the fusion peptide (aa 140 and 144).

These three groups of mutations are a great distance from the 6-HB in the post- triggered F protein and would be unlikely to enlarge the hydrophobic pocket to accommodate binding of a drug to Y198. So in these mutants, the compounds would still be able to bind to Y198, thereby distorting the 6-HB. How, then, could these mutants avoid drug inhibition of F protein function? An alternative explanation is that all of the resistant mutants selected with these compounds destabilize the F protein in some fashion, such that the F protein is triggered 32 randomly, without the need for its normal, specific trigger. This may be the case if the F protein is stabilized in the prefusion form by the Y198-binding compounds. If so, the prefusion form of all of these F mutants would be predicted to be less stable and this possibility could be tested.

Other Compounds Active Against the F Protein

RFI-641. Wyeth-Ayerst Research screened 20,000 compounds in a virus neutralization assay to find novel inhibitors of RSV. They identified two, a stilbene and its more active biphenyl analogue, CL-387626. Both compounds are fluorophores and displayed band narrowing and a blue shift in the presence of the F protein. These changes indicated a less polar environment for the compounds and are evidence of direct binding to the F protein (76). By modifying the biphenyl analog, this group identified a more potent inhibitor, RFI-

641, a biphenyl triazene (130, 215). Both compounds neutralized RSV strain A2 and cp-52, an RSV mutant with F as its only glycoprotein (35, 144), confirming that the target of both drugs is the F protein (244, 245). Although CL-387626 was ineffective when given after infection to cotton rats (315), RFI-641 reduced viral loads in African green monkeys treated by inhalation with the compound even when it was administered 12 and 24 h after RSV inoculation (130, 302). A drug-resistant mutant, G446E, has been isolated in the F protein, confirming its selective pressure on this protein (207) (Table 1.1, Fig. 1.8). RFI-641 is, however, not presently being pursued (207).

33

BTA9881. Biota completed a phase I clinical trial of a small molecule imidazoisoindolone derivative, BTA9881, that inhibited F protein function (20).

But further study was halted due to a poor safety profile in humans (21). Its structure, drug resistant mutants or mechanism of action have not been reported.

Peptide analogues. Trimeris, Inc. showed that peptide analogues of the HIV-1 gp41 protein HRA and HRB domains have antiviral activity (308-310). Their 36 amino acid HRB analog, enfuvirtide fusion inhibitor, was approved by the US

Food & Drug Administration is marketed as Fuzeon®. It is used primarily to treat patients infected with multi-drug-resistant HIV. Trimeris replicated this approach for several paramyxoviruses: RSV, PIV3, and measles virus (168). For RSV, peptide analogues of the HRB domain of the F protein (T-118; aa 488-522) were found to be fusion inhibitors (168, 309), although not as potent as the HIV-1 T-20

(168, 308, 310). A larger version of the T-118 HRB peptide, F478-516, is effective against RSV when present during membrane fusion (184), consistent with the suggestion that this peptide binds to the HRA trimeric coil, preventing intercalation of the protein‟s own HRB helices. The fact that enfuvirtide is an effective drug against HIV infection supports the notion that a peptide derived from the HRB of the RSV F protein could have a therapeutic effect during active infection. However, Trimeris is not presently continuing work on the RSV peptides, perhaps because of the high cost of manufacture, necessity for parenteral (intramuscular) administration (140, 194).

34

The G Protein as a Target for Antivirals

MBX-300. This molecule (originally called NMSO3) was pursued by Microbiotix.

It is a negatively-charged sulfated sialyl lipid with four sulfate groups that is thought to inhibit attachment by inhibiting electrostatic binding (154). RSV mutants resistant to MBX-300 have mutations across the G protein (Table 1.1), making it difficult to determine where it binds. Eight of these mutations are in the two mucin-like regions, and half of these are mutations to Pro, which would change the geometry of these regions, and possibly induce global changes in the conformation of the protein. Three mutations (F165L, F168S, F170S) are in the central conserved region of the G protein, long considered to be its likely receptor binding site, and all three remove a Phe. Another mutation (I189T) is in the heparin binding domain (135, 153). This domain enables attachment to heparan sulfate, a negatively charged molecule on the surface of immortalized, cultured cells (116, 117, 279, 280). MBX-300 has been shown to be less cytotoxic than ribavirin and to have stronger antiviral potency against both laboratory strains and clinical isolates, in both cultured cells and in cotton rats (154). MBX-300 is not specific for RSV as it also inhibits several other viruses, including , human , human immunodeficiency virus 1, and adenovirus (143, 211, 274, 316).

We have found that the G protein is critical for infection of primary well differentiated HAE cultures and that loss of its C-terminus results in the loss of

35 this function (165). We have also found that RSV infects primarily ciliated cells in these cultures and that, during differentiation in culture, susceptibility of these cultures to infection parallels the appearance of cilia (323). Identification of the

RSV receptor on these cells and the site on the G protein that binds these cells will provide a new target for the development of antivirals against RSV.

Compounds Active Against Other Viral Targets

RSV604. Arrow Therapeutics identified several possible RSV antiviral compounds in a screen of 20,000 compounds in viral neutralization and ELISA assays, including 1,4-benzodiazepine (40). RSV604 was developed through chemical optimization of these compounds (126). Resistant mutants had amino acid changes in the N-terminal end of the N protein, K105D, K107N, I129L, and

L139I, indicating that the N protein is the target of these compounds (40, 44).

RSV604 inhibits RSV infection in cultured cells and in an in vitro human airway epithelial model (44, 323). Following Phase I clinical trials (53) Novartis purchased the rights to this compound but is presently not continuing its development (200).

ALN-RSV01. The first virus reported to be susceptible to control by siRNA both in cell culture and in mice was RSV (22, 157, 324). Alnylam Pharmaceuticals has developed an siRNA, ALN-RSV01, directed against a conserved region of

RSV N protein mRNA. It is the first siRNA to be tested in humans against a microbial pathogen (74). This siRNA was determined to be the most potent

36 antiviral among a panel of 70 siRNAs targeting the RSV N, P, and L genes, as evaluated by its ability to inhibit virus production in cultured cells. ALN-RSV01 was also able to inhibit RSV infection in BALB/c mice (7). It has been found to be safe and effective in healthy adults (74, 75) and is currently in Phase II clinical trials (75, 319). ALN-RSV01 was recently considered for support for continued development by two major pharmaceutical companies but was declined by both

(173, 223). However, Alnylam is continuing its partnership with Cubist

Pharmaceuticals to develop siRNA drugs (6, 72), including a second generation siRNA against RSV, ALN-RSV02, intended for use in pediatric patients (6).

Other Promising Developments Towards RSV Antiviral Drugs

Ongoing development of antiviral compounds targeting RSV has yielded some new candidates. In a screen of over 16,000 compounds, two novel benzenesulfonamide-based compounds targeting the F protein, P13 and C15, were discovered. A P13-resistant mutant contained a N197T mutation within the

HRA region (181). N197 is the neighbor of Y198 (Fig. 1.8), the residue shown to bind the F protein inhibitors BMS-433771 and TMC353121, and likely VP-14637, as described above. However, none of those compounds resulted in selection of a mutation close to Y198 in the primary sequence. P13 may bind to the same α- helix but on a neighboring face. In any case, this mutant confirms the importance of this region in HRA, but suggests that N197 is not essential while Y198 is. We have similarly found that N197 (in strain A2) can be mutated to alanine without

37 loss of fusion activity (Chaiwatpongsakorn, S., Ray, W., Costello, H.M., and M.E.

Peeples, manuscript in preparation). Another P13 resistant mutant, T400I, and a

C15 resistant mutant, D489G, were also isolated. These mutants are located in the head of the RSV F protein, in two regions where mutants resistant to the

Y198-binding drugs have also been isolated. This finding indicates further that these two novel compounds may work in a similar, though perhaps not identical, manner to the compounds BMS-433771, VP-14637, and TMC353121.

A less specific approach has been taken by another group that has used palmitoyl-oleoyl-phosphatidylglycerol (POPG) to inhibit both RSV infection and the host inflammatory response in cultured cells and in mice. In cultured cells,

POPG seems to inhibit attachment of the virus to the cells and to inhibit viral spread (219). Dunn, et al., have recently shown that treatment with leflunomide, an immunosuppressive therapeutic, can reduce RSV load in cell culture and in infected cotton rats (79).

Development of new or improved antiviral screening methods has also been a major endeavor, and has been met with some significant success. Park, et al., have developed a peptide binding assay based on the fluorescence polarization caused by an HRB peptide binding to the final available position in an engineered

5-helix bundle of the RSV F protein. The assay could be used to screen for compounds that inhibit 6-helix bundle formation and therefore membrane fusion

(226).

38

Our laboratory has developed a replicon system for screening libraries of chemical compounds to identify those that inhibit the RSV RNA polymerase. We have removed the three glycoprotein genes from the full-length RSV cDNA, replacing them with a blasticidin-resistance gene, and inserted a marker gene that expresses green fluorescent protein (186). To enhance high throughput screening, we have also inserted the Renilla luciferase gene (Malykhina and

Peeples, unpublished data). A compound that inhibits genome replication or mRNA transcription reduces production of luciferase. This RSV replicon system may be able to identify compounds active against the viral polymerase (L), N, P, or M2-1, all of which are involved in viral replication and/or transcription.

Advances in RSV Vaccine Development

There are significant hurdles in the development of a safe, effective RSV vaccine

(112). As described above, the formalin-inactivated vaccine trial of the 1960‟s not only failed to protect, but enhanced disease in many of the recipients (93,

148, 150). This is thought to be largely the result of an exaggerated TH2 inflammatory response upon RSV challenge (169, 239, 299), but other possibilities have been suggested (reviewed in Blanco, et al. (24)). We will review many of the approaches that have been taken toward an effective RSV vaccine, their successes and their failures. We will not spend much time on the immunological concepts and problems with RSV vaccination, or on vaccination of

39 the newborns and infants, in general. Instead, we would refer the reader to several excellent recent reviews of these subjects (24, 107, 255, 258).

Additional challenges in the development of an effective RSV vaccine are the immunogenicity of vaccine components in the very young who have both an immature and the presence of maternal antibodies against RSV

(175, 257, 262, 300). In addition, in the elderly, immune senescence decreases the response to vaccines (33, 201, 202). Furthermore, RSV infection does not induce a particularly effective immune response even in healthy people. RSV is able to infect repeatedly throughout life (115), leading to concerns over the ability of an RSV vaccine to protect. However, the goal is a vaccine that prevents severe disease, not one that prevents RSV infection; the latter is much more difficult, perhaps impossible. Limiting RSV infection to the upper respiratory tract may be sufficient and is a reasonable goal since severe disease in a natural infection is due to lower respiratory tract infection. Finally, an RSV vaccine must not cause symptoms. Our goal here is to review vaccine approaches that have been developed over the years and novel approaches that present new possibilities.

Inactivated RSV vaccines and protein vaccines have generally not been considered for infants and children because of the enhanced disease observed in the original formalin-inactivated RSV vaccine trial and in the mouse model.

Vaccine development has focused instead on live, attenuated viruses for children

40 and subunit vaccines containing one or both of the major RSV glycoproteins, G and F, for adults. Reverse genetics has provided new tools for the construction of live attenuated vaccines with a variety of modifications, as well as live, vectored vaccines.

Live, Attenuated Viruses and Vector-Based Viruses as Vaccine Candidates

Attenuated respiratory vaccines administered intranasally can induce both mucosal and systemic immune responses, as clearly shown by the successful influenza A vaccine (52, 134). Live, attenuated RSV is particularly attractive as a vaccine because, unlike the inactivated RSV vaccine, infection with live virus does not lead to enhanced disease pathology upon challenge (65, 311). The many attenuated RSV viruses that have been developed and tested for their vaccine suitability have recently and thoroughly been reviewed by Schmidt (258).

Here we will just hit the highlights.

