Prevention of Respiratory Syncytial Virus Attachment Protein Cleavage in Vero Cells Rescues Infectivity of Progeny Virions for Primary Human Airway Cultures

Home , Measles hemagglutinin, Mononegavirales, Respirovirus, SV40, U937 (cell line)

DISSERTATION

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

Jacqueline D. Corry, B.A.

Graduate Program in Integrated Biomedical Science Program

The Ohio State University

2015

Dissertation Committee:

Mark E. Peeples, Ph.D.—Advisor

Douglas M. McCarty, Ph.D.

Ian Davis, DVM, Ph.D.

Stefan Niewiesk, DVM, Ph.D.

Jacqueline D. Corry

2015

Abstract

Live attenuated respiratory syncytial virus (RSV) vaccine candidates are produced in

Vero cells, a cell line that cleaves the attachment (G) glycoprotein. As a result, Vero- derived virus is 5-fold less infectious for primary well-differentiated human airway epithelial (HAE) cultures than virus grown in HeLa. HAE cultures are isolated directly from the human airways, so it is likely that Vero-grown vaccine virus would be similarly inefficient at initiating infection of the nasal epithelium following vaccination, requiring a larger inoculum, thereby raising the cost per dose. Using protease inhibitors with increasing specificity, we identified cathepsin L as the responsible protease and confirmed that virus grown in the presence of protease inhibitors was more infectious for

HAE cultures. Our evidence suggests that the G protein interacts with cathepsin L in the late endosome or lysosome via endocytic recycling. While essential for Nipah virus F protein cleavage, endocytic recycling is detrimental to the production of infectious RSV from Vero cells. We found that cathepsin L is able to cleave the G protein in Vero-grown, but not in HeLa-grown virions suggesting a difference in G protein posttranslational modification. Using mutagenesis, we identified a cluster of amino acids that are important for G protein cleavage and they contain a likely cathepsin cleavage site. Virus grown in Vero cells and containing a G protein resistant to cleavage is 5-fold more infectious for HAE than the same virus grown in Vero. Live attenuated RSV vaccine virus containing this mutation would reduce the cost of vaccine production for infants. ii

Dedication

I would like to dedicate this work to my family, especially: my fiancé who has been an

unending source of patience, love, and humor; my mom, whose support has been unflagging in every endeavor; my dad, who is always up for competition or a giant bear

hug; and my grandfather, who challenged me and made me feel that it was okay to be

smart.

iii

Acknowledgments

I would first like to sincerely thank my advisor, Dr. Mark E. Peeples. He has supported me through every step of my Ph.D. Under his tutelage, I have become a more independent scientist, a better mentor, and a better mentee. His open door policy allowed me to share science when it was still fresh and exciting.

I would like to acknowledge my mentor Dr. Jeanette Marketon, who made me think, believed in me and pushed me. It is at her urging that I finally chose to apply to graduate school.

I would also like to thank my lab members, whose zany antics allowed me to be myself and who made the lab a good place to be when science wasn’t working, who were cheerleaders when things got hard, who commiserated over difficult problems, who helped me find solutions, plan experiments, and who talked endlessly with me about food, the future, and of course, kitties. I would also, more seriously, like to thank them for the guidance they have provided to make me a more effective presenter.

I recognize the students I have mentored while in Dr. Peeples’s lab, who helped me to understand the science better, who challenged me with thought provoking questions, and who have joined in on some of the zany antics.

I acknowledge my committee members: Dr. Niewiesk, Dr. Davis, Dr. McCarthy and Dr.

Flano for their support, advice and for challenging me.

Thank you to my classmates who helped me to get through the first couple of years of graduate school relatively unscathed, especially to those that have stayed in touch!

I would like to also thank Dr. Virginia Sanders, a person who so passionately insisted that

I apply to IBGP little more than 2 months before the application deadline.

Finally, and most importantly, I would like to thank my family and friends, without you I would not be here doing something I love.

Vita

May 1999 ...... Abingdon High School

May 2003 ...... B.A. Biochemistry, Knox College

June 2008 to July 2011 ...... Research Assistant I/II

August 2011 to present ...... Graduate Research Associate, Biomedical

Sciences Graduate Program, The Ohio State

University

Publications

Corry J; Johnson SM; Peeples ME. Prevention of Respiratory Syncytial Virus Attachment Protein Cleavage in Vero Cells Rescues Infectivity of Progeny Virions for Primary Human Airway Cultures. Journal of Virology in press.

Webster Marketon JI, Corry J; Teng, M. The respiratory syncytial virus (RSV) nonstructural proteins mediate RSV suppression of glucocorticoid receptor transactivation. Virology. 2014 Jan 20; 449: 62-69.

Webster Marketon JI, Corry J. Respiratory syncytial virus (RSV) suppression of glucocorticoid receptor phosphorylation does not account for repression of transactivation. FEBS Open Bio. 2013 Jul 25; 3:305-309.

Webster Marketon JI; Corry J. 2013. Poly I:C and respiratory syncytial virus (RSV) inhibit glucocorticoid receptor (GR)-mediated transactivation in lung epithelial, but not monocytic, cell lines. Virus Research. Volume 176 no. 1-2. 303-306.

Burnsides C; Corry J; Alexander J; Balint C; Cosmar D; Phillips G; Webster Marketon JI. 2012. Ex vivo stimulation of whole blood as a means to determine glucocorticoid sensitivity. Journal of Inflammation Research. Vol 5. 89-97.

Hinzey A; Alexander J; Corry J; Adams KM; Claggett AM; Traylor ZP; Davis IC; Webster Marketon JI. 2011. Respiratory syncytial virus represses glucocorticoid receptor mediated gene activation. Endocrinology. Vol. 152, no. 2: 483-494.

Fields of Study

Major Field: Integrated Biomedical Science Program

vii

Table of Contents

Abstract ...... ii

Dedication ...... iii

Acknowledgments...... iv

Vita ...... vi

Table of Contents ...... viii

List of Tables ...... xii

List of Figures ...... xiii

Chapter 1: Introduction ...... 1

Chapter 2: Respiratory Syncytial Virus ...... 8

Viral Discovery ...... 8

RSV: The Big Picture ...... 9

CCA-Like Viral Illness ...... 9

Classification & Strains ...... 11

Respiratory Syncytial Virus Spread ...... 13

The Cost of RSV Disease ...... 15

Prophylaxis and Treatment of RSV disease ...... 19

viii

Respiratory Syncytial Virus: Close up ...... 20

Cell Culture...... 20

RNA ...... 23

Viral protein identification ...... 25

Characterization of envelope proteins ...... 30

Attachment...... 34

Heparin/Heparan sulfate ...... 34

Intercellular adhesion molecule 1 ...... 37

Annexin II ...... 38

DC-SIGN and LC-SIGN ...... 38

Nucleolin ...... 38

CX3C chemokine receptor 1 ...... 39

Entry ...... 40

Early characterization of RNA replication and transcription ...... 44

Immune modulation by RSV proteins ...... 49

Viral assembly and egress ...... 67

Respiratory Syncytial Virus: Modeled ...... 72

Mice ...... 73

The cotton rat ...... 79

Ferrets ...... 81

Bovine RSV ...... 82

Neonatal Immunity ...... 83

Formalin-Inactivated Respiratory Syncytial Virus ...... 85

Live Attenuated Vaccine ...... 88

Chapter 3: Cathepsin L, Endocytosis, and Recycling ...... 94

Cathepsin L ...... 94

Membrane Recycling ...... 101

Membrane recycling discovered ...... 104

Sorting motifs ...... 105

Recycling ...... 109

Chapter 4: Gprotein cleavage in Vero cells ...... 111

Introduction ...... 111

Materials and Methods ...... 114

Cell culture ...... 114

Virus infection and drug treatment ...... 114

Biotinylation and immunoblot analysis ...... 116

Cathepsin L treatment ...... 117

PCR ...... 117

Cathepsin activity assays ...... 117

Mutagenesis ...... 118

frG Transfection ...... 118

HAE viral infections ...... 118

Statistical analysis...... 119

Results ...... 119

Protease identification with protease inhibitors ...... 119

Cathepsin L expression and activity ...... 124

Cellular location of cleavage ...... 127

Infectivity for HAE cultures of virus grown in the presence of cathepsin L inhibitor

...... 130

Identification of the G protein cleavage site ...... 131

Infection of HAE cultures with virus containing an uncleavable G protein ...... 134

Discussion ...... 135

Chapter 4: Other Experiments ...... 141

Monkey, Rodent and Human Immortalized Cells ...... 141

Endocytic Recycling ...... 144

Amino Acids Important for G protein Cleavage ...... 149

References ...... 154

List of Tables

Table 1. Order of the genes in the genomes of Paramyxovidae family members...... 13

Table 2. Symptoms of RSV infection...... 15

Table 3. Severe disease risk factors...... 16

Table 4. Protein physical characteristics ...... 27

Table 5. Proteins involved in immunity...... 50

Table 6. Sorting motifs ...... 106

xii

List of Figures

Figure 1. RSV infection of HAE cells...... 5

Figure 2. MRC-5 and WI-38 viral G protein...... 7

Figure 3. Mononegavirales tree...... 12

Figure 4. Causes of death from 1980-2010...... 18

Figure 5. Human airway epithelial cultures...... 22

Figure 6. RSV or Adenovirus infection of HAE cultures...... 23

Figure 7. Localization of heparan sulfate and sialic acid linkages on the surface of HAE cultures...... 36

Figure 8. Virion morphology...... 69

Figure 9. SH protein forms a viroporin...... 71

Figure 10. Control vaccinated monkey lung 7-8 days post-challenge...... 87

Figure 11. FI-RSV vaccinated monkey lung 7-8 days post-challenge...... 88

Figure 12. Cathepsin L processing...... 99

Figure 13. Overview of Rab GTPases on the endocytic pathway...... 103

Figure 14. Protease inhibition of G protein cleavage in Vero cells...... 121

Figure 15. Schematic of the RSV G protein...... 123

Figure 16. Cathepsin L and B expression and activity in HeLa and Vero cells...... 125

Figure 17. Cathepsin L treatment of purified virions that had been grown in the presence of cathepsin L inhibitor...... 127 xiii

Figure 18. Furin-released and membrane-bound G protein processing...... 129

Figure 19. The ability of rgRSV grown in the presence of cathepsin L inhibitor to infect

HAE cultures...... 131

Figure 20. Cartoon displaying the transmembrane RSV G protein...... 132

Figure 21. Mutagenesis of the G protein to identify the cleavage site...... 133

Figure 22. Comparison of rgRSV and rgRSV-L208A infection of HAE cultures...... 135

Figure 23. G protein cleavage by immortalized cell lines...... 142

Figure 24. Cathepsin L treatment of Vero or HAE derived viruses...... 143

Figure 25. Endocytic recycling of the RSV G protein in HeLa and Vero cells...... 145

Figure 26. CaCl2 Treatment...... 146

Figure 27. Effect of endocytosis inhibition on G protein cleavage in Vero cells...... 147

Figure 28. Endocytic recycling motif mutagenesis...... 148

Figure 29. G protein mutagenesis and cleavage...... 150

Figure 30. Comparison of rgRSV and mutant virus infection of HAE cultures...... 152

xiv

Chapter 1: Introduction

In adults and teenagers, most respiratory syncytial virus (RSV) infections produce upper respiratory tract infection and symptoms. However, in infants, the immune-compromised and the elderly, RSV has the potential to cause life-threatening lower respiratory tract infection (1-4). Worldwide, in 2010 alone, over 230,000 children under five years of age died from RSV illness with the majority of these being infants under the age of one (5).

Currently, only supportive care is available to treat individuals with lower respiratory tract disease. A neutralizing mAb, palivizumab, is used prophylactically, but only for infants considered at greatest risk for severe disease. There is a clear need for vaccines to combat this virus.

The attachment (G) glycoprotein and the fusion (F) glycoprotein are important for the initial steps in virus infection, attachment and membrane fusion, respectively. The G protein is a 33 kDa type II membrane protein with a large number of post-translational modifications, including N- and O-glycans, that increase its apparent molecular weight to

90 kDa, when produced in most immortalized cell lines.

Historically, it was difficult to determine if the G protein was a viral or host protein (6-8).

This is at least partially due to the fact that many researchers used African green monkey kidney cells, a cell type that produces a 55 kDa G protein rather than the 90 kDa version produced by other immortalized cells. However, once the G protein was demonstrated to 1 be a virus-associated protein, the difference in size was thought to be due to differences in glycosylation since much of the molecular weight of the G protein is due to O-linked glycans (9). The G protein was known to be the attachment protein, and the virus had been shown to bind glycosaminoglycans (GAG) on the surface of cultured cells (10-15), so a previous graduate student in our laboratory, Steve Kwilas, wanted to understand how this decrease in sugar content might affect binding and subsequent entry of virus grown in Vero cells. To quantify virion attachment to GAGs on the cell surface, he employed the Chinese hamster ovary (CHO) cell pair, K1 and A745 (16). CHO A745 cells were derived from CHO K1 and are deficient in GAGs because they do not produce xylosyl- transferase, an enzyme that links xylose to an S or T residue to initiate GAG synthesis

(965). The GAG dependency of a virus could be determined by titrating the virus on

CHO K1 and CHO A754 cells and dividing the first by the latter. If the resultant number is >1 the virus depends on GAGs for virus entry, and the higher the number the more dependent the virus is (16).

The rgRSV from HEp-2 cells had a GAG dependency of 17 while Vero-produced rgRSV had a GAG dependency of 4. Vero-derived rgRSV GAG dependency was similar to the dependency of the virus grown in HEp-2 that lacked the G protein, suggesting that there is something about the G protein produced in Vero that lowers its ability to bind to

GAGs. To determine if this difference was due to a lower amount of the G protein in virions grown in Vero, a quantitative immunoblot was performed. Sucrose-gradient purified Vero-grown virions contained slightly less G protein, relative to N protein. But

2 more strikingly, the G protein was much smaller, 55 kDa, in Vero-grown virions than the

90 kDa form in HEp-2-grown virions (16).

Using pulse-chase experiments to compare the processing of the G protein in the Vero and HEp-2 cell lines he examined the size of the G protein and its sensitivity to

Endoglycosidase H which removes immature, high mannose N-glycans. The first clearly detectable band to appear was 45 kDa and it was sensitive to Endo H, suggesting that this protein contained immature N-linked glycans. The size of the G protein following EndoH treatment was 36 kDa, close to the expected size of the protein without glycan additions.

The 90 kDa G protein, which appeared at 30 min of chase was not affected by Endo H treatment, suggesting that its N-glycans had been modified and O-glycans had been added, both processes occur in the Golgi. Consistent with this, Monensin treatment, which prevents migration to the Golgi resulted in only the EndoH-sensitive 45 kDa form, confirming the Golgi as the site of glycan addition and/or modification. After 2 h of chase, there was still no 55 kDa G protein visible when produced in Vero, suggesting that the post-translational modification is not differences in glycosylation (16).

The G protein can be detected by immunoblot with mAbs such as 131-2g, L9 or 130-2g.

The 130-2g mAb recognizes an epitope in the C-terminal portion of the protein. If the virus was grown in Vero and detected with the mAb L9, the G protein was primarily 55 kDa, however, if the 130-2g Ab was used, the 55 kDa band was absent. To verify that the

C-terminal portion of the protein is absent when grown in Vero, a 6-His tag was added to the C-terminus of the G protein in the virus. When this virus was grown in Vero cells and probed with mAb L9, the 55 kDa G protein was detected; when probed with a 6-His mAb 3 the G protein was not detected. Taken together these results confirm that the C-terminal portion of the G protein is removed by cleavage when grown in Vero cells (16).

Several cell lines were analyzed to determine the percentage of the 90 kDa compared to the 55 kDa band. For HEp-2, the G was 89% 90 kDa, but all other cell lines had a lower value: MRC-5 produced 72%, A549 43%, PMK (primary monkey kidney) 54%, BSC-1

39%, and Vero 15%. BS-C-1 and Vero are both from African green monkey kidney cells, and PMK cells are from macaque kidney. The other three cell lines are human-derived

(16).

The Vero-derived virus was less able to infect cells containing GAGs (a lower GAG- dependency) than a HEp-2-derived virus, but it was unclear what effect that would have on in vivo infectivity. Primary, well-differentiated human airway epithelial (HAE) cultures are an excellent model of the human respiratory epithelium. The rgRSV was grown in HEp-2 cells or Vero and rgRSV-F was grown in HEp-2 cells. All were titrated on HEp-2 cells and equal HEp-2 infectious units were used to infect HAE cells. The

HEp-2-derived rgRSV infected the HAE cultures significantly better than virus grown in

Vero cells (Figure 1). The Vero-grown virus infected similarly to the rgRSV-F, suggesting that the virus grown in Vero contains a G protein that is not able to perform its attachment function. At 2 dpi, 57% of HAE cells were GFP positive if the wt virus had been grown in HEp-2 and 10% if grown in Vero, and rgRSV-F had infected 5% of the cells. Infection in HAE cultures was delayed by 2 days with the virus from Vero cells.

Taken together these data suggest that the cleaved G protein is not able to perform its attachment function on HAE cells. When HAE-derived viruses, originally grown in Vero, 4 were passaged in HEp-2 cells and the resultant virus was used to reinfect HAE, these viruses were equally infectious to HEp-2-derived virus. This result is important as it confirms that the loss of the C-terminal portion of the protein, when the virus is produced in Vero, is a posttranslational modification and not a genetic change (16).

Figure 1. RSV infection of HAE cells. Infection of primary HAE cell cultures inoculated with rgRSV-SGF grown in HEp-2 and Vero cells and rgRSV-F grown in HEp-2 cells (6.2 × 106 PFU in 100 μl [∼MOI, 0.05]). Reprinted from Journal of Virology, 83 / 20, Kwilas et al., Respiratory Syncytial Virus Grown in Vero Cells Contains a Truncated Attachment Protein That Alters Its Infectivity and Dependence on Glycosaminoglycans, 10710-10718., Copyright (2009), with permission from American Society for Microbiology.

This finding is of interest to the vaccine community because viruses in the initial FI-RSV vaccine (17, 18) and in live attenuated vaccine candidates, tested in clinical trials since 5 the 1990s, have been grown in African green monkey kidney or Vero cells (19-26). The original FI-RSV vaccine induced less neutralizing antibodies than natural infection, especially to the G protein (27). The low induction of neutralizing antibodies could, in part, be due to missing or altered epitopes on the G protein that is lacking its C-terminus.

More importantly, all live-attenuated vaccine candidates have been produced in Vero cells, and it is unclear what effect cleavage might have on neutralizing antibodies, but it is likely that any virus with a cleaved G protein is less likely to infect. This would likely increase the titer of virus required for vaccination, which could increase the cost and increase the likelihood of a reaction to non-viral antigens. The overarching goal of this project is to produce a virus in Vero that is more infectious for HAE that contains genetic mutations that will attenuate the virus, decreasing viral spread. We predict that this will decrease the cost per dose so that ten infants could be immunized with a dose that would be used to vaccinate one infant with the current vaccine candidates.

We could change the cell line used to produce a vaccine, however, currently there are only 3 cell lines approved by the World Health Organization for live attenuated vaccine production, MRC-5, WI-38 and Vero cells (28). The MRC-5 and WI-38 cells grow exceptionally slowly compared to Vero, and the G protein is also cleaved in WI-38 cells

(Figure 2). In addition, Vero cells have the advantage that they do not produce IFN (29), which is especially appealing if a vaccine candidate is deficient in inhibiting the IFN response. We hypothesized that virus produced in Vero cells, with an intact G protein, would more efficiently infect HAE cultures, and, therefore, the cells that line the human airway in vivo. If used in a vaccine, virus that contained an intact G protein would be

6 five-fold more infectious and would enable the use of 5 times less inoculum, allowing for a more economically-feasible vaccine candidate. To test this possibility, we used two approaches. The first involved protease inhibition, we identified the protease as cathepsin

L and demonstrated that cleavage likely occurs during endocytic recycling. The second approach used viral mutagenesis. We used a stop codon mutant to identify the likely area of cleavage, and then used deletion and alanine scanning mutagenesis to identify the cleavage site. Proteins that were cleavage resistant when produced in Vero were built into the virus. Vero produced wtRSV contained an intact G protein when produced in the presence of cathepsin L inhibition as did RSVL208A. Both approaches were tested for the ability to infect HAE cultures to confirm our hypothesis.

Figure 2. MRC-5 and WI-38 viral G protein. Immunoblot of viruses purified from Vero, MRC-5 or WI-38 cells.

Chapter 2: Respiratory Syncytial Virus

Viral Discovery

In 1957, Chanock et al. identified children with bronchopneumonia, bronchiolitis or laryngotracheobronchitis. They were able to obtain throat swabs from some of the patients including 1 patient, Long, with bronchopneumonia and a second, Snyder, with laryngotracheobronchitis. These infectious agents were unfilterable, thus were likely viruses.

The virus has a mean diameter of 106 millimicrons (nanometers), with a range of 90-130 millimicrons, and a density ranging from 1.19 to 1.26. The viruses were cultured in monkey kidney cells and in human amnion cultures. Originally the viruses did not grow very well, but with increasing passages the viruses became more cytopathic, and the

Long virus produced syncytia in the different culture models tested. Prior to this virus isolation only mumps and measles had been demonstrated to cause syncytia formation in cell culture, but unlike mumps and measles, this virus was unable to agglutinate erythrocytes. In addition, the virus was also not able to infect embryonated eggs, confirming that the isolated virus was not influenza. Virus treated with ether was rendered uninfectious (30), and ether treatment had previously been demonstrated to dissolve lipids destroying the infectivity of herpes simplex, influenza, mumps, Newcastle disease virus and others (31). 8

These two isolated viruses and a previously characterized chimpanzee coryza agent

(CCA) had similar characteristics. CCA was isolated from a sick animal (30, 32), other animals in contact with the sick animal developed antibody (Ab) and the handler developed symptoms, but no CCA virus was isolated from the handler or the other chimpanzees. Chimpanzees could be infected with CCA grown in cell culture and respiratory illness was associated with infection (32).

Children with and without respiratory illness were tested for the presence of neutralizing and complement-fixing antibodies to the new virus. Forty-eight percent of the children who exhibited symptoms of respiratory illness and 14% of children who had no respiratory symptoms had a detectable rise in neutralizing and/or complement fixing Ab.

Children under 5 months of age had higher neutralizing Ab levels than children 6-11 months of age, suggesting that the children under 5 months still had maternal Ab. Most children >3 years of age had detectable Ab (32). None of the Ab tested were able to neutralize mumps or measles viruses, further suggesting that these viruses are antigenically distinct (30).

RSV: The Big Picture

CCA-Like Viral Illness

Following the discovery of the CCA-like virus, a study by Beem et al. enrolled children with and without respiratory illness, both hospitalized and treated in the clinic.

Respiratory illness was defined as bronchiolar inflammation, dyspnea, emphysema and

9 expiratory rales. Children were considered free of respiratory illness if they had been free of abnormal respiratory symptoms for 2 weeks prior to testing and 2 days following testing. Swabs were taken from the nose, tonsils and oropharynx of the children, and the swabs were stored in balanced salt solution containing antibiotics and when bacterial contamination was apparent, the virus was frozen, thawed, spun and the supernatant was collected. Blood was also collected for Ab testing. Throat swabs were used to inoculate

HEp-2 cells, which were incubated until cytopathic effect (CPE) was detected. Over the course of the study 48 agents isolated from children were shown to cause extensive syncytia formation. One such agent, named Randall virus, was characterized and was similar to the CCA-like virus. The virus was unable to agglutinate erythrocytes (33).

In order to test if the Randall agent was a CCA-like virus, patient sera were tested for their ability to fix complement when mixed with the Randall agent and two of the CCA- like strains, Long and Sue. The acute phase serum had little to no reactivity for any of the viruses, however, convalescent serum reacted similarly to all three virus strains, suggesting that these three viruses are related. The 48 agents isolated were all shown to form syncytia and failed to agglutinate erythrocytes. Nearly 1/3 of the children hospitalized with acute respiratory illness were positive for a CCA-like virus while none of the children without respiratory illness were positive. Similarly, the virus was only isolated from patients with respiratory illness. Most infants and young children were seronegative near the onset of illness but seroconverted within several weeks. The majority had both complement-fixing and neutralizing antibodies (33).

There was a significant inverse correlation between the age of the child and the likelihood of the child being infected with a CCA-like virus. The child’s age and severity of symptoms were also inversely correlated, and bronchiolitis and pneumonia diagnosis were most often associated with CCA-like viral illness between January and March.

None of the children positive for CCA-like virus had adenovirus, myxovirus (influenza virus), or parainfluenza virus (33).

Classification & Strains

Shortly following the Beem et al. study, the CCA-like virus was named respiratory syncytial virus (RSV). The virus was characterized as a class V virus (34, 35) by the

Baltimore classification. Viruses in class V have a genome that is a single strand of negative-sense RNA, meaning that the genome is complementary to mRNA (34). RSV belongs to the order Mononegavirales, the Paramyxoviridae family, the Pneumovirinae subfamily and the Pneumovirus genus (Figure 3).

Figure 3. Mononegavirales tree. Examples of viruses in the order Mononegavirales. Adapted from Fields Virology ed. 6 (36).

RSV encodes more proteins than any of the other paramyxoviruses with nonstructural protein 1 and 2 (NS1 and NS), and M2-2 found only in RSV, and small hydrophobic

(SH) and M2-1 found only in the Pneumovirinae subfamily (Table 1). In addition, unlike the attachment proteins of the Paramyxovirinae subfamily the attachment proteins of the

Pneumovirinae subfamily are not essential for infection in cell culture (37), and do not have hemagglutinin and/or neuraminidase activity (30).

Table 1. Order of the genes in the genomes of Paramyxovidae family members. Genus Gene order (3’5’) Avulavirus Le N P/V M F H L tr Henipavirus Le N P/C/V M F H L tr Morbillivirus Le N P/C/V M F H L tr Respirovirus Le N P/C/V M F HN L tr Rubulavirus Le N P/V M F HN L tr Metapneumovirus Le N P M F M2 SH G L tr Pneumovirus Le NS1 NS2 N P M SH G F M2-1 L tr /M2-2

There are two subtypes of RSV, A and B, and each subtype has a number of strains. The

A & B subtypes were originally defined by differential neutralization (38-40). The main difference between the subtypes is the attachment (G) protein sequence. While the A strains, Long and A2, share 95% amino acid identity, the 18537 B strain only shares

53% amino acid identity with A2 (41).

Respiratory Syncytial Virus Spread

By the 1960s, it became evident that RSV infection was almost universal, and unlike measles and mumps, but infection did not confer long-lasting immunity (42-46). Further, it was clear that the most severe form of the disease mainly occurs within the first year of life, despite maternal Ab prevalence (17, 44-46). A cohort of infants followed from birth

13 until 2 years of life demonstrated that 68.8/100 children had been infected by 1 year of life, and 22.4/100 of those children had experienced LRTI. By the age of 2, 82.6/100 children had become infected with RSV, and13/100 of these had experienced LRTI in their second year. In addition, >1/2 of the children had experienced a second RSV infection before the age of 2 (47).

In addition to being a common virus, a number of studies demonstrated that RSV spread easily in a hospital setting, where the sickest infants are treated. In one such study, 8 of

42 infants hospitalized for a reason other than RSV infection were infected within the first 17 days of their hospital stay. In addition, 24 of the 43 staff members caring for these infants were infected within 18 days of starting on the ward or 19 days following the first admission of RSV, with 4 staff members having a repeat infection 2-6 weeks later (48).

Fomites were studied to determine how long infectious virus could remain infectious. If secretions were dried the virus had a very short life, however, the virus in liquid secretions remained viable for 7 h on countertops, 5 h on gloves, 2 h on cloth, and 30 minutes on skin or paper tissues (49). To further elucidate how the virus was spread, the interactions between a group of individuals and RSV-infected infants was studied. Each volunteer was allowed to cuddle (n=7), touch (n=10) or sit (n=14) with the sick infants.

Only those that were allowed to touch or cuddle the infants were infected with RSV and the volunteers that cuddled the infants were more likely to become ill and more likely to have severe symptoms (50).

The Cost of RSV Disease

The symptomatology and presentation of RSV are varied (Table 2). The symptoms are defined in Table 2 can include rhinitis, cough, dyspnea, cough, bronchiolitis, and pneumonia. Severe case presentation might include: audible wheeze, prolonged expiratory phase, crackles, fever, difficulty eating, and otitis media. Infants with acute respiratory failure, respiratory acidosis, shock with impending arrest, or apnea require mechanical ventilation (51).

Table 2. Symptoms of RSV infection. Possible symptoms Definition References Rhinitis* Sneezing, congestion, drippy nose (51, 52) Cough* (51) Dyspnea Shortness of breath (51, 53) Croup Loud barking cough (51, 54) Abnormally rapid breathing with expiratory wheezing, prolongation of expiration and evidence of emphysema (51, 55, 56) Bronchiolitis Congestion of the bronchioles

Air sac inflammation in one or both lungs that are (51, 57) Pneumonia filled with fluid *Common symptoms

There have been many studies to assess infants at risk for severe disease. A review of the literature showed that consistently severe disease risk was increased in premature infants, infants with bronchopulmonary dysplasia, infants with chronic lung disease, infants with

15 congenital heart disease, immunodeficient infants and those with Down syndrome (Table

3 (58)). However, there are still an estimated 30/1000 infants that will have severe RSV disease with none of these risk factors (59). In addition, there are also a few host-related factors that can increase risk: male sex, young age, birth during RSV season, overcrowding, siblings, day care exposure (58, 60) and in low-income areas, malnutrition

(58).

However, severe disease risk is not limited to infants. More recently, it has been documented that the elderly are at increased risk for severe disease, 1550-6660 elderly die each year to RSV infection in the U.S. (61). This population has less RSV-related than Influenza-related doctors’ visits, but the hospital stay, rate of intensive care unit

(ICU) admission and mortality rate are all similar between RSV and Influenza disease

(62).

Table 3. Severe disease risk factors. Risk factor # at risk/1000 infants Reference Bronchopulmonary dysplasia 388 Congenital heart disease 92 Premature infants 57-70 No known risk factor 30 (59)

In very severe cases where the infants are no longer able to breathe or eat on their own, they need to be hospitalized. A survey from 1997-2006 studied hospital discharge records and found that for children <5 years old, RSV was indicated in 132,000-172,000

16 hospitalizations/year (63). In 1999, it cost $11,000 per child hospitalized with severe

RSV illness (61). Extrapolating from the data and assuming that only 132,000 infants are hospitalized, the monetary cost would be more than $1 billion in 1999 U.S. dollars (61,

63).

The cost of severe RSV disease is not just in dollars and cents, though. A meta-analysis of death records from 1980-2010 quantify the most frequent causes of infant death in 187 countries (5). In neonates, less than 27 days old, LRTI accounted for 6.8% of the total deaths with RSV being the most frequent at 2.3%, the highest infectious cause of death in this age group (Figure 4). In infants 28-364 days old, 20.1% of deaths were due to LRTI, and RSV was responsible for 6.7% of those, a rate of death second only to malaria among infectious diseases. However, the rate drops off in older children (1-4 years old), where

1.6% of the deaths were attributable to RSV (5).

Figure 4. Causes of death from 1980-2010. Pie chart of global neonatal, post-neonatal, and child deaths in 2010 for children of both sexes combined by cause (A) Age 0–27 days (neonatal); 2 840 157 total deaths. (B) Age 28–364 days (post-neonatal); 2 031 474 total deaths. (C) Age 1–4 years; 1 969 567 total deaths. ETEC=enterotoxigenic Escherichia coli. EPEC=enteropathogenic E coli. Hib=Haemophilus influenzae type B. RSV=respiratory syncytial virus. Reprinted from The Lancet, 380 / 9859, Lozano et al., “Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010”, 2095-2128., Copyright (2012), with permission from Elsevier (5).

Prophylaxis and Treatment of RSV disease

Despite the severity of disease and the economic impact attributable to RSV, there is no vaccine. The lack of a vaccine is at least partially due to the catastrophic failure of the formalin-inactivated RSV (FI-RSV) vaccine trial in 1969. In this trial, vaccinated infants had enhanced rather than attenuated disease upon natural infection and two of the vaccinated infants died as a direct result of this enhanced disease (17, 18). Many studies have tried to elucidate the reason for this failure and a few of the findings will be described in the FI- RSV section.

There are also no medications indicated for treatment outside of salbutamol, which is normally ineffective, and ribavirin, whose use is controversial (51). Asthmatic episodes have similar symptoms to severe RSV disease. Steroids are used to treat asthma.

However, steroids provide little to no benefit in RSV-induced bronchiolitis unless the child has an underlying lung or heart disease (51).

However, Ab can be given to prophylactically prevent or reduce disease severity, although this treatment is very expensive (64). Prophylactic Ab treatment started with a clinical trial where RSV Ig was given monthly to those with bronchopulmonary dysplasia

(65). In children given a high dose of Ab, there were fewer lower respiratory tract infections (LRTI), hospitalizations, days in the ICU, and less ribavirin used (65, 66).

Subsequently RSV-IGIV was tested in children with congenital heart disease, and pre- treatment in this patient group lowered admissions of patients <6 months of age and reduced LRTI. However, there were poor outcomes if the child had to undergo surgery

(67). In 1998, a humanized mouse monoclonal antibody (mAb), Palivizumab, underwent 19 a large clinical trial. The infants in this clinical trial were either high-risk premature infants or infants with bronchopulmonary dysplasia. Every 30 days, 1002 infants were administered Palivizumab and 500 infants were administered a placebo in a double-blind study (68). Outcomes included: a lower RSV-related hospital admissions, fewer days in the hospital and a lower rate of ICU admission (68). Subsequent studies have been performed and a meta-analysis of all of the available data was performed. The available data included more than >72000 subjects from 7 randomized control trials, 4 open-label non-comparative clinical trials, 8 prospective observational studies/registries from 34 countries. This meta-analysis found that palivizumab lowered the rates of LRTI, ICU admission and mechanical ventilation (69). Despite this success, on a population level, there is an argument that this expensive prophylactic treatment is not cost effective because 17 preterm infants need to be administered the mAb to prevent one hospitalization and 59 to prevent 1 ICU admission (64).