The first viable live candidate for infants was cpts248/404, with attenuation gained from mutations that appeared during cold-passage, chemical mutagenesis and high temperature passage (69-71, 149, 305). But this strain caused nasal congestion in recipients (312). In an effort to further attenuate this candidate, the SH gene was deleted and a missense mutation

(ts1030) was included in the L gene, all by reverse genetics, creating rA2cp248/404/1030ΔSH (306). This virus induced a high antibody response in children, but much less of a response in infants. Just as importantly, it did not

41 cause nasal congestion like its predecessor. Furthermore, the infants were protected from a second dose of the vaccine, essentially a challenge RSV infection (145). These ongoing clinical trials are in Phase I/IIa, (MEDI-559).

Other attenuated vaccine candidates have been developed but not yet tested in clinical trials. The rA2ΔM2-2 candidate, lacking its M2-2 gene (47, 283) was immunogenic in African green monkeys and, after two doses, protective against an RSV challenge (131). This attenuated virus, as well as rA2ΔNS1 which is lacking its NS1 gene, were highly attenuated in , a necessity for a pediatric vaccine (283). Candidates rA-GBFB (G and F genes of strain B substituted for the G and F genes of the A2 strain), and rA-GBFBΔM2-2, also missing its M2-2 gene, were both attenuated and protective against an RSV challenge in African green monkeys (47).

Reverse genetics has also enabled the development of vectored vaccine candidates, such as a bivalent viral vaccine designed to protect against both

RSV and human parainfluenza virus type 3 (hPIV3). It is a chimeric, bovine, bPIV3 virus, with the hPIV3 F and hemagglutinin-neuraminidase (attachment protein) genes replacing their bovine virus counterparts, and the addition of the

RSV F gene (MEDI-534, MedImmune) (118, 277, 278). A major advantage of bHPIV3 is that it is naturally attenuated in humans. While MEDI-534 was immunogenic and protective in hamsters and monkeys (276, 277), it was overly

42 attenuated in human adults and children, inducing low antibody titers (103, 278).

Testing in seronegative infants is in Phase I/IIa trials (57, 103).

Subunit Vaccines

RSV subunit vaccines, containing the F or G protein, or both, are being developed to boost immunity in adults and the elderly and protect against RSV disease. As mentioned above, subunit vaccines have not been considered for infants who do not have an established, effective immune response pattern because of the risk of enhanced disease which was seen in the initial inactivated vaccine trial.

Since the F protein is essential for RSV infection and is fairly well conserved between the two subgroups (133) compared to the G protein, which is not (135), the F protein would seem to be ideal for use as a subunit vaccine. Three similar

F subunit vaccines have been developed and tested in clinical trials (85, 108,

208, 230-232, 263). The PFP-1 vaccine developed by Lederle-Praxis Biologicals was composed of the F protein produced from RSV-infected Vero cells and isolated by immunoaffinity chromatography (225, 287). Wyeth-Lederle subsequently developed both the PFP-2 and PFP-3 vaccines composed of the

RSV F protein isolated by ion-exchange chromatography. PFP-3 was produced by a cold-passaged, temperature-sensitive mutant RSV that expressed high levels of the F protein (108, 208, 231). The purity of the F protein in these formulations were all 90% or greater. All three PFP vaccines were administered

43 with an alum adjuvant and all were safe among tested populations of seropositive children (healthy, with cystic fibrosis, or with bronchopulmonary dysplasia), pregnant women, and ambulatory elderly. However, they induced only modest levels of serum neutralizing antibodies or did not prevent lower respiratory tract infection (85, 108, 208, 225, 231, 287).

Novavax, Inc. has recently begun a Phase I clinical trial to investigate the safety of an RSV F protein virus-like particle (VLP) vaccine (55, 217, 218). It was found to be protective in cotton rats with no evidence of enhanced disease (216).

A vaccine developed by Sanofi Pasteur contains a mixture of RSV F, G and M proteins co-purified from infected Vero cells (171). The RSV F and G proteins induce neutralizing antibodies and the RSV M protein may stimulate T cell responses (86, 125, 253, 296, 298). It was found to be safe and immunogenic in a Phase II clinical trial in the elderly (86).

The ideal F protein vaccine would be in the prefusion form because this is the active form of the F protein present on virions and because the most potent neutralizing antibodies are against this conformation (185). Isolation of full-length

F protein from infected or transfected cells by immunoaffinity chromatography and ion exchange chromatography expose this metastable protein to extremes of pH or ionic strength. The simplest method of producing the F protein in an easy to purify form would be in a secreted, soluble (sF) form. However, Yin et al.

(317) found that simply removing the transmembrane and cytoplasmic domains

44 from the PIV3 F protein resulted in the release of postfusion sF, as determined by EM and by X-ray crystallography. In a subsequent report, Yin et al. found that the addition of a self-trimerizing α-helix to the C-terminus of the PIV5 F protein in place of its transmembrane and cytoplasmic domains prevented triggering of the

PIV5 sF protein. They used this protein to solve the structure of the prefusion paramyxovirus F protein (318). We have found that linking this α-helix to the C- terminus of the RSV F protein in place of the transmembrane and cytoplasmic domains also stabilizes it in the prefusion form (Chaiwatpongskorn, S. and

Peeples, M.E., unpublished results). But the addition of this foreign domain was not required to prevent triggering. Simply removing the transmembrane and cytoplasmic domains of the sF protein resulted in the production and release of prefusion, fully cleaved RSV sF protein (43). By adding a 6-His tag to its C- terminus, we were able to isolate this prefusion RSV sF protein on a Ni2+ column.

We have found that the sF protein remains in its prefusion form for at least four months (unpublished data). We have found that the sF protein can be induced to trigger by reducing the buffer molarity (43). It is not clear whether this mode of triggering is physiologically relevant.

The specific component of the formalin-inactivated RSV vaccine that led to enhanced disease upon RSV challenge has not been identified with certainty, but the most likely candidate is the RSV G protein. Two studies have presented particularly strong evidence for the G protein. of mice with G protein purified from RSV-infected Vero cells led to a strong TH2 response upon 45 challenge (120). In addition, vaccination with a recombinant vaccinia virus expressing the RSV G protein also induced a strong TH2 response upon challenge, but removal of amino acids 193-205, part of the mucin-like region II region (Fig. 1.4), avoided the pathogenic response and, in fact induced protective immunity in mice implicating this region as a possible cause of the enhanced pathology (268).

As described in the Introduction, mAb 131-2G targets the central region of the G protein and prevents the CX3C motif from binding to the CX3C receptor but does not neutralize the virus on immortalized cells. This mAb has been shown to prevent enhanced disease in FI-RSV-immunized mice challenged with RSV

(242). In these experiments, the mAb was injected one day before the RSV challenge and may have covered the C3XC site on the G protein of the challenge

RSV, thereby preventing lymphocyte chemotaxis. An alternative possibility is that the mAb neutralized the challenge RSV in vivo, reducing or preventing infection and therefore preventing enhanced disease. The authors did not consider this possibility because mAb 131-2G does not neutralize RSV infection of Vero cells. However, the RSV G protein uses heparan sulfate as its receptor on immortalized cells. It very likely uses a different receptor on fully differentiated airway cells which do not express detectable heparan sulfate on their apical surface (165, 322). Furthermore, the central region of the G protein, where this mAb binds, is the most likely attachment site for its in vivo receptor both because

46 of the conserved nature of much of this region and because it is the only site on the G protein that is not covered with glycans.

A peptide vaccine, BBG2Na (aa 130-230, Fig. 1.4), that contains this central conserved domain of the G protein induces protective immunity in mice (234,

239-241). BBG2Na also includes amino acids 193-205, the sequence suggested to be necessary for enhanced disease (268). But RSV challenge of mice immunized with BBG2Na did not lead to enhanced disease. In human trials,

BBG2Na was well-tolerated and showed a moderate ability to induce neutralizing antibodies in healthy, young adults (240). In infant macaques, BBG2Na did not induce neutralizing antibodies, did not reduce pulmonary viral loads, and resulted in mild eosinophilia upon RSV challenge in two of four animals (73). Following reformulation to remove the BB component, the vaccine was found to be protective for cotton rats and increased the anti-RSV antibody titer in mice that had been previously immunized (214). A similar peptide protected immunized mice from an RSV challenge, with no enhanced disease (152).

Recent Promising Developments Towards an RSV Vaccine

A large number of alternative RSV vaccine approaches are presently being pursued but have not yet reached clinical trials. This vibrant activity has been wide-ranging, including both RSV proteins/peptides produced in vitro for injection and viral vectors that produce RSV proteins in vivo. These multiple approaches are providing further insights into the mechanisms of immunity and the causes of

47 enhanced immunopathology. They are also expanding the possibilities for and the likelihood of an eventual RSV vaccine. We have briefly summarized many, but not all, of these approaches below to enable comparisons.

Recombinant virus vectors expressing RSV proteins in vivo.

Widjojoatmodjo, et al., found that RSV strain A lacking its G gene protected cotton rats from RSV challenge without causing enhanced disease (307), suggesting that this vaccine approach may be promising. This result was surprising because the original G-deleted RSV, a strain B mutant, had not been protective in mice or non-human (144, 145). Perhaps the RSV strain difference is responsible. In our experience, a strain A RSV lacking its G protein is 10-fold less infectious for well differentiated HAE cultures than G-containing virus (165). Poor infectivity in HAE cultures would likely reflect reduced effective titer of an attenuated vaccine in vivo. Furthermore, we found that this same strain, lacking the G gene, produced titers at least 10-fold lower than the parental virus in immortalized cell culture (279), which would reduce the efficiency of vaccine production. However, production of a G-deleted RSV in a cell line expressing the G protein might boost the virus titer and make such a vaccine feasible by enhancing its initial infection in vivo.

A number of novel vectored vaccines have been generated and tested in animals recently. A chimeric measles virus vaccine expressing the RSV F protein, administered intranasally, protected cotton rats and induced only mild

48 inflammation upon RSV challenge; however, vaccine-enhanced eosinophilia was not evaluated (256). A replication-deficient adenovirus expressing aa 155-524 of the RSV F protein, preceded by a signal sequence to enable secretion, administered to the nasal mucosa of mice, protected them against RSV challenge without inducing enhanced pathology (151). This RSV F protein sequence was codon optimized to enable expression from the nucleus, a requirement for the F protein (129). The success of this vaccine in mice is striking because the F protein fragment used excludes F2, pep27, the fusion peptide, and the transmembrane and cytoplasmic domains (151) and therefore much of this F protein fragment would not be expected to be folded properly.

Another adenovirus vector has been constructed with codon-optimized versions of the full-length and an anchorless RSV F protein. Both were immunogenic and protected mice against RSV challenge. BAL fluid collected from immunized and subsequently challenged mice did not indicate eosinophilia (155).

Martinez-Sobrido, et al., created a chimeric Newcastle disease virus (NDV) expressing the RSV F gene. NDV, an avian pathogen, is known to induce a robust innate immune response in infected cells, unlike the weak response induced by RSV. In mice, they found a greater immune response to the RSV F protein expressed from this vector than from RSV infection itself, and it protected from RSV challenge without immunopathology (187). Importantly, NDV is naturally attenuated in humans and high virus titers can readily be produced in embryonated eggs. 49

Sendai virus (SeV), a parainfluenza virus of mice with similarities to human PIV1, engineered to express RSV glycoproteins, is also being developed as a vaccine.

SeV expressing either the RSV G or F protein given intranasally to cotton rats protected them from an RSV challenge with no evidence of pulmonary immunopathology (141, 275, 320, 321). Neither RSV glycoprotein was incorporated into the budding virions (275, 320). Other recombinant SeV-RSV expressing either the complete RSV F protein or a soluble version induced protective immunity against RSV, but these vaccines also elicited slightly enhanced immunopathology (293, 326). These latter SeV vectors contained the

F gene in the third gene position while in the former; the F gene was in the fifth position. A gene in the third position would be expressed at a higher level than a gene in the fifth position because of the polar nature of paramyxovirus gene expression and perhaps the higher level of antigen production led to the mild pathology. Sendai virus, like NDV, has the advantage of stimulating interferon and of high titer production in embryonated eggs.

Mok, et al., has produced nonpropagating Venezuelan equine (VEE) virus replicon particles (VRPs) that contain an RNA genome encoding the VEE replicase proteins and either the RSV F or RSV G protein. Both VRPs protected mice and rats from RSV challenge of the upper respiratory tract without causing enhanced inflammation. The RSV G protein-expressing VPR protected against lower respiratory tract RSV challenge, while the F protein VRP did not (205).

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Wu, et al., have developed a DNA vaccine encoding a fragment of the F protein

(residues 412-524; part of the “head” and HRB, Fig. 1.8) and the same F protein fragment fused to a portion the ctxA2B subunit of the cholera toxin, a potent adjuvant. DNA expressing these proteins was injected intramuscularly in mice, and the mice were subsequently challenged with RSV. Both vaccines were partially protective (264, 313).

Bacterial expression vectors are also being developed for use as T cell vaccines to protect against RSV. Strains of the mycobacterium Bacillus Calmette-Guérin

(BCG) expressing either the RSV N or M2-1 protein were protective against RSV challenge in mice (34). Expression of the N protein elicited TH1 T cell recruitment to the lungs, mediating viral clearance and reducing inflammation upon challenge

(42).

RSV protein vaccines produced in vitro. VLPs are being developed as vaccines and yielding some interesting candidates. Nallet, et al., transiently expressed a codon optimized F protein in Vero cells and purified it by chromatography before inserting it into immunostimulating reconstituted influenza virosomes (IRIV). The IRIV was composed of pure lipids, the RSV F protein and the influenza HA and NA proteins. This IRIV induced the production of neutralizing anti-F antibodies in BALB/c mice, but protection against RSV challenge and vaccine-enhanced immunopathology were not assessed (212).

NDV VLPs containing the NDV NP and M proteins and the ectodomains of the

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RSV F and G proteins fused respectively to the NDV F and HN transmembrane/cytoplasmic domains have been produced from transfected cells and shown to stimulate the production of neutralizing antibodies to both the F and G proteins in mice. These immunized mice were protected from RSV challenge and did not display enhanced disease (193). Stegmann, et al., have produced pure lipid virosomes containing RSV proteins F, G, and, to a lesser extent, M, and have shown that they induce protective neutralizing antibodies in mice and cotton rats. P3CSK4, a synthetic lipopeptide, was included in these liposomes as an adjuvant because it is recognized by TLR-2, and therefore should establish a TH1 response pattern. Immunized mice and cotton rats were protected and showed no indication of vaccine-enhanced disease upon challenge (270).

Trudel, et al., has developed a peptide vaccine composed of 14 amino acids from the central domain of the G protein (Fig. 1.4) (aa 174 to 187, with one mutation,

C186S), cross-linked to keyhole limpet hemocyanin (17, 233, 288). Following mucosal administration, both the hRSV peptide (adjuvanted with cholera toxin) and the bRSV G protein peptide (without adjuvant) protected BALB/c mice from challenge, though the induction of enhanced immunopathology was not determined (17). This peptide includes the CX3C motif except for the Cys mutation and was chosen from a panel of hRSV G protein peptides for its ability to induce a protective response when administered intraperitoneally to mice

(288). 52

Adjuvants. Because of the history of enhanced disease following the formalin- inactivated RSV vaccine, there has been interest in developing adjuvants that promote a TH1 or balanced immune response. CpG oligodeoxynucleotide, indolicidin, and polyphosphazene have been tested as adjuvants. When the soluble version of the bovine RSV F protein (aa 1-522) was mixed with a combination of these adjuvants and administered to mice, the adjuvants enhanced the immune response relative to the soluble bRSV F protein alone, resulting in the production of more neutralizing antibodies and a shift from TH2 to

TH1 response following bRSV challenge (158). After challenge, much less bRSV

RNA was detected in the lungs compared to unimmunized mice. This combination of adjuvants may have potential for future human RSV vaccine formulations with viral subunits. A combination of saponin QS-21 and recombinant interleukin-12 has also been shown to be a strong adjuvant for production of cell-mediated immunity and neutralizing antibodies against the RSV

F protein (119).

NanoBio Corporation recently demonstrated that a nanoemulsion of water-in-oil can both inactivate RSV and function as an adjuvant in the vaccination of mice.

Intranasally vaccinated mice cleared an RSV challenge to either the upper or lower respiratory tract without evidence of enhanced disease (179), suggesting that this approach has promise. NanoBio recently licensed a novel RSV antigen from the NIH for use in the development of this vaccine (213), but the composition of this antigen was not reported. 53

Conclusions

RSV continues to be a major cause of hospitalization for infants and of morbidity and mortality for the elderly. With the prophylactic mAb palivizumab being the only approved, effective compound against RSV, there is a great need for continued intense research into antiviral drugs and effective vaccines. There are significant hurdles to overcome with the development of both. Antiviral compounds must display acceptable safety, half-life, tissue targeting, and bioavailability. While the greatest need is for a drug to treat active infections, antiviral drugs could also play a prophylactic role. Likewise, palivizumab or motavizumab may have a future role in treating established disease in addition to prophylaxis.

Most antiviral drugs developed thus far target the F protein, which is essential for

RSV to infect and to spread within an individual. Interestingly, three of the most thoroughly studied small molecules all bind to the same general site on the F protein that includes Y198, but none of drugs select resistant mutations in Y198.

This amino acid may play a critical role for the F protein, either in initiating its conformational change or in forming its 6-helix bundle, the final step in its fusion function. One of those small molecules, TMC353121, has been shown to distort the 6-helix bundle. This small molecule continues to be pursued and may yet emerge as a useful antiviral drug. Other novel drugs under development also

54 show promise. It is not clear whether the appearance of drug-resistant RSV variants will be a problem once an antiviral is in use. This problem has surfaced with drugs against influenza virus, another acute respiratory virus (98), suggesting that it will likely be a problem with RSV, too. If so, a combination of two or three drugs may be needed to reduce the likelihood of the appearance of drug-resistant RSV.

The publication of the prefusion structure of the RSV F protein represents a significant advancement in terms of understanding the mechanism of the RSV fusion protein function and with the potential to develop targeted small molecule drugs against the protein. However, the structure is not yet perfect, as it is at a

3.6 Å resolution, which is too low for confident prediction of amino acid side chain placement. Additionally, the crystallized protein had a fibritin trimerization attached to its C-terminus and an antibody that rivals the size of the F protein bound to the most dynamic region of the protein. Either of these factors may have constrained the native structure of the protein, or in the case of the antibody, have altered the structure near the epitope for an induced fit. There is significant value in determining the crystal structure of the prefusion RSV F protein at higher resolution, not linked to a trimerization domain, or bound to an antibody. This has proved difficult in the past for paramyxoviruses, but it may be possible with small introduced mutations such as N-glycan deletions, insertion of exogenous intermonomeric disulfide bonds, or using an RSV F protein of a different viral strain. Other options include stabilizing the protein in the prefusion 55 conformation with the addition of cross-linking molecules or with one of the small

Y198-binding drugs (if they do indeed function by preventing triggering). A crystal structure of the uncleaved F protein in its prefusion state would also be helpful to understand the positions of the F2 C-terminus and the F1 N terminus before cleavage.

Attenuated and vectored vaccine development has carefully avoided candidates that cause any significant symptoms following vaccination, and even more carefully avoided vaccine-enhanced disease upon challenge. These twin limitations have made finding a live vaccine that is attenuated while remaining immunogenic very difficult. Presently, the most advanced live vaccine candidates for infants are the attenuated RSV and the chimeric bPIV-3 expressing the RSV F protein. Both should induce antibody and T cell responses to the F glycoprotein. However, only the attenuated RSV will induce antibodies to the F and G proteins as well as T cell responses to the RSV proteins. The antibody response against the G and F glycoproteins is clearly important for protection, but the importance of the cytotoxic T cell response has not been studied in humans. RSV strain A lacking its G gene and vectored RSV vaccines that express the RSV F or G protein from measles virus, NDV, Sendai virus, adenovirus, and VEE have great potential but are earlier in development.

Bacterial T cell vaccines are a novel approach and may allow inoculation and protection early in life.

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Subunit protein vaccines continue to have potential as vaccines for adults in whom a non-pathogenic immune response to RSV has already been established but needs restimulation. It seems that because the most potent antibodies raised against RSV F protein are against the prefusion form (185), there is value is attempting to stabilize this form for use as a subunit vaccine. Introduction of exogenous disulfide bonds, addition of a crosslinker, and stabilization by Y198- binding drugs can also be employed for this objective.

The recent surge in novel RSV vaccine approaches, including the addition of novel adjuvants is encouraging, presenting additional options to the traditional approaches. The road to a protective vaccine against severe RSV disease has not been easy, but many excellent options are being explored and one or more are likely to reach this important goal for human health.

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Chapter 2: The N500 Glycan of the Respiratory Syncytial Virus F (Fusion)

Protein is Required for Fusion, but Not for Stabilization or Triggering of the

Protein

Introduction

Respiratory syncytial virus (RSV) is the most significant cause of serious respiratory infections in infants and young children worldwide (62, 102, 111, 148).

It is also a major cause of serious lower respiratory tract infections in the elderly, second only to influenza in non-pandemic years (82, 84, 115, 284, 295). There are no vaccines or effective antiviral drugs approved for use against RSV, despite intensive research (reviewed in (68)). There is only one prophylactic agent, palivizumab (Synagis), a humanized monoclonal antibody that is administered to at-risk infants during the RSV season (1, 271). There is currently a concentrated effort to learn more about the proteins, replication process, and infection of RSV, in hopes that antiviral drugs and vaccines can be developed.

All enveloped viruses, including paramyxoviruses like RSV, must fuse with a host cell membrane to initiate infection. RSV fuses with the apical surface of ciliated

58 cells in the human respiratory tract using two of its surface proteins: the attachment (G) glycoprotein and the fusion (F) glycoprotein (323). Following the attachment of the G protein to its as yet unknown receptor on the target cell, the

F protein fuses the viral envelope with the host cell membrane, initiating infection

(62).

The F protein is produced as an F0 precursor, trimerizes in the endoplasmic reticulum, and is cleaved in two places during transit through the Golgi. This results in the mature form of the F protein, F1+F2, in which the two subunits are held together by two disulfide bonds. It is the F1+F2 form that accomplishes membrane fusion following a series of dramatic conformational changes. After the virus attaches to its receptor on the host cell, an as-yet undetermined triggering stimulus causes the pre-HRA region of the prefusion form of the each monomer to rearrange into a single, very long α-helix, the HRA region. At the N- terminus of the HRA is the hydrophobic fusion peptide, which is thrust into the target membrane as a consequence of the formation of the HRA domain. At this point, the three long HRA helices of the trimer probably meet, forming a coiled coil structure. The protein next folds in half and the HRB heptad repeat region

(near the C-terminus of the protein) from each monomer in the trimer intercalates into the grooves on the HRA coiled-coil. Because the N-terminus of the HRA trimer is embedded in the target cell membrane and the C-terminus of the HRB helices are embedded in the virion membrane, the folding of the F protein brings the host cell and viral membranes together, mixing the lipids and initiating fusion.

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It is challenging to study the triggering and refolding of the RSV F protein in the context of the complex cellular or viral membrane. In order to biochemically assay for triggering and refolding of the F protein, our laboratory has constructed and characterized a soluble version of the RSV F protein (sF) which is able to be produced and purified in the prefusion form (43). We have also identified a condition that is able to trigger the sF protein, exposure to low molarity (43), and with this, we are able to examine the triggering of the sF protein.

Recently, crystal structures of soluble forms of the prefusion and postfusion RSV

F (sF) protein were solved (197, 198, 273). Both forms were missing their transmembrane and cytoplasmic domains. The postfusion structure was solved by two groups, both of which deleted the 9 or 10 most N-terminal residues of the

18 in the hydrophobic fusion peptide (198, 273). The shortened fusion peptide did not cause the sF protein to aggregate as the full-length fusion peptide does.

Very recently, the prefusion form of the F protein was crystallized and its structure solved by McLellan, et al (197). Both forms were fully cleaved by a furin-like enzyme during transit through the Golgi.