Respiratory Syncytial Virus: Close up

Cell Culture

The majority of the experiments described below, especially the early ones, have used immortalized cell culture. While RSV infects most cell culture lines, in humans, there are very few cell types that are infected. Very early on human tracheas in organ culture were infected with RSV, and only the ciliated epithelial cells were susceptible to infection

(70). Taken together this suggests that immortalized cells might not be an ideal model for

20 studying attachment and entry. As described later, some immortalized cells also have different post-translational modification patterns. These differences can have important effects on infection and spread. Matsui et al. have described an in vitro system to study more physiologically relevant attachment and entry, HAE cultures. To produce these cultures lungs are harvested and the large airways are dissected. Cells are dispersed using enzymatic digestion and plated in a culture medium that enriches for undifferentiated cells. These cells are then plated on filters (Transwells) with medium in both the apical and basal chambers. Once the cells divide and become confluent, tight junctions form. At this point, the apical medium is removed and the cells are allowed to differentiate for 4-8 weeks (71). The cells differentiate into a pseudostratified epithelium, which contains ciliated epithelial cells and mucous producing goblet cells (Figure 5 (71, 72)). RSV only infects via the apical surface and preferentially infects the ciliated cells, while adenovirus only infects when added basally (Figure 6 (72)). A similar procedure is also used to develop cultures from pediatric nasal epithelium and bronchioles. These cultures are called pediatric nasal epithelial cells (PAEC) or pediatric bronchial epithelial cells

(PBEC). These cells also have ciliated and goblet cells and form tight junctions. The

PBEC are more susceptible to RSV infection and produce higher titers. In addition, the

PBEC loses tight junctions and cells are sloughed more often than PAEC. These cells have been shown to secrete a number of cytokines into the basal chamber (73) and are discussed further in a review by Villenave et al. (74).

Figure 5. Human airway epithelial cultures. Cell morphology and KS expression at the apical ciliated surfaces of WD HAE cell cultures. (A) Light micrograph of a cross section of a WD HAE culture grown at an ALI on a semipermeable membrane support for 4 weeks. Under these conditions, pseudostratified mucociliary epithelial cell morphology was generated. The cells were counterstained with hematoxylin and eosin. (B) Confocal fluorescent optical section of a live WD HAE culture exposed to an antibody specific for KS and detected with a secondary antibody conjugated to Texas Red. Note that KS serves as a marker for ciliated columnar epithelial cells at the apical surface of the culture and that the permeable support, a 10-μm-deep layer underlying the basal epithelial cells, displays non-KS-specific autofluorescence. Original magnification, ×100. Reprinted from Journal of Virology, 76/11, Zhang et al., “Respiratory Syncytial Virus Infection of Human Airway Epithelial Cells Is Polarized, Specific to Ciliated Cells, and without Obvious Cytopathology”, 5654-5666., Copyright (2002), with permission from American Society for Microbiology (72).

Figure 6. RSV or Adenovirus infection of HAE cultures. Polarity of rgRSV infection of WD HAE cultures. Shown are confocal fluorescent-optical-section photomicrographs of HAE cultures inoculated via either the apical (Ap) or basolateral (Bl) surfaces with rgRSV or AdVGFP. Twenty-four hours after infection, the cultures were fixed and immunostained with antibody specific for KS and detected by a secondary antibody conjugated to Texas Red. The KS- expressing apical surfaces of ciliated cells are shown in red, and virus-infected cells are shown in green. Original magnification, ×63. Reprinted from Journal of Virology, 76/11, Zhang et al., “Respiratory Syncytial Virus Infection of Human Airway Epithelial Cells Is Polarized, Specific to Ciliated Cells, and without Obvious Cytopathology”, 5654-5666., Copyright (2002), with permission from American Society for Microbiology (72).

RNA

Huang et al. set out to find virally encoded RNA species in RSV-infected cells, however, because RSV does not shut down cellular protein synthesis (6) they needed to treat cells with actinomycin D, a drug that blocks cellular mRNA production from a DNA template

(35). In addition, Davis et al. had previously found that vesicular stomatitis virus, a 23 model negative-sense RNA virus, requires protein synthesis to support the production of negative strand RNA during infection; however, viral mRNA synthesis had no such requirement (75). Huang et al. treated cells with actinomycin D to eliminate cellular mRNA production. RSV-infected cells treated with actinomycin D contained eight RNA species and the largest co-migrated with RNA from purified virions (35). When treated with actinomycin D and cycloheximide only the largest RNA disappeared (35). This finding was in contrast to positive sense virus, mouse hepatitis virus, where cycloheximide treatment blocks the production of all viral RNA species (76). This finding verified that RSV contained a negative-sense genome (35) and demonstrated that the virus coded for at least seven RNA species. These RNA species were subjected to chromatography using an oligo(dT)-cellulose column and bound demonstrating that they are polyadenylated. In addition, were able to anneal to virion RNA suggesting that they were viral mRNA (35).

It was later demonstrated that there are actually 10 mRNA species produced by RSV (77,

78), and the gene order is: NS1-NS2-N-P-M-SH-G-F-M2-L (Table 1) (77). Next, it was recognized that while the intergenic regions of the genome vary from 1-52 nucleotides

(nt), with no conserved features or structure (79), the 5’ and 3’ terminal sequences of the genes are conserved. These sequences are termed gene start signal (GSS) and gene end signal (GES). The GSS signal is 5’-GGGGCAAAU-3’ (79, 80), and the GES Is 5’-

AGU(U/A)UA(U/A)AAAA-3’ for NS2, F, and the M2 gene or 5’-

AGUUA(A/C)(U/A)(U/A)(U/A)AAAA-3’ for NS1, N, P, M, G, and SH (79). If the GSS is deleted, the transcription of that gene and subsequent genes was decreased, and the

24 position in the genome was an important indicator of how great the decrease was.

Deletion of the GES did not affect transcription efficiency, rather a dicistronic mRNA and mRNA that lacked the poly-A tail were produced. Taken together this study demonstrated that the large (L) protein uses GSS and GES as on and off switches (81).

There are two remaining conserved untranslated RNA regions located at the beginning

(leader) and end (trailer) of the genome, the leader precedes the NS1 gene, and the trailer follows the L protein gene in the antigenome (82, 83). The leader is the genomic promoter important for regulating transcription (81) while the trailer region is the antigenomic promoter that influences genome production and replication (81, 84).

Viral protein identification

RSV has 10 genes that encode 11 proteins. In this section, I will describe the history, discovery and analysis of the viral proteins and their functions. Though RSV is eventually fatal to a cell, the cellular DNA and RNA synthesis continues for at least 18 hpi (85). In addition, the virus does not appear to have a mechanism for shutting off protein synthesis (6). In the late 1970s through the 1980s, before mAbs and immunoblots were available for identifying viral proteins, cells were fed radioactive (3H, 14C, 35S) substrates to label RNA, DNA, proteins and glycans. Radioactively labeled viral proteins were synthesized in infected cells, but because RSV did not shut down protein synthesis, it was difficult to detect viral proteins above the cellular protein background. Instead, the virus was purified from the medium of infected cells, compared to similar uninfected cell

25 preparations. Components present in both preparations were ignored (6). Taking this approach, 7 proteins were identified with molecular weights of 79, 56, 44, 32, 28, 25, and

22 kDa (6). The use of different buffer systems or molecular weight markers for electrophoresis complicated the identification of proteins, for example in a different buffer the 79 kDa protein was 90 kDa, so different labs would call the proteins they identified by different nomenclature (Table 4 (6, 77, 80, 86-88)). Once the RNA had been characterized it was clear that there were more proteins in RSV than in any other paramyxovirus (77). The RSV proteins are listed in Table 4, along with their physical characteristics.

Table 4. Protein physical characteristics

Protein nomenclature Size Protein used RNA (aa*) Special characteristics Ref. First gene to be transcribed; closest to the leader Nonstructural Unique to RSV (41, 77, protein 1d,e NS1 1c 139 Well conserved between subtypes 78, 89) Nonstructural Unique to RSV protein 2d,e NS2 1b 124 Well conserved between subtypes (77, 90) Nucleocapsid protein (6, 41, 77, Nucleoproteind,e,f 44 kDa 4 391 The most conserved RSV protein across subtypes 91)

27 Acidic Devoid of C and Y residues Constitutively phosphorylated Phosphoproteind, No overlapping protein as with other paramyxovirus P e P 2a 241 proteins (77, 91) Two hydrophobic clusters, associated with membranes Two nuclear localization signals M Two nuclear export signals (6, 8, 77, Matrix proteind,f 28 kDa 3a 256 Zinc finger domain 92, 93) Continued

Table 4 continued Only present in some paramyxoviruses Type II transmembrane protein Non-glycosylated form (7.5 kDa) Small Glycosylated with 1-2 N-linked sugars (13-15 kDa) hydrophobic SH Polylactosaminoglycan modified (21-30 kDa) (6, 41, 77, proteina,c,e,f 7.5 kDa 1a 64 2 related products, differing at their N-termini 94-96) Type II transmembrane protein 2 start codons: both membrane-bound and soluble forms as 84-90 kDa Contains 4 C residues that form a Cys noose CX3C chemokine motif 1/10 amino acids are Prolines

28 1/3 are Serine or Threonine, mucin-like

4 to 6 N-glycans G Heavily O-glycosylated (50% of its molecular weight) Major surface Central conserved region (HFEVFNFVPCSIC) partially glycoprotein overlapping a 4-cysteine defined noose GP1 Least conserved of the viral proteins with features rather than 79 kDa residues conserved across subtypes (6, 41, 77, Attachment GP90 Completely unrelated to the Paramyxovirinae subfamily 79, 86, 87, proteina,b,f GP84 2b 298 attachment protein 97-102) Continued

Table 4 continued F 56 kDa Well conserved, type I membrane protein VPM27 F0 cleaved by furin at two sites, KKRKRR and RARR, VGP48 releasing 27 amino acid peptide with 2-3 N-linked glycans (6, 41, 77, Fusion GP26 F1 and F2 fragments held together by 2 disulfide bonds 79, 80, 86, proteina,b,c,e,f VPM27 5 574 3 N-linked glycans in mature protein 87) Only present in Pneumovirinae M2-1 Overlapping open reading frame with M2-2 M2 Gene overlaps with L protein gene 22 kDa, Contains a zinc finger domain (6, 77, M2-1 proteind M2(ORF1) 3b 194 Phosphorylated 103-106) M2-2 Only present in Pneumovirnae

29 M2 Overlapping open reading frame with M2-1 (77, 103, d M2-2 protein M2(ORF2) 3b 90 Gene overlaps with L protein gene 106) Large polymerase L Gene overlaps with M2 protein gene (77, 107- proteind Large protein 7 2165 Polymerase needs to backtrack to transcribe the L protein 109) * Amino acids a Further discussed in “Characterization of envelope proteins” b Further discussed in “Attachment” c Further discussed in “Entry” d Further discussed in “Early characterization of RNA replication and transcription” e Further discussed in “Immune modulation by RSV proteins” f Further discussed in “Viral assembly and egress”

Characterization of envelope proteins

From the first experiment used to identify proteins, it was clear that the 79 kDa and 56 kDa proteins were glycoproteins (6). I will refer to the 79 kDa and 56 kDa proteins as G and F respectively. The F protein was observed as 56 kDa when run under non-reducing conditions or 2 smaller proteins if under reducing conditions, suggesting that the protein is cleaved and held together by a disulfide bond consistent with other paramyxovirus’ fusion proteins (86).

Initially, the G protein was controversial, some labs observed this protein while other labs did not. Differences in the cell line, the buffer type, and the virus strain used, all had an effect on whether the protein was present (6, 7, 86). If HEp-2 cells were used to produce virus the protein was present regardless of strain. However, if BSC-1 cells were used to propagate subtype A virus, the 79 kDa band was absent from purified virions, despite the fact that the protein was present in these cells. To resolve if the G protein was actually a viral protein, Gruber, et al. mock or RSV-infected cells labeled with 3H-glucosamine, and the surface of both uninfected and infected cells were trypsinized. Trypsin did not affect the banding pattern or intensity in the area that the G protein had been observed.

However, in infected cells that were trypsinized, there was a difference in band intensity between the cells that were and were not trypsin treated, demonstrating that there are two proteins, one a cellular protein that is unaffected by trypsin (inside the cell), and the G protein which is removed by trypsin. To further confirm that there were two different proteins Gruber et al. used two different buffer types for their electrophoresis, a neutral phosphate buffer described by Weber and Orson (110) and a second buffer described by 30

Laemmli (111), when separated with the phosphate buffer, the host protein and the G protein no longer migrated at the same rate (86, 110, 111).

In early studies, both the F and G proteins were recognized as important proteins for virus neutralization in cell culture (87), mice (112), and cotton rats (88). Not only did Abs against the F protein neutralize virus in the presence and absence of complement, the Abs also blocked fusion of infected cells, confirming the role of the F protein in membrane fusion (87). In a cell culture study, Abs to the G protein only blocked infection of cells in the presence of complement (87). Subsequently, Walsh et al. demonstrated that hyperimmune rabbit serum that specifically recognized the G protein was able to neutralize viral infection in the absence of complement. In addition, purified G protein was able to bind to HEp-2 cells and the immune serum was able to block binding, suggesting that the G protein is important for binding the virus to the host cell (113).

Because this method does not directly test if the G protein is the attachment protein,

Levine et al. incubated the virus with antibodies to the G protein and allowed the virus to attach (but not penetrate) at 4°C. Anti-G mAb were able to block attachment, but mAb to the F protein did not block attachment as efficiently, demonstrating that the G protein is the primary protein responsible for attachment (114).

Hendricks et al. demonstrated that there was a second, smaller G protein form that was secreted from HEp-2 cells (100). The virions in the cell culture medium were removed by sucrose gradient centrifugation, and the top fractions were immunoprecipitated so that the glycoproteins could be identified, the only viral protein detected in these top fractions was G, suggesting that this protein is a soluble version, and not secreted from degraded 31

virions (100). In addition, this secreted G is 6-9 kDa smaller and is evident in the medium as early as 6 hpi, 6 h earlier than the first virion-associated G protein appeared (99).

Soluble G secretion was not dependent on N-linked glycosylation as tunicamycin-treated cells also secreted the smaller G protein (99). Amino-terminal sequencing demonstrated that there are two different secreted forms of G, one missing amino acids 1-66 and the other 1-75 (99). The alternate start codon is at amino acid 67, suggesting that this the full-length secreted protein and that the protein missing the first 75 amino acid is a cleaved version of the secreted protein.

Using wt virus and recombinant vaccinia expressing the RSV SH protein was found to accumulate within the cell in multiple structurally distinct forms that remain membrane- bound (94). The first form is labeled only with 35S-methionine and is 4.8 kDa, this is a truncated form that is missing a portion of the N-terminal amino acids (96). Next is a 7.5 kDa full-length protein that also is only labeled with 35S-methionine (non-glycosylated).

The next band, 15 kDa, is a mix of two proteins that contain 35S-met and 3H-glucosamine, glycosylated with immature sugars. A pulse-chase experiment demonstrated that a 13 kDa protein appears later, suggesting trimming of the glycans. This is normally displayed as a 13-15 kDa smear, reminiscent of other glycosylated proteins. The largest form is 21-

30 kDa and is strongly 3H-glucosamine labeled. Tunicamycin, a drug that blocks the addition of N-linked sugars, reduced the production of the 13-15 and 21-30 kDa forms demonstrating that the sugars are N-linked glycans. The same pattern is observed with an anti-SH Ab (94).

Sucrose gradient analysis demonstrated the F, G, and SH proteins are all oligomers (96,

101, 115). Collins et al used carbonylcyanide m-chlorophenylhydrazine, Brefeldin A, and monensin, all of which inhibit protein transport at different points in the ER and Golgi, to determine if the mature and unglycosylated G protein had similar sedimentation rates.

The mature G protein, the G protein with only N-linked glycans, and the G protein with no glycans all had similar sedimentation rates, demonstrating that glycosylation does not affect this rate and verifying that the G protein is likely an oligomer (101, 115).

Using chemical crosslinking and electrophoresis, the F protein was demonstrated to migrate as apparent dimers and tetramers though the possibility of it crosslinking with proteins other than SH and G could not be ruled out (115). Subsequently, using a number of biochemical techniques, the F protein was demonstrated to be a trimer (116). The G &

SH proteins sediment similarly to the F protein (96, 101), if the F protein is a trimer and the sedimentation rate of G is independent of sugar content this would suggest that the G protein would also be a trimer. However, the SH protein is more difficult to interpret because all of the SH forms were found to sediment in the same fraction. Despite this, SH co-migrated with the F protein, which migrated at 280K (suggesting an F protein tetramer), approximately 19-24 individual SH proteins would be needed to allow for a similar sedimentation rate (96).

Viruses that are missing the SH and/or G proteins show that neither protein is absolutely required for infection of immortalized cells (37, 117) though virus lacking G has a 10- fold lower titer, smaller plaques, and slower attachment and entry (37). Virus without SH is able to replicate as efficiently as wt, however, the G, F, and M2 mRNA are increased 33

(117), likely because they are one gene closer to the promoter because the SH gene is missing. Virus without SH actually produced larger plaques and grew better in some cell lines (117).

The Paramyxovirinae subfamily viruses are non-infectious without their attachment protein, as the attachment protein is required for the triggering of the F protein in addition to attachment (118). However, the fact that the RSV G protein is not essential for infection and the fact that cells expressing the F protein without the G protein fuse with their neighboring cells show that the RSV F protein is able to trigger independently of G

(119). The fully cleaved, mature F protein contains three N-linked glycans, but only the glycan at N500 is required for fusion (120). When fully glycosylated the F protein is metastable and, similarly to HIV gp41, forms a 6 helix bundle in the stable post-fusion form (121).

Attachment

To study attachment, most laboratories use immortalized cells. Molecules that have been described to interact with the F or G protein are described below though most have not been confirmed using HAE cultures.

Heparin/Heparan sulfate

Glycosaminoglycans decorate the surface of immortalized cells and the G protein contains a region just past the cysteine noose that is rich in positively charged K residues and has few S and T residues, and, therefore, few O-linked glycans. Heparin, a negatively charged glycan, is able to neutralize RSV infection of immortalized cells and peptides

from the K-rich region on the G protein bind to heparin, identifying it as a “heparin- binding domain” (HBD) (122). Heparin is released from basophil and mast cell granules when these cells are stimulated, and as such could not be an RSV receptor. Nevertheless,

G protein binds to immobilized heparin (13) and enzymatic removal of heparan sulfate and chondroitin sulfate lowered RSV infection of immortalized cells (12) Taken together, these data suggest that heparin blocks the interaction between G and a negatively charged viral receptor on the cell surface. Subsequently both the F and G proteins were shown to interact with heparan sulfate, a glycan similar to heparin but linked to particular cell surface proteins on immortalized cells (10, 11, 123). Feldman, et al. generated a number of biotinylated peptides and demonstrated that amino acids 184-198 were important for attachment to cells, as heparin was able to block peptide binding. G peptide 184-198 preincubated with cells prevented virus infection confirming its ability to bind to the RSV receptor (122). However, the area just downstream of 184-198 is also rich in K residues.

Shields et al. synthesized multiple peptides that included amino acids 143-231 with and without 184-198 and concluded that the K residues from 184-231 are important for G protein binding to immortalized cells (124).

Despite its role in the entry of immortalized cultures, heparan sulfate is not present on the apical surface of HAE cultures (Figure 7B (125)), suggesting that RSV utilizes a different molecule for attachment and entry of these cells. There have been many potential cell- surface proteins that can act as binding partners with the G protein, many of which need to be followed up in HAE cells, these are elucidated below.

Figure 7. Localization of heparan sulfate and sialic acid linkages on the surface of HAE cultures. Localization of cell surface HS and sialic acid linkages on PD airway cells, viewed en face (A, C, and E) and HAE, viewed in histological cross-section (B, D, and F). (A) Representative fluorescent photomicrographs of HS localization, detected with antibody F58-10E4, on the cell surface of a subpopulation of PD cells (red). (B) In HAE, HS localization was restricted to basal epithelial cells (red, arrowheads). (C and D) Sialic acid residues with α2-3 linkages, detected with MAA lectin, were present on the cell surface of a subpopulation of PD cells (C) (red), whereas in HAE (D) they were localized on the apical surface (green), viewed against a counterstain of antibody to β-tubulin IV (red). Staining with MAA was predominantly on ciliated cells (arrows in panel D) but was also detected on some nonciliated cells (arrowhead in panel D). (E and F) Sialic acid residues with α2-6 linkages, detected with SNA lectin, were present on the cell surface of PD cells (E) (red), whereas in HAE (F) localization was predominately detected on the apical surface (green), viewed against a counterstain of antibody to β-tubulin IV (red). In panel F, SNA lectin was detected on ciliated (arrows) and nonciliated (arrowheads) cells. Bar, 10 μm. Reprinted from Journal of Virology, 79/2, Zhang et al., “Infection of Ciliated Cells by Human Parainfluenza Virus Type 3 in an In Vitro Model of Human Airway Epithelium”, 1113-1124., Copyright (2005), with permission from American Society for Microbiology.

Intercellular adhesion molecule 1

Intercellular adhesion molecule 1 (ICAM-1) has been demonstrated as a receptor for rhinovirus. RSV infection upregulates ICAM-1 on the surface of epithelial cells and an anti-ICAM-1 Ab reduces RSV infection of HEp-2, NHBE, and A549 cells. In addition,

RSV binds to ICAM-1 using an ELISA-like assay, where RSV was incubated on plates coated with ICAM-1 and an Ab to RSV was used to detect binding. Preincubation with either an anti-F or G Ab blocked binding of the virus, while an isotype control did not.

Similarly, if F protein produced in baculovirus was used to coat plates, ICAM-1 bound, and increasing the concentration of ICAM-1 increased the ELISA output signal, suggesting that F and ICAM-1 interact (126).

Surfactant protein A

Surfactant protein A (SP-A) is a molecule in the lung that is thought to have a role in immunity (127, 128), it is an opsonin for pulmonary pathogens like influenza (128) and has been shown to increase the uptake of RSV by peripheral blood mononuclear cell

(PBMC) and U937 cells (127). SP-A levels are decreased in the lung in pneumonia and bronchiolitis (127), however, SP-A remains on type II epithelial cells throughout the lung. SP-A and the G protein were shown to interact using a modified ELISA (128) performed similarly to the ICAM-1 – F assay (126), and both EDTA and mannan blocked

SP-A – G protein interaction suggesting that the binding is between the carbohydrate-rich region on G and carbohydrate recognition domain on SP-A. Serially diluted purified SP-

A or mannan were added to viral inoculum which was then used to infect Hep-2C cells.

The addition of SP-A increased RSV infection of Hep-2C cells while mannan had no 37

effect on viral infection. (128). SP-A also seems to change the immune response to

RSV, as SP-A – G binding results in increased secretion of interleukin (IL) -8 (128) and tumor necrosis factor (TNF) -α and decreased secretion IL-10 (Table 5) secretion induced by infected PBMC (127, 128).

Annexin II

A pan-selectin inhibitor (TBC1269) reduced infection of HEp-2 cells and mice. Annexin

II, a selectin, interacted with RSV, and RSV infection increased annexin II expression.

Annexin II inhibition reduced the susceptibility of HEp-2 cells to RSV infection, but much less so than TBC1269 did (129).

DC-SIGN and LC-SIGN

The G protein has been shown to interact with both cell surface lectins: DC-SIGN and L-

SIGN, which are only present on a few cell types. Blocking the interaction does not block infection of dendritic cells (DC), but it does increase secretion of interferon (IFN) –α

(Table 5), MIP-1α, MIP1-β to encourage maturation of DCs (130). This result suggests that the G protein might modify the immune response in important ways.

Nucleolin

RSV was found to bind nucleolin (129). A second group used a virus overlay protein binding assay (VOPBA) to identify RSV surface protein binding partners. VOPBA involves lysis of cells that are susceptible to RSV infection, electrophoresis of the cellular lysate and blotting similar to a Western blot, but instead of using an Ab as a probe, the virus is used. The bound virus is detected with a virus-specific Ab. Areas that permitted

virus binding are then eluted and their proteins analyzed. Cells were biotinylated and cell surface molecules were used in this VOPBA. A 100 kDa protein, nucleolin, was shown to interact with both A2 and B subtypes of RSV, and the G protein was not required for this interaction. Subsequently, confocal microscopy showed that nucleolin and the F protein colocalized in 1HAEo- cells (131), a cell line that is transformed by SV40 antigen, but retains tight junctions and ion transport (132). Nucleolin knockdown in 1HAEo- cells decreased infection, and nucleolin expression in an insect cell line, Sf9, rendered these cells permissive to RSV infection (131). Nucleolin surface expression is indicative of highly proliferating cells, as such it is a cancer cell marker and multiple therapies have been developed to use cell-surface nucleolin expression to selectively kill cancer cells

(133).

CX3C chemokine receptor 1

The G protein binds to the CX3C chemokine receptor 1 (CX3CR1) (134). CX3CR1 is a chemokine receptor on immune cells and its ligand, fractalkine, has a CX3C motif as does the RSV G protein. The G protein and fractalkine can both mediate leukocyte chemotaxis in vitro to similar degrees (134). Mice administered purified G produced in

Vero cells show signs of apnea. If mice are administered an anti-G antibody, an anti-

CX3CR1 antibody, or fractalkine (to a lesser extent) this symptom is alleviated (135).

Vaccinating mice with G protein peptides that contain the CX3C motif, induces antibodies and these antibodies block the interaction between the G protein and CX3CR1

(136). These observations were thought to be due to an interaction on immune cells, and that by blocking this interaction there was less inflammation, less weight loss, and less 39

infection. However, an alternative explanation would be that the G protein – CX3CR1 interaction is important for infection of the target cell in the lung, as a decrease in infection would also lead to less inflammation, less weight loss, and less viral yield.

Consistent with this alternative explanation, our laboratory and 2 others have recently shown that CX3CR1 is expressed on the cilia of the ciliated epithelial cells in HAE cultures, the cell type that RSV infects. An anti-CX3CR1 antibody incubated with HAE cultures prior to infection is able to inhibit RSV infection of these cells, while an isotype control is not. In addition, CX3CR1-/- are less susceptible to RSV infection than are

CX3CR1+/+ mice. Taken together this data suggests that CX3CR1 – G interaction may not just be modifying the immune response, but might also be blocking infection (137-

139).

Entry

As with attachment, entry studies have been primarily performed with immortalized cells.

This work started in 1991 when Srinivasakumar et al. utilized ammonium chloride, a weak base, to increase the pH of the late endosomal/lysosomal compartments. While vesicular stomatitis virus, a virus that requires low pH for entry, infection was inhibited in cells treated with ammonium chloride, this weak base had no effect on RSV infection, suggesting that RSV does not require low pH to infect. Next a quenching assay was used to determine if RSV could fuse with the target cell plasma membrane. In the quenching assay, the RSV membrane contained a fluorescent lipid that was present at self- quenching concentrations, if the virus fuses at the viral membrane the fluorophore will be

diluted allowing for fluorescence. Fluorescence was detected, suggesting that the virus fuses at the plasma membrane.

Next the group tested treated cells with sodium azide, a metabolic inhibitor, and observed the effect of the drug on the rate of infection. This is thought to work through inhibition of active entry processes, but sodium azide had no effect on entry, suggesting that the infection was at the cell surface. If labeled virus was heated at 75°C prior to infection or treated with trypsin, the level of dequenching was lowered, suggesting that a functional protein is required for this dequenching, in this case, likely the fusion protein. In addition, this fusion did not occur readily at low temperatures. Taken together a viral protein is responsible for RSV fusion at the plasma membrane and this fusion does not require energy expenditure by the cell (140).

More recently a number of studies have brought the question of RSV entry back to the forefront. Using siRNA screening 6 proteins involved in clathrin-coated pit formation, 7 in endocytosis and 3 in actin polymerization were identified as being important for early events in RSV infection. Moreover, both dominant negative forms of proteins involved in clathrin-mediated endocytosis and chlorpromazine, an inhibitor of clathrin endocytosis, inhibited RSV infection, without affecting RSV attachment (141).

Following this study, San-Juan-Vergara et al. used viruses labeled with R18 and DiOC, at levels where R18 (red) quenched the fluorescence of DiOC (green). Using these viruses they found no orange or yellow signal inside of the cells, suggesting that fusion was occurring at the cell surface though hemifusion could not be ruled out by this experiment.

A dynamin inhibitor had no effect on viral infection, but Pak1 inhibition did block 41

infection suggesting that cytoskeletal rearrangement and not dynamin-dependent endocytosis was important for viral entry. The authors also labeled lipid rafts with cholera toxin subunit B and found that RSV colocalized with these lipid rafts in the cellular membrane. If cells were depleted of cholesterol RSV infection was blocked, and reconstituting cholesterol enabled RSV infection (142).

Krzyzaniak et al. were able to build on the previous work. They allowed the virus to adsorb to cells that were subsequently treated with trypsin at 0, 15, 30 and 60 minutes post-infection. At 0 and 15 minutes, there was no labeling of the F protein on trypsin- treated cells, but by 30 minutes F could be visualized in cells treated with trypsin. By 60 minutes, the F protein was equally resistant and susceptible to trypsin digestion, indicating that some of the F protein had been internalized. Next, viruses were labeled with R18 and DiOC, at levels where R18 (red) quenched the fluorescence of DiOC

(green) (143). While San-Juan-Vergara et al. observed virus internalization for 37 min

(142), Krzyzaniak et al. followed viral internalization for 120 min post attachment at 4C

(143). At 0 min post-infection, red fluorescence was detected on the cell surface and by

60 min green fluorescence was detected, and the number of green foci increased over time, suggesting that the red and green lipids are being diluted. If treated with trypsin, red areas that did not also fluoresce green were susceptible to trypsin, however, the green fluorescence was not, suggesting that this fluorescence dilution is occurring inside of the cell and is protected from the protease. Because fusion is likely occurring after the virus is endocytosed, they used Bafilomycin A and monensin to demonstrate that viral infection was independent of pH. Cells treated with a number of clathrin-mediated

endocytosis inhibitors, all failed to reduce RSV infection. Once it was clear that endocytosis was involved in some way in early stages of RSV infection, the cells were labeled with phalloidin to observe actin, and RSV binding was found to induce a cascade of events that culminated in actin rearrangement, cell morphology change, and fluid uptake by the cell. If actin rearrangements were inhibited with cytochalasin D and latrunculin A, infection was inhibited. In addition, dextran uptake was increased with

RSV infection. All of these observations suggested that RSV might be taken up through macropinocytosis. (143).

Extracellular signal-regulated kinase (ERK) 1 and 2, components of the mitogen- activated protein kinase (MAPK (Table 5)) pathway are activated in A549 cells within minutes of viral infection (130, 144, 145). As the MAPK pathway is important in regulating a number of cellular activities, including stress and antiviral responses, Kong et al wanted to understand what role this upregulation played in infection. They first pretreated cells or virus with heparin and this pre-treatment inhibited ERK1/2 activation and subsequent RSV infection in both cases, suggesting that attachment of the virus to the host membrane was important for activating ERK1/2 (144, 145). If prior to infection, cells were treated with inhibitors that block the activation of the MAPK pathway, RSV infection is decreased compared to controls, suggesting that MAPK activation and subsequent signaling is important for RSV infection (144, 145). The MAPK pathway can also induce cytoskeletal rearrangements (146), and as discussed cytoskeletal rearrangements are required for entry of RSV particles into cells (143), suggesting that the MAPK pathway likely plays a role in the entry of RSV into cells.

One question that was still left unanswered, if RSV does not require an acidic environment for entry, why would this massive rearrangement of the cytoskeleton and activation of a pathway that is known to trigger immune factors be important for entry?

To answer this question, Krzyzaniak et al. treated cells with a cell permeable furin inhibitor, which reduced infection of both wt virus and virus lacking SH and G. Cells treated at 1 hpi with the furin inhibitor no longer inhibited RSV infection, suggesting that furin is required early in viral entry. These results are exciting because they may give us new insights into how RSV infection might occur in vivo and give us new targets for

RSV infection inhibition. However, these experiments need to be repeated in a more physiologically relevant context.

Early characterization of RNA replication and transcription

One striking feature of RSV-infected cells is the inclusion bodies. These were noted in the original Chanock et al. paper (30), and many papers since then. A number of stains were used to determine what the inclusion bodies contained. Feulgen, Periodic acid-

Schiff, and Oil Red O stains did not react with the inclusion bodies (147) suggesting that they do not contain: chromosomal or cellular DNA, polysaccharides, triglycerides or lipids (148). Interestingly the inclusion bodies stained green with acridine orange (147), which would be indicative of DNA or double-stranded (ds) RNA (148) and pink when stained with methyl green-pyronin (147), which would stain RNA (148). This suggests that there is RNA present in the inclusion bodies, to confirm this, cells treated with

RNase lessened staining with methyl green-pyronin, but DNase did not have the same 44

effect (147). However the green staining of the acridine orange stained samples was not lessened by RNase treatment (147), further suggesting that there is dsRNA present in these inclusions, and the dsRNA is likely a replicative intermediate. The cytoplasmic inclusions also include the L protein (149), the N protein, the P protein and the 22 kDa

(M2-1) protein, but cells expressing only the N and P proteins also have cytoplasmic inclusions demonstrating that N and P are sufficient for their formation (149).

The L protein is an RNA-dependent RNA polymerase. It is responsible for genome replication, antigenome production, mRNA transcription, ATP binding, and mRNA capping, cap methylation at two sites and polyadenylation. The open reading frame of the

L protein and the M2 gene overlap, so that the first 68 nt of L are also the last 68 nt of the

M2 gene (150). Both the GES of M2 and the GSS of L are required for L transcription.

However moving the L GSS after the GES of M2 does not block L transcription, demonstrating that the overlap is not necessary for transcription. In order for both genes to be transcribed, the polymerase, after finishing M2 transcription, must scan backward to start the L gene. In addition, the polymerase must ignore the M2 GES in order to transcribe the L mRNA (108).