In addition to disulfide bond formation, the F protein has several notable post- translational modifications. It oligomerizes into a homotrimer in the endoplasmic reticulum shortly after or concurrently with translation (64, 172, 189, 197, 198,

273). In the Golgi, each monomer is cleaved in two places by a furin-like protease, releasing a 27-amino acid peptide (pep27). The resulting F1 and F2 fragments are held together by two disulfide bonds (109, 110). The F protein is 60 palmitoylated at residue C550 in the cytoplasmic tail during transport through the

ER and Golgi (13).

In addition to these modifications, the RSV F protein is N-glycosylated in several positions. High mannose N-glycans are attached in the endoplasmic reticulum to asparagine residues in the consensus sequence N-X-S/T. As the protein travels through the Golgi apparatus, the glycan chains are trimmed back and elongated with various other sugars.

The RSV strain A2 F protein is N-glycosylated at 5 residues: N27, N70, N116,

N126, and N500. An additional N-glycan is found at N120 on the F protein of the

Long strain (Fig. 1.5). Glycans at N116, N120, and N126 are linked to pep27, which appears to be released, and thus are unlikely to be of consequence in the mature, cleaved protein. The remaining three N-glycans, N27, N70, and N500, are linked to asparagines present in the F1+F2 mature form of the F protein. N- glycans may play roles in the initial intracellular protein folding in the ER, transport through the cell, cleavage of the protein, or F protein refolding following triggering, all of which are important for RSV infection.

Prior studies of the RSV F protein N-glycans have determined that the glycan structures are heterogeneous and that the F protein is present in multiple glycosylated forms (191, 246). When the maturation of the N-glycans of the virus were inhibited by alpha mannosidase inhibitors, 100-fold less infectious virus was produced (190). The N-glycan maturation inhibition had no effect on the morphology of the virus or on the processing of the G protein, leading to the 61 conclusion that maturation of the N-glycans of the F protein is essential for viral infectivity. In two very similar studies, it was determined through mutating the asparagine residues of the N-glycosylation motifs to glutamine that the N500 glycan is essential for efficient fusion, while the others seem to be largely unimportant (177, 328). None of these glycosylations seem to be important for initial protein folding or cell surface expression, as mutations of the individual glycan sites did not affect either (177, 328). It has also been shown that when all

N-glycosylation is inhibited by tunicamycin, the F protein is still transported to the cell surface efficiently, indicating that this transport does not rely on the presence of the glycans (64).

Although it seems clear that the N500 glycan is important for the F protein to perform its role in membrane fusion, it is not known how it contributes to F protein function. It may serve to keep the protein stabilized in its prefusion form, play a role in triggering, or be essential to some step in protein refolding from the prefusion to the postfusion form. In this study, we examined the importance and roles of the N27, N70, and N500 glycans in RSV F protein-induced fusion and refolding following a triggering stimulus.

Materials and Methods

Cells. Human Embryonic Kidney (HEK) 293T adherent cells were grown in

Dulbecco‟s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine

62

® serum (FBS) in a humidified chamber at 37°C and 5% CO2. HEK 293 Freestyle cells (Life Technologies, Carlsbad, CA), a suspension line developed for protein expression in animal component and protein free medium, were grown in

Freestyle 293 Expression Medium® (Life Technologies, Carlsbad, CA) on a shaker platform rotating at 125 RPM in a humidified chamber at 37°C and 8%

CO2.

Cloning and plasmids. A codon-optimized version of the full-length RSV F protein gene from strain D53 (MP340) was used to construct the N27Q, N70Q,

N500Q, N27/70Q, and N27/70/500Q genes by site-directed mutagenesis (38).

These same mutations were moved to a soluble version of the same codon- optimized RSV F protein gene that was constructed and characterized previously

(43). The full-length F protein and soluble F protein WT and mutant genes were expressed from the pcDNA3.1 plasmid. The empty pcDNA3.1 plasmid was used as an experimental negative control. Each construct was tested in triplicate. The pFR-Luc and pBD-NFκB constructs were a generous gift of Alfred Del Vecchio

(Centocor, Inc.) (28).

Transfection. HEK293T cells were seeded onto poly-d-lysine coated plates and transfected per manufacturer protocol using Lipofectamine 2000 transfection reagent (Life Technology, Carlsbad, CA). HEK293 Freestyle cells were transfected per manufacturer protocol using 293fectin transfection reagent (Life

Technology, Carlsbad, CA).

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Determination of cell surface expression by biotinylation and immuno- precipitation. At 12 h post transfection, HEK 293T cells expressing full-length

RSV F WT or mutant proteins were rinsed with phosphate buffered saline (PBS) and incubated for 30 min with 0.5 mg/mL Thermo Scientific EZ-Link Sulfo-NHS-

LC-Biotin® in Hanks Balanced Salt Solution (HBSS) supplemented with Mg2+ and

Ca2+. The biotin solution was removed and the cells were rinsed twice with 0.5 M glycine in PBS. The cells were lysed in a tris-buffered saline solution (50 mM tris, 150 mM NaCl, pH 7.5) containing 1% Triton-X-100 and clarified by a 10 min centrifugation at 14 krpm. The supernatant was incubated overnight at 4°C, tipping, with Protein G-sepharose beads that had been preincubated with the anti-RSV F protein monoclonal antibody L4 (297). The beads were rinsed six times with cell buffer and boiled in SDS-PAGE sample buffer with 10% 2- mercaptoethanol. The samples were electrophoresed on a 4-12% bis-tris gradient gel (NuPage gel, Novex system, Life Technologies) and transferred to a nitrocellulose membrane. The membrane was blocked with 1% bovine serum albumin (BSA) blocking diluent for 1 h at room temperature and probed with a

1:6000 dilution of horseradish peroxidase-conjugated streptavidin in 1% BSA blocking diluent for 1 h at room temperature. The blot was rinsed five times for

20 min each with PBS containing 0.05% Tween-20. The blot was bathed in

Roche Lumi-Light® chemiluminescent substrate and subsequently exposed to x- ray film to visualize.

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Determination of cell surface expression by flow cytometry. At 12 h post transfection, HEK 293T cells expressing full-length RSV F WT or mutant proteins were washed with PBS and lifted from the plate with Versene. An excess of

DMEM + 10% FBS was added to quench the Versene, and the cells were pipetted up and down to ensure a single cell suspension. Each replicate was moved to the well of a non-tissue-culture coated 96-well plate and pelleted at

1200 rpm for 5 min at 4°C. The cells were washed twice with FACS Wash Buffer

(PBS + 10% FBS + 0.1% NaN3), with centrifugations of 1200 rpm for 5 min at

4°C following each wash. The cells were resuspended in FACS Fixation Buffer

(PBS + 10% FBS + 0.1% NaN3 + 2% paraformaldehyde) and incubated while tipping for 20 min at 4°C. Cells were washed two times with FACS Wash Buffer and resuspended in 1 μg/mL of a monoclonal antibody against the RSV F protein diluted in FACS Wash Buffer and incubated while tipping for 30 min at 4°C. Cells were washed three times with FACS Wash Buffer and resuspended in a 1:500 dilution (in FACS Wash Buffer) of a goat anti-human antibody conjugated to

DyLight® 633 and incubated while tipping for 30 min at 4°C. Cells were washed three times with FACS Wash Buffer and resuspended in a final volume of 200 μL

FACS Wash Buffer. An isotype control (IgG1 human), a control with secondary antibody only, and an unstained sample were all included. Flow cytometry was performed using the appropriate channels for the fluorescent dye using

FACSDiva software. Data were analyzed using Flow Jo software.

Cell-to-cell fusion assay. To establish effector cells, plasmids expressing F

65 protein mutants were transfected into HEK 293T cells with Lipofectamine 2000

(Invitrogen) per the manufacturer‟s protocol along with pFR-Luc, a plasmid containing a luciferase gene driven by a Gal4 promoter. A second set of HEK

293T cells, the target cells, was transfected with pBD-NFκB, a plasmid expressing a Gal4 binding domain fused to the NFκB transcription factor. At 12 h post transfection, the target cells were suspended with Versene, washed in PBS and resuspended in DMEM-10% FBS and an equal number were placed directly onto the effector cells. The cells were incubated together for 8 h, washed and lysed in Steady-Glo Luciferase Assay Substrate (Promega, Madison, WI). The relative luciferase levels were determined using a luminometer plate reader.

Producing and purifying sF protein. Plasmids containing the WT sF and mutant sF genes were transfected into HEK 293 Freestyle cells. The sF protein was purified from the medium harvested between 48 and 72 h post transfection by passage over a Ni2+ column (Probond® purification system; Life Technologies,

Carlsbad, CA) and eluted with 100 mM imidazole in a 50 mM Hepes buffer with

500 mM NaCl. Samples were analyzed by Western blotting with a monoclonal antibody against the RSV F protein, goat anti-human antibody conjugated to horseradish peroxidase (KPL, Gaithersburg, MD), and Lumi-Light Western blotting substrate (Roche, Basel, Switzerland).

Velocity sedimentation gradient. Purified sF protein was layered over a 5 mL

5 – 20% linear sucrose gradient (in 30 mM Hepes buffer + 170 mM NaCl) and centrifuged at 37.5 krpm for 17 h at 4°C with an SW 55 rotor in an ultracentrifuge 66

(Beckman Coulter Inc., Brea, CA). Gradients were fractionated into 0.5 mL fractions by pipetting from the top surface. Aliquots of each fraction were analyzed by Western blotting with a monoclonal antibody against the RSV F protein.

Dialysis. A dialysis cassette (Slide-A-Lyzer®, Thermo Scientific) of appropriate capacity was loaded with WT or mutant sF protein and promptly dialyzed against an excess of chosen buffer (250 times the volume of the sample). The sample was dialyzed with fresh buffer twice for two hours and again overnight, all at 4°C.

The dialyzed samples were analyzed by velocity sedimentation gradient.

Results

Mutagenic inhibition of N500 glycosylation results in lowered fusion. We mutated each of the three asparagines (N) within an N-glycosylation consensus sequence that would be present in the cleaved F protein (Fig. 1.5) to glutamine

(Q) by reverse PCR. Glutamine was chosen because its structure is very similar to asparagine, but will disrupt the N-glycosylation consensus sequence of N-X-

S/T, preventing glycosylation. In addition to the single mutants of N27Q, N70Q, and N500Q, we also constructed the combination mutants N27/70Q and

N27/70/500Q.

To determine the role of these glycans in fusion, we transiently transfected each of the three single mutants into HEK 293T cells and examined the cells microscopically for presence of syncytia. N27Q, N70Q, and N27/70Q produced

67 syncytia at similar levels to WT (Fig. 2.1). N500Q and N27/70/500Q, however, appeared to have completely lost their ability to fuse, as the level of syncytial formation is similar to the empty vector negative control. It is not clear, however, if the lowered fusion is a result of a lack of cell surface expression of the mutant or a deficiency in function of the mutant.

RSV F protein N-linked glycans are not required for cell surface expression.

To compare the cell surface expression of these mutants to WT, we transiently transfected each mutant into HEK 293T cells and incubated at 37°C for 12 h to allow expression of the F protein and its presentation on the surface of the cell.

Twelve hours is adequate to assess the amount of the protein transported to the cell surface, but is not long enough for large syncytia to form. The cells were then probed with an anti-F monoclonal antibody and a goat anti-human secondary antibody conjugated to the DyLight 633 fluorophore. Median fluorescent intensity of each sample was analyzed via flow cytometry. Each mutant was compared to the intensity of the WT F protein fluorescence. The single mutants (N27Q, N70Q, and N500Q) and the double mutant (N27/70Q) were expressed on the cell surface at near WT levels (Fig. 2.2, blue columns).

The triple mutant, N27/70/500Q, was expressed at about 45% of the level of WT.

A similar mutant was reported previously to have an expression level near WT

(328). These findings were confirmed in a cell surface protein biotinylation/ immunoprecipitation experiment (data not shown). The expression of the mutants on the cell surface signifies that these proteins are processed and folded 68 correctly. This indicates that none of the three glycans in the RSV F protein are required for the folding or transport activities of the F protein.

The N500 glycan is

critical for cell-to-

cell fusion. To

determine the

importance of the

N500 glycan in

fusion, we employed

a quantitative fusion

assay with two sets

of transfected cells,

an effector set

cotransfected with Figure 2.1. Syncytia formation of HEK 293T cells transiently co- transfected with WT RSV F protein or glycosylation mutant and the F protein WT or pTracer, a plasmid containing GFP. pcDNA3.1 is empty vector as negative control. mutant plasmid and

a luciferase plasmid

(pFR-Luc) and a target set transfected with a transcription activator plasmid

(pBD-NFκB). The target cells are suspended at 12 h post transfection and overlayed onto the effector cells. After 8 h incubation, the cells are lysed and the level of luciferase quantified. Luciferase is only produced if target cells and effector cells had fused.

69

The single mutants N27Q and N70Q and the double mutant N27/70Q, have fusion levels similar to WT F protein, indicating that these two N-glycans are not involved in the fusion process (Fig. 2.2, green columns). However, similar to what was observed microscopically, N500Q fuses at ~5% the level of WT. The cell surface expression of the triple mutant, N27/70/500Q, was about half that of

WT and its fusion level was almost completely ablated. The N500 glycan of the

RSV F protein clearly plays a critical role in the process of fusion.

Figure 2.2. Cell surface expression and fusion of N-glycosylation mutants. Cell surface expression (blue) as determined by flow cytometry as median fluorescent intensity of stained transfected HEK 293T cells. WT RSV F protein and each glycosylation mutant are represented, calculated as a percentage of WT expression. Cell-to-cell fusion (green) as determined by a quantitative luciferase fusion assay in transfected HEK 293T cells. The fusion assay was performed in parallel with cell surface expression determination. Each value is presented as a percentage of WT fusion.

70

Using the soluble F protein to analyze the role of its glycans. It is clear from the results in

Figures 2.1 and 2.3 that the N500 glycan is critical for the fusion activity of the F protein. The next Figure 2.3. A soluble version of the RSV F protein (sF) was generated by replacing the transmembrane (TM) and question is what role cytoplasmic domains with FLAG and 6-His tags. The asparagine to glutamine mutations were introduced into the sF for biochemical testing. this glycan plays. The

N500 glycan could be involved in stabilizing the prefusion form, in triggering the F protein, or in enabling it to refold properly. Analysis of these possibilities requires the ability to distinguish between the prefusion and postfusion forms of the F protein, which has not been possible in the context of a cell. However, using a soluble form of the RSV F protein we have been able to distinguish its forms, and we have identified a condition which causes the prefusion form to trigger, resulting in the postfusion form (43). To take advantage of this system, we moved each of the glycosylation mutants into a soluble version of the RSV F protein (Fig. 2.3). The

71 soluble F (sF) protein is lacking its transmembrane and cytoplasmic domains, containing FLAG and 6-HIS tags in their place.

Figure 2.4. WT and mutant sF protein transfected Freestyle 293 cell lysates and cell supernatants as examined by Western blotting for the F protein. For each, 10 times more cell lysate than supernatant was loaded. Migration positions of the WT F1 and F0 protein are denoted. Samples were run in both reducing (A) and non-reducing (B) conditions. “X” indicates a fragment of the F1 protein that is detected by the mAb within the cells but is not released into the medium.

Plasmids expressing the soluble F protein (sF) of WT and the glycan mutants

(sN27Q, sN70Q, sN500Q, sN27/70Q, and sN27/70/500Q) were transiently transfected into HEK 293T cells. At 48 h post transfection, the medium was collected and the cells were lysed. Samples were prepared in both reducing and non-reducing conditions, electrophoresed, and analyzed by Western blotting. All of the mutant sF proteins were expressed and secreted efficiently (Fig. 2.4A).

Similar amounts of sF protein appear in the cell lysate (C) lanes as in the medium (M) lanes, but 10 times more cell equivalents were loaded in the C lanes

72 than in the M lanes, Therefore, 90% of the sF protein produced by these cells was released into the medium. In all mutants, only the fully cleaved sF protein is secreted, since only the sF1 protein (50 kDa) was present in the M lanes, while its precursor sF0 (~70 kDa) was present only in the cell lysate, indicating that the sF protein that is secreted following transfection with all of these mutants is efficiently cleaved.

The F2 (19 kDa) is not detected in Fig. 2.4A because the blot was developed with an antibody specific for the F1 protein. The sF1 and F2 subunits are linked together by disulfide bonds but were dissociated by the reducing conditions of the sample buffer. As expected, under the reducing conditions, only sF proteins containing the N500Q mutation migrated differently from WT because this is the only mutation in the sF1 protein.

However, when the samples were treated with non-reducing sample buffer instead, the sF1 and F2 subunits remained associated and migrated together on the gel as a 70 kDa species when fully glycosylated (WT) (Fig. 2.4B). The sizes of the sF1+F2 proteins varied from mutant to mutant, as expected for mutants missing one or more glycans, confirming that the asparagine to glutamine mutations have prevented the glycosylation at that position.

N-glycans are not necessary for maintaining the sF protein in its prefusion form. One hypothesis that could explain why the N500Q mutant is fusion- deficient is that the N500 glycan is important for maintaining the prefusion conformation of the F protein. If this were the case, the sF protein secreted from 73 cells might already be in the postfusion form. This appears to be the case with a mutant lacking the first 9 – 10 amino acids of the fusion peptide of the N-terminus of the F1 protein (198, 250, 273).

To examine this possibility for all the glycan mutants, we transiently expressed them from Freestyle 293 cells, purified them by passage over a nickel column and elution with a buffer containing imidazole (50 mM Hepes + 500 mM NaCl +

100 mM imidazole). The purified sF proteins were analyzed by velocity gradient sedimentation. The sF proteins were layered on top of linear sucrose gradients and centrifuged at 37.5 krpm overnight at 4°C. Proteins migrate into the gradient at a rate that depends on their mass.

Individual trimer molecules do not migrate far into the gradient. Their highly hydrophobic fusion peptides are hidden within a channel in the side of the trimer

(197). However, once the F protein is triggered, it exposes its fusion peptide. In the absence of a membrane to insert into, the hydrophobic fusion peptides aggregate, resulting in „rosettes‟ of two to 8 or more postfusion sF trimers. Due to its larger mass, this postfusion rosette form migrates much further into the gradient.

Each mutant sF protein migrated near the top of the gradient, in fractions 2-4, as did the WT sF protein (Fig. 2.5A). That is, all of these mutant sF proteins were produced and secreted in the prefusion form. Therefore, the decreased ability of the N500Q mutant to fuse is not due to the production of an unstable, prematurely triggered F protein. 74

N-glycans are not necessary for triggering the sF protein. Our next hypothesis was that the N500Q mutation may prevent sF protein triggering. Our group has recently found that the sF protein can be triggered by exposure to low molarity buffers (43). To test this hypothesis, we dialyzed the purified WT and each mutant sF protein into a buffer of 10 mM Hepes over 20 h and analyzed the aggregation state of each by velocity sedimentation gradient. The majority of the

WT and mutant sF proteins migrated to fraction 10, at the bottom of the gradient, indicating that the exposure to this low molarity buffer triggered all of these sF proteins (Fig. 2.5B). The N500Q mutant is, therefore, able to be triggered.

Figure 2.5. Velocity sedimentation gradients of WT and mutant sF proteins. Gradients were fractionated from the top, with fraction 1 being the least dense sucrose and fraction 10 being the densest sucrose. Proteins were examined immediately following purification into 650 mM buffer from transfected Freestyle 293 cells (A) and following dialysis to 10 mM Hepes buffer (B).

75

Discussion

N-glycans serve a variety of purposes in proteins including taking part in folding by binding to chaperone proteins, aiding in transport to the cell surface, contributing to the activity of proteins, and shielding of immunological epitopes

(reviewed in (272)).

N-glycosylation has been found to be important in the life cycle of many viruses.

N-glycans of glycoproteins have also been described as shielding many viruses from neutralizing antibodies, including human immunodeficiency virus, virus, Nipah virus and influenza virus (4, 45, 174, 265, 301). The N-glycan attached to N414 on the Hendra virus F protein has been shown to be important in protein folding and transport (39). The N-glycans of some viruses play a role in their entry into the target cell. For Nipah virus, the N-glycans on its attachment protein modulate the fusion activity of the F protein (19), dampening its hyperfusogenicity (4). The F protein of a virus similar to RSV, metapneumovirus, has three N-glycans that have been reported as being involved in cell fusion to different degrees, although it has no N-glycans in its HRB domain (259).

The roles of the glycans of the RSV F protein are poorly defined. It has been previously determined, and confirmed in our experiments on the anchored protein here, that the N-glycans of the RSV F protein are uninvolved in cleavage, folding of the native form, and transport of the protein (177, 328). An N-glycan in the HRB domain of F protein has been shown to play a role in fusion (328).

76

We have found here that none of the three glycans of the RSV F protein seem to be involved in initially stabilizing the prefusion form of the protein, as all soluble glycan mutants were secreted from transfected cells in the prefusion state.

When dialyzed into a low molarity buffer the majority of the trimers formed aggregates and migrated to the bottom of a velocity sedimentation gradient, indicating that these proteins had triggered.

Dialysis of the protein from 650 mM to 10

mM is an admittedly drastic and rapid

change in molarity, and it is possible that

any moderate changes in the stabilization

or “triggerability” caused by deleting the

N500 glycan may not have been evident.

This experiment will need to be repeated

with less drastic, incremental changes in Figure 2.6. Cartoon representing the 6- HB of the RSV F protein in its molarity to determine if any of the glycan postfusion conformation. The membranes from the target cell and virion have been brought together. Below, a mutants trigger more or less readily than view of the 6-HB from one end, with N- glycans represented as yellow cones. the WT sF.

Particularly interesting is that both sF proteins that contained the N500Q mutation were produced in the prefusion form and were able to be triggered. In the anchored form, the N500Q mutation had no effect on cleavage, folding, or transport to the surface of transfected cells, but it did drastically inhibit fusion to neighboring cells. If the N500 glycan is not 77 involved in processing, stabilization, or triggering, then why is it required for the function of the protein? The most likely possibility that remains is that the N500 glycan is involved in refolding of the F protein into the postfusion form.

The N500 residue is present near the middle of HRB, an α-helix near the extreme

C-terminus of the F protein ectodomain. The side chain of the asparagine, and therefore the glycan chain points away from the six-helix bundle (6-HB) of the postfusion structure of the RSV F protein (198, 273). The structure of its N- glycan is large and branching, attached to the nitrogen atom of the side chain of asparagine by an N-acetylated glucosamine molecule. The three-dimensional shape of the glycan can be described as a cone or a pyramid, with the point attached to the protein (Fig. 2.6). With one of these attached to each of the three

HRB helices, it is possible that their bulk may “guide” the HRB helices into the correct orientation for binding to the HRA coiled coil by sterically hindering the faces of HRB with the side chain from approaching. Understanding the refolding events and particularly the assembly of the 6-HB will be valuable for the targeting of antiviral agents.

Interestingly, the F protein of Newcastle disease virus also includes an N-glycan on its HRB domain and this glycan is important for its fusion activity (192).

However, an N-glycan on the HRB domain of the F protein of the Hendra and

Nipah viruses actually dampens fusion (39). Furthermore, the F proteins of human metapneumovirus, measles virus, canine distemper virus, and Sendai virus do not have N-glycans in their HRB domains (259, 260, 294). It appears 78 that some paramyxoviruses, like RSV and NDV, have evolved to use the glycan on their HRB domain to enhance the activity of their F protein while others have not,

79

Chapter 3: Anti-RSV Compounds R170591 and TMC353121 Function by

Stabilizing the Prefusion Form of the F Protein and Inhibiting Triggering

Introduction

RSV causes many of the serious respiratory infections that lead to hospitalizations, and in some cases, death (67, 84, 102, 111, 148, 284, 295).