The P protein is also essential for transcription. Dupuy et al. demonstrated in an in vitro transcription assay, using modified P proteins, if the P protein is not phosphorylated by casein kinase 2 at S232, transcription efficiency is reduced by 50%. Casein kinase 2 can also phosphorylate amino acid 215, but this phosphorylation has no effect on transcription. RSV-infected cells treated with D609, a casein kinase 2 inhibitor, produce less virus than untreated cells (151). 45

In order to understand transcription and replication of RSV, plasmids containing the L, P, and N genes were transfected into cells along with a mini-antigenome (positive sense) or minigenome (negative-sense). Plasmids expressing the N, P and L proteins driven by a

T7 promoter were infected with a modified vaccinia virus Ankara expressing the T7 polymerase. Using this system, the L protein was not well expressed and the L protein that was expressed was a range of sizes. Titrating the amounts of the P and N plasmids showed that the most efficient protein production was achieved when the P and N plasmids were in equal concentration. Excess P protein expression was inhibitory to RNA synthesis (152).

Once the transfection conditions were optimized, the mini-antigenome and the mini- genome, which encodes chloramphenicol acetyltransferase (CAT), to investigate transcription and replication. When the mini-antigenome was provided the mini-genome, and more mini-antigenome were produced. When the mini-genome was provided the mini-antigenome and a smear of CAT mRNA was produced, as well as, more mini- genome (152).

The smear of CAT mRNA was of interest because it indicated that the L protein was not producing full transcripts. This suggested that although the N, P, and L proteins are necessary for transcription, the L polymerase requires another protein to produce full transcripts. Consistent with this, if cells are also infected with RSV, the mini-genome- generated mRNA are completed (152). To delineate what protein might be important for transcription elongation, a plasmid expressing a dicistronic mini-genome containing both a CAT gene and a luciferase gene, was co-transfected with a series of plasmids 46

expressing other RSV proteins. A low concentration of plasmid expressing M2 resulted in the production of mini-antigenome and complete mRNA for CAT from the mini- genome. At some concentrations of M2, the luciferase mRNA was also expressed.

However, if M2 expression was too high, both replication and transcription were inhibited. The M2 gene has two open reading frames. When they were separated, and the first open reading frame, M2-1, was expressed, the transcripts were complete, and the amount of mRNA increased (103). M2-1 enhances transcription of mono- and polycistronic mRNA while M2-2 is inhibitory to transcription. This result suggests that

M2-1 is a co-factor that somehow helps the L protein to complete the process of mRNA transcription. Once an L protein leaves the genomic template, it cannot reinitiate downstream. Therefore, M2-1 is a limiting factor for transcription. Fearns and Collins, using the mini-genome expressing NS1, NS2, and CAT, found that in the absence of M2-

1 there is a large amount of NS1 and NS2 mRNA, but very little of the CAT mRNA expressed. (153). Since M2-1 is the next to the last gene of the genome, these results suggest that the M2-1 protein must be packaged in the virion, consistent with this, microscopic evidence shows M2-1 within viral filaments (154).

M2-1 has a Cys3-His1 motif (CX7CX5CX3H), reminiscent of zinc finger motif, near the amino terminus (104). Zinc finger domains bind zinc, coordinating the zinc by contact with all four of the critical amino acids and are often involved in gripping DNA, RNA and proteins. Proteins with zinc finger domains are usually associated with transcription complexes or are transcription factors (104, 155). Consistent with this M2-1 increases L protein processivity: finishing the synthesis of mRNA, but also promoting the read-

through of intergenic regions. If the cysteine or histidine residues are mutated so that the protein can no longer bind zinc, the read-through/anti-termination effect of M2-1 is blocked, as is the protein’s ability to interact with the N protein. However, the N protein and M2-1 interaction are not essential for processive transcription because a mutation,

E10G, which destroys the M2-1 – N interaction does not alter L protein processivity

(104).

While M2-1 is needed for efficient transcription, M2-2 inhibits transcription. To further delineate the role of M2-2, virus lacking the M2-2 gene was generated. Cells infected with this mutant produced much less genomic RNA and a surplus of viral mRNA. Taken together, M2-1 is important for complete mRNA elongation early in infection, and as viral proteins accumulate, M2-2 inhibits mRNA production while, and perhaps by, increasing replication (36, 153).

Using the mini-genome system, it was demonstrated that the NS1 protein also has an inhibitory effect on viral mRNA synthesis. In the mini-genome system also transfected with an NS1 expressing plasmid, there was a large reduction in CAT and luciferase mRNA and mini-antigenome production, independent of M2 inclusion. In addition, in the absence of NS1 protein, cells treated with nuclease contained intact antigenome.

However, if nuclease was added in the presence of NS1, the antigenome was degraded, but the genome remained intact, suggesting that NS1 did not prevent antigenomic RNA synthesis but instead blocked its encapsidation and, since encapsidation is required for the viral polymerase to produce antigenome or mRNA, blocked replication, and transcription (89). 48

Immune modulation by RSV proteins

Both melanoma differentiation associated protein 5 (MDA5) and retinoic acid-inducible gene I protein (RIG-I (Table 5)) are pattern recognition receptors (PRR) that launch an antiviral response. The ligand or pathogen-associated molecular pattern (PAMP) for RIG-

I is dsRNA with a 5’ di- or triphosphate and small (<200 bp) RNA (156-159), while

MDA5 recognizes genomic RNA and replication intermediates (158). When MDA5 or

RIG-I encounter their ligand, the proteins undergo a conformational change, which unmasks the domain that interfaces with mitochondrial antiviral-signaling protein

(MAVS (Table 5 (157))). MAVS then activates a chain of events that leads to IFN regulatory factor (IRF) -3 activation (Table 5 (157)). IRF3 activates genes that have antiviral activity or cytokines that can induce an antiviral state, such as type I IFN and nuclear factor κ-B (NFκB (157, 158)). MAVS is anchored in membrane-bound organelles including peroxisomes, mitochondria and the mitochondrion-associated membrane

(MAM) a specialized area of ER (156).

Table 5. Proteins involved in immunity. Molecule Alternate names Purpose Ref. Bap31 ER protein that when activated associates with Fis1 and B-cell receptor- Protein CDM serves as a bridge between ER and Golgi, causes Ca2+ associated protein 31 p28 release from ER and recruits procaspase 8 (160, 161) Produced as a zymogen CASP-8 TNF-α activates Casp-8, which cleaves caspases involved Apoptotic cysteine in apoptosis to activate them, leading to apoptosis protease Cleaves BID, which is also pro-apoptotic FADD homologous Restricts necrotic cell death ICE/ced-3-like protease Restricts inflammation through: necrosis block, apoptosis ICE-like apoptotic induction, and restricts IRF3 activation by RIG-I Caspase 8 protease 5 Induces inflammation through: activation of proIL-1β (161, 162)

50 CCL5

Regulated upon Activation, Normal T- cell Expressed and Secreted (RANTES) C-C motif chemokine Eosinophil chemotactic Chemokine for T-cells, monocytes, eosinophils and 5 cytokine basophils (159) CCL11 C-C motif chemokine 11 Eosinophil chemotactic protein Chemoattractant for eosinophils, basophils, monocytes, and Eotaxin Small-inducible cytokine lymphocytes (163, 164) Continued

Table 5 continued Ligand for Flt3 Required for myeloid and lymphoid DC maturation Increases IgG1 titers somewhat, large increase in IgG2a titers Inhibits canonical NFκB signaling Fms-related tyrosine Flt3L Promotes anti-inflammatory response: IL-10 kinase 3 ligand Flt3 ligand Induces Th1 type T-cells in the lung (165) Regulating non-classical NFκB activation via LPS, or cytokines Activate the type I IFN response by activating IRF3 and 7 Inhibitor of NFκB Activates STAT1 kinase subunit ε IKKε Suppression of canonical NFκB activation (166, 167)

51 Constitutively expressed, shuttles in and out of the nucleus,

but primarily in the cytoplasm, until it is stimulated to dimerize and enter nucleus Stimulation from TLR3, 7, 9, RIGI, or type I IFN Induces First IFN production Transcription factor that activates antiviral factors such as IFIT, IFNα and β Transcribes RANTES and IL-15 Upregulates genes important in recruiting immune cells Interferon regulatory Known to interact with NFκB, IRF7 or itself to form dimers (157, 158, factor 3 IRF3 Can be triggered by RIG-I to induce apoptosis 168-173) Continued

Table 5 continued Only expressed in B-cells, pDC, monocytes in the spleen, thymus and blood lymphocytes; and expression is transient Inactive in cytoplasm, dimerizes and translocates to nucleus upon stimulation from TLR3, 7, 9, RIGI, or type I IFN via a number of kinases including IKKε Regulates transcriptional activation of IFNα and β, NFκB, IRF7 Interferon regulatory Positive feedback and amplification of immune response factor 7 IRF7 Known to interact with NFκB, IRF3 or itself to form dimers (168-170, 172) Type I IFN Secreted Binds to IFN receptor that activates Janus

52 kinasesactivates STAT1/2 and 1/1 proteinstranslocates

to nucleus and associates with IFN regulatory factor 9leads to transcription of IFN stimulated genes (ISG) ISG can lead to apoptosis, antiviral effects, immune modulation, host defense, transcription factor upregulation, cell cycle arrest, suppression of angiogenesis Activates MAPK and other signaling pathways that do not IFN α IFN-α lead to STAT activation (174, 175) Continued

Table 5 continued Type I IFN Secreted Binds to IFN receptor that activates Janus kinasesactivates STAT1/2 proteinstranslocates to nucleus and associates with IFN regulatory factor 3leads to transcription of ISG ISG with antiviral, antiproliferative and antitumor properties, Th1 cell development, increases plasma cell differentiation, increases Ab class switching and Interferon β IFN-β production, promotes DC maturation and activation (175, 176) Type II IFN Secreted

53 Binds to IFN receptor that activates Janus kinases--

>activates STAT1 dimerizationtranslocates to nucleusleads to transcription of ISG ISGs that promote Th1 and CD8+ T-cell differentiation and proliferation, activate NK cells and macrophage activate proliferation, or activate apoptosis Interferon γ IFN-γ Inhibits production of IL-9 (170, 176-178) Potent inflammatory cytokine Fever, myalgia, headache, fatigue, all associated with IL-1 Stimulates release of IL-6, IL-17a, TNFα, nitric oxide, reactive oxygen species and cortisol release Th1, Th17, CD8, neutrophil, NK cell, B-cell, and DC inducing cytokine Interleukin-1β IL-1β Can lead to activation of MAPK, NFκB (177, 179-185) Continued

Table 5 continued Synergistic and antagonistic effects of other cytokines shape response Cytokine that activates STAT6, which stimulates Th2 responses Regulates mast cell growth and maturation enhances degranulation Triggers histamine release Increase smooth muscle contraction Promotes B-cell survival and stimulates production of IgG, IgE and IgM antibodies Stimulates production and release of IL-4, IL-5, IL-6, IL-9 IL-4 and IL-13

54 B-cell stimulatory factor Recruits eosinophil to site of inflammation (170, 184,

Interleukin-4 1 Decrease chloride secretion 186-189) Th2 type cytokine, but does not induce Th2 differentiation IL-5 Stimulates production and recruitment of eosinophils B-cell differentiation Stimulates STAT1/5 and MAPK to induce survival, factor I division, and differentiation of basophils and eosinophils Eosinophil Induces proliferation, class switching, terminal differentiation factor differentiation, survival and memory of B-cells Interleukin-5 T-cell replacing factor Induces NFκB in B-cells (187, 190-192) Continued

Table 5 continued Activates STAT1/3 and to some extent 5 Activates NFκB and MAPK Promotes T cell expansion and activation Stimulates B-cell differentiation Increases hematopoiesis, body temperature, glucose metabolism, fatigue, appetite loss Induces granulopoiesis, but inhibitory to neutrophil chemotaxis Induces alternatively activated macrophages, which is important in wound healing and in inhibiting microbicidal and proinflammatory effector release Co-stimulates Tfh cells with IL-21, stimulating B-cell and

55 plasma cell survival, differentiation and division

IL-6 Stimulates survival and expansion of T-cells B-cell stimulatory factor In coordination with TGFβ induces differentiation of Th17 2 cells through induction of STAT3, but promotes IL-10 CTL differentiation production factor Blocks Th17 differentiation into Treg Hybridoma growth Promotes Th22 cells in combination with TNF (170, 177, Interleukin-6 factor Inhibits IL-8 and fractalkine production 181, 184, 193) Continued

Table 5 continued IL-8/CXCL8 Granulocyte chemotactic protein 1 Monocyte-derived Chemokine that primarily recruits neutrophils, and neutrophil chemotactic induces phagocytic and respiratory burst phenotypes factor Recruits T-cells and basophils Neutrophil-activation CXC chemokine family member protein 1 Proangiogenic (163, 164, 177, Interleukin-8 T-cell chemotactic factor Activates NFκB and MAPK 194, 195) Often seen in Th2 inflammation, but is not induced by nor does it induce Th2 cells Activates MAK, STAT1 3 and 5 in multiple combinations

56 to drive expression of: cell survival, inflammatory

mediators and proliferation Promotes hematopoietic stem cell differentiation and maturation Stimulates mast cell, goblet cell, and mucin-producing cell division Promotes CD4+ T-cell growth Enhances IL-4 induced IgG and IgE production Th17 differentiation Stimulates Gob-5, IL-6, IL-8, IL-13 and eotaxin Interleukin-9 IL-9 production (188, 196-198) Continued

Table 5 continued Stimulates Gob-5 production Stimulates STAT3/3 Suppresses TLR signaling through miRNA degradation, direct suppression, and TLR signaling component ubiquitination Blocks NFκB activation and/or nuclear localization Reduces pro-inflammatory cytokine production via miR, Interleukin-10 IL-10 and suppressing APC activation (196, 199) Th2 related cytokine, but does not induce Th2 phenotype Stimulates Gob-5, IL-4 and IL-13 production Activates STAT6 for downstream immune regulation Stimulates goblet cell hyperplasia, airway

57 hyperresponsiveness, smooth muscle alterations, and

fibrosis Increase mucosal permeability, smooth muscle contraction, mucus production Recruits eosinophil to site of inflammation (175, 184, 186, Decrease chloride secretion 187, 189, 192, Interleukin-13 IL-13 Stimulates production of IgG1 and IgM 196) Continued

Table 5 continued Activates STAT3/5 and MAPK Promotes division of neutrophils and mast cells Anti-apoptotic effects on neutrophil Activates DC to activate CD8+ and NK cells NK cell survival are IL-15 dependent Promotes macrophage phagocytosis, and production of IL-1, TNF, IL-8, RANTES/CCL5 Promotes proliferation of CD4+, CD8+ and CD4- and CD8- T-cells, including Th1 and Th17 cells, and promotes maintenance of CD8 memory Promotes B-cell division leading to synthesis of IgG1, Interleukin-15 IL-15 IgM and IgA (200)

58 Proangiogenic, stimulates erythrocyte production in

spleen Stimulates granulopoiesis and trafficking to site of inflammation Stimulates MUC5AC production Activates STAT3, NFκB and MAPK Leads to germinal center formation, and increasing somatic hyper-mutation (164, 170, 178, Interleukin-17 IL-17 Induces IL-1β, IL-6, IL-8, antimicrobial peptides 181, 201-204) Continued

Table 5 continued Activates STAT3 Induces and maintains Th17 cells Induces Th2 cell and eosinophil activity Important for memory T-cell proliferation Stimulates DC cytokine release Induces IL-17 production (178, 185, 193, Interleukin-23 IL-23 Induces antimicrobial peptide production 204-206) Key kinases that lead to STAT phosphorylation in (174, 176, 177, Janus kinase JAK response to different receptor/ligand binding 194, 200) MDA5 Melanoma IFN-induced helicase C differentiation domain-containing PRR that recognizes genomic RNA and replication

59 associated protein 5 protein 1 intermediates (158)

MAVS Mitochondrial IFN beta promoter antiviral-signaling stimulator protein 1 protein (IPS-1) Interfaces with MDA5 or RIG-I to activate IRF3 (157) Signaling molecules in this pathway Important for activating signaling molecules that in turn lead to activation of a variety of transcription factors and as such a variety of cellular responses including stress and inflammatory responses All of the TLR, RIG-I-like receptors, NOD-like receptors, and C-type lectin receptors signal through MAPK and Mitogen-activated MAPK NFκB (146, 190, 191, protein kinase 1 MAP kinase Important for cytoskeletal rearrangements 207) Continued

Table 5 continued Defense barrier to traps particles, pathogens and chemicals at mucosal membranes Cilia beat the mucous and accompanying debris away MUC-5AC from the airways Mucin-5 subtype AC Promotes healing of mucosal injury Major airway Contributes to tear fluid stability glycoprotein Lubrication of mucosal surfaces (188, 198, 208, Mucin-5AC Tracheobronchial mucin Absorbs water 209) Inactive in cytoplasm, activated by IKK complex Transcription factor Canonical activation is quick and transient, non-canonical activation is slow but increased in duration

60 Activation and repression can lead to activation of genes

that control the stress response, proliferation, apoptosis, Nuclear factor kappa- cell adhesion, innate immunity, adaptive immunity (163, 172, 177, B NFκB Known to interact with IRF3 or IRF7 198) Enzyme involved in induction of signal transduction pathways, leading to transcription, translation and stress O-linked N- responses acetylglucosamine OGT Acts as a rheostat with small activation leading to transferase O-GlcNAc transferase proliferationdifferentiationstressapoptosis (210) Continued

Table 5 continued Receptor expressed on T-cells, B-cells, and NK cells Important for lowering inflammation that will result in injury of cells and tissues Helps to downregulate proinflammatory cytokines Controls activation of lymphocytes and immune tolerance Tfh cell PD-1 interacts with PDL-1 on germinal center B- Programmed cell cells, important for balance and composition of Ab death protein 1 PD-1 response (211, 212) PD-1 ligand Expressed on a subset of macrophages constitutively IFNγ induces expression on other cell types Upregulated on germinal center B-cells and engages PD-1

61 Programmed cell on Tfh cells, important for balance and composition of

death protein-ligand 1 PDL-1 Ab response (211, 212) PRR that recognizes dsRNA with a 5’ di- or triphosphate Retinoic acid- and RNA <200 bp inducible gene I Activates IKKε protein RIG-I Activation unmasks the MAVS interacting domain (156-158, 166) Transcription factor that bind to IFN-stimulated response Signal transducer and elements to transcribe protein-coding genes, miRNA, and activator of long noncoding RNA transcription 1 or 2 STAT 1 or 2 Critical for viral clearance (174, 177, 213) Opsonin Enables more ready uptake of pathogens by phagocytes Surfactant protein A SP-A Lipoprotein that reduces ALI surface tension in the lung (127, 128, 214) Continued

Table 5 continued B-cell survival and in Ab class switching, has a potential TNF receptor- role in T-cell development and signaling associated factor 3 TRAF3 Activates IRF3 (166, 215) APC expression on cellular surface Activated by bacterial lipoproteins Can activate NFκB and MAPK Toll-like receptor 2 TLR2 Not a polarized T-cell response, instead variable (216, 217) PRR that induces a strong Th1 and CTL responses Activated by dsRNA Activates IRF3, IKKε and NFκB Can directly induce cell migration and adhesion w/o Toll-like receptor 3 TLR3 transcription factor activation (166, 173, 216)

62 Activated by LPS

Activates MAPK, IKKε, NFκB Toll-like receptor 4 TLR4 Leads to inflammatory cytokine release (166, 217) Activates NFκB by the canonical pathway, leading to inflammation, oxidative stress Acts on endothelial cells to promote vasodilation and edema to allow for immune cell extravasation through blood vessel Induces nitric oxide synthase to promote smooth muscle relaxation in blood vessels and apoptosis in endothelial cells Induces angiogenesis Tumor necrosis factor Stimulates release of the chemokine IL-8 and adhesion α TNFα molecules like ICAM-1 (177, 204, 218)

By 6 hpi, inclusion bodies have begun to form in the cytoplasm. These inclusion bodies are the site of viral mRNA transcription and replication, so the N, P, L and M2-1 proteins are all localized to these inclusion bodies. The antiviral proteins MDA5, RIG-I and later

MAVS, a protein on the surface of mitochondria, also co-localize with these inclusions.

In a transfection model, where the N and P proteins are co-expressed, MAVS and the mitochondria are By 24 hpi, MAVS co-localizes with the viral filaments at the plasma membrane, Lifland et al. did not test if MAVS was still associated with the mitochondrion. Transfection studies have demonstrated that N and P protein expression was sufficient to change the localization of MDA5 and MAVS. In addition, proximity ligation assays demonstrated that independent of infection, the N protein is in very close proximity to both MDA5 and MAVS, but not to RIG-I (219). These results suggest that the viral proteins localized to the inclusion bodies are not only masking the RNA from the PRRs but are also sequestering antiviral proteins.

Late in infection, both p38, a molecule critical to the MAPK signaling pathways, and O- linked N-acetylglucosamine transferase (Table 5) also localize to the inclusion bodies

(145). Stress granules are aggregations of ribonucleoproteins that form under environmental stress or pathogen insult to free up the transcription machinery for antimicrobial or stress response factors (220). The trigger for stress granules formation is the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), a protein that regulates initiator tRNAi-Met loading onto the ribosome to initiate protein synthesis (221,

222) in an energy dependent manner. When phosphorylated, eIF2α acts as an inhibitor of eIF2B the GTP/GDP exchange factor that activates the translation complex (223). This

inhibition reduces the amount of eIF2-GTP- tRNAi-Met when translation is started without this complex, the initiation complex stalls, and these stalled complexes are assembled into cytoplasmic foci that are also known as stress granules (222, 224, 225).

Protein kinase R (PKR) is the kinase that phosphorylates eIF2α and this kinase is increased by RSV infection (226). An increase in PKR should be followed by an increase in eIF2α phosphorylation, but in RSV-infected cells there is much less eIF2α phosphorylation than expected because RSV also induces a phosphatase, protein phosphatase 2a, which removes the phosphate on eIF2α. In addition, the RSV N protein is able to bind PKR, suggesting that this protein is also sequestered in the inclusion bodies (226).

The NS1 and NS2 proteins in bovine RSV had also been shown to suppress innate immunity. These proteins suppress IFN induction (Table 5), and as a result, the downstream IFN stimulated genes (227). RSV induces IFN-α & -β production in A549 cells (228) and viruses lacking the NS1 and/or NS2 are do not grow well in immortalized cells that produce and respond to IFN-α or -β, nor do they infect mice, monkeys or chimpanzees well (228-231).

To understand the role of NS1 and NS2 in immune suppression, a method to detect these proteins was required. The NS1 and NS2 plasmids were modified to contain FLAG-tags for detection and purification. The plasmids were co-expressed, and the NS1 and NS2 proteins purified and analyzed by size-exclusion chromatography. Surprisingly, the NS1 and NS2 complexes eluted in the size range from 200 kDa to greater than 600 kDa. The purified complexes were subsequently incubated with different proteins known to be 64

involved in immunity and/or mitochondria (232). IRF3 (233), IRF7, RIG-I, TRAF3,

IKKε, and STAT2, (Table 5), all proteins involved in IFN activation, were degraded by the purified NS1/2 complexes in conjunction with mitochondrial proteins, however, mitochondria alone were not sufficient to degrade the proteins (232, 233). In mouse cells expressing NS1, NS2, and MAVS were present, NS1, NS2, RIG-I, RAF3, IRF3 and

STAT2 all localize to the mitochondria while in the absence of MAVS only IRF3 localized to the mitochondria. In these experiments, greater degradative power was correlated with more confluent cells, shorter mitochondrial length, and mitochondrial movement (232).

Goswami et al. did not test if NFκB (Table 5) was degraded by NS1 and NS2. NFκB is another transcription factor that can lead to IFN stimulation and a potent antiviral state

(163, 172, 177, 198). In RSV-infected cells, NFκB is activated within 6 hpi, and unexpectedly, if the virus used to infect is lacking the NS1 or NS2 genes there was less

NFκB activation. This result suggests that one of the NS proteins induces NFκB, however, because NS1 deletion affects the level of NS2 expression it is difficult to determine which protein is responsible for the NFκB inducing effect (233).

It seems counterintuitive that NS1 and NS2, potent immunomodulatory proteins, would increase activation and translocation of a transcription factor, NFκB, that regulates genes involved in both innate and adaptive immune responses (233, 234). One possible explanation is that NFκB activates transcription of a number of genes that are important for proliferation (235) and apoptosis inhibition (236). Consistent with this notion, following NFκB activation a number of anti-apoptotic factors are activated, and if NS1 65

and NS2 are knocked down by siRNA the cells undergo apoptosis much earlier. RSV- induced NFκB activation is independent of IFN stimulation because Vero cells are also sensitive to apoptosis earlier if NS1 and NS2 are not expressed. Finally, both NS1 and

NS2 transfected immortalized cells suppressed TNF-α induced apoptosis (234).

The SH protein of parainfluenza virus V (PIV5) also has a role in blocking apoptosis.

Infection by wt PIV5 does not induce apoptosis of MDBK or L929 cells, but infection with PIV5-ΔSH has a potent killing effect on these cells. If the SH protein from RSV is used to replace the PIV5 SH, this CPE phenotype is lost, suggesting that the RSV SH protein is also able to block apoptosis. Unlike PIV5-ΔSH, RSV lacking the SH protein produced enhanced CPE in some cell lines, L929 at 1 dpi and A549 at 3 dpi, but reduced

CPE in others (237). In a yeast 2-hybrid assay, the SH protein was shown to interact with

Bap31 (238). Bap31 is a substrate of caspase-8, a key regulator of apoptosis (160-162).

In addition to the SH, NS1, NS2, N, and P proteins having a role in suppressing immunity, a number of studies have demonstrated that an interaction between the F protein and TLR4 somehow plays an important role in RSV infection (239-243). The first showed that purified F protein alone was sufficient to induce IL-6 production in PBMCs and that the response was blocked by a CD14 Ab; CD14 is a TLR4 co-receptor.

Polymyxin B was able to block IL-6 secretion, in the presence of LPS or the F protein suggesting that CD14 and/or TLR4 activation is F protein-mediated. IL-6 secretion was induced in PBMCs isolated from CD14+/- mice, but not from CD14-/- mice. Similarly,

PBMC from mice without TLR4 expression, or with an inactive TLR4 were also unable to produce IL-6 in response to F protein stimulation. In PBMC of mice containing a 66

deletion of TLR4 or a non-responsive TLR4, IL-6 was not induced by the F protein. Mice with this defect had increased RSV replication and decreased viral clearance (239), as well as impaired natural killer (NK) cells and CD14+ cell recruitment than their wt counterparts (240). In addition, clinically TLR4 polymorphisms have been associated with severe disease at some positions (241, 243) while others are conditionally associated with severe disease (242).

Viral assembly and egress

At 20 hpi, the M protein localizes to the cytoplasmic face of the plasma membrane at lipid rafts with the F, N and G proteins (93, 244). Although virus lacking G assembles and releases less virus, virus lacking the M or F proteins do not produce infectious virus

(245). In cells infected with RSV lacking the M protein gene, the large inclusion bodies do not resolve and the plasma membrane has concentrated sections of short filaments, suggesting that the M protein is important in transferring the ribonucleoprotein complex to the cell surface for viral assembly, release, and filament formation (92, 246). But the

M protein is not sufficient to form mature filaments, as filaments also do not form in the absence of the F protein, or if the cytoplasmic tail of the F protein is altered so that M cannot interact with it. In fact, in the absence the F protein’s cytoplasmic tail, the F and

M proteins are retained at inclusion bodies (244).

Not all RSV particles are filamentous; they can also be spherical and asymmetric (Figure

8 (154)), and the amount of M protein is important for the shape of the resultant particle.

The more M, the less curvature to the membrane, meaning that the particle has more surface area to volume ratio and as such the particles contain fewer genome copies. In spherical particles M covers ~20% of the inner membrane, asymmetric particles have

60% of the inner membrane covered, and filamentous particles have ~80% of the membrane covered (154).

Figure 8. Virion morphology. Basic morphological characterization of the three hRSV morphologies. Representative particles of the three morphology categories found in the RSV sample: spherical (A to C), asymmetric (D to F), and filamentous (G to I). Common structural features present in the 3D reconstructions of all hRSV morphologies include the surface glycoproteins, the viral membrane, the matrix protein layer, and RNP. Images are 7.5-nm central slices from tomographic reconstructions. Scale bars, 100 nm. Reprinted from Journal of Virology, 88/13, Kiss et al., “Structural analysis of respiratory syncytial virus reveals the position of M2-1 between the matrix protein and the ribonucleoprotein complex”, 7602-7617., Copyright (2014), American Society of Microbiology.

Filament formation is not enough for viral release. For this, many enveloped viruses have been demonstrated to bud through endosomal sorting complexes required for transport

(ESCRT) dependent mechanisms. The plasma membrane is deformed as the viral

components are assembled and the ESCRT machinery induces the fission of the two membranes using Vps4 induced ATP hydrolysis (247). However, influenza (248, 249),

RSV (250), and Sendai virus (251) exit cells in a Vps4 independent manner (248-251), suggesting that they do not use the ESCRT machinery. Both influenza (252) and RSV

(253) encode for SH proteins that have viroporin-like structure and activity (252, 253).

Previously the SH protein was demonstrated to be a multimer by sucrose gradient separation (96). More recently molecular modeling (254) and ER images (Figure 9 (255)) of the SH protein have revealed that there are 5 to 6 subunits that surround a hollow core

(254, 255). To demonstrate that the SH protein could have viroporin functionality, the SH gene was expressed transiently in bacteria that were treated with hygromycin B. This antibiotic blocks protein synthesis, but it is unable to enter Escherichia coli. However when the SH protein is produced by the bacteria, protein synthesis is decreased, as detected by 35S-methionine incorporation, suggesting that hygromycin was able to permeate the cell. In addition, 3H-uridine was able to leak out of the cell. This data suggested that SH was affecting the permeability of the bacterial membrane (256). This result is consistent with other SH viral proteins, a class of proteins known as viroporins

(253).

Figure 9. SH protein forms a viroporin. (A) Direct EM observation of FLAG-SH using negative staining, with individual representatives of putative pentamer (5) and hexamer (6) classes shown at higher magnification. (B) Putative pentamer and hexamer class-averages. (C) Molecular model of full-length SH protein as a hexamer. W15/H22 residues within the trans-membrane domain are shown within the putative channel lumen. (D) Model of the SH hexamer inserted into a PAPC bilayer, as used in the liposome assay. Reprinted from FEBS Letters, 584/90, Carter et al., “Direct visualization of the small hydrophobic protein of human respiratory syncytial virus reveals the structural basis for membrane permeability”, 2786-2790., Copyright (2010), Elsevier. 71

Viroporins have been shown to allow passive membrane crossing of molecules up to 1.5 kDa. This means that ions can freely leave and enter cells, disrupting the membrane potential. The change in membrane potential could provide the energy for budding to occur. Calcium ions are also able to move freely through the pore, resulting in leakage from the extracellular environment and intracellular stores. Calcium plays a pivotal role in enzyme activity, gene expression, and post-translational modifications, so viroporins are likely to have a pathogenic effect on cells. Finally, as intracellular calcium increases apoptosis is triggered, in fact, expression of some viroporins in the absence of other viral proteins induces apoptosis (253).

Respiratory Syncytial Virus: Modeled

Early on, Coates and Chanock tried to infect a number of animal species and found that ferrets, mink, chinchillas, marmosets, African rats, guinea pigs, and mice are susceptible to RSV infection with ferrets and mink being the most susceptible (257). Animal models are important for research as they can give us a glimpse into what is happening during the course of infection. In addition, parameters can be changed, and proper controls can be used. However, every model has limitations. All data gathered from models needs to be reviewed thoroughly because viral strain, propagating cell type, purification method, and breed of animal can all have an effect on the experimental outcome. Unfortunately, not all of these parameters are provided in every paper. I will point out some findings and

characterize a few models of RSV infection, but will refer the reader to other reviews

(215, 258, 259) for a more extensive look at the cotton rat model and (260) for a review of pneumonia virus of mice (PVM), a related pneumovirus that can be used in it native host animal to study replication and pathogenesis.

Mice

Important factors to consider

BALB/c mice are the inbred mouse strain that is most susceptible to RSV infection.

Immunohistochemistry of the lungs of these animals demonstrates that the cells infected in mice are the alveolar cells, and not the ciliated epithelium. Histologically, airway epithelial cell shedding occurs, along with infiltration of small lymphocytes in perivascular tissue and mononuclear infiltrates in the peribronchiolar and perivascular tissue (261).

Despite the advances in RSV immunity in the mouse model, it has a few key limitations.

The first being the extremely poor infection and replication of the virus in the upper and lower respiratory tract, 1 X 107 pfu are required to generate an infection in both airways, as a result, almost every vaccine or drug tested in these animals results in protection.

Conversely, PVM, another paramyxovirus can cause severe pathology in BALB/c mice with as few as 200 infectious particles (262). There are also some important differences in the immune system and lung structure between mice and humans. The innate and adaptive immune differences have been recently reviewed (263). Unlike RSV in humans,

where the target cells are the ciliated epithelium (264), pneumocytes are the primary target in mice (265).

The G protein related RSV animal data is difficult to interpret. As discussed previously the G protein binds to a different cell receptor in immortalized cell culture and HAE cultures (137-139). In addition, the virus in mouse infects a different cell type (261). It is unknown how the virus enters these cells. This is further complicated by the fact that the

G protein is cleaved in African green kidney monkey cells (16), which a number of groups use to grow their virus. This cleavage renders the virus less infectious for HAE cultures, but it is unclear what effect cleavage might have on the infection of mice.

The mAb, 131-2g, binds to the G protein, but this Ab does not neutralize viral infection of immortalized cells (266). This antibody blocks the interaction between CX3CR1 and the G protein (267, 268), and is able to reduce inflammation in RSV infected mice. This anti-inflammatory effect was thought to be due to 131-2g Ab blocking the interaction between the G protein and CX3CR1 on immune cells. However, our lab and other have shown that CX3CR1 is also present on the cilia of HAE cells (137-139). With this new knowledge, it is likely that the Ab is not only interfering with the CX3CR1 on immune cells (267, 268) but also on the ciliated epithelium (137-139). This could mean that the anti-inflammatory effect is due to decreased immune activation and decreased infection.

In these experiments, PCR was used to test viral load, which does not indicate that infectious virus is present (267).

As a result of the issues enumerated above, for a number of years there has been a debate in the field about the G protein and its effects on the immune system. Openshaw et al. set 74

out to test and expand on a previous study, where mice sensitized to the G protein had

CD4+ T-cell dependent eosinophilia upon challenge (269). They wanted to sensitize DCs to the G protein, and then transfer these DCs into the mice and determine if this could prevent the challenge induced eosinophilia. In doing so, the group found that DCs actually aggravated the eosinophilia and induced greater weight loss upon challenge. In fact, CD4 T-cells were secreting more IL5 in animals in which DCs had been transferred, and this was independent of the stimulus with which the DCs were activated. They tested whether growing the DCs and virus in the presence of mouse serum blocked this eosinophilic response, and it did (270). This may be a cautionary tale that all animal studies looking at immune modulation need to have stringent controls for all components added in a given treatment, and the serum used for growing the virus should be from the same type of animal. Unfortunately, this information is not always easily accessible from the literature.

Lessons on immunity from RSV-infected knockout and wt mice

A recombinant RSV that expresses only the membrane-bound G protein (mG RSV) was used to infect BALB/c or JHD mice, which are deficient in B-cells. In BALB/c mice, viral load was increased when treated with clodronate (271), which depletes phagocytic cells. JHD mice produced a lower viral titer than BALB/c; however, phagocyte depletion did raise viral titer, suggesting that phagocytic cells are important for clearing RSV infection. There was no difference in the viral load if BALB/c mice were infected with either the wt RSV or mG RSV, however, if animals were given F-specific antiserum IP 75

one day prior to inoculation, the mG RSV was cleared much more effectively than wt

RSV. If these animals were depleted of phagocytes the anti-F Ab was not able to reduce the titer in animals infected with the mG RSV virus, suggesting that the anti-F Ab is opsonizing the virus and phagocytes are phagocytosing the virus. This further suggests that somehow the secreted form of G is having an inhibitory effect on this opsonization and subsequent phagocytosis of the virus. Neutrophil depletion had no effect on viral clearance, and neutrophil depletion did not inhibit anti-F mediated decrease in viral replication. Complement inactivation also had no effect on viral load, but complement inactivation in combination with the anti-F Ab increased viral load over anti-F Ab alone

(271).

As discussed previously, SP-A is a molecule in the lung that is an opsonin for pathogens

(127, 128, 214). SP-A-/- mice were used to test the role of SP-A in RSV infection. In low dose inoculation, there were more infiltrating immune cells in the BAL on 1 dpi, but no significant difference on subsequent days. However, animals given a high dose (107 pfu) of RSV had greater infiltration on all days tested (125, 272-274). There were significantly more PMNs at days 3, 5 and 7 in the SP-A-/- mice though percentages of lymphocytes were similar between the wt and SP-A-/- mice, SP-A-/- mice had higher titers at day 3, 5, and 7 post-infection. However, if SP-A-/- mice were treated with SP-A, their viral loads, and immune infiltration were decreased (275) demonstrating that SP-A is critical for clearance and immunomodulation.

Another immunomodulatory molecule is TLR3, a PRR that is localized to the endosomal membrane and is activated by dsRNA (Table 5). TLR3 is important for regulating the 76

maturation of DCs and promotes an antigen-specific T-cell response (reviewed in (166,

173, 216)). TLR3-/- mice in a black 6 background were used to determine the role of

TLR3 in immune response and resolution of RSV infection (276). TLR3 deficient mice did not have a higher viral load than wt mice but instead had an exaggerated inflammatory response. The mice had greater mucous secretion and higher levels of both

IL-13 and IL-5 (276), cytokines that drive a Th2 based response. IL-13 drives immunoglobulin (Ig) E isotype switching, mucous production, and smooth muscle contractions, and recruits innate immune cells (reviewed in (175, 184, 186, 187, 189, 192,

196)), while IL-5 drives activation of Th2 cells, and the survival, development and recruitment of eosinophils (187, 190-192). From this experiment, Rudd et al. learned that while TLR3 stimulation may not be beneficial for viral clearance, it is likely important for the shaping of the subsequent immune response to RSV.

The TLR proteins are upstream of a number of transcription factors. One such transcription factor, STAT1 is known to have a role in activating an antiviral response

(Table 5 (174, 177, 213)), but it was not clear what role this molecule might have in RSV clearance and immune shaping. STAT1-/- or wt BALB/c mice were infected and the animals observed. Like TLR3-/- mice, there was no consistent difference in lung titers or

RSV clearance between wt and STAT1-/- BALB/c mice, however, there was increased disease. Wild-type mice lost weight initially but were back at their starting weight by 3 dpi, however, STAT-/- mice, lost nearly 3 g of their weight by 7 dpi and did not reach their starting weight until 12 dpi. Consistent with this profound weight loss, at day 8 post- infection when immune cells were few in number in wt mice, the lung tissue of STAT1-/-

mice displayed high levels of lung infiltrates including neutrophils (277), macrophages, eosinophils and lymphocytes, leaving very little open air space (277, 278), and leading to much higher lung resistance in STAT1-/- mice after methacholine challenge (278). In addition to the increased infiltrate, IL-13, IFN-γ, eotaxin (277, 278), IL-17, Muc5ac, and

Gob-5 (278), all proinflammatory cytokines and molecules (Table 5), were also greatly increased.

DCs are APCs that migrate from the site of infection/inflammation to the lymph node where they can interact with immature T and B-cells. DCs in the mouse come in two types, conventional and plasmacytoid. Conventional DCs have been implicated in allergic and Th2 type responses while plasmacytoid DC are implicated in suppressing the Th2 response (279). BALB/cByJ mice were treated with Flt3L (280), a growth factor that can stimulate hematopoietic cell proliferation (281), and infected with RSV. Treated animals had decreased IL-4, IL-13, IL-5, IFN-γ, and mucous production, as well as decreased airway hyper-responsiveness. In addition, the treated animals had an increase in CD8+ T- cell responses. These differences in cytokine and cellular responses resulted in significantly reduced lung pathology and could be reversed by plasmacytoid DC depletion. These results suggest that the balance of DC populations are important for positive outcomes in RSV illness (280).

PD-1 is an important regulatory molecule on a number of cell types including NK, T- and

B-cells. In both mice and human lungs PD-1 is upregulated on T-cells of the respiratory tract. PD1 and its ligand PDL1 are vital to restrict inflammation and injury of cells and tissues. When PD-1 and PDL-1 interactions were blocked, animals lost on average 27% 78

of their body weight following RSV infection, while animals treated with an isotype control lost only 18% of their body weight. This weight loss was likely due in part to the massive inflammation seen in animals where the PD-1:PDL-1 interaction was blocked, including increases in IL-1β, IL-6, IL-15, IL-17, RANTES, MIG, IL-4, IL-5, IL-9, MCP-

1, MIP1α and β, IFN γ, TNFα, IP10, and eotaxin (211).

The cotton rat

Cotton rats are susceptible to RSV infection of the upper and lower respiratory tract. Age at time of infection has no effect on titer or duration of RSV infection of the lung or trachea, however, replication was higher and shedding duration increased in the upper respiratory tract of younger animals. Animals that were inoculated as adults or at 28 days of life had a robust neutralizing Ab response by 9 dpi while the animals inoculated at day

14 hit the same peak titer around day 12. The adult animals had 16 times more neutralizing Ab on day 9 than the animals that were 3 days of age at the time of inoculation, and the young cotton rats were not able to produce that level of Ab until day

20 post-infection. Immunohistochemistry revealed that viral infection was limited to the epithelium, and the histology of the alveolar cells appeared normal, but the bronchus and bronchiolar epithelium showed signs of shedding and patches of the epithelium that were highly proliferative. The upper respiratory tract showed destruction and shedding of columnar epithelium. In addition, eosinophils were commonly observed, with neutrophils being less common (282).

Early studies using cotton rats demonstrated that infection protected the cotton rat nose from a second infection for up to 8 months and protected the lung from infection for the length of the study (>18 months). In order to determine what was protecting the animals from reinfection, animals were joined in one of three combinations so that the animals shared a blood supply: two naïve paired animals, two immune paired animals, and a naïve paired to an immune cotton rat. As expected when challenged with RSV the naïve pair had full RSV infection and immune paired cotton rats were protected from infection. The immune to naïve paired animals shared neutralizing antibodies so that the immune animal had half of the antibodies it had prior to the pairing. While the immune animal was protected from infection in the upper and lower respiratory tract, the naïve animal was susceptible to infection in the upper respiratory tract, but not the lung. If young naïve animals were treated with immune serum from cotton rats that had been infected 21 days previously, the animals’ lungs were protected from infection but again their upper respiratory tract was not, suggesting that neutralizing antibodies are not sufficient to protect against RSV challenge in the nose (283). Consistent with this, while the monoclonal antibodies that have been tested in clinical trials do not seem to decrease infection rate, they do protect against more severe LRTI (64-67, 69, 284).

The cotton rat model also has a few disadvantages, most importantly the cost and the relative scarcity of reagents. However, with increased interest, more reagents for studying their immune responses are becoming available (259, 285). In addition, with new technologies knockout animals should be more easily produced. Cotton rats are also more difficult to handle than mice as they are larger, easily agitated and fragile; however, with

training and the proper equipment they are manageable (285). In addition, studies that require a blood draw or intravenous drug administration are best performed in the retro- orbital plexus, which requires training to avoid damage (259). Despite all of the interest, little is known about cotton rat immunology and how it relates to human immunology.

Ferrets

In ferrets the cells that are the primary target of RSV infection are the fibroblasts of the lamina propria and serosa, no viral antigen is detected in the ciliated epithelium (70).

Despite this, the neonatal ferret model does provide some useful information about infection in the lung and the nose. In an experiment in which pups were infected on the day of birth, RSV could be detected in nasal and lung tissue the following day. While

RSV was only detectable in the lungs for two days, it was detectable in the nasal tissue for 8-9 days. If pups were infected at 3, 7, or 14 days of age the nasal infection was the same titer and duration of detection. However, the peak titer in the lungs decreased in animals if inoculated later in life. If infected at 28 days of life, no virus could be detected in the lung tissue following infection, though, in the upper respiratory tract, the titer and shedding were the same. This suggests that the difference in age-related immune differences may be due to maturation in the lung rather than in the upper respiratory tract

(286).

Guinea pig

Pregnant guinea pigs are being developed as a model for transplacental Ab transmission.

Buraphacheep and Sullender infected guinea pigs with RSV at 25-35 days gestation or at

35-45 days of gestation. Within 72 h of being born infant pups were challenged with

RSV, and 4 days post-challenge the pups were sacrificed. Regardless of the timing of the maternal RSV infection, pups produced lower lung titers if the mother had been infected with RSV during pregnancy. They also had serum antibodies that neutralized RSV (287).

Bovine RSV

In one experiment, bovine RSV was rescued from cDNA (288) and characterized in calves that were 8-16 weeks old. These calves had similar symptomatology to infants including increased temperature, cough, harsh lung sounds and dyspnea. On day 7, the pulmonary resistance was significantly higher in infected calves, and 3 of the calves were euthanized on day 7 due to extreme respiratory distress. The calves were evaluated, 1 calf had a fluid-filled sac in the left lung and another had air in the space between the lung and the chest wall, and all had bronchiolitis and alveolitis (289). Understanding of BRSV infection and immunity may translate to an understanding of HRSV; however, cows are not susceptible to HRSV, so vaccines and drugs cannot be directly tested in this model.

Neonatal immunity

Prior to birth, fetal immune systems are occupied with three tasks: protection against pathogens from mother to child; avoidance of pro-inflammatory Th1 immune responses that might induce alloimmunity, preterm labor, and fetal abortion; and transitioning from mother’s womb to an environment rich in pathogens and new antigenic stimuli (193). The placenta produces TGFβ, progesterone, and prostaglandin E2, all of which lower Th1 responses (290).

Neonates’ skin is thin but is covered in waxy coat called the vernix caseosa. This coat protects the infant and contains more antimicrobial proteins and peptides than adult skin

(193). In addition in the gastrointestinal tract of infants, TLR4, and MD2, both of which are stimulated by LPS a bacterial PAMP, are at increased levels, and when stimulated show exaggerated NFκB responses. However, in the neonatal intestine LPS activation leads to decreased LPS responsiveness and down-regulation of key molecules in the

TLR4 signaling cascade. This decreased LPS responsiveness likely blunts the inflammatory response that would otherwise ensue given the plethora of microbes the newborn gastrointestinal tract would see for the first time. While being colonized with

“good” bacteria, the infant’s intestines develop Paneth cells which secrete many antimicrobial peptides and proteins to protect from pathogenic bacteria (193).

Prior to birth, fetal lungs are devoid of TLR2 and TLR4 but they rapidly increase after birth. However, TLR2 and TLR4 responsiveness do not reach adult levels until 4 weeks after birth. Exposure to TLR agonists can induce the expression of antimicrobial peptides

and proteins through IL-1β activation. Prior to birth, TLR stimulation can lead to preterm birth and abnormal lung development and to bronchopulmonary dysplasia (193).

IL-2 production is low in neonates compared to adults, as a result, there is limited T-cell proliferation. This affects both Th1 and Th2 responses (291). In neonates, there is a high concentration of adenosine that acts at adenosine receptors to induce cAMP which in turn inhibits Th1 cytokines (290). However, CD8+ and Th1 responses can be induced by congenital cytomegalovirus infection, live vaccines, adult DCs, DNA vaccines, and CpG motifs (291).

T-cell dependent Ab responses in neonates develop less quickly, reach a lower peak level and do not last as long as adult antibodies. IgG2 levels are lower in neonates and the affinity and repertoire are lower in neonates than in adults. B-cells are immature, and this immaturity lessens the ability of costimulatory molecule and MHCII upregulation, as a result, the B-cells are not able to interact with T cells as effectively. In addition, for high affinity and repertoire Ab responses, lymphoid follicles, follicular-DC networks and germinal centers are required, none of which are present at birth. In fact, germinal centers develop at 4 months after birth (291). For long-lived Ab responses, memory B-cells need to be generated, and neonates seem to have the capacity for developing memory responses, however, at least in mice these memory B-cells do not migrate to the bone marrow.

T-cell independent antibodies are produced in B cells that are in the marginal zone of the spleen. This region develops later in life as well, around 1-2 years. In addition, these B-

cell responses have some reliance on complement (291), which is present in smaller quantities in neonates (193, 291).

Neonatal neutrophils show defects in the level of antimicrobial peptides and proteins, chemotaxis, rolling adhesion, transmigration, and lamellipodia formation. In addition, neonatal antigen-presenting cells tend to secrete large quantities of IL-6, which is inhibitory to neutrophil migration (193). Neonatal APCs show decreased response to

TLR activation with respect to IFN and TNF production, but secrete IL-6, IL-10 and IL-

23 at higher levels than adults, so, it is not a lack of immunity, but different immunity that leaves infants susceptible to bacterial and viral pathogens. IL-23 is of importance because it can induce production of antimicrobial proteins and peptides. Decreased TNF is important because over-production can lead to CNS damage that can lead to cerebral palsy (193).

Formalin-inactivated respiratory syncytial virus

The FI-RSV vaccine enhancement of disease has been extensively studied. Human infants (27), African green monkeys (292), infant macaques (293) and BALB/c mice

(294) had very low levels of RSV-specific neutralizing antibodies induced following FI-

RSV vaccination compared to natural infection, suggesting that formalin fixation may have altered neutralizing epitopes on the virus (27, 292-294). The human infants also had a much lower response to the G protein post vaccination (27), this will become important later as the G protein is cleaved in the cell line used to propagate FI-RSV (268). In

addition, in African green monkeys the FI-RSV did reduce the peak titer and delay the onset of viral shedding (292). African green monkeys that were sham vaccinated and challenged with RSV had patchy inflammation in the lungs, mostly in the bronchioles, interstitium, and alveoli (Figure 10). The animals that were FI-RSV vaccinated and subsequently challenged were euthanized and histology was performed to examine the pathology. The animals’ lungs had so many infiltrating immune cells that they no longer resembled lungs (Figure 11). The lung inflammatory score was 2 for sham vaccinated animals, 4 if the animal was vaccinated with FI-herpes simplex virus prior to challenge, and 9 for low and high dose FI-RSV vaccinated animals (292). FI-RSV with or without G has a similar phenotype to wt virus when challenged with RSV suggesting that the G protein is not responsible for the increased inflammatory response (295).

Figure 10. Control vaccinated monkey lung 7-8 days post-challenge. Representative lesions in lung of adjuvant control-vaccinated monkeys 7 or 8 days after RSV challenge. Note moderate patchy inflammatory response in terminal bronchioles, interstitium, and alveoli. Hematoxylin phloxine-eosin stain; bar = 65 µm. Kakuk et al., “A Human Respiratory Syncytial Virus (RSV) Primate Model of Enhanced Pulmonary Pathology Induced with a Formalin-Inactivated RSV Vaccine but Not a Recombinant FG Subunit Vaccine”, Journal of Infectious Diseases, 1993, Vol. 167, No. 3, 553-561, by permission of Oxford University Press.

Figure 11. FI-RSV vaccinated monkey lung 7-8 days post-challenge. Representative lesions in lung of formalin-inactivated RSV-vaccinated monkey. Note severe peribronchiolar and parenchymal inflammatory involvement obliterating pulmonary structures. Hematoxylin phloxine-eosin stain; bar = 65 µm. Kakuk et al., “A Human Respiratory Syncytial Virus (RSV) Primate Model of Enhanced Pulmonary Pathology Induced with a Formalin-Inactivated RSV Vaccine but Not a Recombinant FG Subunit Vaccine”, Journal of Infectious Diseases, 1993, Vol. 167, No. 3, 553-561, by permission of Oxford University Press.

Live-attenuated vaccine

A study in 1991 demonstrated the difficulty in identifying an RSV vaccine that produces sterilizing immunity. In this study 15 adults were challenged with RSV 2, 4, 8, 14, 20 and

26 months after natural infection. By 2 months, half of the subjects could be reinfected, and by 8 months 2/3 of the subjects could be reinfected. Seventy-three percent of participants had two or more infections and 47% had three or more infections. It appears

that the immune response does not protect against reinfection of the upper respiratory tract, however, higher neutralizing Ab did correlate with reinfection resistance (20).

Sterilizing immunity is the goal with any vaccine, however, it is not required for a successful vaccination program, and as a result of the failed FI-RSV trial, much effort has been put into the development of a live attenuated vaccine. Live attenuated vaccines are known to induce better immunity than FI-RSV unless an adjuvant is added, but for any live attenuated vaccine the “cold chain” is important. The cold chain is the storage and handling of vaccines prior to use. This is especially important given that RSV is unstable when exposed to freeze-thaws (33, 296) and in media with pH less than or greater than

7.5. More importantly, however, the virus infectivity decreases by 2 logs after 2 days at

37°C, by day 4 when stored at 25°C, and by day 8 when stored at 4°C (296). Storing a live vaccine at -80C during transport and before vaccination will add to the cost per dose.

A live vaccine is needed throughout the world, particularly in developing countries, so cost is important. There have been a number of reports identifying parameters that increase viral stability. One method is to grow the virus under conditions where a baculovirus glycoprotein, GP64, could be incorporated into the virion. This incorporation increased viral stability at 22°C by 104 fold when stored for six weeks (297), however, the post-translational modifications would not be present on the proteins, and it is unclear that this virus would be infectious. Another study looked at stabilizers that the virus could be suspended in. Temperature, secondary and tertiary structures, aggregation and dissociation of proteins, in the presence of these stabilizers, were observed using circular dichroism, light scattering, and spectroscopy. Sugars, amino acids, polyols (non-sugar

carbohydrates), and polyanions all lowered aggregation, but while polyols stabilized secondary and tertiary structures, polyanions destabilized the structure. The best Tm

(melting temperature) stabilizing compound was the sugar trehalose (298). This information will be important for wide-spread dissemination of a vaccine, however, we do not currently have a candidate that is safe and immunogenic.

Shortly after the FI-RSV vaccine trial, Kim et al. tested a temperature sensitive RSV mutant of the A2 strain as a vaccine in infants and children (6 months – 13 years). This virus was under-attenuated for RSV naïve individuals as it produced mild symptoms in those under the age of 8 and in some very young infants it was associated with fever, rhinitis, pharyngitis, bronchitis and otitis media. Vaccinees did seroconvert, suggesting that a live attenuated vaccine could be functional and protective (299). A 1976 trial further demonstrated that the temperature sensitive and cold-passaged viruses are not genetically stable when given to seronegative children (300). A 1982 trial showed promise in that the vaccine was immunogenic and did not elicit symptoms, but it also did not protect from disease upon natural infection (301). Next, a B strain of the virus, cp-

52/2B5, in which most of the SH and G genes were deleted was grown in Vero and tested as a vaccine. Vaccinated children had an increased risk of upper respiratory symptoms after vaccination compared to controls, and there was no subsequent rise in neutralizing

Ab titer (19).

A cpts530/1009 and cpts248/955 were developed using traditional mutagenesis strategies and grown in Vero. When used to vaccinate adults, they were overly attenuated, and there was no increase in serum Ab titer. Cpts530/1009 was more attenuated in seropositive 90

children and in infants than cpts248/955. Infants vaccinated with cpts248/955 shed more virus than was given as vaccine, 100% of the infants had an RSV-like illness and 67% had a fever or upper respiratory tract infection (URTI). The children were observed following vaccination on a number of visits. During these subsequent visits, both vaccine viruses spread to control sham vaccinated infants. A number of these shed viruses were tested to determine if they retained their temperature sensitive phenotype, and they did, suggesting that though the viruses were not yet attenuated enough for infants, they had not reverted to wt virus (21).

In 1996, Conzelmann published a paper demonstrating a method to genetically manipulate the genome of non-segmented negative-sense RNA viruses (302). As a result, he was able to selectively modify the cpts530/1009 virus to generate a less attenuated virus cpts530/1030. The cpts530/1009, cpts530/1030, or cpts248/955 were evaluated in seropositive infants. There was a greater incidence of shedding, of fever, and of URTI in the cpts530/1030 group than in the other two, suggesting that this virus is not attenuated enough to serve as a vaccine for seronegative infants (26).

The cpts248/955 virus was modified to cpts-248/404, and this virus was tested in both seropositive and negative children. The cpts-248/404 virus was immunogenic in infants greater than 6 months of age; however, very young infants (1-2 months of age) only secreted IgA Ab, the response was primarily toward the G protein and the antibodies were non-neutralizing. Despite this profile, the vaccine did protect against shedding when the infants were challenged with a second dose of the vaccine. Importantly the vaccine did not cause fever or LRTI in young infants, but vaccination with cpts-248/404 did 91

correlate with more upper respiratory symptoms than placebo in all seronegative infants up to 24 months old. The conclusion from this study is that this vaccine showed promise but needed to be further attenuated for very young infants (22).

Karron et al.further mutagenized this vaccine candidate by deleting the SH gene and/or by adding another mutation. Both rA2cp248/404ΔSH and the rA2cp248/404/1030ΔSH viruses were attenuated in adults and seropositive children. rA2cp248/404/1030ΔSH was more attenuated in seronegative infants than its counterpart, but less than half of the infants with the higher dose of the virus had a subsequent rise in Ab titer (23). This vaccine was later demonstrated to be unstable and the amino acid that reverts is one that confers temperature sensitivity. Changing the amino acid, to further stabilize the change, resulted in a secondary mutation that also reduced the temperature-sensitive phenotype

(303), this was eventually further stabilized with silent mutations and trialed in 2010

(discussed below (304)).

A number of the viruses tested were next modified to delete the NS2 gene: rA2cpΔNS2, rA2cp248/404ΔNS2 and rA2cp530/1009ΔNS2. All viruses were tested in adults and seropositive children and only 13% of adults had an increase in Ab titer at the 107pfu dose of rA2cpΔNS2. None of the other candidates induced Ab in these groups. In addition, less than half of the seronegative infants had an increased neutralizing Ab response when immunized with the rA2cp248/404ΔNS2 candidate. No infants responded to rA2cp530/1009ΔNS2 (24).

The most recent live attenuated vaccine candidate tested in clinical trials was MEDI-559

(25, 304). This was administered to seronegative infants on three occasions with the 92

vaccinations occurring two months apart, and symptoms, adverse events, and safety were observed for 365 days. During the study, nasal washes and serum antibodies were tested at multiple time points. There was a slight increase in vaccine-associated URTI and a higher incidence of LRTI in the first 28 days following dosing in the vaccine arm.

Vaccinees had a greater neutralizing Ab response than placebo vaccinated infants. Taken together MEDI-559 is immunogenic, but further safety trials are required to ensure that the vaccine does not contribute to higher LRTI risk (25).

My particular project has very important vaccine implications. This will be discussed more thoroughly in Chapter 3. As I have mentioned in the preceding text, the G protein is cleaved in Vero cells. This cleavage renders the virus less infectious for HAE cultures. In

Chapter 2, I will identify the protease responsible for cleaving the G protein and the role of membrane recycling which appears to be important for cleavage.

Chapter 3: Cathepsin L, Endocytosis, and Recycling

Cathepsin L

The initial discovery and much of the early work with cathepsin L was performed with a mouse protease, major excreted protein (MEP), which is now called cathepsin L. The two proteins are almost identical. However, MEP is actually more similar to human cathepsin

V in sequence. However, cathepsin V expression is very limited and cathepsin L and

MEP have been shown to be expressed more broadly and to work in very similar ways

(305).

Proteolytic cleavage is a posttranslational modification that is critical for cell survival.

The human genome encodes over 550 proteases (305). Proteolytic cleavage is a very powerful function and must be tightly controlled because, unlike other posttranslational modifications such as palmitoylation or phosphorylation, cleavage is permanent. Cells use proteolytic cleavage for protein turnover, but a more important role is in protein maturation (306). Many proteins that would have a deleterious effect on normal cellular functions are produced as zymogens, or inactive precursor proteins, which are activated by cleavage but only in the proper environment and one of the cleavage events liberates the protease from a pro-piece, which can act as a potent protease inhibitor (306). Another cellular mechanism for protecting cells against aberrant protease activity is endogenous protease inhibitors, of which, our genome encodes at least 200 (305). 94

Proteases use a nucleophilic attack on the carbonyl side of an amide bond to effectively destroy the covalent bond. Serine, cysteine, and aspartic acid can all be at the active site of proteases. In addition, metalloproteases require a metal ion at the active site. Cysteine proteases, such as cathepsin L, have a thiol group in close proximity to a basic amino acid that can remove the hydrogen from the sulfur. This sulfur now has a negative charge and can attack the covalent bond as described. These proteins all have a similar structure, two globular heads and in between a small cleft or pocket, in order to be cleaved, a protein must fit into the cleft of the protease (306).

Acid proteases are bound for the lysosome; however, some fraction of lysosomal proteases are secreted from immortalized cells. When this occurs there are signals on the protein that signal the pinocytosis of the protein. Much of the work described was performed using β-glucuronidase, but the concepts are translatable to other acid hydrolases. Protein pinocytosis is blocked by the addition of free sugar phosphates to the culture (307, 308). Acidification of the media can also decrease pinocytosis, and chloroquine-treated cells have much more enzyme in the extracellular space than non- treated cells as a result of increased enzyme secretion and decreased receptor expression on the cell surface (307). Endoglycosidase H (Endo H) is an enzyme that digests high mannose sugars, and cells treated with Endo H had decreased uptake of the protein via pinocytosis, but proteolytic activity was unaffected, suggesting that the sugar present on the lysosomal proteases is mannose-6 phosphate, consistent with increasing mannose-6 phosphate receptor levels on the surface of the cell, increasing protease pinocytosis (309).

Cathepsin L, an acidic protease, was identified using fractionated lysate enriched for lysosomes. Enzyme activity was greatest at pH 5 and was inhibited by leupeptin, suggesting that the protein was a cysteine protease. This enzyme lacked amino- or carboxypeptidase activity and it did not hydrolyze esters (310). Other enzymes identified in the lysosomes were called cathepsin proteases from the Greek word kathépsein, meaning to digest or boil down (305), this particular enzyme would later be called cathepsin L.

Two years later, a protein of low molecular weight was found to be secreted from murine fibroblasts, but secretion was dependent on transformation. This protein was described as

35 kDa and was named the major excreted protein (MEP). The protein was then shown to be secreted by a number of transformed mouse cell lines and characterized for how much protein was secreted. Cells stained with a mAb to MEP demonstrated a large amount of cell-associated MEP (311), the mouse version of cathepsin L (312-314).

Cathepsin L is a ~36 kDa protein with a 17 amino acid pre-region (312) that functions as a signal peptide to direct the protein into the ER, this peptide is cleaved during translation to liberate cathepsin L from the membrane. The protein is N-glycosylated in the ER, where the glycosylations are high in mannose, but in the Golgi these sugars are processed into mannose-6-phosphate (305), a molecule that will target the cathepsin L proform to the lysosome. The mannose-6-phosphate receptor binds to the mannose-6-phosphate and this complex is taken to the endosome. However, if there is limited mannose-6-phosphate receptor present the protease is secreted from the cell. For an unknown reason, cathepsin

L does not bind with high affinity to the mannose-6-phosphate receptor as other 96

lysosomal proteases do (315). Cathepsin L is upregulated by PDGF, and as a result,

PDGF treatment results in an increase in the secretion of cathepsin L from mouse cells.

This suggested that the low-affinity nature of the mannose-6-phosphate receptor may serve to selectively secrete cathepsin L when the receptor is the limiting factor in protease transport (316).

Cathepsin L is transported to the lysosome where the proform is further processed by cleavage to its mature form. Protein processing was demonstrated using an in vitro assay and proform cathepsin L. If this proform was purified and suspended in buffer at pH 5.5 the protease was larger and 10 times less active than the purified mature cathepsin L. If the proform was incubated on ice for 30 or 60 minutes some of the cathepsin L had undergone cleavage and is a mixture of 39 and 31 kDa protein. Despite the proteolytic processing of cathepsin L, the enzymatic activity is actually less active than the proform that was not incubated on ice. If the cathepsin L was instead incubated at 37°C with dextran sulfate and dithiothreitol, procathepsin L was almost completely cleaved to the

31 kDa form, which was ~25% less active than the purified mature cathepsin L (317).

The different protein mixtures were sequenced to determine what amino acids were included in the fully active vs. the partially active forms. The mature cathepsin L terminates with an isoleucine [Ile]PKS. The protein incubated on ice and cleaved had the following sequence: EPLMLK[Ile]PKS and the protein incubated at 37°C terminated with LK[Ile]PKS. This suggests that not only does the protease need to be activated to produce the proform, but in order to obtain full functionality the pro-protein must be cleaved two more times, one cleavage product is 6 amino acids larger than the fully

active mature protein, and the second cleavage product is 2 amino acids larger than the fully active mature protein (317). The mature protein is actually made up of a heavy and light chain that are held together by disulfide (Figure 12 (305)).

Cathepsin L has a number of start codons (305, 318), one of these start codons results in a cathepsin L isoform that is lacking the signal sequence. This cathepsin L isoform has a nuclear localization signal and it plays a critical role in processing a transcription factor that transitions the cell from the G1 phase of cell division to the S phase. In cathepsin L-/- cells, there is very little cellular proliferation (318).

Figure 12. Cathepsin L processing. Alternative translation from downstream AUG sites produces cytoplasmic and nuclear cathepsin L. (A) Alternative translation from downstream AUG sites produces cathepsin L that is devoid of the leader sequence and that can be present outside the lysosome, including in the cytosol and the nucleus. (B) Alternative translation leads to peptides that lack the leader sequence and thus are targeted to the cytosol or nucleus. The initial folding of the protein requires an intact proregion, and the mature protein is stabilized by three disulfide bonds. Thus, this process may require the presence of yet to be identified chaperones to generate a functional enzyme. Reprinted from The Journal of Clinical Investigation, 120/10, Reiser et al., “Specialized roles for cysteine cathepsins in health and disease”, 3421-3431., Copyright (2010), with permission from American Society for Clinical Investigation.

Cathepsin B and L have very similar functions, and there are very few if any substrates that cathepsin L is able to cleave that cathepsin B cannot although the converse does not appear to be true. As a result, a cathepsin L deficiency is not lethal in knockout animals,

but significant symptoms are observed (319) because cathepsin L has many critical functions, including intracellular protein turnover, bone resorption, cartilage proteoglycan breakdown, antigen processing, follicular wall degradation during oogenesis, MHCII antigen presentation in some cell types, and prohormone processing (319). In addition, cathepsin L can cleave elastase and collagenase which enhances cancer metastasis but in development this function is critical for maturation and integrity of the central nervous system (319). Animals deficient in cathepsin L have brain atrophy (320), hair loss, epidermal hyperplasia (321), lower production of thyroid hormone (322), dilated cardiomyopathy (323), and a number of other heart-related issues (305). This is likely due to the fact that cathepsin L attenuates a cascade that initiates apoptosis in multiple cell types (324, 325).

Lysosomal degradation of cellular components is critical for homeostasis, without it cellular components that need to be degraded accumulate in the lysosomal compartments.

If this is not rectified, it can lead to cell death and more importantly tissue failure. This is evidenced most clearly in a number of lysosomal storage disorders. These diseases are wide-ranging and result from a number of defects (326).

Despite all of the measures to curtail unwanted proteolysis, sometimes cathepsin L can cause or is associated with disease states. Elevated cathepsin L expression has been associated with a variety of human inflammatory skin disorders such as atopic eczema, and psoriasis (166). In addition, cathepsin L is elevated in the synovial fluids of patients with rheumatoid arthritis (320, 327), a debilitating autoimmune disease that results in inflammation of the tissue around joints. In a mouse model of rheumatoid arthritis, 100

cathepsin L-/- mice have reduced disease severity, characterized by reduced swelling, inflammation and tissue destruction. Animals also had lower levels of both the cell- mediated and Ab-mediated immune responses (328).