RSV is able to infect an individual repeatedly throughout life, indicating that RSV infection does not elicit lasting immunity (115). The only effective antiviral agent available commercially is a humanized mouse monoclonal antibody, palivizumab, administered prophylactically to at-risk infants (1). For these reasons, there have been numerous endeavors to discover and develop small molecule compounds with anti-RSV activity (145), the most successful of which are outlined in Table

1.1. However, despite these intensive efforts, none have yet been approved for public use.

Most of these anti-RSV compounds have been found to act against the fusion (F) protein. Many of these compounds have been used to select viruses with drug resistant mutations in an attempt to determine where they bind. Interestingly, three of the best-characterized anti-F compounds, BMS-233675, VP-14637, and

R170591/TMC353121, have each selected mutations in some of the same 80 regions of the protein (Fig. 1.8) (12, 51, 77, 78, 207, 249). For instance, each of these compounds selected mutations in the 486-489 amino acid (aa) region of the protein, near the middle of the HRB α-helix, suggesting that each of these compounds may bind this region and may therefore have a similar mode of action.

However, a photoreactive, radiolabeled analog of BMS-433771 cross-linked to tyrosine-198 (Y198) in the HRA helix of the F protein (49). In the postfusion conformation of the F protein, the 486-489 aa region of the HRB α-helix and the

Y198 of the HRA α-helix are next to each other in opposite helices, together forming a hydrophobic pocket (Fig. 1.8B). In a crystal structure of the RSV 6-HB,

TMC353121 was found attached to Y198 in this hydrophobic pocket, distorting the bundle at the membrane-distal end of the 6-HB (249). VP-14637 and BMS-

433771 have also been suggested to bind to this hydrophobic pocket (49, 77, 78,

249).

The proximity of Y198 in HRA and these mutations in HRB in the postfusion form has given rise to the current hypothesis of the mechanisms of action of these

Y198-binding drugs: the compound binds to Y198 inside the hydrophobic pocket in the 6-HB, and the resulting distortion inhibits fusion between the viral and host cell membranes (249). These compounds would likely bind to Y198 during refolding, probably following the extension and trimerization of HRA but preceding or concomitant with intercalation of the HRB helices that complete 6-

HB formation (184, 249). 81

There are some puzzles, however, regarding this hypothesis. For instance, many of the resistant mutations selected by these drugs are in locations distant from Y198 in the postfusion F protein. It seems unlikely that such distant mutations would prevent these compounds from inserting into the 6-HB, distorting it and preventing fusion initiation.

It is possible that these anti-RSV compounds bind to the prefusion F protein and affect its ability to trigger. They could induce premature triggering, before the F protein is adjacent to the target cell membrane, they could prevent the F protein from triggering, or they could prevent one of the movements during refolding.

Any of these activities could prevent membrane fusion and the initiation of infection.

Here, we sought to determine the mechanisms of action of five of the antiviral compounds that act against the RSV F protein: R170591, TMC353121, BMS-

433771, a compound developed by Biota, and a compound developed by

Trimeris. Three of these compounds, R170591, TMC353121, and BMS-433771, have been shown in previous studies to bind to Y198. We found that none of the compounds caused premature triggering of the F protein but that TMC353121 and its predecessor R170591 are both able to inhibit triggering of the F protein.

We suggest that the mechanism of action of these drugs is to stabilize the F protein in its prefusion conformation.

82

Materials and Methods

Cells. HeLa and Human Embryonic Kidney (HEK) 293T adherent cells were grown in Dulbecco‟s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified chamber at 37°C and 5% CO2. HEK

293 Freestyle® cells, a suspension line developed for protein expression in animal component and protein free medium, were grown in Freestyle 293

Expression Medium® on a shaker platform rotating at 125 RPM in a humidified chamber at 37°C and 8% CO2.

Plasmids. Codon-optimized versions of both the full-length RSV F protein gene from strain D53 and a soluble version of the same RSV F protein gene that was constructed and characterized previously (43) were used. The full-length F protein was expressed from the pcDNA3.1 plasmid. The soluble F protein was expressed from the pcDNA3.1 plasmid and also from the higher-expressing pCAGGs plasmid. The pFR-Luc and pBD-NFκB constructs were a generous gift of Alfred Del Vecchio (Centocor, Inc.) (28).

Cloning. Drug-resistant mutations were introduced into the codon-optimized full- length RSV F protein by site-directed mutagenesis (38). The mutations were confirmed by Sanger sequencing.

Transfection. HEK293T cells were seeded onto poly-d-lysine coated plates and transfected per manufacturer protocol using Lipofectamine 2000 (Life

Technology, Carlsbad, CA) transfection reagent. HEK293 Freestyle cells were

83 transfected per manufacturer protocol using 293fectin (Life Technology,

Carlsbad, CA) transfection reagent.

RSV neutralization assay. Hela cells were infected with 200 PFU/well of GFP- expressing rgRSV224 in presence of dilutions of anti-RSV compounds ranging from 0 to 30 μM. Foci were counted at 48 h.

Cell-to-cell fusion assay. Plasmids expressing F protein mutants were transfected into HEK 293T cells with Lipofectamine 2000 (Invitrogen) per the manufacturer‟s protocol along with pFR-Luc, a plasmid containing a luciferase gene behind a Gal4 promoter to establish the effector cells. A second set of HEK

293T cells, the target cells, was transfected with pBD-NFκB, a plasmid containing a fusion protein with a Gal4 binding domain and the NFκB transcription factor. At

24 h post transfection, the target cells were suspended with Versene, washed in

PBS and resuspended in DMEM-10% FBS and placed directly onto the effector cells in equal numbers to the effector cells. At 48 h post transfection (allowing 24 h for the cells to fuse), luciferase levels were quantified using the Steady-Glo

Luciferase Assay System (Promega) and a luminometer plate reader. Each construct was tested in triplicate.

Producing and purifying soluble F protein. Plasmids containing the sF WT and mutant genes were transfected into HEK 293 Freestyle cells. The sF protein was purified from the medium harvested between 48 and 72 h post transfection by passage over a Ni2+ column (Probond® purification system; Life Technologies) and eluted with 100 mM imidazole in a 50 mM Hepes buffer with 500 mM NaCl. 84

Samples were analyzed by Western blotting with motavizumab (Numax®), goat anti-human antibody conjugated to horseradish peroxidase (KPL), and Lumi-

Light Western blotting substrate (Roche).

Velocity sedimentation gradient. Purified sF protein was layered over a 5 mL

10 – 55% linear sucrose gradient (in 30 mM Hepes buffer + 170 mM NaCl) and centrifuged at 37.5 krpm for 17 h at 4°C with an SW 55 rotor in a Beckman ultracentrifuge (Beckman Instruments). Gradients were fractionated into 0.5 mL fractions by pipetting from the top surface. Aliquots of each fraction were analyzed by Western blotting with a monoclonal antibody against the RSV F protein.

Dialysis. A dialysis cassette (Slide-A-Lyzer®, Thermo Scientific) of appropriate capacity was loaded with WT or mutant sF protein and promptly dialyzed against an excess of chosen buffer (250 times the volume of the sample). The sample was dialyzed with fresh buffer twice for two hours and once for overnight, all at

4°C. The dialyzed samples were analyzed by velocity sedimentation gradient.

Liposome co-flotation assay. An anti-RSV compound was added at a final concentration of 30 μM in a solution of sF in a buffer containing of total molarity of 400 μM (30 mM Hepes, 310 mM NaCl, 60 mM imidazole) in 100 µL and incubated at 37°C for 1h.

Liposomes were prepared from an 8:2:5 molar ratio of 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-

85 phosphoethanolamine (POPE), and cholesterol (Avanti Polar Lipids) dissolved in chloroform-methanol (2:1). Lipid films were layered into the interior of glass tubes by removing most of the solvent with a stream of argon gas and subsequent lyophilization under vacuum for 4 h (Freezone 2.5; Labconco). Lipid films were stored at -20°C under argon gas for up to one month. Before use, the lipid film was resuspended in 1 mL of DMEM or Hepes buffer (varied concentration based on target final concentration of reaction), freeze-thawed with liquid N2 and tepid, running water to create multilayered lipidic vesicles. The solution was extruded 40 times through a 100 μm filter to remove the outer layers of the vesicles and stored on ice.

Following incubation of solution containing sF and an anti-RSV compound, 20 μL was added to the liposome solution (final concentration: 50 mM liposomes, 30μM anti-RSV compound, 400 mM total molarity) and incubated on ice for 30 min. An equal volume of 100 mM sodium carbonate (pH 11) was added and incubated at

RT for 10 min. The reaction was mixed with 60% sucrose (in 50 mM Hepes and

750 mM NaCl) to a final volume of 1 mL and a final concentration of 50% sucrose and overlaid with 1 mL each of 40% sucrose (in 10 mM Hepes), 30% sucrose (in

10 mM Hepes), 20% sucrose (in 10 mM Hepes), and 10 mM Hepes. The gradients were centrifuged at 55 krpm for 2 h, 20 min at 4°C. The gradients were fractionated in 1.2 mL fractions from the bottom with a peristaltic pump and fraction collector. To dissolve the liposomes, 40 μL of 10% Triton X-100 was added to each fraction. Two μg bovine serum albumin and 12 uL of 2%

86 deoxycholate were added to each fraction, vortexed, and incubated on ice for 30 min. 250 μL 100% trichloroacetic acid was added to each fraction, vortexed, and incubated at 4°C overnight. The fractions were pelleted by centrifuging at 18 kxg for 1 h and were washed twice with acetone. The fractions were analyzed by

Western blotting with a monoclonal antibody against the RSV F protein.

Results

Anti-RSV compounds neutralize RSV with different efficiencies. HeLa cells were inoculated with 200 PFU of

GFP-expressing

RSV (rgRSV) in the presence of increasing concentrations of each anti-RSV compound.

Green (infected) Figure 3.1. RSV neutralization by anti-RSV compounds. rgRSV224, a foci were GFP-producing recombinant RSV, was used to inoculate HeLa cells in the presence of increasing concentrations of R170591, BMS443771, counted at 48 h TMC353121, a compound made by Biota, and a compound made by Trimeris. Infected foci were counted at 48 h postinfection. and plotted (Fig.

3.1). All of these compounds were able to neutralize RSV. TMC353121 and

87

R170591 were the most potent, each with an IC50 near 5 nM. The IC50 of BMS-

443771 was 14 nM, the IC50 of the Trimeris compound was 41 nM. The Biota compound was the least potent with an IC50 near 1000 nM.

TMC353121 and R170591 inhibit cell-to-cell fusion. We examined the ability of the two most potent compounds, TMC353121 and R170591, to inhibit the fusion activity of the F protein. We employed a quantitative cell-to-cell fusion assay with two sets of transfected HEK 293T cells, a target set transfected with a transcription activator plasmid (pBD-NFκB) and an effector set cotransfected with a luciferase plasmid (pFR-Luc) and the F protein WT or mutant plasmid. The target cells were suspended by EDTA rather than trypsin at 24 h post transfection, washed and added to the effector cell monolayer. After 24 h incubation, the cells are lysed and the level of luciferase quantified. Luciferase is only produced in an effector cell that fused with a target cell. Both of the compounds inhibited cell-to-cell fusion, although TMC353121 was more potent at higher concentrations (Fig. 3.2).

The IC50 values of the compounds are higher when tested by cell-to-cell fusion, at about 1000 nM, than when tested by viral neutralization, at about 5 nM. These assays clearly differ in sensitivity, perhaps due to differences in membrane curvature and F protein density. Regardless, R170591 and TMC353121 are inhibitory towards both viral infection and cell-to-cell fusion.

88

Anti-RSV compounds do not cause premature triggering of the F protein.

An anti-RSV compound targeting the F protein could function in one of at least three ways. It could cause the F protein to prematurely trigger, it could prevent the F protein from triggering, or it could prevent a step in refolding following the

Figure 3.2. Cell-to-cell fusion in the presence of R170591 and TMC353121. Fusion was quantified in transfected HEK 293T cells by luciferase expression.

triggering event. A compound that successfully inhibits any one of these mechanisms would render the virus noninfectious, since F protein refolding to bring the virion and target cell membranes together for fusion is essential for the initiation of infection. Analysis of these possibilities requires the ability to distinguish between the prefusion and postfusion forms of the F protein. This is a difficult proposition in the context of a cell. However, using a soluble form of the

RSV F protein, the prefusion and postfusion forms can be distinguished by 89 velocity sedimentation in a sucrose gradient. The prefusion form is an independent trimer with its fusion peptide hidden. Once the F protein has converted to the postfusion form, it exposes its highly hydrophobic fusion peptide and these peptides interact to form “rosettes” as the exposed fusion peptides interact. The unaggregated prefusion trimers do not migrate far into the gradient during centrifugation compared to the postfusion rosettes which do because of their higher molecular mass. Our laboratory previously identified a condition, exposure to low molarity, that causes the prefusion form to trigger and refold into the postfusion form (43).