A number of proteases have been implicated in cancer metastasis. Tumors result from unchecked cell division. As tumor size increases vascularization is poor, and as a result, the interior of the tumor is not supplied with oxygen as readily as the outside edges. This oxygen-poor environment turns acidic due to excess CO2 production, glycolysis and ion transfer pumps (329). As discussed, a number of proteases are regulated by acidic pH, and cancer cells secrete a number of growth factors, IFNγ, and IL-6, all of which induce the production of cathepsin L. Cathepsin L upregulation has been reported in many different types of cancers and expression levels correlate with grade and stage of cancer.

Coincident with cathepsin L upregulation is a downregulation of both cystatins and stefins, potent cysteine protease inhibitors (330). In addition, cathepsin L also likely has an anti-apoptotic role as cells deficient in cathepsin L are increasingly sensitive to apoptotic signals (324, 325). Cathepsin L is an important target for treatment and for inhibition of metastasis.

Membrane Recycling

The information presented below on membrane recycling and endocytic motifs is the best estimations of what is going on. There is not one path for any of these proteins because organelles, especially sorting organelles are dynamic. We think of them as discrete

101

entities when in reality they are ever changing, so we have to make an educated guess on the organelle in question based on markers, or on functionality. If a marker protein is in a position that has not previously been described, our educated guess may be incorrect.

Figure 13 is a nice example of the types of routes that proteins can take as they traffic through the cell, and it highlights some of the seminal research on these pathways (Figure

13 (331)).

102

Figure 13. Overview of Rab GTPases on the endocytic pathway. Overview of Rab GTPases on the endocytic pathway. Rab GTPases function in internalization and transport to degradation, as well as recycling to the plasma membrane and the Golgi. For details regarding individual Rab GTPase function. Reprinted from Cold Spring Harbor Perspectives in Biology, 6/11, Wandinger- Ness A, Zerial M, “Rab proteins and the compartmentalization of the endosomal system”, Copyright (2014), with permission from Cold Spring Harbor.

103

Membrane recycling discovered

An observation was made that macrophages and mouse L cells rapidly internalized their membrane, with at least 125 pinocytic vesicles each minute, suggesting that these cells interiorize the entire plasma membrane surface area in 33 and 125 minutes respectively.

Inconsistent with this, the volume and surface area of the lysosome stays relatively constant. This suggests that there must be a mechanism where some of the pinocytosed vesicles were recycling back to the cell surface (332).

A receptor internalization study was performed to study the metabolism of cholesterol. In doing so, Brown et al. discovered that the low-density lipoprotein receptor and ligand complex were taken into cells in coated pits. Despite this, the receptor was not degraded in the lysosome of the cell, and if protein synthesis was blocked by cycloheximide, the rate of internalization stayed constant for many hours. This further suggested that there was some level of recycling of the pinocytosed membrane back to the surface of the cell

(333).

Coated pits were seen in a variety of cell lines derived multiple tissues. The protein that coated the pits was clathrin, which was estimated to be approximately 180 kDa, and formed coats with a number of clathrin monomers assembling into a cage of hexagon and pentagons (334). We now know that there are also several types of clathrin-independent endocytosis (review: (335)).

Macrophages phagocytize latex spheres, transferring them to the lysosome.

Lactoperoxidase covalently coupled to latex spheres was used to add 125I to the proteins

104

in the lysosome of living macrophages. Less than half of the label was rapidly digested

(t1/2 ~1 h) and the product was recovered in the media. However, the remainder was lost slowly (t1/2 ~24-30 h). Label was detected at the cell surface within 15 to 30 min of labeling the lysosome. In addition, these labels continued to be taken up and re-presented on the cell surface and some even traversed the Golgi during their recycling (336).

Sorting motifs

In a set of experiments, the hemagglutinin of influenza virus was mutagenized to study export, folding, and antigenicity. None of the mutations made affected any of these parameters, but Lazarovits et al. made a serendipitous discovery. It was known that hemagglutinin recycles but at a very low rate. However, when a cysteine at position 543 was changed to a tyrosine, the protein began recycling rapidly. This change was specific to this position and to tyrosine. A phenylalanine substitution did not demonstrate the same phenotype (337). This was the first paper to demonstrate that cytoplasmic amino acids, and, in particular, tyrosine, were important for internalization and recycling of the plasma membrane (Table 6 (338)).

105

Table 6. Sorting motifs

Motifs in this and other tables are denoted using the PROSITE syntax (http://www.expasy.ch/prosite/). Amino acid residues are designated according to the single letter code as follows: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan, and Y, tyrosine. X stands for any amino acid and Ø stands for an amino acid residue with a bulky hydrophobic side chain. Abbreviations: PTB, phosphotyrosine-binding; Dab2, disabled-2; AP, adaptor protein; VHS domain present in Vps27p, Hrs, Stam; GGAs, Golgi-localized, -ear- containing, ARF-binding proteins; PACS-1, phosphofurin acidic cluster sorting protein 1; TIP47, tail-interacting protein of 47 kDa; SHD1, Sla1p homology domain 1; UBA, ubiquitin associated; UBC, ubiquitin conjugating; UIM, ubiquitin interaction motif. Reprinted from Annual Review of Biochemistry, 72, Bonifacino and Traub, “Signals for sorting of transmembrane proteins to endosomes and lysosomes”, 395-447., Copyright (2003), with permission from Annual Reviews.

In a subsequent study, DNA from a patient with a family history of hypercholesterolemia was sequenced. From his mother, the patient had inherited an LDL receptor gene that produced no detectable protein. From his father, he had inherited a gene with a mutation that does not allow the LDL receptor to be internalized. This had been observed previously, but in the previous patients the cytoplasmic tail was shortened. This young 106

boy’s mutation was a single base substitution in the cytoplasmic tail that caused an amino acid substitution at Y807A. To determine what effect this substitution had on the receptor, the gene was cloned and expressed in cell culture. Cells were fed medium depleted of lipoprotein or with increasing concentrations of LDL. There was very little difference in the surface binding of the LDL to the mutant receptor, however, the intracellular trafficking and degradation were attenuated in the mutant. Using different combinations of mutations, it was demonstrated that any amino acid with a phenol ring

(Y or F) at position 807 was enabled endocytosis of the receptor (339). Shortly after this discovery, Chen et al. set out to determine which of the amino acids were important in trafficking of the protein. The motif that was important was NPXY where X stands for any amino acid (340).

Another internalization motif, YXRF, was identified in the cytoplasmic tail of the transferrin receptor using mutagenesis (341). In a review of this and other papers, it was later shown that a number of sorting motifs fall into the YXXΦ motif, where X stands for any amino acid and Φ stands for V, Y, F, I, or L, in other words, any hydrophobic amino acid. This is an over-simplification because amino acids outside of this region also clearly play a role in internalization and externalization of many of these proteins, however, the generalization allows for grouping and comparison of similar motifs (342, 343).

In certain contexts, this YXXΦ motif can target proteins to the lysosome (344) while in others, the YXXΦ motif actually targets the protein to the basolateral surface of MDCK cells. One YXXΦ containing protein, Igp120, normally shuttles between the lysosome and the basolateral membrane. If the Y in this motif is replaced, the protein is taken to the 107

apical membrane where it is not rapidly internalized (345). Interestingly in MDCK cells a trans-Golgi protein, TGN38, contains the YXXΦ motif and it recycles between the basolateral membrane and the Golgi, further suggesting that the Golgi also has a role in protein sorting within the cell. Mutations in the cytoplasmic tail of TGN38 block this recycling pathway and the protein accumulates on both the apical and basolateral surfaces of the cell (346). In light of these outliers, as a general rule, proteins that contain the

YXXΦ motifs within 10-40 amino acids of the transmembrane domain are often endocytosed and recycled, while YXXΦ motifs that target to the lysosome are 6-9 amino acids from the plasma membrane, but in neither case have any these motifs been found at the C-terminus of the protein (338).

Another motif that is important in protein sorting is a di-leucine motif. This comes in a number of varieties. One example is the mannose-6 phosphate receptor, which has an

HLL motif in the cytoplasmic tail that is required for direct targeting to the lysosomal compartment from the Golgi (347, 348). The mannose-6-phosphate receptor is responsible for lysosomal protease targeting to the lysosome, so this is a very important motif in this protein (347, 348). Deleting or changing residues resulted in the protein being trafficked to the cellular membrane. However, this protein also contains another motif in the cytoplasmic tail (YKYSKV) that can serve as an endocytic motif (348). The di-leucine motif in the cytoplasmic tails of E-cadherin (349) and the Fc receptor (350) allow for trafficking of these proteins to the basolateral plasma membrane of MDCK cells (349, 350). In addition, charged residues upstream of the di-leucine motif can serve

108

as a signal for cellular trafficking. If the charged residues are changed, the protein traffics incorrectly, although this change has no effect on protein endocytosis (351).

Recycling

Recycling to the plasma membrane is important in my project, so here I will quickly go through the steps of endocytosis and different fates of the resultant proteins (Figure 13

(322, 331, 352)). Endocytosis initiates when a small region of the plasma membrane is invaginated by a clathrin-dependent or clathrin-independent mechanism. This vesicle pinches off from the plasma membrane and fuses with a sorting endosome. The pH of the sorting endosome is 5.5-6 (322, 352), a pH at which ligands dissociate from their receptors (322). Approximately two minutes after entry, the protein can be rapidly recycled back to the plasma membrane (331, 352). Alternatively the protein can be trafficked to the Golgi, the recycling endosome, or the late endosome. From the Golgi, the protein could be trafficked to the ER, the lysosome or back to the cell surface. From the recycling endosome, proteins can be taken to the plasma membrane or the Golgi.

Finally, proteins that are trafficked to the late endosome can either be taken to the Golgi or the lysosome. In the lysosome, the protein can stay, return to the late endosome or be degraded (331, 352).

All protein sorting is energy dependent, as such Rab GTPases are critical players in the sorting pathways. The Rab proteins are on the cytoplasmic interface of membranes and rely on GTP/GDP cycling for assembly of machinery within the cell. The machinery is

109

dynamic and regulated. Rab proteins undergo conformational changes upon GTP binding and hydrolysis that enable their association with other proteins. There are 66 known Rab proteins encoded in the human genome, including isoforms that have distinct functions.

For a review of Rabs and their associated proteins see (331).

In the next chapter, I will be discussing the key experiments that led up to my project, as well as the key pieces of data that have allowed me to discover that the G protein is cleaved by cathepsin L and that it does so during endocytic recycling.

110

Chapter 4: Gprotein cleavage in Vero cells

Introduction

90 kDa, when produced in most immortalized cell lines. Historically, it was difficult to determine if the G protein was a viral or host protein (6-8) because differences in the cell line, virus strain, and method of protein detection, all had an effect on the apparent size and presence of the G protein (6-8). While HEp-2 cells produced a 90 kDa G protein, BS- 111

C-1 cells, an African green monkey kidney cell line, produced a 55 kDa G protein, and the size discrepancy was assumed to be due to differences in glycosylation (9).

More recently, in the G protein produced by another African green monkey kidney cell line, Vero, this size difference was found to be caused by cleavage, a different post- translational modification. Although the G protein is not absolutely essential for infection of immortalized cells (19), virus lacking the G protein is 10-fold less infectious for these cells (37). Virus lacking the G protein is a further 10-fold less infectious for HAE cultures (16), which are an excellent in vitro model for the natural target cells in the human respiratory tract (353, 354). Similarly, virions produced by Vero cells, which contain primarily the cleaved G protein, are 5-fold less infectious for HAE cultures, suggesting that cleavage severely compromises the attachment function of the G protein

(16).

This finding is of particular importance for live attenuated vaccine development because viruses in the initial formalin-inactivated FI-RSV vaccine tested in the 1960s (17, 18) and in live attenuated vaccine candidates, tested in clinical trials since the 1990s, have been grown in African green monkey kidney or Vero cells (19-23, 26, 300, 355).

There are currently only 3 cell lines approved by the Food and Drug Administration for live attenuated vaccine production, MRC-5, WI-38 and Vero cells. The MRC-5 and WI-

38 cells are much less proliferative compared to Vero and the G protein is also cleaved in

MRC-5 (16) and WI-38 cells (unpublished data). In addition, Vero cells do not produce

IFN (29), which is especially appealing if a vaccine candidate is deficient in inhibiting the IFN response. 112

We hypothesized that virus produced in Vero cells, with an intact G protein, would more efficiently infect HAE cultures, and in vivo, the cells that line the human airway. To test our hypothesis, we used protease inhibitors to identify cathepsin L as the protease responsible for cleaving the G protein in Vero cells and demonstrated that cleavage likely occurs during endocytic recycling. Others have shown that the Nipah virus fusion protein is activated by cathepsin L during recycling (356, 357). In addition, endocytic recycling is important in the envelopment of RSV (250, 358). However, our work demonstrates for the first time that endocytic recycling and cathepsin L can be detrimental to the production of infectious viral progeny in one cell type but not another.

In addition, we demonstrated that growing the virus in Vero cells in the presence of cathepsin L inhibitor resulted in RSV with an intact G protein that was 5-fold more infectious for HAE cultures. We also identified the amino acids that are important for G protein cleavage in Vero cells and built the mutation that inhibited cleavage most efficiently into the viral genomic cDNA, rescued the modified virus and demonstrate that this Vero-derived virus had an intact G protein and was 5-fold more infectious for HAE cultures, without the addition of a protease inhibitor.

113

Materials and methods

Cell culture

HeLa and Vero cells (ATCC, Manassas, VA) were cultured in DMEM (Corning

Incorporated, Corning, NY) supplemented with 10% FBS (Atlanta Biologicals, Norcross,

GA), described throughout as medium. Cells were incubated at 37°C and 5% CO2. HAE cultures were generated from human airway tissue as previously described (359) and grown on collagen coated Trans-well inserts (Corning Incorporated). Upon reaching confluency and forming tight junctions, the apical medium was removed and cultures were maintained at the air-liquid interface for 6 to 8 weeks to form well-differentiated, polarized cultures before use. The basal medium was changed three times weekly and the apical surface was washed for 2 h once weekly with D-PBS.

Virus infection and drug treatment

HeLa or Vero cells were inoculated with recombinant green fluorescent protein (GFP)- expressing recombinant RSV (rgRSV), the D53 variant of the A2 strain (360, 361) or a mutant virus, diluted in the medium. Cells were tipped at 37°C for 2 h and the inoculum was replaced with fresh medium. At 48 h post inoculation (hpi) fresh medium was added and at 72 hpi cells were scraped, medium containing cells was collected and pulse vortexed. Cells were pelleted at 1200 xg for 5 min in a Megafuge (Baxter Scientific

Products) and the supernatant was divided into equal aliquots, snap frozen on dry ice, and stored at -80°C. All viruses were titrated on HeLa cells. Inoculated HeLa or Vero cells as

114

above with rgRSV or rgRSV-L208A. For protease inhibitor experiments, at 7 hpi medium containing 0.5 μM cathepsin l inhibitor III (Calbiochem, San Diego, CA) or an equal volume of vehicle (DMSO) was added to the cells. 72 hpi virus was harvested as described above and partially purified by layering over 35% sucrose in 1X Hanks

Balanced Salt Solution, with calcium and magnesium, and centrifuged overnight at 4°C and 26,000 xg in an F14-14x50cy rotor for the Sorvall Lynx 6000 (Thermo Fisher

Scientific). A portion of these virus preparations was further purified through a sucrose gradient by centrifugation in a TH-641 rotor (Thermo Fisher Scientific) and Beckman

Ultracentrifuge at 287,000 x g for 20 h. Gradient fractions were examined by immunoblot stained with a mAb to the N protein (362). Fractions containing virus were separated by

SDS-PAGE and G protein detected by immunoblot using a mAb, L9 (363).

The full-length RSV cDNA construct, RW30 (36), representing the D53 variant of the A2 strain was used as the backbone for a modification of the G protein gene at amino acid

208 from leucine to alanine. Synthetic double-stranded DNA gBlock (Integrated DNA

Technologies, Coralville, IA) containing the G gene with the L208A mutation was inserted into RW30 plasmid using restriction sites that flank the G protein gene and the

Gibson Assembly kit (New England Biolabs, Ipswich, MA). The G protein mutant virus, rgRSV-L208A, was rescued from this plasmid as previously described (361).

HeLa and Vero cells were inoculated with rgRSV (MOI: 1) or mock inoculated, 2 hpi the inoculum was replaced with fresh medium, and at 4 hpi cells the medium was again replaced with fresh medium containing 2-fold dilutions of these protease inhibitors

(Sigma-Aldrich, St. Louis, MO) dissolved in water: Aprotinin (3.125 to 50 μg/ml), 115

Leupeptin (6.25 to 100 μg/ml), E-64 (6.25 to 100 μg/ml); or equal volumes of water in medium. In other experiments, cells were treated at 6 hpi with drugs that blocked calpains and cathepsins; 10-fold dilutions (0.1 to 100 µM) of: Cathepsin inhibitor I (Calbiochem),

ALLM (Santa Cruz, Dallas, TX), Chloroquine diphosphate salt (Sigma-Aldrich) or equivalent volumes of vehicle (water), or of CA-074 (Calbiochem), Cathepsin L inhibitor

III (Calbiochem), or an equal volume of vehicle (DMSO) in medium. ALLM is a calpain and cathepsin inhibitor, CA-074 is a cathepsin B inhibitor.

Biotinylation and immunoblot analysis

At 24 hpi cells were biotinylated with Ez-link Sulfo-NHS-LC-Biotin (Thermo Fisher

Scientific, Waltham, MA). Cells were lysed with lysis buffer containing 150 mM NaCl,

1% Triton X-100, 50 mM Tris, 0.1% SDS, and 1X Halt protease cocktail inhibitor

(Thermo Fisher Scientific). Proteins were quantified using BCA protein assay kit (Pierce,

Waltham, MA), and equal amounts of protein were added to high capacity streptavidin agarose beads (Thermo Fisher Scientific). The mixtures were rotated for 1 h at 4°C, the beads were pelleted and washed with lysis buffer (without protease cocktail inhibitor), and the proteins separated by NuPAGE Novex 4-12% bis-tris protein gels (Life

Technologies, Carlsbad, CA) and immunoblots were probed with mouse mAb L9 to the

RSV G protein or D14 to the RSV N protein (Ed Walsh, University of Rochester) or a polyclonal rabbit Anti-CTSL antibody that recognizes cathepsin L (Sigma) followed by the appropriate human serum-adsorbed and peroxidase labeled secondary antibody: anti- mouse IgG (H+L) antibody or anti-rabbit IgG (H+L) antibody (KPL, Inc. Gaithersburg,

MD). 116

Cathepsin L treatment

Viruses grown in the presence of vehicle or cathepsin L inhibitor were pelleted through a sucrose cushion and resuspended in citric acid-sodium phosphate buffer at pH5.5. Active cathepsin L enzyme (Sigma) or vehicle was added to a final concentration of 50 ng/μl.

Samples were incubated for 2 h at 37°C and the G protein was assayed by immunoblot with L9 Ab.

PCR

Primers against cathepsin L were designed to amplify a 294 bp product and to cross exon

– exon boundaries to decrease the chance of amplifying genomic DNA (Forward: gaggcaacagaagaatcc, Reverse: cccagctgttcttcacc). Total mRNA was isolated from uninfected cells at 24 hpi, reverse transcribed, and amplified by PCR. PCR products were separated by 2% agarose gels and visualized with Ethidium Bromide.

Cathepsin activity assays

At 24 hpi, inoculated or mock infected cells were treated with lysis buffer without protease inhibitors and maintained on ice. Protein concentrations were determined with a

BCA protein assay (Pierce) and assayed by the InnoZyme Cathepsin L activity kit,

Fluorogenic (Calbiochem). Results were normalized for protein added and displayed as

Amido-4-methylcoumarin (AMC) released/g protein. Protein from HeLa or Vero was similarly assayed using InnoZyme Cathepsin B activity kit, Fluorogenic (Calbiochem).

117

Mutagenesis

A soluble version of the A2 strain G protein was constructed by replacing its cytoplasmic tail and transmembrane domain with the Schwarz measles virus H protein cytoplasmic tail, transmembrane and a portion of the stalk and inserting a furin cleavage site, 6-His tag and Factor Xa site between the MV stalk and the G protein.

For all other G protein mutants, a codon optimized, strain A2 G protein gene was mutagenized using synthetic gBlock DNA (Integrated DNA Technologies) inserted into pcDNA3.1

frG Transfection

Wild-type G or frG proteins were expressed in HeLa or Vero cells following plasmid transfection with FuGene HD (Promega) or Lipofectamine LTX (Life Technologies), respectively, in medium containing 2% FBS. In frG experiments, the medium was collected and concentrated using Ultracel – 10 K centrifugal filters (EMD Millipore,

Billerica, MA). For other transfection experiments, Vero cells were transfected using

Lipofectamine 3000 (Life Technologies) in DMEM containing 10% FBS. For all transfection experiments, cells were lysed and protein quantified by BCA assay.

Equivalent HeLa or Vero cell lysate protein and equivalent volumes of concentrated proteins from the medium were analyzed by immunoblot.

HAE viral infections

Partially purified virions were titrated on HeLa cells. The apical surface of well- differentiated HAE cultures in Transwells was washed with DPBS for 2 h and the basal

118

medium changed before equivalent pfu, between 2,000 and 10,000 pfu (HeLa-infectious units) of the appropriate RSV stock, were diluted in HAE medium and added to the apical chamber of the Transwell. In parallel, HeLa cells were inoculated with 200 pfu. At 2 hpi, the inoculum was removed from HeLa cells and replaced with fresh medium only.

Fluorescent (364) cells were visualized with an EVOS fl inverted fluorescence microscope (Life Technologies) and counted in HeLa cultures at 24 hpi and on HAE cultures at 48 hpi.

Statistical analysis

All data are one experiment representative of three unless otherwise noted. Data from three to six replicates of each experimental condition were expressed as the mean ± standard deviation. All experiments were repeated > 3 times. A 2-tailed student’s t-test was employed to determine the significance of differences between experimental conditions. A p-value of <0.05 was considered to be statistically significant.

Results

Protease identification with protease inhibitors

We hypothesized that Vero-derived virus containing an intact G protein would be more infectious for HAE cultures. To test this, we set out to identify the protease responsible for cleavage. Vero cells were inoculated with recombinant green fluorescent protein expressing RSV (rgRSV) and treated with protease inhibitors: aprotinin (a serine protease inhibitor); leupeptin (a serine/cysteine protease inhibitor); or E-64 (a cysteine protease

119

inhibitor). Since glycoproteins on the cell surface are the most likely to be incorporated into the virion, at 24 hr post inoculation (hpi) the cell surface proteins were labeled with biotin. Biotinylated proteins were pulled-down with streptavidin beads and the G protein was detected by immunoblot with an antibody to the G protein (Figure 14A). As expected, in the untreated and vehicle-treated samples most of the cell surface G protein was cleaved (~55 kDa), leaving only a small portion of full length (~90 kDa) G protein.

Aprotinin had no effect on cleavage. However, both leupeptin and E-64 prevented G protein cleavage suggesting that a cysteine protease is responsible for cleavage of the G protein in Vero cells.

120

Figure 14. Protease inhibition of G protein cleavage in Vero cells. Immunoblot detection of biotinylated cell surface RSV G protein (55 or 90 kDa) produced in rgRSV-inoculated Vero cells treated with protease inhibitors: (A) 2-fold dilutions of Aprotinin (3.125 to 50 μg/ml), Leupeptin or E-64 (6.25 to 100 μg/ml), (B) 10-fold dilutions of inhibitor ALLM (0.1 to 100 μM), (C) 10- fold dilutions of (0.1 to 100 μM) CA-074 (cathepsin B inhibitor) or cathepsin L inhibitor III. The immunoblots were probed with mAb L9. Data are representative of three independent experiments.

To narrow the field of cysteine proteases, rgRSV-infected Vero cells were treated with

ALLM, a protease inhibitor that blocks the activity of cathepsin B, cathepsin L, calpain I, and calpain II. While nearly all of the cell surface G protein in Vero cells was cleaved in the absence of the inhibitor, ALLM inhibited G protein cleavage in a concentration- dependent manner (Figure 14B).

The G protein is a type II membrane protein, whose N-terminal signal sequence is not cleaved, serving also as its anchor in cell and virion membranes (Figure 15). During transit to the cell surface the C-terminal, ectodomain, of the protein would be inside 121

vesicles and only the N-terminal cytoplasmic tail would be exposed to the cytoplasm.

Since calpains are exclusively cytoplasmic proteases, it is unlikely that they would be responsible for cleavage. Cathepsins B and L, however, reside inside vesicles and organelles or are secreted from cells and, therefore, would be more likely to have access to the G protein.

122

Figure 15. Schematic of the RSV G protein. The RSV A2 strain G protein is 298 amino acids long and consists of two heavily glycosylated mucin-like regions, separated by a central conserved, unglycosylated cysteine noose (yellow-green loop at the top) that is stabilized by a pair of disulfide bonds. The unglycosylated N terminus (blue) includes the cytoplasmic and transmembrane domains. To approximate the structure, a linear a-helical prototype of the G protein was subjected to steered molecular dynamics (NAMD), pulling the central noose perpendicular to the backbone until mucin-like region 1 (green) and 2 (orange) arrived in a near- parallel arrangement. This simulation resulted in the loss of a-helical structure over much of each mucin domain, primarily due to the abundant prolines, without affecting the helical structure in the TM and Nterminal domains. Complex glycans were positioned at each of the four N-linked sites (yellow side chains), and simple glycans (gray) were positioned at each O-linked site predicted by NetOGlyc3.1. The glycans in this representation have slightly higher-than-biological mass to reflect the probable space they would occupy. Springer Science + Business Media, Current topics in Microbiology and Immunology, Challenges and Opportunities for Respiratory Syncytial Virus Vaccines, 2013, page 93, Structure and Function of Respiratory Syncytial Virus Surface Glycoproteins, Jason S. McLellan, William C. Ray and Mark E. Peeples, figure number 3, reprinted with kind permission from Springer Science and Business Media. 123

To test the possibility of cathepsin cleavage of the G protein, RSV-infected Vero cells were treated with a cathepsin B inhibitor (CA-074) or a cathepsin L inhibitor (cathepsin L inhibitor III). Vehicle treated RSV infected cells displayed a mixture of cleaved and uncleaved G protein and the cathepsin B inhibitor did not change this pattern (Figure

14C). However, the cathepsin L inhibitor prevented G protein cleavage strongly even at the lowest concentration tested, suggesting that cathepsin L is the protease that cleaves the G protein.

Cathepsin L expression and activity

Both Vero and HeLa cells expressed cathepsin L mRNA in rgRSV-infected and uninfected cells (Figure 16A). However, only Vero cells contained detectable cathepsin L protein, and expression was independent of infection (Figure 16B). Cathepsin L mRNA is present in HeLa, but the protein is undetectable, suggesting that there could be a block in protein synthesis, but the mechanism of this inhibition was not pursued.

124

Figure 16. Cathepsin L and B expression and activity in HeLa and Vero cells. (A) Cathepsin L mRNA from mock-treated or rgRSV-infected HeLa and Vero cells was reverse transcribed, PCR amplified and the specific 294 bp product displayed by agarose gel electrophoresis. (B) Immunoblot detection of cathepsin L protein species in mock or rgRSV infected cell lysates. (C- D) Enzymatic activity of (C) cathepsin L and (D) cathepsin B in lysates. (A & B) Data are representative of three independent experiments. (C & D) Data from four independent experiments were combined. *p<0.05, **p<10-7 (unpaired, 2-tailed t-test, unequal variance)

Though cathepsin L is present in Vero cells, the protein may not be active. Cathepsin L has multiple splice variants (365-367) and at least two isoforms (318, 368). Like other

125

acid hydrolases, cathepsin L is a zymogen that needs to be cleaved three times for full protease activity (364, 369-372). Mock or rgRSV-infected cells were harvested at 24 hpi in the absence of protease inhibitors and assayed for cathepsin L activity. Vero cell cathepsin L was 100-fold more active than HeLa cell cathepsin L (Figure 16C). However, a comparable cathepsin B assay found a much smaller difference in activity in infected

HeLa and Vero cells (Figure 16D). These data support the inhibitor data suggesting that cathepsin L is the protease that cleaves the G protein in Vero cells.

To directly determine if cathepsin L is sufficient to cleave the G protein, we incubated G protein produced in Vero cells with pure, active cathepsin L protein. To generate the intact G protein substrate, HeLa and Vero cells were infected with rgRSV and treated with vehicle or cathepsin L inhibitor during virus production. Progeny virions were purified to remove the cathepsin L inhibitor and incubated with cathepsin L or vehicle.

As expected, virus grown in Vero without the protease inhibitor contained a mix of cleaved and intact G protein, while virus grown in the presence of inhibitor contained primarily intact G protein (Figure 17A, lanes 1 and 2). Exogenously added cathepsin L cleaved most of the G protein that had remained uncleaved in virions grown in vehicle- treated Vero cells. It also cleaved the intact G protein produced in Vero cells that were treated with cathepsin L inhibitor (Figure 17A, lanes 3 and 4). In both cases, the size of the cathepsin L cleaved G protein was 55 kDa, the same as the G protein cleaved in Vero cells. Interestingly, the G protein in virus grown in HeLa cells was not cleaved when incubated with exogenous cathepsin L (Figure 17B) suggesting a post-translational

126

modification of the G protein in HeLa cells that prevents cleavage by cathepsin L; this posttranslational modification does not occur in Vero cells.

Figure 17. Cathepsin L treatment of purified virions that had been grown in the presence of cathepsin L inhibitor. (A) Vero or (B) HeLa cells were inoculated with rgRSV and treated with medium containing vehicle (lane 1 and 3) or 0.5 μM cathepsin L inhibitor III (lane 2 and 4). Virus produced from these cells was partially purified and incubated with vehicle (lane 1 and 2) or 50 ng/μl cathepsin L (lane 3 and 4). G protein was detected by immunoblot. Data are representative of three independent experiments.

Cellular location of cleavage

Cathepsin L is found in the nucleus, in the late endosomes/lysosomes and can be secreted. While there is little likelihood and no evidence that the membrane-anchored G glycoprotein would transit through the nucleus, the G protein is transported through the cell in vesicles though no evidence for transit through the late endosomes/lysosomes has been presented. The G protein might also contact secreted cathepsin L at the cell surface.

A soluble G protein would allow us to test if the G protein is cleaved during transit to the cell surface or after it is secreted into the medium.

127

The second methionine in the G protein gene can also be used as a start codon, producing a secreted version of the G protein in some cell lines (99, 100, 373). However, this G protein is released from the cell as a monomer whereas cell associated G appears to be in a tetramer form (374). In the hope of producing a soluble tetramer, we constructed a gene that expresses a furin-releasable (fr) version of the G protein. The construct (Figure 18A) contains the G ectodomain attached to the measles virus hemagglutinin (H) protein stalk, transmembrane, and cytoplasmic domains. A furin cleavage site was inserted between the measles virus H stalk and the RSV G protein ectodomain. Our hope was that the measles virus stalk would stabilize the G protein in its tetramer form until it reached the trans-

Golgi where it would be released as frG by furin cleavage and secreted into the medium.

The H stalk was utilized as a spacer to separate the furin cleavage site from the plasma membrane so that furin could more easily access this cleavage site, resulting in the efficient release of the frG protein from the H stalk. This frG protein is in a tetramer form

(Capella, Johnson, Mejias, Ramilo and Peeples, manuscript in preparation).

128

Figure 18. Furin-released and membrane-bound G protein processing. (A) A chimeric protein that contains the N-terminus of the measles virus hemagglutinin, including its cytoplasmic tail, its transmembrane domain, and a portion of its stalk, followed by the RSV G-protein ectodomain. The ectodomain contains two hypervariable mucin-like regions flanking a central conserved, cysteine noose and heparin binding domain (HBD) [44]. Amino acid numbering is based on the complete G protein from the A2 lab strain without the MV stalk. (B) Membrane-bound (mG) or frG from HeLa and Vero cells detected by immunoblot. (C) Immunoblot of biotinylated cell surface RSV G protein (55 or 90 kDa) on Vero cells inoculated with rgRSV and treated with vehicle or 10-fold dilutions of chloroquine (0.1 to 100 µM). Data are representative of three independent experiments.

HeLa and Vero cells were transfected with plasmids expressing either full-length, membrane-bound G protein (mG) or frG protein. 48 h post transfection, cell culture medium was collected and concentrated. While cleaved mG was detected in Vero cell lysates, medium from both cell types contained only intact, 90 kDa frG protein (Figure

18B). This result indicates that the released frG protein is not cleaved during transit to the cell surface or while exposed to the medium, strongly suggesting that the full-length G

129

protein is unlikely to be cleaved on its way to or at the cell surface. The 90 kDa size of the released frG protein also indicates that the protein is fully glycosylated.

If the G protein is not cleaved during transit to the cell surface, the next most likely possibility would be during endocytic recycling. As cathepsin L is present primarily in the interior of the late endosome/lysosome and is optimally active at acidic pH, we treated rgRSV-infected Vero cells with chloroquine to raise the pH of acidic organelles.

Chloroquine treated Vero cells produced primarily a full-length G protein unlike the control treated samples (Figure 18C). Taken together these data suggest that the G protein encounters cathepsin L during endocytic recycling.

Infectivity for HAE cultures of virus grown in the presence of cathepsin L inhibitor

We used the virus produced and assayed in Figure 17 to test if virus grown in the presence of protease inhibitor is more infectious for HAE cultures than the control-treated virus. First, we determined the infectious titer of these viruses on HeLa cells and based on those titers, HAE cultures were inoculated with equivalent “HeLa-infectious units” of all viruses. The same dilution series from each virus stock that was used to inoculate the

HAE cultures was used to inoculate HeLa cells. Infected cells in both cell cultures were counted and data displayed as the ratio of HAE to HeLa infectious units (Figure 19).