We produced sF protein in transiently transfected Freestyle 293 cells and purified it from the cell culture medium by its ability to bind nickel-coated beads via the

6His tag at its C-terminus. After dialysis into 150 mM Hepes buffer and subsequent centrifugation through a sucrose velocity gradient, the sF protein remained near the top of the gradient (Fig. 3.3). This confirms that the purified protein is in the prefusion form. However, after dialysis into buffers of decreasing molarity, more of the sF protein migrated further into the gradient, signifying that it had aggregated. The low molarity treatment had triggered the refolding of the sF prefusion form into the postfusion form, exposing its fusion peptide and aggregating by its hydrophobic nature.

To determine if the sF protein is triggered by any of these anti-RSV compounds, the protein was first dialyzed into 70 mM Hepes buffer, as it remained largely in the prefusion form at this concentration (Fig. 3.3). The protein was incubated 90 with 30 μM of anti-RSV compound for 2 h at 37°C and subsequently loaded atop a sucrose velocity gradient and centrifuged for 17 h. The sF protein remained near the top of the gradient for all of compounds tested, as it did for the DMSO negative control (Fig. 3.4), signifying that these compounds do not cause premature triggering of the sF protein.

TMC353121 and R170591

prevent triggering of the RSV

sF protein. We next examined if

any of the compounds inhibit the

triggering of the sF protein using a

liposome flotation assay. After the

sF protein was pre-incubated with

30 μM of each anti-RSV

compound for 1 h at 37°C, it was

mixed with liposomes Figure 3.3. Velocity sedimentation gradients of sF protein following dialysis into buffers of (POPC/POPE/cholesterol 8:2:5) various molarities. Gradients were fractionated from the top, with fraction 1 being the least dense sucrose and fraction 10 being the densest. while keeping the compound

concentration constant at 30 μM and incubated for 30 min on ice. When the sF protein is triggered in the presence of liposomes, it associates with a liposome via its hydrophobic fusion peptide. The sF protein is mixed with liposomes and added to dense sucrose. A sucrose gradient is layered over it and centrifuged. The liposomes float to the

91 top of the gradient, along with any associated sF protein. Any sF protein that did not trigger does not associate with the liposomes and remains at the bottom of the gradient. The gradients are fractionated and analyzed by Western blot.

Following incubation with a nonrelevant control compound, an HIV-inhibitory peptide, the

RSV sF protein associated and co-floated with the liposomes, as indicated by its presence near the top of the gradient,

(Fig. 3.5), indicating that it was triggered under these Figure 3.4. Velocity sedimentation gradients of sF conditions. The Biota and protein following incubation with anti-RSV compounds at 37°C for 2 h. Gradients were BMS-443771 compounds were fractionated from the top, with fraction 1 being the least dense sucrose and fraction 10 being the densest. able to be triggered when exposed to liposomes at lowered molarity. However, sF protein incubated with

TMC353121 or R170591 remained mostly at the bottom of the gradient, indicating that these compounds prevented triggering of the sF protein and its association with the liposomes under these conditions.

DMSO-treated sF protein dialyzed against a low molarity buffer containing DMSO migrated to the bottom of the velocity gradient, while a discernible fraction of the

92 sF protein that was both pretreated and dialyzed against buffers containing

TMC353121 did not trigger and remained higher in the gradient (Fig. 3.6).

Figure 3.5. sF protein and liposome co-flotation following incubation with and in the presence of various anti-RSV compounds. An unrelated anti-HIV peptide was used as a negative control. Reactions were performed in a 400 mM Hepes/NaCl buffer. Gradients were fractionated from the bottom, with fraction 4 being the bottom, densest sucrose and fraction 1 being the top, least dense sucrose.

sF protein incubated with BMS-443771, another Y198 binding compound, was able to trigger (Fig. 3.5). It is possible that its lower effectiveness in inhibiting virus infection (Fig. 3.1: IC50 14 nM vs. 5 nM for TMC353121 and R170591) indicates lower avidity for the sF protein. It is also possible that BMS-443771 inhibits RSV infection by a mechanism different from that used by TMC353121 and R170591.

Figure 3.6. Velocity sedimentation gradients of sF protein following incubation with TMC353121 for 2 h at 37°C and dialysis against a 50 mM Hepes buffer containing 30 μM TMC353121 at 21°C. Gradients were fractionated from the top, with fraction 1 being the least dense sucrose and fraction 10 being the densest.

93

Discussion

The finding that TMC353121 and R170591 prevent triggering of the RSV sF protein presents an alternative explanation for the mechanisms of action of these compounds. The most recent hypothesis is that these compounds bind to residues in a hydrophobic pocket formed between the HRA and HRB helices in the 6-HB of the postfusion form of the F protein, distorting the 6-HB and preventing fusion between the viral and host cell membranes (249). This distortion is localized to the membrane-distal end of the bundle. The membrane- proximal end of the 6-HB that is bound to the compound remains unchanged

(249) and it is unclear how a local alteration at the distal end of the 6-HB could inhibit membrane fusion, since fusion is driven by the other, unchanged end of the bundle. It is also unclear how the drug-resistant mutations in four consecutive aa of the HRB helix (aa 486-489) would all accommodate these small molecule inhibitors bound to Y198 on HRA. As an α-helix has about 3.5 amino acids per complete turn, mutation of at most two of these four residues (Fig. 1.8), the ones facing the hydrophobic pocket (aa 488 and aa 489), might accommodate a compound bound to the 6-HB pocket, but the other two could not because they would be facing away from HRA.

Drug resistant viral mutants in two additional regions of the “head” of the F protein (aa 398-400) were selected by both TMC353121 and VP-14637 (Fig. 1.8)

(12, 51, 77, 78, 207, 249). BMS-433771 also selected mutations, within aa 392-

394 and within the fusion peptide (aa 140 and aa 144) (51). These mutations are 94 far from the 6-HB in the postfusion F protein. It would seem highly unlikely that these mutations could affect the hydrophobic drug-binding pocket, enabling the drug to remain bound to Y198, as suggested for the 486-489 aa mutations.

Therefore, it remains unexplained how these distant mutations could cause the F protein to resist inhibition of proper 6-HB inhibition by these drugs.

These compounds have been demonstrated to bind to Y198, a contributing residue of the 6-HB hydrophobic pocket. In the prefusion F protein conformation, however, Y198 is in the pre-HRA domain, the site of dramatic protein refolding following triggering (Fig. 1.8A). Because of the dynamics of this region, we have examined in a separate study if it is involved in triggering the F protein. We have found that the Y198 residue is essential for protein triggering, as the Y198A mutation results in an F protein incapable of fusion, even though it reaches the plasma membrane (Chaiwatpongsakorn, S., Ray, W., Costello, H.M., and M.E.

Peeples, manuscript in preparation). Further emphasizing the importance of this of this residue, none of the RSV mutants selected by these Y198-binding drugs were in Y198 (Table 1.1) even though these molecules clearly bind to this residue.

In the prefusion conformation of the F protein, the aromatic side group of Y198 projects toward the aromatic side group of residue F223, another residue of the pre-HRA domain. The structures of each of these Y198-binding compounds have at least two aromatic rings (Fig. 1.10). It is conceivable that the aromatic rings from the drug stack on the aromatic rings of Y198 and F223, stabilizing the 95 pre-HRA domain and preventing either triggering or refolding of the HRA helix following triggering. Interestingly, Y198W and Y198F mutants, both of which function in cell-to-cell fusion, are inhibited by TMC353121 and R170591 to similar degrees as the WT F protein when tested in a cell-to-cell fusion assay (Costello,

H.M., and M.E. Peeples, unpublished data), indicating that the compounds may be able to bind to any amino acid containing an aromatic ring.

If TMC353121 and R170591 stabilize the F protein and prevent fusion as we have demonstrated and if many of the drug-resistant mutants would not affect drug binding, how could a resistant mutant avoid this mechanism of drug action?

A possible alternative is that these drug-resistant mutations destabilize the prefusion F protein such that the F protein is triggered spontaneously, even in the presence of the stabilization due to drug binding.

Interestingly, the four domains of mutations selected by the Y198-binding drugs localize to two sites on the prefusion F protein: in the fusion peptide (aa mutations 140 and 144) and the channel (aa mutations 398-400) in which it resides in the prefusion conformation and an area at the base of the head of the protein, at the intermonomeric interface of the prefusion conformation (aa mutations 392-394 and 486-489). These mutations allowing for infection despite the stabilization of the F protein suggests that these two regions may be important for maintaining the stability of the F protein and that mutations in these regions may destabilize the protein.

96

In summary, our model for the mechanism of action of the Y198-binding compounds is that the F protein is stabilized in the prefusion form by stacking of the aromatic rings of the compound on Y198 and on F223, an aromatic ring on a neighboring α-helix. The prefusion form of the mutants resistant to these compounds would be less stable and able to be triggered randomly. This is a testable hypothesis, and we have constructed each of the drug-resistant mutations selected by the Y198-binding drugs listed in Table 1.1 into a full-length codon-optimized RSV F gene. We will use these mutants to test our hypothesis that these mutants trigger more readily in a future study, described further in

Chapter 4.

97

Chapter 4: Relevance and Future Directions

Role of N-glycans on the RSV F protein

Chapter 2 outlined our finding that while the N500 glycan of the RSV F protein is essential for fusion, it is not important in either stabilizing the prefusion form or in triggering. The N500 glycan is most likely involved, therefore, in the activity of the F protein following triggering, but before fusion of the host cell and viral membranes: the refolding of the protein to the postfusion form following triggering.

The N500 glycan is positioned in the middle of the HRB α-helix, and in the postfusion form, the glycan projects away from the 6-HB. Two faces of each

HRB α-helix associate with the two HRA α-helices that form the coiled-coil core of the 6-HB (198, 273, 325). The N500 glycan may provide a sort of barrier to prevent incorrect contacts so that the 6-HB bundle is efficiently assembled, without having to “sample” incorrect orientations of the HRB α-helices.

To test this hypothesis, we will take advantage of a recent discovery in our laboratory. We have recently discovered that the BS3 crosslinker differentially crosslinks the prefusion and postfusion forms of the sF protein

98

(Chaiwatpongsakorn, et al., manuscript in preparation). Electrophoretic analysis of the crosslinked protein demonstrates that the sF1 and F2 subunits of the monomer are crosslinked in the prefusion protein. In a similar analysis of the postfusion sF protein, the F1 and F2 crosslink is absent, and instead the dimer and trimer forms are crosslinked. We are using this finding and our soluble glycosylation mutants to determine if mutants without the N500 glycan less readily form a 6-HB, indicating that the glycan is important for 6-HB formation.

We are also using biotinylated synthetic peptides of the HRB domain in surface plasmon resonance and in pull-down assays to probe for the prehairpin intermediate form. If successful, we will determine if mutants without the N500 glycan remain in the prehairpin intermediate form instead of locking into the 6-

HB.

We have yet to identify the N27 and N70 glycans‟ roles in RSV F protein function.

Neither was important for protein folding, for cleavage, or for cell surface expression. Mutants of these glycosylation sites did not result in notable differences in cell-to-cell fusion as compared to WT, so these glycans are seemingly nonessential to fusion. It is possible that they may have minor roles in either enhancing or tempering triggering or fusion. To test this hypothesis, we will decrease the time allowed for fusion in our cell-to-cell fusion assay to tease out slight differences. In addition, we will determine the molarity of the point of triggering of the soluble mutants as compared to the WT. If the triggering molarities differ from WT, then we can determine if these glycans enhance or

99 temper triggering. If the N27 and N70 mutants trigger at similar molarities and fuse to similar levels as the WT, then we can consider the glycans for deletions for future crystal structure studies (described below).