130

Figure 19. The ability of rgRSV grown in the presence of cathepsin L inhibitor to infect HAE cultures. The infectivity of partially purified rgRSV virions that were produced in HeLa or Vero cells in the presence of cathepsin L inhibitor or DMSO (Figure 17), the left two lanes of each panel) was tested on HAE cultures. HAE cultures were inoculated with equal “HeLa-infectious units” of these 4 viruses, as were HeLa cells, on the same day and from the same dilution series for comparison. The ratio of HAE-infected cells to HeLa-infected cells is plotted. Data from three experiments were combined. *p<0.01, **p<10-4 (unpaired, 2-tailed t-test).

Cathepsin L inhibitor provided during viral growth increased the infectivity of Vero- derived virus for HAE cultures by 6-fold, such that there was no significant difference in the infectivity of virus derived from cathepsin L treated Vero cells and HeLa cells. Virus produced in HeLa cells treated with cathepsin L inhibitor displayed 30% lower infectivity for HAE cultures than virus grown in the same conditions but without the inhibitor. This relatively minor inhibitory effect on virus production may reflect the loss of cathepsin L activity on one or more of its many cellular functions.

Identification of the G protein cleavage site

Another, more permanent way to reduce G protein cleavage during virus growth in Vero cells would be to identify the site of cleavage using mutagenesis and insert the mutation

131

that most efficiently prevents cleavage into the viral cDNA and rescue virus. Such a virus produced in Vero cells should display full infectivity for HAE cultures.

To estimate the position of cleavage we took into account the positions of the 4 N-linked glycans in the strain A2 G protein and the predicted positions of the many O-linked glycan sites using NetOGlyc software (375). We predicted that the G protein is cleaved in

Vero cells around amino acid 210 (Figure 20). To test this estimate, we mutated codon

211 to a stop codon. The resultant protein was 50-60 kDa (Figure 21A), similar to the size of the cleaved G protein in Vero cells.

Figure 20. Cartoon displaying the transmembrane RSV G protein. The RSV G protein is a type II membrane protein with two hypervariable mucin-like regions flanking a conserved cysteine noose and heparin binding domain. Amino acid numbers are based on the A2 lab strain.

132

Figure 21. Mutagenesis of the G protein to identify the cleavage site. Immunoblot analysis of transiently expressed RSV G protein: (A) with a premature stop codon at amino acid 211, in HeLa; (B) with overlapping deletion mutations in this region, in Vero; and (C) with alanine mutations in this region, in Vero. Data are representative of three independent experiments.

To identify amino acids important for G protein cleavage in Vero cells, we deleted overlapping regions around amino acid 210 (200-211, 204-213, 206-215, 208-217, or

209-213), with the expectation that deletion of the cleavage site would prevent G protein cleavage in Vero cells. All of these deletion mutants reduced cleavage when compared to the wild-type G protein (Figure 21B), confirming that this region contains the protease site. In addition, all deletion mutants were missing amino acids 209-211, suggesting that cleavage requires these amino acids but not ruling out the importance of other amino acids in this region. Surprisingly, Δ206-210 and Δ206-215, both of which delete all or the

133

majority of 209-211, only partially inhibited cleavage. However, deletions have the potential of bringing together amino acids that might serve as a proteolytic site and this is likely the case here.

To further delineate amino acids that are important for cleavage, we individually mutated amino acids around 210 to alanine. Alanine substitutions at L208, K209, K212, K213, and D214 were resistant to cleavage, consistent with our hypothesis that amino acids 209-

211 are important for cleavage. Amino acid substitutions at positions 197,199, 200, 201,

204, 206, 210 and 211 had little or no effect on cleavage.

Cathepsin L is able to cleave substrates with a variety of sequences but has preferential cleavage sites. Importantly, the L208/K209 motif is consistent with a known cathepsin L cleavage preference, a hydrophobic amino acid in the P2 position and a basic residue in

P1 (376) with cleavage occurring between P1 and P1’. In this case, cleavage would be between amino acid 209 and 210.

Infection of HAE cultures with virus containing an uncleavable G protein

We built the L208A mutation into the rgRSV genomic cDNA and rescued the virus

(rgRSV-L208A). Both rgRSV and rgRSV-L208A were grown in HeLa or Vero cells and sucrose gradient purified (Figure 22A). As expected, HeLa-derived rgRSV contained intact G protein, and Vero-derived rgRSV contained a mix of cleaved and uncleaved G protein. However, consistent with the transiently expressed modified G protein, Vero- grown rgRSV-L208A virions contained almost exclusively uncleaved G protein. Partially purified viruses were titrated on HeLa cells and equivalent HeLa-infectious units were

134

used to inoculate HAE or HeLa cultures. Infected cells were counted and data displayed as the ratio of HAE to HeLa infectious units (Figure 22B). Consistent with our hypothesis, rgRSV-L208A is 5-fold more infectious for HAE cultures than rgRSV when grown in Vero cells.

Figure 22. Comparison of rgRSV and rgRSV-L208A infection of HAE cultures. HeLa-derived (H-D) and Vero-derived (V-D) rgRSV and rgRSV-L208A were produced in HeLa or Vero cells. (A) The virus was purified by sucrose gradient, and G protein detected by immunoblot. (B) Viruses were partially purified by centrifugation through a sucrose cushion and titrated on HeLa cells. Equal “HeLa-infectious units” were used to inoculate HAE and HeLa cells on the same day, as described in the legend of Figure 19. Data from 4 experiments were combined. *p<10-8 (unpaired, 2-tailed t-test, unequal variance).

Discussion

Cells use endocytic recycling to transport proteins from the trans-Golgi, turn over their membranes, bring in nutrients from the extracellular space (377) and process proteins for loading into MHC-I complexes (378). All enveloped viruses have hijacked this system to deliver their glycoproteins to the correct membranes for enveloping their capsid 135

structures (379-390). In at least one instance, the Nipah virus F protein requires endocytic recycling to activate its F protein in the late endosome/lysosome by either cathepsin B or

L (357, 391, 392).

Unlike these examples where recycling is required for the production of infectious virus, we have found that RSV G protein recycling in Vero cells is detrimental to the infectivity of progeny virus for its in vivo target, the ciliated airway epithelial cell. In this context, cathepsin L encountered during endocytic recycling serves as a viral restriction factor.

Similar restriction factors could reduce the production of other enveloped viruses, in particular, cell lines, animals or organs.

Cathepsins are critically important for lysosomal degradation and a number of other cellular processes and, as such, many are ubiquitously expressed (319). However, the G protein is only cleaved in a few cell lines (16). One reason for this difference is that cathepsin L activity is much lower in HeLa cells than in Vero cells (Figure 16), another is that the mature HeLa-derived G protein is not susceptible to cathepsin L cleavage while the Vero-derived G protein is (Figure 17). This result suggests that there is something fundamentally different about the G protein when it is produced in HeLa cells as compared to Vero cells, most likely a difference in post-translational modification. Each

G protein is modified by the addition of an estimated 35 O-glycans, but their specific locations on the G protein have not been determined for any cell line. In any case, these results demonstrate that not all immortalized cell lines are equivalent for viral production.

The immortalized cell receptor for the G protein is heparan sulfate (HS) (10-12, 122-

124), a long chain glycan modification found on a specific subset of cell surface 136

glycoproteins on many cell types (88). The first indication that Vero-derived RSV was different from HEp-2-derived RSV came when we found that HEp-2-derived virus was

20-fold more infectious than Vero-derived virus for CHO cells that express HS relative to

CHO cells that are deficient in HS production (16). This finding prompted us to examine the G protein in virions and we found that most of it was 55 kDa rather than 90 kDa. We went on to demonstrate that its smaller size was due to cleavage. We concluded that the loss of the G protein C-terminus reduced the ability of the Vero-derived virus to infect cells expressing HS, assuming that the HS binding domain had been lost when the C- terminus was removed by cleavage.

Originally, a highly basic peptide from the G protein representing amino acids 184-198 was shown to bind to cells and to inhibit infection (90). This region of the G protein was dubbed the “heparin binding domain” (HBD). In the current report, we identified the site of cleavage in Vero cells as between amino acids 209 and 210. This cleavage site is C- terminal to amino acids 184-198, the originally defined HBD, so cleavage at this site does not remove these amino acids from the 55 kDa G protein. However, in another study,

Shields et al. found that basic amino acids between 198 and 231 are also important for G protein binding to immortalized cells (124). Taken together these results support an extension of the HBD to encompass amino acids 184-231. Furthermore, from our current study the most important region for HS binding would seem to be the portion of the 183-

231 region that is lost by cleavage in Vero cells: amino acids 210-231.

Though important for entry into immortalized cells, HS is only detectable on the basal surface of HAE cultures (125). RSV only infects via the apical surface, and more 137

specifically, targets the ciliated epithelial cells (72), strongly suggesting that there is a different virus receptor on HAE cells. Consistent with this hypothesis, we have found that soluble HS readily inhibits RSV infection of HeLa cells, but not HAE cells (139). In addition, we and others have recently shown that a molecule known to interact with the G protein, CX3CR1 (134), is present on the cilia of HAE cultures (137, 139), and co- localizes with bound RSV virions on these cultures (137). Further, we have shown that the RSV G protein CX3C motif (amino acids 182-186) is important for infection of HAE cultures (139).

If amino acids 182-186 are important for attachment to the HAE cellular receptor

CX3CR1 and these amino acids are retained in the Vero-cleaved G protein, as they would be with cleavage taking place between amino acid 209 and 210, it is not clear why HAE infection would be impaired (Figure 21 and Figure 22). This result suggests that the

CX3C motif is not the only portion of the G protein that is important for infection of

HAE cultures. The C-terminal domain of the G protein, which is lost due to cleavage in

Vero cells, might also be involved in binding to the CX3CR1 receptor, to a second receptor, or in some other aspect of infection initiation. However, it is also possible that protein cleavage changes the structure of the G protein such that its CX3C motif is no longer able to bind CX3CR1.

An important role for the C-terminal portion of the G protein is consistent with the appearance, in 2003, of an RSV B strain virus in Buenos Aires containing a 60 nucleotide duplication in the C-terminal third of the G protein (393). Since then, these BA strain viruses have become the prominent circulating B strains (394-400). More recently, an A 138

strain with a 72 nucleotide duplication in the C-terminal third of its G protein has become a predominant circulating A strain (394-396, 398, 400-402). The duplicated regions in these A and B strains do not overlap, but the fact that these viruses have arisen independent of one another and become the predominant circulating strains also suggests that this C-terminal region of the G protein may play an important functional role.

We identified key residues in the C-terminal third of the G protein that are important for cathepsin L cleavage and chose the most efficient cleavage-inhibiting modification,

L208A, for inclusion in the virus. The rgRSV-L208A virus was equally infectious regardless of the cell type that produced the virus. The rgRSV-L208A virus grown in

Vero and rgRSV grown in HeLa were also equally infectious, confirming the importance of the full-length G protein for RSV infection of HAE cultures. Importantly, the comparable infectivity of Vero-derived rgRSV-L208A and HeLa-derived rgRSV indicates that the L208A mutation does not affect G protein function during virus entry into HeLa or HAE cells.

If a mutation that prevents G protein cleavage in Vero cells is incorporated into a live attenuated RSV vaccine candidate, the virus produced would have an intact G protein and be 5-fold more infectious for the nasal epithelium, the site of vaccine inoculation. As a result, 5 times less inoculum could be used for each vaccination, resulting in a more economically feasible vaccine candidate. Because cleavage is a posttranslational modification, in vivo infected airway epithelial cells should spread within the airway epithelium similarly to the wild-type virus because both viruses would produce an intact, functional G protein. While the work presented here appears to solve the problem of G 139

protein cleavage in Vero cells for live attenuated vaccine production, efforts to reach the right balance between attenuation and immunogenicity continue.

Hotard et al. recently set out to identify the purpose of this duplication. To do this, they built the duplication into the line 19 strain of the virus. The viruses had similar growth curves in BEAS-2B cells. In addition, there is a slight increase in the GAG dependence and binding to immortalized cells. However, in BALB/c mice there was no difference in the pfu/g of lung between the two viruses. A large caveat is, immortalized cells and mice are the worst model systems to look at entry of RSV: RSV infects pneumocytes in mice

(265) and ciliated epithelium in humans (264), attachment in immortalized cells requires heparan sulfate (10-15), which is not present on the surface of HAE cultures (125), where the virus infects the ciliated epithelium (446). The virus was also shown to infect the ciliated epithelium in human trachea organ culture, and in human lungs of a patient who died of RSV disease (70, 264). Viruses with the duplication should be tested in HAE cultures and other animal models to before saying that the duplication has no effect on viral entry in vivo.

140

Chapter 5: Other Experiments

Monkey, rodent, and human immortalized cells

G protein cleavage results in a virus that is less infectious for HAE cultures, but we do not know what other cells might cleave the G protein and, as such, we do not know what cells to avoid using in viral production. I infected a range of mouse, hamster, monkey and human cell lines and found that the G protein is cleaved in a variety of cell types including B82, BHK, Vero, BS-C-1, CV-1, all of which produced a mixture of cleaved and uncleaved G protein (Figure 23). But the G protein remained uncleaved in HeLa,

L929, 3T3, and LLC-MK-2. The only common theme I observed is that the African green monkey kidney cell lines, BS-C-1, CV-1, and Vero, cleaved the G protein to varying degrees. I hypothesize that the G protein is also cleaved in other African green monkey cells, such as the airways during infection and this could explain the inefficient spread within and between these animals. To test this hypothesis, I would obtain trachea and/or lung from one of these animals and produce well-differentiated AGM airway epithelial cultures, infect, and observe the size of the G protein in the released virions.

141

Figure 23. G protein cleavage by immortalized cell lines. Immunoblot of a cell lysate from a number of immortalized cell lines infected with RSV.

We have performed this experiment with human, mouse and macaque airways and demonstrated that the G protein is not cleaved in any of these cultures. In fact, the G protein produced by these cells is larger, 180 kDa as opposed to 90 kDa, a size difference that is a result of a covalently but not disulfide linked dimer or increased sugar content

(16). We sometimes do see some minor amount of 90 kDa G produced by the HAE cultures after two to four weeks of infection. To test if this was a cleaved G protein and to determine if the G protein produced by these cells was able to be cleaved, I incubated virus from HAE cultures with cathepsin L as I did with the HeLa and Vero cells in Figure

17. The G protein produced by HAE was not cleaved by cathepsin L.

142

Figure 24. Cathepsin L treatment of Vero or HAE derived viruses. The first four lanes were shown in Figure 17. Cathepsin L treatment of purified virions that had been grown in the presence of cathepsin L inhibitor. The virus was purified from infected HAE cells and incubated with vehicle (lane 5) or 50 ng/μl cathepsin L (lane 6). G protein was detected by immunoblot.

The original experiment where virus was grown in HeLa or Vero and then purified virions were treated with cathepsin L, the HeLa-derived G protein was resistant to cathepsin L-mediated cleavage (Figure 17). I hypothesized that this resistance could be due to a sugar that is blocking the cleavage site when the protein is produced in this cell line and not in Vero. I constructed and transiently expressed the T210A or the T211A mutant in HeLa cells and demonstrated that the G protein remained intact. At the time I had performed the experiment, I had assumed that the lack of cleavage was due to the low cathepsin L activity in HeLa cells (Figure 16), so I produced a cathepsin L gene in a pcDNA3.1 plasmid. However, more recently frG has been produced in 293F cells and this protein was demonstrated to be glycosylated at both T210 and T211 (preliminary data, Capella, Peeples, and Gorman). While the frG from HeLa cells has not yet been examined, we would predict that HeLa-derived frG would also contain glycans on both T residues. The next step will be to test a singly-mutated T210A or T211A, and a doubly

143

mutated T210/211A with and without cathepsin L overexpression. I would anticipate that the double mutant, and not the single mutants, will be cleaved only when cathepsin L is over-expressed.

If there is a different pattern of glycosylation in HeLa versus Vero cells, this may be of therapeutic interest, especially if there are different glycosyltransferases that affect this change. One way to approach this problem would be to look at the expression profiles of glycosyltransferases, or other enzymes important in sugar processing, between cells that cleave the G protein and those that do not.

Endocytic recycling

After determining that the G protein was cleaved by cathepsin L (Figure 14) and that the frG protein was secreted from Vero cells intact, we were left with a conundrum: if cathepsin L is not cleaving G on its way to the cell surface or after it is released into the medium, cleavage in the lysosome, the primary location of cathepsin L in the cell would be the most likely location for cleavage. How might G get to the lysosome? Dr.

Maisner’s group had found that Nipah virus F protein is cleaved by cathepsin L and that cleavage takes place in during endocytic recycling (357, 383, 391, 403). We hypothesized that the G protein was undergoing endocytosis as well and that the late endosome or lysosome was the location that the G protein and cathepsin L interact. Dr. Maisner supplied us with details of the method their lab had used and we followed them. All reagents and cells are ice-cold throughout the process to prevent movement of proteins

144

within the cell, except when entry is being tested by a brief incubation at 37C. The surface proteins of infected cells are labeled with an NHS-ester linked to biotin. The linker contains a disulfide bond, which when reduced releases the biotin label by a cell- impermeable reducing agent, MESNA, from the surface of cells. After biotinylation, the linking process is quenched with TBS or glycine. The cells are then incubated for 0, 5,

10, or 15 min at 37°C and returned to the cold environment for processing. The cell surface biotin is cleaved with MESNA and the cells are lysed. Biotinylated proteins are pulled-down using streptavidin beads and examined by immunoblot. We found that both

HeLa and Vero cells endocytosed a small amount of the biotinylated G protein after incubation at 37C, suggesting that this hypothesis is feasible (Figure 25, compare 0 to 5 min).

Figure 25. Endocytic recycling of the RSV G protein in HeLa and Vero cells. Immunoblot detection of endocytosed and biotinylated G protein from HeLa or Vero cells. HeLa or Vero cells were infected with RSV. In the walk-in refrigerator, 24 hpi cells were labeled with cleavable biotin, the biotin reaction was quenched with glycine, and cells were incubated at 37°C for 0, 5, 10, 15 minutes. The cells were then treated with MESNA to reduce the disulfide-linked biotin and cells were lysed. Proteins that were labeled and endocytosed were protected from reduction and were pulled-down with streptavidin beads.

145

If both HeLa and Vero cells endocytosed the G protein, why did HeLa cells not cleave the G protein when Vero cells did? It seemed unlikely that increasing endocytosis might increase cleavage of G by HeLa cells. Calcium treatment has been shown to increase the rate of endocytosis and size of the endocytic vesicles (404, 405). I treated HeLa or Vero cells with calcium chloride 7 hpi. Increasing calcium concentration did not increase G protein cleavage by HeLa cells (Figure 26).

Figure 26. CaCl2 Treatment. Immunoblot detection of RSV G protein (55 or 90 kDa) produced in rgRSV-inoculated HeLa or Vero cells treated 7 hpi with CaCl2 (2.6 to 20 mM). The immunoblots were probed with mAb L9.

To further demonstrate that the G protein is cleaved in Vero cells during endocytic recycling, I used a number of endocytosis inhibitors at 8.5 hpi: Cytochalasin D, an F- actin depolymerizer that should inhibit all endocytosis; 5-(N,N-Dimethyl) amiloride hydrochloride (DMA), which inhibits the sodium hydrogen ion exchanger and, as a

146

result, blocks macropinocytosis; and Dynasore, which blocks dynamin a molecule important in fission of endocytic vesicle from the plasma membrane in a number of endocytic pathways. DMA and Cytochalasin D both had an effect highest doses tested, however, there is also less G protein present (Figure 27). To rule out these pathways further I will need to try each of these drugs in a time course with a smaller range of doses or I will need to refresh the drug during infection because it is possible that the drug is used up and I am observing the cells too long after the drugs are added.

Figure 27. Effect of endocytosis inhibition on G protein cleavage in Vero cells. Immunoblot detection of biotinylated cell surface RSV G protein produced in rgRSV-inoculated Vero cells treated with DMA (20.8 to 250 μM), Cytochalasin D (50 to 50000 μM), or Dynasore (1 to 100 μM) 9 hpi. The immunoblots were probed with mAb L9.

Another way to slow endocytosis is to mutate any endocytosis motifs present in the cytoplasmic tail of the protein. However, the G protein does not have the traditional endocytic motifs. There are two motifs that could potentially serve as endocytic motifs, a

YXXXΦ instead of YXXΦ motif and DXXXXXLL instead of DXXXLL. To test the

147

importance of these motifs in G protein cleavage, I singly-mutated the L27A or Y31A or doubly-mutated L27A and Y31A. None of the mutations blocked cleavage of the G protein (Figure 28). To ensure that the G protein really does not contain an endocytic motif I deleted most of the cytoplasmic tail of the G protein (amino acid 2-36) and transiently expressed it in Vero cells. The G protein was still cleaved, confirming that its cytoplasmic tail was not needed for cleavage.

Figure 28. Endocytic recycling motif mutagenesis. Immunoblot analysis of transiently expressed RSV G protein with L25A, Y31A, L25A/Y31A or deletion of the cytoplasmic tail (amino acids 2-36). All mutants were transiently expressed in Vero cells.

Another way to confirm that G protein cleavage is dependent on endocytic recycling is to replace the cytoplasmic tail of the G protein with the cytoplasmic tail of a rapid-recycling protein like the transferrin receptor (406). For this experiment, I would remove the cytoplasmic tail (amino acid 1-36) and add the transferrin receptor cytoplasmic tail only or its cytoplasmic tail and transmembrane domain. In addition, I would insert the YXRF motif between amino acids 12 and 13 of the G protein. As a control, I will do the same thing with the Nipah virus F protein transmembrane, cytoplasmic tail, and endocytic 148

motif because we know that this protein is cleaved by cathepsin L and likely is sorted to the late endosome/lysosome, however, this protein is oriented in the opposite orientation to the G protein.

Amino acids important for G protein cleavage

My mutagenesis of the G protein to determine the amino acids important for cleavage was not limited to deletion, alanine-scanning and single-point mutations. I also generated a number of protein genes with multiple mutations and others with more conservative substitutions. The region of the G protein, as I have discussed previously, that is important for cleavage is also important for binding of the virus to the cellular GAGs.

The L208A mutant that we have built into the virus does function in infection of HAE cells and, therefore, seems to use the in vivo receptor. But it is possible that this mutation might reduce the immunogenicity of the G protein.

L208I substitution allowed more cleavage of G than the L208A mutation, however, the

L208I was not cleaved as readily as the wt (Figure 29). The K209R mutant was also more resistant to cleavage than the wt protein but less than the K209A mutation. These results are consistent with the preference of a hydrophobic followed by a basic amino acid motif that was discussed previously (376). As expected the L208A/K209A double mutant was completely resistant to cleavage while the L208I/K209R double mutant was not.

Changing all amino acids from 208-213 to A also completely blocked cleavage as did mutating K at 209/212/213 to A. Mutating amino acids 211-213 to A did not have a

149

major inhibitory effect on cleavage, indicating that the K209A mutation is important for cleavage inhibition.

Figure 29. G protein mutagenesis and cleavage. Immunoblot analysis of transiently expressed RSV G protein in Vero cells.

I chose 5 of these mutants to build into the viral genome: K209A, L208A/K209A, Δ208-

217, X208-213A, and K209/212/213A. The K209A mutant virus grew to very low titers and did not infect HAE cells well, but because that result was unexpected, I will re-rescue that virus before drawing any conclusion. However, the other viruses all infected HeLa and Vero cells well. These viruses were produced in HeLa or Vero, and as before, titrated on HeLa. HeLa and HAE cells were infected in parallel to control for any vial-to-vial variability.

As demonstrated above, in Figure 22, the rgRSVL208A mutant grown in Vero cells was significantly more infectious for HAE cultures than the wt virus also grown in Vero cells.

Interestingly, all of the new mutants infected HAE cells better than the wt, independent of the cell type the virus was grown in (Figure 30). There are a number of possible explanations for the increased infectivity of these viruses on HAE cells compared to 150

HeLa. The most logical explanation is that these mutations all reduce the ability of the virus to infect HeLa cells and this number is the denominator in the HAE/HeLa ratio. A reduction in the denominator would result in an increase in the HAE/HeLa ratio. The area of the protein we are modifying is within the “heparin binding site” that is known to be important for binding to heparan sulfate, the immortalized cellular receptor. More specifically, clusters of lysines in the G protein bind to heparan sulfate and replacing lysines that are important in this binding site would weaken it. The rgRSVL208A mutation did not affect the ratio of HAE/HeLa probably because it did not replace a lysine.

It is unlikely that these changes would increase infectivity for HAE cultures due to better viral entry because the HAE cell receptor is different from heparan sulfate and a different region of the G protein is involved in binding to that receptor (139). To ensure that this explanation is correct, the GAG dependency of these viruses needs to be tested. In addition, to rule out unexpected mutations, these viruses need to be sequenced. It would be of interest to compare the maximum titers of these viruses because a reduced ability to bind to the HeLa receptor may well reduce the ability of these viruses to grow in HeLa cells, and to be detected by infection of HeLa cells. The relative number of genome copies, determined by qPCR will also be compared to the HeLa infectious units. Once these viruses have been characterized it will be important to test them for their ability to infect cotton rats and to determine if these changes affect the immune response to the virus.

151

1.8 rgRSV224 rgRSVL208A 1.6 rgRSVL208A/K209A rgRSVΔ208-217 1.4 rgRSVX208-213A 1.2 rgRSVK209/212/213A

0.8

0.6

0.4 HAE/HeLa units infectious HAE/HeLa 0.2

0 HeLa-derived virus Vero-derived virus

Figure 30. Comparison of rgRSV and mutant virus infection of HAE cultures. HeLa-derived and Vero-derived rgRSV, rgRSVL208A, rgRSVL208A/K209A, rgRSVΔ208-217, rgRSVX208- 213A, or rgRSVK209/212/213A were produced in HeLa or Vero cells. Viruses were partially purified by centrifugation through a sucrose cushion and titrated on HeLa cells. Equal “HeLa- infectious units” were used to inoculate HAE and HeLa cells on the same day, as described in the legend of Figure 19. Data from 4 experiments were combined.

rgRSV Containing the G protein from Clinical Isolates

It is clear from the data I have presented that the C-terminal third of the G protein is important in some way for infection of HAE cells and likely the human airways, and this is not simply the presence of the CX3C motif, as this motif has not been removed in the

Vero cell cleaved G protein. To probe the role of this region of the protein I propose a number of mutants. The first would be to replace the lab adapted G protein gene with a

152

clinical isolate sequence. I have been able to move GA2-CH28 (407), GA5-A/WI/629-

4285/98 (408), ON67-1210A (409), or BA4128/99B (393)G protein genes into the full- length genome plasmid. These G genes are representatives of the major currently circulating RSV strains. The G proteins of two of these stains have duplications in the C- terminal portion of the protein and these strains have been growing in prominence since their appearance, possibly suggesting a gain of efficiency. I would also like to make a number of overlapping deletions within the C-terminus of the protein to replace conserved amino acids in the C-terminal third (210-298) of the protein to identify the domain that is important for infection of HAE cells.

153

References

1. Obata K, Mukai K, Tsujimura Y, Ishiwata K, Kawano Y, Minegishi Y, Watanabe N, Karasuyama H. 2007. Basophils are essential initiators of a novel type of chronic allergic inflammation. Blood 110:913-920. 2. Falsey AR, Hennessey PA, Formica MA, Cox C, Walsh EE. 2005. Respiratory syncytial virus infection in elderly and high-risk adults. N Engl J Med 352:1749- 1759. 3. Falsey AR, Walsh EE. 2005. Respiratory syncytial virus infection in elderly adults. Drugs Aging 22:577-587. 4. Falsey AR, Cunningham CK, Barker WH, Kouides RW, Yuen JB, Menegus M, Weiner LB, Bonville CA, Betts RF. 1995. Respiratory syncytial virus and influenza A infections in the hospitalized elderly. J Infect Dis 172:389-394. 5. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, Abraham J, Adair T, Aggarwal R, Ahn SY, Alvarado M, Anderson HR, Anderson LM, Andrews KG, Atkinson C, Baddour LM, Barker-Collo S, Bartels DH, Bell ML, Benjamin EJ, Bennett D, Bhalla K, Bikbov B, Bin Abdulhak A, Birbeck G, Blyth F, Bolliger I, Boufous S, Bucello C, Burch M, Burney P, Carapetis J, Chen H, Chou D, Chugh SS, Coffeng LE, Colan SD, Colquhoun S, Colson KE, Condon J, Connor MD, Cooper LT, Corriere M, Cortinovis M, de Vaccaro KC, Couser W, Cowie BC, Criqui MH, Cross M, Dabhadkar KC, et al. 2012. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 380:2095-2128. 6. Levine S. 1977. Polypeptides of respiratory syncytial virus. J Virol 21:427-431. 7. Pringle CR, Shirodaria PV, Gimenez HB, Levine S. 1981. Antigen and polypeptide synthesis by temperature-sensitive mutants of respiratory syncytial virus. J Gen Virol 54:173-183. 8. Kingsbury DW. 1991. The Paramyxoviruses. Plenum Press, New York. 9. Elango N, Prince GA, Murphy BR, Venkatesan S, Chanock RM, Moss B. 1986. Resistance to human respiratory syncytial virus (RSV) infection induced by immunization of cotton rats with a recombinant vaccinia virus expressing the RSV G glycoprotein. Proc Natl Acad Sci U S A 83:1906-1910. 10. Feldman SA, Audet S, Beeler JA. 2000. The fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate. J Virol 74:6442-6447.

154

11. Hallak LK, Spillmann D, Collins PL, Peeples ME. 2000. Glycosaminoglycan sulfation requirements for respiratory syncytial virus infection. J Virol 74:10508- 10513. 12. Hallak LK, Collins PL, Knudson W, Peeples ME. 2000. Iduronic acid- containing glycosaminoglycans on target cells are required for efficient respiratory syncytial virus infection. Virology 271:264-275. 13. Krusat T, Streckert HJ. 1997. Heparin-dependent attachment of respiratory syncytial virus (RSV) to host cells. Arch Virol 142:1247-1254. 14. Martinez I, Melero JA. 2000. Binding of human respiratory syncytial virus to cells: implication of sulfated cell surface proteoglycans. Journal of General Virology 81:2715-2722. 15. Techaarpornkul S, Collins PL, Peeples ME. 2002. Respiratory syncytial virus with the fusion protein as its only viral glycoprotein is less dependent on cellular glycosaminoglycans for attachment than complete virus. Virology 294:296-304. 16. Kwilas S, Liesman RM, Zhang L, Walsh E, Pickles RJ, Peeples ME. 2009. Respiratory syncytial virus grown in Vero cells contains a truncated attachment protein that alters its infectivity and dependence on glycosaminoglycans. J Virol 83:10710-10718. 17. Kim HW, Canchola JG, Brandt CD, Pyles G, Chanock RM, Jensen K, Parrott RH. 1969. Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. Am J Epidemiol 89:422-434. 18. Kapikian AZ, Mitchell RH, Chanock RM, Shvedoff RA, Stewart CE. 1969. An epidemiologic study of altered clinical reactivity to respiratory syncytial (RS) virus infection in children previously vaccinated with an inactivated RS virus vaccine. Am J Epidemiol 89:405-421. 19. Karron RA, Buonagurio DA, Georgiu AF, Whitehead SS, Adamus JE, Clements-Mann ML, Harris DO, Randolph VB, Udem SA, Murphy BR, Sidhu MS. 1997. Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication in vitro: clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant. Proc Natl Acad Sci U S A 94:13961-13966. 20. Hall CB, Walsh EE, Long CE, Schnabel KC. 1991. Immunity to and frequency of reinfection with respiratory syncytial virus. J Infect Dis 163:693-698. 21. Karron RA, Wright PF, Crowe JE, Jr., Clements-Mann ML, Thompson J, Makhene M, Casey R, Murphy BR. 1997. Evaluation of two live, cold- passaged, temperature-sensitive respiratory syncytial virus vaccines in chimpanzees and in human adults, infants, and children. J Infect Dis 176:1428- 1436. 22. Wright PF, Karron RA, Belshe RB, Thompson J, Crowe JE, Jr., Boyce TG, Halburnt LL, Reed GW, Whitehead SS, Anderson EL, Wittek AE, Casey R, Eichelberger M, Thumar B, Randolph VB, Udem SA, Chanock RM, Murphy BR. 2000. Evaluation of a live, cold-passaged, temperature-sensitive, respiratory syncytial virus vaccine candidate in infancy. J Infect Dis 182:1331-1342.

155

23. Karron RA, Wright PF, Belshe RB, Thumar B, Casey R, Newman F, Polack FP, Randolph VB, Deatly A, Hackell J, Gruber W, Murphy BR, Collins PL. 2005. Identification of a recombinant live attenuated respiratory syncytial virus vaccine candidate that is highly attenuated in infants. J Infect Dis 191:1093-1104. 24. Wright PF, Karron RA, Madhi SA, Treanor JJ, King JC, O'Shea A, Ikizler MR, Zhu YW, Collins PL, Cutland C, Randolph VB, Deatly AM, Hackell JG, Gruber WC, Murphy BR. 2006. The interferon antagonist NS2 protein of respiratory syncytial virus is an important virulence determinant for humans. Journal of Infectious Diseases 193:573-581. 25. Malkin E, Yogev R, Abughali N, Sliman J, Wang CK, Zuo FR, Yang CF, Eickhoff M, Esser MT, Tang RS, Dubovsky F. 2013. Safety and Immunogenicity of a Live Attenuated RSV Vaccine in Healthy RSV- Seronegative Children 5 to 24 Months of Age. PLoS One 8. 26. Whitehead SS, Firestone CY, Karron RA, Crowe JE, Elkins WR, Collins PL, Murphy BR. 1999. Addition of a missense mutation present in the L gene of respiratory syncytial virus (RSV) cpts530/1030 to RSV vaccine candidate cpts248/404 increases its attenuation and temperature sensitivity. J Virol 73:871- 877. 27. Murphy BR, Prince GA, Walsh EE, Kim HW, Parrott RH, Hemming VG, Rodriguez WJ, Chanock RM. 1986. Dissociation between Serum Neutralizing and Glycoprotein Antibody-Responses of Infants and Children Who Received Inactivated Respiratory Syncytial Virus-Vaccine. J Clin Microbiol 24:197-202. 28. Anonymous. 2015. WHO Reference Cell Banks (RCBs), on World Health Organization. http://www.who.int/biologicals/areas/vaccines/WHO_reference_cell_banks/en/. Accessed 29. Desmyter J, Melnick JL, Rawls WE. 1968. Defectiveness of interferon production and of rubella virus interference in a line of African green monkey kidney cells (Vero). J Virol 2:955-961. 30. Chanock R, Roizman B, Myers R. 1957. Recovery from infants with respiratory illness of a virus related to chimpanzee coryza agent (CCA). I. Isolation, properties and characterization. Am J Hyg 66:281-290. 31. Andrewes CH, Horstmann DM. 1949. The susceptibility of viruses to ethyl ether. J Gen Microbiol 3:290-297. 32. Chanock R, Finberg L. 1957. Recovery from infants with respiratory illness of a virus related to chimpanzee coryza agent (CCA). II. Epidemiologic aspects of infection in infants and young children. Am J Hyg 66:291-300. 33. Beem M, Wright FH, Hamre D, Egerer R, Oehme M. 1960. Association of the chimpanzee coryza agent with acute respiratory disease in children. N Engl J Med 263:523-530. 34. Baltimore D. 1971. Expression of animal virus genomes. Bacteriol Rev 35:235- 241.

156

35. Huang YT, Wertz GW. 1982. The genome of respiratory syncytial virus is a negative-stranded RNA that codes for at least seven mRNA species. J Virol 43:150-157. 36. Knipe DM, Howley PM. 2013. Fields virology, 6th ed. Wolters Kluwer/Lippincott Williams & Wilkins Health, Philadelphia, PA. 37. Techaarpornkul S, Barretto N, Peeples ME. 2001. Functional analysis of recombinant respiratory syncytial virus deletion mutants lacking the small hydrophobic and/or attachment glycoprotein gene. J Virol 75:6825-6834. 38. Coates HV, Kendrick L, Chanock RM. 1963. Antigenic differences between two strains of respiratory syncytial virus. Proc Soc Exp Biol Med 112:958-964. 39. Anderson LJ, Hierholzer JC, Tsou C, Hendry RM, Fernie BF, Stone Y, McIntosh K. 1985. Antigenic characterization of respiratory syncytial virus strains with monoclonal antibodies. J Infect Dis 151:626-633. 40. Mufson MA, Orvell C, Rafnar B, Norrby E. 1985. Two distinct subtypes of human respiratory syncytial virus. J Gen Virol 66 ( Pt 10):2111-2124. 41. Johnson PR, Spriggs MK, Olmsted RA, Collins PL. 1987. The G glycoprotein of human respiratory syncytial viruses of subgroups A and B: extensive sequence divergence between antigenically related proteins. Proc Natl Acad Sci U S A 84:5625-5629. 42. Beem M. 1967. Repeated infections with respiratory syncytial virus. J Immunol 98:1115-1122. 43. Kravetz HM, Knight V, Chanock RM, Morris JA, Johnson KM, Rifkind D, Utz JP. 1961. Respiratory syncytial virus. III. Production of illness and clinical observations in adult volunteers. JAMA 176:657-663. 44. Gardner PS, McQuillin J, Court SD. 1970. Speculation on pathogenesis in death from respiratory syncytial virus infection. Br Med J 1:327-330. 45. Jacobs JW, Peacock DB, Corner BD, Caul EO, Clarke SK. 1971. Respiratory syncytial and other viruses associated with respiratory disease in infants. Lancet 1:871-876. 46. Parrott RH, Kim HW, Arrobio JO, Hodes DS, Murphy BR, Brandt CD, Camargo E, Chanock RM. 1973. Epidemiology of respiratory syncytial virus infection in Washington, D.C. II. Infection and disease with respect to age, immunologic status, race and sex. Am J Epidemiol 98:289-300. 47. Glezen WP, Taber LH, Frank AL, Kasel JA. 1986. Risk of primary infection and reinfection with respiratory syncytial virus. Am J Dis Child 140:543-546. 48. Hall CB, Geiman JM, Douglas RG, Jr., Meagher MP. 1978. Control of nosocomial respiratory syncytial viral infections. Pediatrics 62:728-732. 49. Hall CB, Douglas RG, Jr., Geiman JM. 1980. Possible transmission by fomites of respiratory syncytial virus. J Infect Dis 141:98-102. 50. Hall CB, Douglas RG, Jr. 1981. Modes of transmission of respiratory syncytial virus. J Pediatr 99:100-103. 51. Simoes EA. 1999. Respiratory syncytial virus infection. Lancet 354:847-852. 52. Staff MC. 2013. Nonallergic rhinitis. http://www.mayoclinic.org/diseases- conditions/nonallergic-rhinitis/basics/definition/con-20026910. Accessed 157

53. Staff MC. 2013. Shortness of breath. http://www.mayoclinic.org/symptoms/shortness-of-breath/basics/definition/sym- 20050890. Accessed 54. Staff MC. 2013. Croup. http://www.mayoclinic.org/diseases- conditions/croup/basics/definition/con-20014673. Accessed 55. Staff MC. 2013. Bronchiolitis. http://www.mayoclinic.org/diseases- conditions/bronchiolitis/basics/definition/con-20019488. Accessed 56. Kojima T, Go M, Takano K, Kurose M, Ohkuni T, Koizumi J, Kamekura R, Ogasawara N, Masaki T, Fuchimoto J, Obata K, Hirakawa S, Nomura K, Keira T, Miyata R, Fujii N, Tsutsumi H, Himi T, Sawada N. 2013. Regulation of tight junctions in upper airway epithelium. Biomed Res Int 2013:947072. 57. Staff MC. 2015. Pneumonia. http://www.mayoclinic.org/diseases- conditions/pneumonia/basics/definition/con-20020032. Accessed 58. Sommer C, Resch B, Simoes EA. 2011. Risk factors for severe respiratory syncytial virus lower respiratory tract infection. Open Microbiol J 5:144-154. 59. Boyce TG, Mellen BG, Mitchel EF, Jr., Wright PF, Griffin MR. 2000. Rates of hospitalization for respiratory syncytial virus infection among children in medicaid. J Pediatr 137:865-870. 60. Simoes EA. 2003. Environmental and demographic risk factors for respiratory syncytial virus lower respiratory tract disease. J Pediatr 143:S118-126. 61. Han LL, Alexander JP, Anderson LJ. 1999. Respiratory syncytial virus pneumonia among the elderly: an assessment of disease burden. J Infect Dis 179:25-30. 62. Falsey AR. 2005. Respiratory syncytial virus infection in elderly and high-risk adults. Exp Lung Res 31 Suppl 1:77. 63. Stockman LJ, Curns AT, Anderson LJ, Fischer-Langley G. 2012. Respiratory syncytial virus-associated hospitalizations among infants and young children in the United States, 1997-2006. Pediatr Infect Dis J 31:5-9. 64. Isaacs D. 2013. Should respiratory care in preterm infants include prophylaxis against respiratory syncytial virus? The case against. Paediatr Respir Rev 14:128- 129. 65. Groothuis JR, Simoes EA, Levin MJ, Hall CB, Long CE, Rodriguez WJ, Arrobio J, Meissner HC, Fulton DR, Welliver RC, et al. 1993. Prophylactic administration of respiratory syncytial virus immune globulin to high-risk infants and young children. The Respiratory Syncytial Virus Immune Globulin Study Group. N Engl J Med 329:1524-1530. 66. Anonymous. 1997. Reduction of respiratory syncytial virus hospitalization among premature infants and infants with bronchopulmonary dysplasia using respiratory syncytial virus immune globulin prophylaxis. The PREVENT Study Group. Pediatrics 99:93-99. 67. Simoes EA, Sondheimer HM, Top FH, Jr., Meissner HC, Welliver RC, Kramer AA, Groothuis JR. 1998. Respiratory syncytial virus immune globulin for prophylaxis against respiratory syncytial virus disease in infants and children with congenital heart disease. The Cardiac Study Group. J Pediatr 133:492-499. 158

68. Anonymous. 1998. Palivizumab, a Humanized Respiratory Syncytial Virus Monoclonal Antibody, Reduces Hospitalization From Respiratory Syncytial Virus Infection in High-risk Infants. Pediatrics 102:531-537. 69. Wegzyn C, Toh LK, Notario G, Biguenet S, Unnebrink K, Park C, Makari D, Norton M. 2014. Safety and Effectiveness of Palivizumab in Children at High Risk of Serious Disease Due to Respiratory Syncytial Virus Infection: A Systematic Review. Infect Dis Ther doi:10.1007/s40121-014-0046-6. 70. Henderson FW, Hu SC, Collier AM. 1978. Pathogenesis of respiratory syncytial virus infection in ferret and fetal human tracheas in organ culture. Am Rev Respir Dis 118:29-37. 71. Matsui H, Grubb BR, Tarran R, Randell SH, Gatzy JT, Davis CW, Boucher RC. 1998. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 95:1005- 1015. 72. Zhang L, Peeples ME, Boucher RC, Collins PL, Pickles RJ. 2002. Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology. J Virol 76:5654-5666. 73. Guo-Parke H, Canning P, Douglas I, Villenave R, Heaney LG, Coyle PV, Lyons JD, Shields MD, Power UF. 2013. Relative respiratory syncytial virus cytopathogenesis in upper and lower respiratory tract epithelium. Am J Respir Crit Care Med 188:842-851. 74. Villenave R, Shields MD, Power UF. 2013. Respiratory syncytial virus interaction with human airway epithelium. Trends Microbiol 21:238-244. 75. Davis NL, Wertz GW. 1982. Synthesis of vesicular stomatitis virus negative- strand RNA in vitro: dependence on viral protein synthesis. J Virol 41:821-832. 76. Sawicki SG, Sawicki DL. 1986. Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis. J Virol 57:328-334. 77. Collins PL, Huang YT, Wertz GW. 1984. Identification of a tenth mRNA of respiratory syncytial virus and assignment of polypeptides to the 10 viral genes. J Virol 49:572-578. 78. Collins PL, Wertz GW. 1983. cDNA cloning and transcriptional mapping of nine polyadenylylated RNAs encoded by the genome of human respiratory syncytial virus. Proc Natl Acad Sci U S A 80:3208-3212. 79. Collins PL, Dickens LE, Buckler-White A, Olmsted RA, Spriggs MK, Camargo E, Coelingh KV. 1986. Nucleotide sequences for the gene junctions of human respiratory syncytial virus reveal distinctive features of intergenic structure and gene order. Proc Natl Acad Sci U S A 83:4594-4598. 80. Collins PL, Huang YT, Wertz GW. 1984. Nucleotide sequence of the gene encoding the fusion (F) glycoprotein of human respiratory syncytial virus. Proc Natl Acad Sci U S A 81:7683-7687. 81. Kuo L, Grosfeld H, Cristina J, Hill MG, Collins PL. 1996. Effects of mutations in the gene-start and gene-end sequence motifs on transcription of monocistronic and dicistronic minigenomes of respiratory syncytial virus. J Virol 70:6892-6901.

159

82. Collins PL, Mink MA, Stec DS. 1991. Rescue of synthetic analogs of respiratory syncytial virus genomic RNA and effect of truncations and mutations on the expression of a foreign reporter gene. Proc Natl Acad Sci U S A 88:9663-9667. 83. Mink MA, Stec DS, Collins PL. 1991. Nucleotide sequences of the 3' leader and 5' trailer regions of human respiratory syncytial virus genomic RNA. Virology 185:615-624. 84. Hanley LL, McGivern DR, Teng MN, Djang R, Collins PL, Fearns R. 2010. Roles of the respiratory syncytial virus trailer region: effects of mutations on genome production and stress granule formation. Virology 406:241-252. 85. Levine S, Peeples M, Hamilton R. 1977. Effect of respiratory syncytial virus infection of HeLa-cell macromolecular synthesis. J Gen Virol 37:53-63. 86. Gruber C, Levine S. 1983. Respiratory syncytial virus polypeptides. III. The envelope-associated proteins. J Gen Virol 64 (Pt 4):825-832. 87. Walsh EE, Hruska J. 1983. Monoclonal antibodies to respiratory syncytial virus proteins: identification of the fusion protein. J Virol 47:171-177. 88. Walsh EE, Schlesinger JJ, Brandriss MW. 1984. Protection from respiratory syncytial virus infection in cotton rats by passive transfer of monoclonal antibodies. Infect Immun 43:756-758. 89. Atreya PL, Peeples ME, Collins PL. 1998. The NS1 protein of human respiratory syncytial virus is a potent inhibitor of minigenome transcription and RNA replication. J Virol 72:1452-1461. 90. Collins PL, Wertz GW. 1985. Nucleotide sequences of the 1B and 1C nonstructural protein mRNAs of human respiratory syncytial virus. Virology 143:442-451. 91. Collins PL, Anderson K, Langer SJ, Wertz GW. 1985. Correct sequence for the major nucleocapsid protein mRNA of respiratory syncytial virus. Virology 146:69-77. 92. Satake M, Venkatesan S. 1984. Nucleotide sequence of the gene encoding respiratory syncytial virus matrix protein. J Virol 50:92-99. 93. Marty A, Meanger J, Mills J, Shields B, Ghildyal R. 2004. Association of matrix protein of respiratory syncytial virus with the host cell membrane of infected cells. Arch Virol 149:199-210. 94. Olmsted RA, Collins PL. 1989. The 1a-Protein of Respiratory Syncytial Virus Is an Integral Membrane-Protein Present as Multiple, Structurally Distinct Species. J Virol 63:2019-2029. 95. Rixon HWM, Brown G, Murray JT, Sugrue RJ. 2005. The respiratory syncytial virus small hydrophobic protein is phosphorylated via a mitogen- activated protein kinase p38-dependent tyrosine kinase activity during virus infection. Journal of General Virology 86:375-384. 96. Collins PL, Mottet G. 1993. Membrane orientation and oligomerization of the small hydrophobic protein of human respiratory syncytial virus. J Gen Virol 74 ( Pt 7):1445-1450. 97. Wertz GW, Collins PL, Huang Y, Gruber C, Levine S, Ball LA. 1985. Nucleotide sequence of the G protein gene of human respiratory syncytial virus 160

reveals an unusual type of viral membrane protein. Proc Natl Acad Sci U S A 82:4075-4079. 98. Satake M, Coligan JE, Elango N, Norrby E, Venkatesan S. 1985. Respiratory syncytial virus envelope glycoprotein (G) has a novel structure. Nucleic Acids Res 13:7795-7812. 99. Hendricks DA, McIntosh K, Patterson JL. 1988. Further characterization of the soluble form of the G glycoprotein of respiratory syncytial virus. J Virol 62:2228- 2233. 100. Hendricks DA, Baradaran K, McIntosh K, Patterson JL. 1987. Appearance of a soluble form of the G protein of respiratory syncytial virus in fluids of infected cells. J Gen Virol 68 ( Pt 6):1705-1714. 101. Collins PL, Mottet G. 1992. Oligomerization and post-translational processing of glycoprotein G of human respiratory syncytial virus: altered O-glycosylation in the presence of brefeldin A. J Gen Virol 73 ( Pt 4):849-863. 102. Taylor G, Stott EJ, Bew M, Fernie BF, Cote PJ. 1983. Monoclonal antibodies protect against respiratory syncytial virus. Lancet 2:976. 103. Collins PL, Hill MG, Cristina J, Grosfeld H. 1996. Transcription elongation factor of respiratory syncytial virus, a nonsegmented negative-strand RNA virus. Proceedings of the National Academy of Sciences of the United States of America 93:81-85. 104. Hardy RW, Wertz GW. 2000. The Cys(3)-His(1) motif of the respiratory syncytial virus M2-1 protein is essential for protein function. J Virol 74:5880- 5885. 105. Lambert DM, Hambor J, Diebold M, Galinski B. 1988. Kinetics of Synthesis and Phosphorylation of Respiratory Syncytial Virus Polypeptides. Journal of General Virology 69:313-323. 106. Elango N, Satake M, Venkatesan S. 1985. mRNA sequence of three respiratory syncytial virus genes encoding two nonstructural proteins and a 22K structural protein. J Virol 55:101-110. 107. Dyer KD, Percopo CM, Fischer ER, Gabryszewski SJ, Rosenberg HF. 2009. Pneumoviruses infect eosinophils and elicit MyD88-dependent release of chemoattractant cytokines and interleukin-6. Blood 114:2649-2656. 108. Fearns R, Collins PL. 1999. Model for polymerase access to the overlapped L gene of respiratory syncytial virus. J Virol 73:388-397. 109. Stec DS, Hill MG, 3rd, Collins PL. 1991. Sequence analysis of the polymerase L gene of human respiratory syncytial virus and predicted phylogeny of nonsegmented negative-strand viruses. Virology 183:273-287. 110. Weber K, Osborn M. 1969. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406- 4412. 111. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.

161

112. Taylor G, Stott EJ, Bew M, Fernie BF, Cote PJ, Collins AP, Hughes M, Jebbett J. 1984. Monoclonal antibodies protect against respiratory syncytial virus infection in mice. Immunology 52:137-142. 113. Walsh EE, Schlesinger JJ, Brandriss MW. 1984. Purification and characterization of GP90, one of the envelope glycoproteins of respiratory syncytial virus. J Gen Virol 65 ( Pt 4):761-767. 114. Levine S, Klaiber-Franco R, Paradiso PR. 1987. Demonstration that glycoprotein G is the attachment protein of respiratory syncytial virus. J Gen Virol 68 ( Pt 9):2521-2524. 115. Collins PL, Mottet G. 1991. Post-translational processing and oligomerization of the fusion glycoprotein of human respiratory syncytial virus. J Gen Virol 72 ( Pt 12):3095-3101. 116. Mathews JM, Young TF, Tucker SP, Mackay JP. 2000. The core of the respiratory syncytial virus fusion protein is a trimeric coiled coil. J Virol 74:5911- 5920. 117. Bukreyev A, Whitehead SS, Murphy BR, Collins PL. 1997. Recombinant respiratory syncytial virus from which the entire SH gene has been deleted grows efficiently in cell culture and exhibits site-specific attenuation in the respiratory tract of the mouse. J Virol 71:8973-8982. 118. Morrison TG. 2003. Structure and function of a paramyxovirus fusion protein. Biochim Biophys Acta 1614:73-84. 119. Barretto N, Hallak LK, Peeples ME. 2003. Neuraminidase treatment of respiratory syncytial virus-infected cells or virions, but not target cells, enhances cell-cell fusion and infection. Virology 313:33-43. 120. Zimmer G, Trotz I, Herrler G. 2001. N-glycans of F protein differentially affect fusion activity of human respiratory syncytial virus. J Virol 75:4744-4751. 121. Zhao X, Singh M, Malashkevich VN, Kim PS. 2000. Structural characterization of the human respiratory syncytial virus fusion protein core. Proc Natl Acad Sci U S A 97:14172-14177. 122. Feldman SA, Hendry RM, Beeler JA. 1999. Identification of a linear heparin binding domain for human respiratory syncytial virus attachment glycoprotein G. J Virol 73:6610-6617. 123. Bourgeois C, Bour JB, Lidholt K, Gauthray C, Pothier P. 1998. Heparin-like structures on respiratory syncytial virus are involved in its infectivity in vitro. J Virol 72:7221-7227. 124. Shields B, Mills J, Ghildyal R, Gooley P, Meanger J. 2003. Multiple heparin binding domains of respiratory syncytial virus G mediate binding to mammalian cells. Arch Virol 148:1987-2003. 125. Zhang L, Bukreyev A, Thompson CI, Watson B, Peeples ME, Collins PL, Pickles RJ. 2005. Infection of ciliated cells by human parainfluenza virus type 3 in an in vitro model of human airway epithelium. J Virol 79:1113-1124. 126. Behera AK, Matsuse H, Kumar M, Kong X, Lockey RF, Mohapatra SS. 2001. Blocking intercellular adhesion molecule-1 on human epithelial cells

162

decreases respiratory syncytial virus infection. Biochem Biophys Res Commun 280:188-195. 127. Barr FE, Pedigo H, Johnson TR, Shepherd VL. 2000. Surfactant protein-A enhances uptake of respiratory syncytial virus by monocytes and U937 macrophages. Am J Respir Cell Mol Biol 23:586-592. 128. Hickling TP, Malhotra R, Bright H, McDowell W, Blair ED, Sim RB. 2000. Lung surfactant protein A provides a route of entry for respiratory syncytial virus into host cells. Viral Immunol 13:125-135. 129. Malhotra R, Ward M, Bright H, Priest R, Foster MR, Hurle M, Blair E, Bird M. 2003. Isolation and characterisation of potential respiratory syncytial virus receptor(s) on epithelial cells. Microbes Infect 5:123-133. 130. Johnson TR, McLellan JS, Graham BS. 2012. Respiratory syncytial virus glycoprotein G interacts with DC-SIGN and L-SIGN to activate ERK1 and ERK2. J Virol 86:1339-1347. 131. Tayyari F, Marchant D, Moraes TJ, Duan W, Mastrangelo P, Hegele RG. 2011. Identification of nucleolin as a cellular receptor for human respiratory syncytial virus. Nat Med 17:1132-1135. 132. Cozens AL, Yezzi MJ, Yamaya M, Steiger D, Wagner JA, Garber SS, Chin L, Simon EM, Cutting GR, Gardner P, Friend DS, Basbaum CB, Gruenert DC. 1992. A Transformed Human Epithelial-Cell Line That Retains Tight Junctions Post Crisis. In Vitro Cellular & Developmental Biology-Animal 28A:735-744. 133. Koutsioumpa M, Papadimitriou E. 2014. Cell surface nucleolin as a target for anti-cancer therapies. Recent Pat Anticancer Drug Discov 9:137-152. 134. Tripp RA, Jones LP, Haynes LM, Zheng H, Murphy PM, Anderson LJ. 2001. CX3C chemokine mimicry by respiratory syncytial virus G glycoprotein. Nat Immunol 2:732-738. 135. Tripp RA, Dakhama A, Jones LP, Barskey A, Gelfand EW, Anderson LJ. 2003. The G glycoprotein of respiratory syncytial virus depresses respiratory rates through the CX3C motif and substance P. J Virol 77:6580-6584. 136. Zhang W, Choi Y, Haynes LM, Harcourt JL, Anderson LJ, Jones LP, Tripp RA. 2010. Vaccination to induce antibodies blocking the CX3C-CX3CR1 interaction of respiratory syncytial virus G protein reduces pulmonary inflammation and virus replication in mice. J Virol 84:1148-1157. 137. Jeong KI, Piepenhagen PA, Kishko M, DiNapoli JM, Groppo RP, Zhang L, Almond J, Kleanthous H, Delagrave S, Parrington M. 2015. CX3CR1 Is Expressed in Differentiated Human Ciliated Airway Cells and Co-Localizes with Respiratory Syncytial Virus on Cilia in a G Protein-Dependent Manner. PLoS One 10:e0130517. 138. Chirkova T, Boyoglu-Barnum S, Gaston KA, Malik FM, Trau SP, Oomens AG, Anderson LJ. 2013. Respiratory syncytial virus G protein CX3C motif impairs human airway epithelial and immune cell responses. J Virol 87:13466- 13479.

163

139. Johnson S, Peeples, ME. 2015. Respiratory Syncytial Virus Uses CX3CR1 as a Receptor on Primary Human Airway Epithelial Cultures. PloS Pathogens In press. 140. Srinivasakumar N, Ogra PL, Flanagan TD. 1991. Characteristics of fusion of respiratory syncytial virus with HEp-2 cells as measured by R18 fluorescence dequenching assay. J Virol 65:4063-4069. 141. Kolokoltsov AA, Deniger D, Fleming EH, Roberts NJ, Jr., Karpilow JM, Davey RA. 2007. Small interfering RNA profiling reveals key role of clathrin- mediated endocytosis and early endosome formation for infection by respiratory syncytial virus. J Virol 81:7786-7800. 142. San-Juan-Vergara H, Sampayo-Escobar V, Reyes N, Cha B, Pacheco-Lugo L, Wong T, Peeples ME, Collins PL, Castano ME, Mohapatra SS. 2012. Cholesterol-Rich Microdomains as Docking Platforms for Respiratory Syncytial Virus in Normal Human Bronchial Epithelial Cells (vol 86, pg 1832, 2012). J Virol 86:5408-5408. 143. Krzyzaniak MA, Zumstein MT, Gerez JA, Picotti P, Helenius A. 2013. Host cell entry of respiratory syncytial virus involves macropinocytosis followed by proteolytic activation of the F protein. PLoS Pathog 9:e1003309. 144. Kong XY, San Juan H, Behera A, Peeples ME, Wu J, Lockey RF, Mohapatra SS. 2004. ERK-1/2 activity is required for efficient RSV infection. FEBS Lett 559:33-38. 145. Fricke J, Koo LY, Brown CR, Collins PL. 2013. p38 and OGT Sequestration into Viral Inclusion Bodies in Cells Infected with Human Respiratory Syncytial Virus Suppresses MK2 Activities and Stress Granule Assembly. J Virol 87:1333- 1347. 146. Pullikuth AK, Catling AD. 2007. Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: A perspective. Cellular Signalling 19:1621- 1632. 147. Corbitt G, Beswick TS. 1972. The cytochemistry of inclusion bodies in cell cultures into which respiratory syncytial virus has been inoculated. J Med Microbiol 5:144-147. 148. Jocelyn H. Bruce-Gregorios MD. 2006. Histopathological Techniques, 2nd edition. Goodwill trading co, 149. Carromeu C, Simabuco FM, Tamura RE, Arcieri LEF, Ventura AM. 2007. Intracellular localization of human respiratory syncytial virus L protein. Arch Virol 152:2259-2263. 150. Collins PL, Olmsted RA, Spriggs MK, Johnson PR, Bucklerwhite AJ. 1987. Gene Overlap and Site-Specific Attenuation of Transcription of the Viral Polymerase L-Gene of Human Respiratory Syncytial Virus. Proceedings of the National Academy of Sciences of the United States of America 84:5134-5138. 151. Dupuy LC, Dobson S, Bitko V, Barik S. 1999. Casein kinase 2-mediated phosphorylation of respiratory syncytial virus phosphoprotein P is essential for the transcription elongation activity of the viral polymerase; Phosphorylation by

164

casein kinase 1 occurs mainly at Ser(215) and is without effect. J Virol 73:8384- 8392. 152. Grosfeld H, Hill MG, Collins PL. 1995. RNA replication by respiratory syncytial virus (RSV) is directed by the N, P, and L proteins; transcription also occurs under these conditions but requires RSV superinfection for efficient synthesis of full-length mRNA. J Virol 69:5677-5686. 153. Fearns R, Collins PL. 1999. Role of the M2-1 transcription antitermination protein of respiratory syncytial virus in sequential transcription. J Virol 73:5852- 5864. 154. Kiss G, Holl JM, Williams GM, Alonas E, Vanover D, Lifland AW, Gudheti M, Guerrero-Ferreira RC, Nair V, Yi H, Graham BS, Santangelo PJ, Wright ER. 2014. Structural Analysis of Respiratory Syncytial Virus Reveals the Position of M2-1 between the Matrix Protein and the Ribonucleoprotein Complex. J Virol 88:7602-7617. 155. Klug A, Schwabe JWR. 1995. Protein Motifs .5. Zinc Fingers. Faseb Journal 9:597-604. 156. Vazquez C, Horner SM. 2015. MAVS Coordination of Antiviral Innate Immunity. J Virol 89:6974-6977. 157. Weber F. 2015. The catcher in the RIG-I. Cytokine 76:38-41. 158. Reikine S, Nguyen JB, Modis Y. 2014. Pattern Recognition and Signaling Mechanisms of RIG-I and MDA5. Front Immunol 5:342. 159. Gyoneva S, Ransohoff RM. 2015. Inflammatory reaction after traumatic brain injury: therapeutic potential of targeting cell-cell communication by chemokines. Trends in Pharmacological Sciences 36:471-480. 160. Grimm S. 2012. The ER-mitochondria interface: The social network of cell death. Biochimica Et Biophysica Acta-Molecular Cell Research 1823:327-334. 161. Kepp O, Senovilla L, Galluzzi L, Panaretakis T, Tesniere A, Schlemmer F, Madeo F, Zitvogel L, Kroemer G. 2009. Viral subversion of immunogenic cell death. Cell Cycle 8:860-869. 162. Salvesen GS, Walsh CM. 2014. Functions of caspase 8: The identified and the mysterious. Semin Immunol 26:246-252. 163. Jundi K GC. 2015. Transcription of Interleukin-8: How Altered Regulation Can Affect Cystic Fibrosis Lung Disease. Biomolecules 5:1386-1398. 164. Zarogoulidis P, Katsikogianni F, Tsiouda T, Sakkas A, Katsikogiannis N, Zarogoulidis K. 2014. Interleukin-8 and Interleukin-17 for Cancer. Cancer Investigation 32:197-205. 165. Ramos MI, Tak PP, Lebre MC. 2014. Fms-like tyrosine kinase 3 ligand- dependent dendritic cells in autoimmune inflammation. Autoimmun Rev 13:117- 124. 166. Verhelst K, Verstrepen L, Carpentier I, Beyaert R. 2013. IkappaB kinase epsilon (IKKepsilon): a therapeutic target in inflammation and cancer. Biochem Pharmacol 85:873-880. 167. Amaya M, Keck F, Bailey C, Narayanan A. 2014. The role of the IKK complex in viral infections. Pathog Dis 72:32-44. 165

168. Honda K, Yanai H, Negishi H, Asagiri M, Sato M, Mizutani T, Shimada N, Ohba Y, Takaoka A, Yoshida N, Taniguchi T. 2005. IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature 434:772-777. 169. Hiscott J. 2007. Triggering the innate antiviral response through IRF-3 activation. Journal of Biological Chemistry 282:15325-15329. 170. Sin WX, Li P, Yeong JP, Chin KC. 2012. Activation and regulation of interferon-beta in immune responses. Immunol Res 53:25-40. 171. Servant MJ, Grandvaux N, Hiscott J. 2002. Multiple signaling pathways leading to the activation of interferon regulatory factor 3. Biochem Pharmacol 64:985-992. 172. Ning S, Pagano JS, Barber GN. 2011. IRF7: activation, regulation, modification and function. Genes Immun 12:399-414. 173. Chattopadhyay S, Sen GC. 2014. dsRNA-Activation of TLR3 and RLR Signaling: Gene Induction-Dependent and Independent Effects. Journal of Interferon and Cytokine Research 34:427-436. 174. Talpaz M, Mercer J, Hehlmann R. 2015. The interferon-alpha revival in CML. Ann Hematol 94 Suppl 2:S195-207. 175. O'Shea JJ, Schwartz DM, Villarino AV, Gadina M, McInnes IB, Laurence A. 2015. The JAK-STAT pathway: impact on human disease and therapeutic intervention. Annu Rev Med 66:311-328. 176. Kasper LH, Reder AT. 2014. Immunomodulatory activity of interferon-beta. Ann Clin Transl Neurol 1:622-631. 177. Wang H, Yang YG. 2014. The complex and central role of interferon-gamma in graft-versus-host disease and graft-versus-tumor activity. Immunol Rev 258:30- 44. 178. Isailovic N, Daigo K, Mantovani A, Selmi C. 2015. Interleukin-17 and innate immunity in infections and chronic inflammation. J Autoimmun 60:1-11. 179. Piccioli P, Rubartelli A. 2013. The secretion of IL-1 beta and options for release. Semin Immunol 25:425-429. 180. Ruscitti P, Cipriani P, Carubbi F, Liakouli V, Zazzeroni F, Di Benedetto P, Berardicurti O, Alesse E, Giacomelli R. 2015. The role of IL-1beta in the bone loss during rheumatic diseases. Mediators Inflamm 2015:782382. 181. Krishnan SM, Sobey CG, Latz E, Mansell A, Drummond GR. 2014. IL-1beta and IL-18: inflammatory markers or mediators of hypertension? Br J Pharmacol 171:5589-5602. 182. Ben-Sasson SZ, Wang K, Cohen J, Paul WE. 2013. IL-1beta strikingly enhances antigen-driven CD4 and CD8 T-cell responses. Cold Spring Harb Symp Quant Biol 78:117-124. 183. Dinarello CA. 2011. A clinical perspective of IL-1beta as the gatekeeper of inflammation. Eur J Immunol 41:1203-1217. 184. McLeod JJ, Baker B, Ryan JJ. 2015. Mast cell production and response to IL-4 and IL-13. Cytokine 75:57-61. 185. Hunter CA, Jones SA. 2015. IL-6 as a keystone cytokine in health and disease. Nat Immunol 16:448-457. 166