R170591 and TMC353121 resistant mutations of the RSV F protein

Our finding that R170591 and TMC353121 inhibit RSV sF protein triggering is novel and requires further investigation. We hope to find the mechanism of action that leads to stabilization of the prefusion F protein. Is it, as we suspect, stacking of the aromatic rings of the compounds with both Y198 and F223, linking them together and stabilizing the prefusion form of the F protein or are the compounds binding to other amino acids in the F molecule?

As mentioned in Chapter 3, we have found that Y198 is essential for fusion.

When mutated to alanine (A), the RSV F protein loses nearly all of its ability to fuse, while maintaining proper protein folding and export to the cell surface. In the course of this separate study, we constructed both full-length and soluble mutant RSV F proteins with Y198 mutated to alanine, isoleucine (I), tryptophan

(W), phenylalanine (F), and histidine (H). All of these mutants are processed and exported to the cell surface efficiently. We plan to use these mutants to probe the interaction between R170591 and TMC353121 and Y198. We will test our hypothesis that these compounds bind to Y198 through aromatic ring stacking, stabilizing the protein in the prefusion form. If this is the case, then these compounds would be unable to inhibit triggering of Y198A, Y198I, and F223A

100 mutants. Conversely, if the aromatic ring stacking hypothesis is true, Y198W,

Y198F, F223W, and F223Y mutants may also be stabilized by R170591 and

TMC353121, because they contain aromatic rings in this position.

Table 4.1. Mutations Selected by Y198-binding RSV Antivirals

Drug Company/Developer Resistant Mutants Ref. F Drugs Fusion HRA (392-394) (398 -400) (486-489) (Y198 binders) Peptide BMS-433771 Bristol-Myers F140I D392G D489Y (51) Squibb V144A K394R VP-14637 ViroPharma; T400A D486E (78) (MDT-637) RSVCo F488I, V, Y, L (207) (+N517I) D489Y TMC353121 Janssen Research S398L D486N (12) (Improved upon Foundation; S398L E487D (249) JNJ2408062/R1 Tibotec/Johnson & (+K394R) D489Y (207) 70591) Johnson K399I (77) P13 Lundin, et al. N197T T400I (181) C15 Lundin, et al. D489G (181)

We also plan to test our hypothesis that the resistant mutations selected by the

Y198-binding drugs work by making the prefusion F protein less stable and more

“triggerable”. We have constructed and fully sequenced each of the mutations found in Table 4.1 into our codon-optimized full-length RSV F protein in a pcDNA3.1 plasmid. The mutants will be tested for their fusion activity in the cell- to-cell fusion assay. Hyperfusogenicity as compared to the WT F protein will indicate that a mutant is less stable and triggers more easily than the WT. We can also determine which mutations bypass the triggering inhibition induced by

R170591 and TMC353121 by performing the cell-to-cell fusion assay with the mutants in the presence of each compound. We also have several of these

101 drug-resistant mutations built into the infectious virus and have the ability and tools to construct the rest of the panel. We plan to test the infectivity of these viruses in the absence of the drugs to determine if these drug resistant mutations make the virus infectivity less stable at various temperatures, as would be predicted for an F protein with a higher rate of spontaneous triggering.

We will also move any interesting drug-resistant mutations into the sF protein to determine if sF triggering is enhanced. This information could be valuable for developing novel, targeted small molecule drugs against RSV. A small molecule drug that destabilizes one of the regions required for F protein stabilization could lead to premature triggering, rendering the virus noninfectious.

It is possible that our in vitro, biochemical method of triggering the sF protein, exposure to low molarity, may not reflect physiological triggering. The monomer

HRB domains comprising the stalk of the full-length F protein are likely stabilized by the membrane in which they are anchored. However, the sF protein is produced and studied outside of the context of the membrane, and the HRB domains of the sF protein are not stabilized. This may result in a “loosening” of the F protein trimer, initiating triggering that would not otherwise happen in the full-length protein. If this is the case, we will use a soluble F protein that has been trimerized with a GCNt or fibritin domain C-terminal to the HRB domain to mimic the structure of the full-length F protein. We have already constructed and characterized the GCNt-clamped sF and found that it is triggered in the liposome flotation assay similarly to the unclamped sF. Additionally, we can construct a sF 102 protein with the HRB stalk deleted, removing the possibility that movement of the

HRB domains causing triggering.

Disulfide bond mutations to stabilize the prefusion form

Beyond learning about RSV F protein triggering and refolding, one of the goals of our lab is to develop an RSV vaccine. As described in

Chapter 1, previous RSV F protein subunit vaccines have failed (85, 108, 208, 230-232,

263). This is most likely because the isolated protein was in the postfusion form, and the most potent neutralizing antibodies are against the prefusion form (185). We have attempted to construct a stabilized prefusion RSV F protein using introduced intermonomeric disulfide bonds Figure 4.1. Trimer model of the RSV F protein. Protein was for potenial use as a better subunit vaccine. modeled by Will Ray. Two monomers are space-filling models in pink and grey. The Prior to the recent publication of the prefusion third monomer is a cartoon to illustrate positions of α-helices RSV F protein (197), we relied on molecular and β-sheets. models of the RSV F protein to make predictions about the protein. Our lab used a model constructed by our collaborator Dr. Will

Ray (Fig. 4.1) based on the published crystal structure of the prefusion form of the F protein of a related virus, parainfluenza virus 5. Using this model, we

103 identified amino acid pairs that were Table 4.2. Introduced Disulfide Bond Constructs Attempted Construct Residues Soluble/ Strain predicted to be close enough and in mutated to Anchored cysteine the correct orientation that, if mutated HC-18 N524 526 Soluble D53 HC-19 M525 527 Soluble D53 HC-20 T523 525 Soluble D53 to cysteine, may form disulfide bonds. HC-21 T522 524 Soluble D53 HC-22 S521 523 Soluble D53 We targeted mostly intermonomeric HC-23 K520 522 Soluble D53 HC-24 G519 521 Soluble D53 potential disulfide bonds because HC-39 Q81 K272 Anchored D53 HC-41 D194 N254 Anchored D53 HC-43 R235 N276 Anchored D53 they could be tested easily for HC-45 K77 N262 Anchored D53 HC-47 K85 S362 Anchored D53 successful linkage by SDS-PAGE gel HC-48 Q81 Q270 Anchored D53 HC-50 P480 F488 Anchored D53 HC-52 P376 D486 Anchored D53 electrophoresis both with and without HC-53 E487 F488 Anchored D53 HC-55 K394 S485 Anchored D53 2-mercaptoethanol. If a mutant HC-67 E487C - Anchored D53 HC-69 L481C D489C Anchored D53 successfully formed intermonomeric HC-70 S509C D510C Anchored D53 HC-75 S485C V482C Anchored D53 HC-91 S509C D510C Soluble D53 disulfide bonds, it would run as a HC-100 S509C D510C Anchored D53 F488C D489C D53 dimer or trimer in nonreducing HC-103 S509C D510C Soluble D53 F488C D489C D53 conditions, depending on how efficiently the disulfide bond was formed. However, adding the reducing agent 2- mercaptoethanol causes disulfide bonds to break, and under these conditions such a disulfide-linked trimer would run as the F1 monomeric species.

We identified and constructed 22 potential disulfide bonding pairs (Table 4.2).

We also reproduced a published, successful disulfide bonded trimer with two potential disulfide bonds that had been built into the RSV F protein (185).

However, we separated the bonding pairs into two separate mutants in order to test the ability of each to form disulfide bonds (HC-69 and HC-70). Of these 24

104 mutants, the only success we had was with HC-70 (Fig. 4.2), and that success was not complete: about 30% of the protein was linked as a dimer or trimer.

However, when the HC-70 mutations were introduced into the sF protein, the protein was not linked into oligomers. This is likely because the mutated amino acids, S509C and D510C are in the HRB α-helix, and in the soluble version of the protein, the HRB α-helices are not anchored in the membrane by the attached transmembrane domain. They may not have been held near enough to the other HRB α-helices to allow disulfide bond formation.

Our inability to create successful disulfide bonded F protein constructs is due to the differences between our F protein model and the actual structure of the F protein. Because we predicted disulfide bonds on a model that differed from the actual structure of the RSV F Figure 4.2. Cell surface expression of two protein, most of the predicted bonding potentially disulfide bonded full-length RSV F proteins, modeled after two pairs were either not in the optimal published disulfide bond partners (185) . Samples were electrophoresed in non- reducing sample buffer (A) and sample buffer proximity or ideal angle to form containing 2-mercaptoethanol (B). RSV F protein monomers are designated by the disulfide bonds. We, however, do plan labels F1 and F1+F2. The sizes of dimer and trimer RSV F proteins are noted. in the future to generate other disulfide bonding pairs using the published prefusion F protein structure. Our goal is to have a successfully disulfide-bonded sF protein with which to begin immunization

105 studies in mice. We also aim to probe separate movements in the refolding process of the F protein by locking certain regions of the sF protein together with disulfide bonds and studying protein folding steps in isolation.

Determining the structure of the native RSV F prefusion protein

The past several years have been very fruitful for the study of the RSV F protein.

Both the prefusion and postfusion forms of the F protein have been solved and published (197, 198, 273). These have been arguably the biggest advance in our understanding the F protein in recent years, allowing more accurate predictions concerning the refolding of the protein and its function. With the knowledge of the prefusion structure also comes the potential for understanding the mechanism of action of the small molecule inhibitors that have been developed against the RSV F protein, and the development of new inhibitors.

Although these publications represent major advances in the field, there are still some lingering questions regarding the pretriggered structure. Firstly, the crystal was resolved at 3.6 Å, which does not allow for confident prediction of side group orientation of the amino acids. This would be an important determinant in developing targeted fusion inhibitors, as the inhibitor most likely would interact with specific amino acids.

A fibritin trimerization domain was cloned into the C-terminus of the F protein to assist in maintaining the pretriggered structure of the protein, as the GCNt trimerization domain was added to the PIV5 F protein prior to its successful

106 crystallization (197, 318). There is a possibility that this trimerization by an introduced sequence induced a constrained or artificial conformation to the HRB helices and/or the neighboring structure.

To crystallize the F protein in the pretriggered conformation, the authors added an antibody specific for the pretriggered form and crystallized the complex (197).

The D25 antibody binds to the newly-defined antigenic site Φ, which includes

Y198 and is the most dynamic region of the protein. It neutralizes RSV with 100- fold higher efficiency than palivizumab, perhaps by locking the protein into the pretriggered state. However, it is also possible that this binding by the D25 antibody distorts the local region at/near the epitope through an induced-fit binding. Because of this site‟s radical reorganization following triggering, it stands to reason that it would be an ideal site for a small molecule inhibitor to bind and inhibit triggering and/or refolding. In terms of in silico development of a targeted small molecule inhibitor, it would be ideal to use the native form of the pretriggered F protein structure.

There is definite value in determining the structure of the pretriggered RSV F protein unconstrained by trimerization domains and antibodies and at a higher resolution. We have been working towards such a crystal structure for several years, approaching the problem with different strategies. We have greatly increased the yield and purity of our sF preparations. We also intend to improve the crystal packing by removing any nonessential N-glycans from the F protein, as glycans tend to hamper proper crystal packing. We also will attempt to 107 stabilize the prefusion form of the sF protein with the BS3 crosslinker, the Y198- binding compounds, or with an intermonomeric disulfide bond.

We also have the novel crystallization goals of determining the structure the uncleaved RSV F protein to determine where pep27 associates prior to cleavage and of the prehairpin intermediate form of the protein. We have several sF proteins with mutations in the cleavage sites to examine the uncleaved F protein.

If the N500 glycan is indeed involved in the formation of the 6-HB, the N500Q mutant would be ideal for attempting this crystal structure because of its inability to fold back into the postfusion form. Also, an introduced disulfide bond that allows for triggering, but not fold-back of the protein into the 6-HB, would be a possibility for this structure. These are lofty goals, certainly, and unlikely to be accomplished in the near future. However, the groundwork for approaching these goals has been and is currently being laid by the work discussed in this dissertation and by other work being performed in our laboratory.

108

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