186. Suzuki A, Leland P, Joshi BH, Puri RK. 2015. Targeting of IL-4 and IL-13 receptors for cancer therapy. Cytokine 75:79-88. 187. Bao K, Reinhardt RL. 2015. The differential expression of IL-4 and IL-13 and its impact on type-2 immunity. Cytokine 75:25-37. 188. Goswami R, Kaplan MH. 2011. A brief history of IL-9. J Immunol 186:3283- 3288. 189. Gour N, Wills-Karp M. 2015. IL-4 and IL-13 signaling in allergic airway disease. Cytokine 75:68-78. 190. Broughton SE, Dhagat U, Hercus TR, Nero TL, Grimbaldeston MA, Bonder CS, Lopez AF, Parker MW. 2012. The GM-CSF/IL-3/IL-5 cytokine receptor family: from ligand recognition to initiation of signaling. Immunol Rev 250:277- 302. 191. Takatsu K. 2011. Interleukin-5 and IL-5 receptor in health and diseases. Proc Jpn Acad Ser B Phys Biol Sci 87:463-485. 192. Wynn TA. 2015. Type 2 cytokines: mechanisms and therapeutic strategies. Nature Reviews Immunology 15:271-282. 193. Levy O. 2007. Innate immunity of the newborn: basic mechanisms and clinical correlates. Nat Rev Immunol 7:379-390. 194. Singh JK, Simoes BM, Howell SJ, Farnie G, Clarke RB. 2013. Recent advances reveal IL-8 signaling as a potential key to targeting breast cancer stem cells. Breast Cancer Research 15. 195. Gales D, Clark C, Manne U, Samuel T. 2013. The Chemokine CXCL8 in Carcinogenesis and Drug Response. ISRN Oncol 2013:859154. 196. Long AJ, Sypek JP, Askew R, Fish SC, Mason LE, Williams CM, Goldman SJ. 2006. Gob-5 contributes to goblet cell hyperplasia and modulates pulmonary tissue inflammation. Am J Respir Cell Mol Biol 35:357-365. 197. Lv X, Wang X. 2013. The role of interleukin-9 in lymphoma. Leuk Lymphoma 54:1367-1372. 198. Zhao P, Xiao X, Ghobrial RM, Li XC. 2013. IL-9 and Th9 cells: progress and challenges. Int Immunol 25:547-551. 199. Mittal SK, Roche PA. 2015. Suppression of antigen presentation by IL-10. Curr Opin Immunol 34:22-27. 200. Yang XK, Xu WD, Leng RX, Liang Y, Liu YY, Fang XY, Feng CC, Li R, Cen H, Pan HF, Ye DQ. 2015. Therapeutic potential of IL-15 in rheumatoid arthritis. Hum Immunol doi:10.1016/j.humimm.2015.09.041. 201. Hashimoto K, Graham BS, Ho SB, Adler KB, Collins RD, Olson SJ, Zhou WS, Suzutani T, Jones PW, Goleniewska K, O'Neal JF, Peebles RS. 2004. Respiratory syncytial virus in allergic lung inflammation increases Muc5ac and Gob-5. Am J Respir Crit Care Med 170:306-312. 202. Sparber F, LeibundGut-Landmann S. 2015. Interleukin 17-Mediated Host Defense against Candida albicans. Pathogens 4:606-619. 203. Mojsilovic S, Jaukovic A, Santibanez JF, Bugarski D. 2015. Interleukin-17 and its implication in the regulation of differentiation and function of hematopoietic and mesenchymal stem cells. Mediators Inflamm 2015:470458. 167

204. Shabgah AG, Fattahi E, Shahneh FZ. 2014. Interleukin-17 in human inflammatory diseases. Postepy Dermatol Alergol 31:256-261. 205. Anonymous. !!! INVALID CITATION !!! 206. Li Y, Hua S. 2014. Mechanisms of pathogenesis in allergic asthma: role of interleukin-23. Respirology 19:663-669. 207. Arthur JS, Ley SC. 2013. Mitogen-activated protein kinases in innate immunity. Nat Rev Immunol 13:679-692. 208. Tiffany JM. 2003. Tears in health and disease. Eye (Lond) 17:923-926. 209. Kashima T, Itakura H, Akiyama H, Kishi S. 2014. Rebamipide ophthalmic suspension for the treatment of dry eye syndrome: a critical appraisal. Clin Ophthalmol 8:1003-1010. 210. Nagel AK, Ball LE. 2014. O-GlcNAc transferase and O-GlcNAcase: achieving target substrate specificity. Amino Acids 46:2305-2316. 211. Yao S, Jiang L, Moser EK, Jewett LB, Wright J, Du J, Zhou B, Davis SD, Krupp NL, Braciale TJ, Sun J. 2015. Control of pathogenic effector T-cell activities in situ by PD-L1 expression on respiratory inflammatory dendritic cells during respiratory syncytial virus infection. Mucosal Immunology 8:746-759. 212. Yao S, Chen LP. 2014. PD-1 as an Immune Modulatory Receptor. Cancer Journal 20:262-264. 213. Au-Yeung N, Mandhana R, Horvath CM. 2013. Transcriptional regulation by STAT1 and STAT2 in the interferon JAK-STAT pathway. JAKSTAT 2:e23931. 214. Goto H, Mitsuhashi A, Nishioka Y. 2014. Role of surfactant protein A in non- infectious lung diseases. J Med Invest 61:1-6. 215. Grieves JL YZ, Durbin RK, Durbin JE. 2015. Acute and Chronic Airway Disease After Human Respiratory Syncytial Virus Infection in Cotton Rats (Sigmodon hispidus). Comparative Medicine 65:315-326. 216. Basto AP, Leitao A. 2014. Targeting TLR2 for vaccine development. J Immunol Res 2014:619410. 217. Liu Y, Yin H, Zhao M, Lu Q. 2014. TLR2 and TLR4 in autoimmune diseases: a comprehensive review. Clin Rev Allergy Immunol 47:136-147. 218. Ferrari G, Bignami F, Rama P. 2015. Tumor necrosis factor-alpha inhibitors as a treatment of corneal hemangiogenesis and lymphangiogenesis. Eye Contact Lens 41:72-76. 219. Lifland AW, Jung J, Alonas E, Zurla C, Crowe JE, Jr., Santangelo PJ. 2012. Human respiratory syncytial virus nucleoprotein and inclusion bodies antagonize the innate immune response mediated by MDA5 and MAVS. J Virol 86:8245- 8258. 220. Panas MD, Kedersha N, McInerney GM. 2015. Methods for the characterization of stress granules in virus infected cells. Methods doi:10.1016/j.ymeth.2015.04.009. 221. Dever TE. 2002. Gene-specific regulation by general translation factors. Cell 108:545-556. 222. Kedersha N, Chen S, Gilks N, Li W, Miller IJ, Stahl J, Anderson P. 2002. Evidence that ternary complex (eIF2-GTP-tRNA(i)(Met))-deficient preinitiation 168

complexes are core constituents of mammalian stress granules. Mol Biol Cell 13:195-210. 223. Krishnamoorthy T, Pavitt GD, Zhang F, Dever TE, Hinnebusch AG. 2001. Tight binding of the phosphorylated alpha subunit of initiation factor 2 (eIF2alpha) to the regulatory subunits of guanine nucleotide exchange factor eIF2B is required for inhibition of translation initiation. Mol Cell Biol 21:5018- 5030. 224. Kedersha NL, Gupta M, Li W, Miller I, Anderson P. 1999. RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2 alpha to the assembly of mammalian stress granules. J Cell Biol 147:1431-1442. 225. Kedersha N, Cho MR, Li W, Yacono PW, Chen S, Gilks N, Golan DE, Anderson P. 2000. Dynamic shuttling of TIA-1 accompanies the recruitment of mRNA to mammalian stress granules. J Cell Biol 151:1257-1268. 226. Groskreutz DJ, Babor EC, Monick MM, Varga SM, Hunninghake GW. 2010. Respiratory syncytial virus limits alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) phosphorylation to maintain translation and viral replication. J Biol Chem 285:24023-24031. 227. Schlender J, Bossert B, Buchholz U, Conzelmann KK. 2000. Bovine respiratory syncytial virus nonstructural proteins NS1 and NS2 cooperatively antagonize alpha/beta interferon-induced antiviral response. J Virol 74:8234- 8242. 228. Whitehead SS, Bukreyev A, Teng MN, Firestone CY, St Claire M, Elkins WR, Collins PL, Murphy BR. 1999. Recombinant respiratory syncytial virus bearing a deletion of either the NS2 or SH gene is attenuated in chimpanzees. J Virol 73:3438-3442. 229. Jin H, Zhou H, Cheng X, Tang R, Munoz M, Nguyen N. 2000. Recombinant respiratory syncytial viruses with deletions in the NS1, NS2, SH, and M2-2 genes are attenuated in vitro and in vivo. Virology 273:210-218. 230. Teng MN, Collins PL. 1999. Altered growth characteristics of recombinant respiratory syncytial viruses which do not produce NS2 protein. J Virol 73:466- 473. 231. Teng MN, Whitehead SS, Bermingham A, St Claire M, Elkins WR, Murphy BR, Collins PL. 2000. Recombinant respiratory syncytial virus that does not express the NS1 or M2-2 protein is highly attenuated and immunogenic in chimpanzees. J Virol 74:9317-9321. 232. Goswami R, Majumdar T, Dhar J, Chattopadhyay S, Bandyopadhyay SK, Verbovetskaya V, Sen GC, Barik S. 2013. Viral degradasome hijacks mitochondria to suppress innate immunity. Cell Res 23:1025-1042. 233. Spann KM, Tran KC, Collins PL. 2005. Effects of nonstructural proteins NS1 and NS2 of human respiratory syncytial virus on interferon regulatory factor 3, NF-kappaB, and proinflammatory cytokines. J Virol 79:5353-5362. 234. Bitko V, Shulyayeva O, Mazumder B, Musiyenko A, Ramaswamy M, Look DC, Barik S. 2007. Nonstructural proteins of respiratory syncytial virus suppress

169

premature apoptosis by an NF-kappaB-dependent, interferon-independent mechanism and facilitate virus growth. J Virol 81:1786-1795. 235. Kaltschmidt B, Kaltschmidt C, Hehner SP, Droge W, Schmitz ML. 1999. Repression of NF-kappaB impairs HeLa cell proliferation by functional interference with cell cycle checkpoint regulators. Oncogene 18:3213-3225. 236. Baichwal VR, Baeuerle PA. 1997. Activate NF-kappa B or die? Curr Biol 7:R94-96. 237. Fuentes S, Tran KC, Luthra P, Teng MN, He B. 2007. Function of the respiratory syncytial virus small hydrophobic protein. J Virol 81:8361-8366. 238. Li Y, Jain N, Limpanawat S, To J, Quistgaard EM, Nordlund P, Thanabalu T, Torres J. 2015. Interaction between human BAP31 and respiratory syncytial virus small hydrophobic (SH) protein. Virology 482:105-110. 239. Kurt-Jones EA, Popova L, Kwinn L, Haynes LM, Jones LP, Tripp RA, Walsh EE, Freeman MW, Golenbock DT, Anderson LJ, Finberg RW. 2000. Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus. Nat Immunol 1:398-401. 240. Haynes LM, Moore DD, Kurt-Jones EA, Finberg RW, Anderson LJ, Tripp RA. 2001. Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus. J Virol 75:10730-10737. 241. Puthothu B, Forster J, Heinzmann A, Krueger M. 2006. TLR-4 and CD14 polymorphisms in respiratory syncytial virus associated disease. Disease Markers 22:303-308. 242. Lofgren J, Marttila R, Renko M, Ramet M, Hallman M. 2010. Toll-Like Receptor 4 Asp299Gly Polymorphism in Respiratory Syncytial Virus Epidemics. Pediatric Pulmonology 45:687-692. 243. Awomoyi AA, Rallabhandi P, Pollin TI, Lorenz E, Sztein MB, Boukhvalova MS, Hemming VG, Blanco JCG, Vogel SN. 2007. Association of TLR4 polymorphisms with symptomatic respiratory syncytial virus infection in high- risk infants and young children. Journal of Immunology 179:3171-3177. 244. Baviskar PS, Hotard AL, Moore ML, Oomens AG. 2013. The respiratory syncytial virus fusion protein targets to the perimeter of inclusion bodies and facilitates filament formation by a cytoplasmic tail-dependent mechanism. J Virol 87:10730-10741. 245. Teng MN, Collins PL. 1998. Identification of the respiratory syncytial virus proteins required for formation and passage of helper-dependent infectious particles. J Virol 72:5707-5716. 246. Mitra R, Baviskar P, Duncan-Decocq RR, Patel D, Oomens AGP. 2012. The Human Respiratory Syncytial Virus Matrix Protein Is Required for Maturation of Viral Filaments. J Virol 86:4432-4443. 247. Votteler J, Sundquist WI. 2013. Virus budding and the ESCRT pathway. Cell Host Microbe 14:232-241. 248. Bruce EA, Medcalf L, Crump CM, Noton SL, Stuart AD, Wise HM, Elton D, Bowers K, Digard P. 2009. Budding of filamentous and non-filamentous

170

influenza A virus occurs via a VPS4 and VPS28-independent pathway. Virology 390:268-278. 249. Watanabe R, Lamb RA. 2010. Influenza virus budding does not require a functional AAA+ ATPase, VPS4. Virus Res 153:58-63. 250. Utley TJ, Ducharme NA, Varthakavi V, Shepherd BE, Santangelo PJ, Lindquist ME, Goldenring JR, Crowe JE, Jr. 2008. Respiratory syncytial virus uses a Vps4-independent budding mechanism controlled by Rab11-FIP2. Proc Natl Acad Sci U S A 105:10209-10214. 251. Gosselin-Grenet AS, Marq JB, Abrami L, Garcin D, Roux L. 2007. Sendai virus budding in the course of an infection does not require Alix and VPS4A host factors. Virology 365:101-112. 252. Rossman JS, Jing X, Leser GP, Lamb RA. 2010. Influenza virus M2 protein mediates ESCRT-independent membrane scission. Cell 142:902-913. 253. Nieva JL, Madan V, Carrasco L. 2012. Viroporins: structure and biological functions. Nat Rev Microbiol 10:563-574. 254. Kochva U, Leonov H, Arkin IT. 2003. Modeling the structure of the respiratory syncytial virus small hydrophobic protein by silent-mutation analysis of global searching molecular dynamics. Protein Sci 12:2668-2674. 255. Carter SD, Dent KC, Atkins E, Foster TL, Verow M, Gorny P, Harris M, Hiscox JA, Ranson NA, Griffin S, Barr JN. 2010. Direct visualization of the small hydrophobic protein of human respiratory syncytial virus reveals the structural basis for membrane permeability. FEBS Lett 584:2786-2790. 256. Perez M, Garcia-Barreno B, Melero JA, Carrasco L, Guinea R. 1997. Membrane permeability changes induced in Escherichia coli by the SH protein of human respiratory syncytial virus. Virology 235:342-351. 257. Coates HV, Chanock RM. 1962. Experimental infection with respiratory syncytial virus in several species of animals. Am J Hyg 76:302-312. 258. Boukhvalova MS, Blanco JCG. 2013. The Cotton Rat Sigmodon Hispidus Model of Respiratory Syncytial Virus Infection. Challenges and Opportunities for Respiratory Syncytial Virus Vaccines 372:347-358. 259. Green MG, Huey D, Niewiesk S. 2013. The cotton rat (Sigmodon hispidus) as an animal model for respiratory tract infections with human pathogens. Lab Animal 42:170-176. 260. Dyer KD, Garcia-Crespo KE, Glineur S, Domachowske JB, Rosenberg HF. 2012. The Pneumonia Virus of Mice (PVM) model of acute respiratory infection. Viruses 4:3494-3510. 261. Taylor G, Stott EJ, Hughes M, Collins AP. 1984. Respiratory syncytial virus infection in mice. Infect Immun 43:649-655. 262. Cook PM, Eglin RP, Easton AJ. 1998. Pathogenesis of pneumovirus infections in mice: detection of pneumonia virus of mice and human respiratory syncytial virus mRNA in lungs of infected mice by in situ hybridization. Journal of General Virology 79:2411-2417. 263. Mestas J HC. 2004. Of mice and not men: differences between mouse and human immunology. Journal of Immunology 172:2731-2738. 171

264. Johnson JE, Gonzales RA, Olson SJ, Wright PF, Graham BS. 2007. The histopathology of fatal untreated human respiratory syncytial virus infection. Modern Pathology 20:108-119. 265. Martinez-Sobrido L, Gitiban N, Fernandez-Sesma A, Cros J, Mertz SE, Jewell NA, Hammond S, Flano E, Durbin RK, Garcia-Sastre A, Durbin JE. 2006. Protection against respiratory syncytial virus by a recombinant Newcastle disease virus vector. J Virol 80:1130-1139. 266. Anderson LJ, Bingham P, Hierholzer JC. 1988. Neutralization of respiratory syncytial virus by individual and mixtures of F and G protein monoclonal antibodies. J Virol 62:4232-4238. 267. Boyoglu-Barnum S, Todd SO, Chirkova T, Barnum TR, Gaston KA, Haynes LM, Tripp RA, Moore ML, Anderson LJ. 2015. An anti-G protein monoclonal antibody treats RSV disease more effectively than an anti-F monoclonal antibody in BALB/c mice. Virology 483:117-125. 268. Miao C, Radu GU, Caidi H, Tripp RA, Anderson LJ, Haynes LM. 2009. Treatment with respiratory syncytial virus G glycoprotein monoclonal antibody or F(ab')2 components mediates reduced pulmonary inflammation in mice. J Gen Virol 90:1119-1123. 269. Openshaw PJM, Clarke SL, Record FM. 1992. Pulmonary Eosinophilic Response to Respiratory Syncytial Virus-Infection in Mice Sensitized to the Major Surface Glycoprotein-G. Int Immunol 4:493-500. 270. Ostler T, Ehl S. 2002. A cautionary note on experimental artefacts induced by fetal calf serum in a viral model of pulmonary eosinophilia. J Immunol Methods 268:211-218. 271. Lotz MT, Peebles RS. 2012. Mechanisms of Respiratory Syncytial Virus Modulation of Airway Immune Responses. Curr Allergy Asthma Rep 12:380- 387. 272. Sacco RE, Durbin RK, Durbin JE. 2015. Animal models of respiratory syncytial virus infection and disease. Curr Opin Virol 13:117-122. 273. You D, Siefker DT, Shrestha B, Saravia J, Cormier SA. 2015. Building a better neonatal mouse model to understand infant respiratory syncytial virus disease. Respir Res 16:91. 274. Moore ML, Peebles RS, Jr. 2006. Respiratory syncytial virus disease mechanisms implicated by human, animal model, and in vitro data facilitate vaccine strategies and new therapeutics. Pharmacol Ther 112:405-424. 275. LeVine AM, Gwozdz J, Stark J, Bruno M, Whitsett J, Korfhagen T. 1999. Surfactant protein-A enhances clearance of respiratory syncytial virus in vivo. Am J Respir Crit Care Med 159:A18-A18. 276. Rudd BD, Smit JJ, Flavell RA, Alexopoulou L, Schaller MA, Gruber A, Berlin AA, Lukacs NW. 2006. Deletion of TLR3 alters the pulmonary immune environment and mucus production during respiratory syncytial virus infection. Journal of Immunology 176:1937-1942.

172

277. Durbin JE, Johnson TR, Durbin RK, Mertz SE, Morotti RA, Peebles RS, Graham BS. 2002. The role of IFN in respiratory syncytial virus pathogenesis. J Immunol 168:2944-2952. 278. Hashimoto K, Durbin JE, Zhou WS, Collins RD, Ho SB, Kolls JK, Dubin PJ, Sheller JR, Goleniewska K, O'Neal JF, Olson SJ, Mitchell D, Graham BS, Peebles RS. 2005. Respiratory syncytial virus infection in the absence of STAT1 results in airway dysfunction, airway mucus, and augmented IL-17 levels. Journal of Allergy and Clinical Immunology 116:550-557. 279. de Heer HJ, Hammad H, Soullie T, Hijdra D, Vos N, Willart MAM, Hoogsteden HC, Lambrecht BN. 2004. Essential role of lung plasmacytoid dendritic cells in preventing asthmatic reactions to harmless inhaled antigen. Journal of Experimental Medicine 200:89-98. 280. Smit JJ, Lindell DM, Boon L, Kool M, Lambrecht BN, Lukacs NW. 2008. The Balance between Plasmacytoid DC versus Conventional DC Determines Pulmonary Immunity to Virus Infections. PLoS One 3. 281. Wodnar-Filipowicz A. 2003. Flt3 ligand: Role in control of hematopoietic and immune functions of the bone marrow. News in Physiological Sciences 18:247- 251. 282. Prince GA, Jenson AB, Horswood RL, Camargo E, Chanock RM. 1978. The pathogenesis of respiratory syncytial virus infection in cotton rats. Am J Pathol 93:771-791. 283. Prince GA, Horswood RL, Camargo E, Koenig D, Chanock RM. 1983. Mechanisms of immunity to respiratory syncytial virus in cotton rats. Infect Immun 42:81-87. 284. Anonymous. 1998. Palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. The IMpact-RSV Study Group. Pediatrics 102:531- 537. 285. Niewiesk S, Prince G. 2002. Diversifying animal models: the use of hispid cotton rats (Sigmodon hispidus) in infectious diseases. Laboratory Animals 36:357-372. 286. Prince GA, Porter DD. 1976. The pathogenesis of respiratory syncytial virus infection in infant ferrets. Am J Pathol 82:339-352. 287. Buraphacheep W, Sullender WM. 1997. The guinea pig as a model for the study of maternal immunization against respiratory syncytial virus infections in infancy. Journal of Infectious Diseases 175:935-938. 288. Buchholz UJ, Finke S, Conzelmann KK. 1999. Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. J Virol 73:251-259. 289. Woolums AR, Anderson ML, Gunther RA, Schelegle ES, LaRochelle DR, Singer RS, Boyle GA, Friebertshauser KE, Gershwin LJ. 1999. Evaluation of severe disease induced by aerosol inoculation of calves with bovine respiratory syncytial virus. Am J Vet Res 60:473-480.

173

290. Sanchez-Schmitz G, Levy O. 2011. Development of Newborn and Infant Vaccines. Science Translational Medicine 3. 291. Adkins B, Leclerc C, Marshall-Clarke S. 2004. Neonatal adaptive immunity comes of age. Nature Reviews Immunology 4:553-564. 292. Kakuk TJ SK, Brideau RJ, Zaya RM, Cole SL, Zhang JY, Roberts ED, Wells PA, Wathen MW. 1993. A human respiratory syncytial virus (RSV) primate model of enhanced pulmonary pathology induced with a formalin- inactivated RSV vaccine but not a recombinant FG subunit vaccine. Journal of Infectious Disease 167:553-561. 293. de Swart RL, Kuiken T, Timmerman HH, van Amerongen G, van den Hoogen BG, Vos HW, Neijens HJ, Andeweg AC, Osterhaus ADME. 2002. Immunization of macaques with formalin-inactivated respiratory syncytial virus (RSV) induces interleukin-13-associated hypersensitivity to subsequent RSV infection. J Virol 76:11561-11569. 294. Delgado MF, Coviello S, Monsalvo AC, Melendi GA, Hernandez JZ, Batalle JP, Diaz L, Trento A, Chang HY, Mitzner W, Ravetch J, Melero JA, Irusta PM, Polack FP. 2009. Lack of antibody affinity maturation due to poor Toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease. Nat Med 15:34-41. 295. Johnson TR TM, Collins PL, Graham BS. 2004. Respiratory syncytial virus (RSV) G glycoprotein is not necessary for vaccine-enhanced disease induced by immunization with formalin-inactivated RSV. Journal of Virology 78:6024-6032. 296. Hambling MH. 1964. Survival of the Respiratory Syncytial Virus during Storage under Various Conditions. Br J Exp Pathol 45:647-655. 297. Sastre P, Oomens AGP, Wertz GW. 2007. The stability of human respiratory syncytial virus is enhanced by incorporation of the baculovirus GP64 protein. Vaccine 25:5025-5033. 298. Ausar SF, Espina M, Brock J, Thyagarayapuran N, Repetto R, Khandke L, Middaugh CR. 2007. High-throughput screening of stabilizers for respiratory syncytial virus: identification of stabilizers and their effects on the conformational thermostability of viral particles. Hum Vaccin 3:94-103. 299. Kim HW, Arrobio JO, Pyles G, Brandt CD, Camargo E, Chanock RM, Parrott RH. 1971. Clinical and immunological response of infants and children to administration of low-temperature adapted respiratory syncytial virus. Pediatrics 48:745-755. 300. Wright PF, Karron RA, Madhi SA, Treanor JJ, King JC, O'Shea A, Ikizler MR, Zhu Y, Collins PL, Cutland C, Randolph VB, Deatly AM, Hackell JG, Gruber WC, Murphy BR. 2006. The interferon antagonist NS2 protein of respiratory syncytial virus is an important virulence determinant for humans. J Infect Dis 193:573-581. 301. Belshe RB, Van Voris LP, Mufson MA. 1982. Parenteral administration of live respiratory syncytial virus vaccine: results of a field trial. J Infect Dis 145:311- 319.

174

302. Conzelmann KK. 1996. Genetic manipulation of non-segmented negative-strand RNA viruses. J Gen Virol 77 ( Pt 3):381-389. 303. Luongo C, Winter CC, Collins PL, Buchholz UJ. 2012. Increased genetic and phenotypic stability of a promising live-attenuated respiratory syncytial virus vaccine candidate by reverse genetics. J Virol 86:10792-10804. 304. Schickli JH, Kaur J, Tang RS. 2012. Nonclinical phenotypic and genotypic analyses of a Phase 1 pediatric respiratory syncytial virus vaccine candidate MEDI-559 (rA2cp248/404/1030DeltaSH) at permissive and non-permissive temperatures. Virus Res 169:38-47. 305. Reiser J, Adair B, Reinheckel T. 2010. Specialized roles for cysteine cathepsins in health and disease. Journal of Clinical Investigation 120:3421-3431. 306. Chapman HA, Riese RJ, Shi GP. 1997. Emerging roles for cysteine proteases in human biology. Annu Rev Physiol 59:63-88. 307. Gonzaleznoriega A, Grubb JH, Talkad V, Sly WS. 1980. Chloroquine Inhibits Lysosomal-Enzyme Pinocytosis and Enhances Lysosomal Enzyme-Secretion by Impairing Receptor Recycling. Journal of Cell Biology 85:839-852. 308. Ullrich K, Mersmann G, Weber E, Vonfigura K. 1978. Evidence for Lysosomal Enzyme Recognition by Human Fibroblasts Via a Phosphorylated Carbohydrate Moiety. Biochemical Journal 170:643-650. 309. Natowicz MR, Chi MM, Lowry OH, Sly WS. 1979. Enzymatic identification of mannose 6-phosphate on the recognition marker for receptor-mediated pinocytosis of beta-glucuronidase by human fibroblasts. Proc Natl Acad Sci U S A 76:4322-4326. 310. Kirschke H, Langner J, Wiederanders B, Ansorge S, Bohley P. 1977. Cathepsin L. A new proteinase from rat-liver lysosomes. Eur J Biochem 74:293- 301. 311. Gottesman MM. 1978. Transformation-dependent secretion of a low molecular weight protein by murine fibroblasts. Proc Natl Acad Sci U S A 75:2767-2771. 312. Portnoy DA, Erickson AH, Kochan J, Ravetch JV, Unkeless JC. 1986. Cloning and characterization of a mouse cysteine proteinase. J Biol Chem 261:14697-14703. 313. Mason RW, Gal S, Gottesman MM. 1987. The Identification of the Major Excreted Protein (Mep) from a Transformed Mouse Fibroblast Cell-Line as a Catalytically Active Precursor Form of Cathepsin-L. Biochemical Journal 248:449-454. 314. Troen BR, Gal S, Gottesman MM. 1987. Sequence and Expression of the Cdna for Mep (Major Excreted Protein), a Transformation-Regulated Secreted Cathepsin. Biochemical Journal 246:731-735. 315. Dong JM, Prence EM, Sahagian GG. 1989. Mechanism for Selective Secretion of a Lysosomal Protease by Transformed Mouse Fibroblasts. Journal of Biological Chemistry 264:7377-7383. 316. Prence EM, Dong JM, Sahagian GG. 1990. Modulation of the Transport of a Lysosomal-Enzyme by Pdgf. Journal of Cell Biology 110:319-326.

175

317. Ishidoh K, Kominami E. 1994. Multi-step processing of procathepsin L in vitro. FEBS Lett 352:281-284. 318. Goulet B, Baruch A, Moon NS, Poirier M, Sansregret LL, Erickson A, Bogyo M, Nepveu A. 2004. A cathepsin L isoform that is devoid of a signal peptide localizes to the nucleus in S phase and processes the CDP/Cux transcription factor. Mol Cell 14:207-219. 319. Guha S, Padh H. 2008. Cathepsins: fundamental effectors of endolysosomal proteolysis. Indian J Biochem Biophys 45:75-90. 320. Weitoft T, Larsson A, Manivel V, Lysholm J, Knight A, Ronnelid J. 2014. Cathepsin S and cathepsin L in serum and synovial fluid in rheumatoid arthritis with and without autoantibodies. Scandinavian Journal of Rheumatology 43:53- 53. 321. Roberts R. 2005. Lysosomal cysteine proteases: structure, function and inhibition of cathepsins. Drug News Perspect 18:605-614. 322. Muller S, Dennemarker J, Reinheckel T. 2012. Specific functions of lysosomal proteases in endocytic and autophagic pathways. Biochimica Et Biophysica Acta- Proteins and Proteomics 1824:34-43. 323. Victor BC, Sloane BF. 2007. Cysteine cathepsin non-inhibitory binding partners: modulating intracellular trafficking and function. Biol Chem 388:1131-1140. 324. Wille A, Gerber A, Heimburg A, Reisenauer A, Peters C, Saftig P, Reinheckel T, Welte T, Buhling F. 2004. Cathepsin L is involved in cathepsin D processing and regulation of apoptosis in A549 human lung epithelial cells. Biol Chem 385:665-670. 325. Tang Q, Cai J, Shen D, Bian Z, Yan L, Wang YX, Lan J, Zhuang GQ, Ma WZ, Wang W. 2009. Lysosomal cysteine peptidase cathepsin L protects against cardiac hypertrophy through blocking AKT/GSK3beta signaling. J Mol Med (Berl) 87:249-260. 326. Parkinson-Lawrence EJ, Shandala T, Prodoehl M, Plew R, Borlace GN, Brooks DA. 2010. Lysosomal Storage Disease: Revealing Lysosomal Function and Physiology. Physiology 25:102-115. 327. Ikeda Y, Ikata T, Mishiro T, Nakano S, Ikebe M, Yasuoka S. 2000. Cathepsins B and L in synovial fluids from patients with rheumatoid arthritis and the effect of cathepsin B on the activation of pro-urokinase. J Med Invest 47:61- 75. 328. Schurigt U, Eilenstein R, Gajda M, Leipner C, Sevenich L, Reinheckel T, Peters C, Wiederanders B, Brauer R. 2012. Decreased arthritis severity in cathepsin L-deficient mice is attributed to an impaired T helper cell compartment. Inflammation Research 61:1021-1029. 329. Kato Y, Ozawa S, Miyamoto C, Maehata Y, Suzuki A, Maeda T, Baba Y. 2013. Acidic extracellular microenvironment and cancer. Cancer Cell International 13. 330. Sudhan DR, Siemann DW. 2015. Cathepsin L targeting in cancer treatment. Pharmacol Ther doi:10.1016/j.pharmthera.2015.08.007.

176

331. Wandinger-Ness A, Zerial M. 2014. Rab proteins and the compartmentalization of the endosomal system. Cold Spring Harb Perspect Biol 6:a022616. 332. Steinman RM, Brodie SE, Cohn ZA. 1976. Membrane flow during pinocytosis. A stereologic analysis. J Cell Biol 68:665-687. 333. Brown MS, Goldstein JL. 1979. Receptor-mediated endocytosis: insights from the lipoprotein receptor system. Proc Natl Acad Sci U S A 76:3330-3337. 334. Pearse BM. 1976. Clathrin: a unique protein associated with intracellular transfer of membrane by coated vesicles. Proc Natl Acad Sci U S A 73:1255-1259. 335. Mayor S, Pagano RE. 2007. Pathways of clathrin-independent endocytosis. Nat Rev Mol Cell Biol 8:603-612. 336. Muller WA, Steinman RM, Cohn ZA. 1980. The membrane proteins of the vacuolar system. II. Bidirectional flow between secondary lysosomes and plasma membrane. J Cell Biol 86:304-314. 337. Lazarovits J, Roth M. 1988. A Single Amino-Acid Change in the Cytoplasmic Domain Allows the Influenza-Virus Hemagglutinin to Be Endocytosed through Coated Pits. Cell 53:743-752. 338. Bonifacino JS, Traub LM. 2003. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annual Review of Biochemistry 72:395-447. 339. Davis CG, Lehrman MA, Russell DW, Anderson RG, Brown MS, Goldstein JL. 1986. The J.D. mutation in familial hypercholesterolemia: amino acid substitution in cytoplasmic domain impedes internalization of LDL receptors. Cell 45:15-24. 340. Chen WJ, Goldstein JL, Brown MS. 1990. Npxy, a Sequence Often Found in Cytoplasmic Tails, Is Required for Coated Pit-Mediated Internalization of the Low-Density-Lipoprotein Receptor. Journal of Biological Chemistry 265:3116- 3123. 341. Collawn JF, Stangel M, Kuhn LA, Esekogwu V, Jing SQ, Trowbridge IS, Tainer JA. 1990. Transferrin Receptor Internalization Sequence Yxrf Implicates a Tight Turn as the Structural Recognition Motif for Endocytosis. Cell 63:1061- 1072. 342. Jadot M, Canfield WM, Gregory W, Kornfeld S. 1992. Characterization of the Signal for Rapid Internalization of the Bovine Mannose 6-Phosphate Insulin-Like Growth Factor-Ii Receptor. Journal of Biological Chemistry 267:11069-11077. 343. Canfield WM, Johnson KF, Ye RD, Gregory W, Kornfeld S. 1991. Localization of the signal for rapid internalization of the bovine cation- independent mannose 6-phosphate/insulin-like growth factor-II receptor to amino acids 24-29 of the cytoplasmic tail. J Biol Chem 266:5682-5688. 344. Williams MA, Fukuda M. 1990. Accumulation of Membrane-Glycoproteins in Lysosomes Requires a Tyrosine Residue at a Particular Position in the Cytoplasmic Tail. Journal of Cell Biology 111:955-966. 345. Hunziker W, Harter C, Matter K, Mellman I. 1991. Basolateral Sorting in Mdck Cells Requires a Distinct Cytoplasmic Domain Determinant. Cell 66:907- 920.

177

346. Rajasekaran AK, Humphrey JS, Wagner M, Miesenbock G, Lebivic A, Bonifacino JS, Rodriguezboulan E. 1994. Tgn38 Recycles Basolaterally in Polarized Madin-Darby Canine Kidney-Cells. Mol Biol Cell 5:1093-1103. 347. Johnson KF, Kornfeld S. 1992. A His-Leu-Leu sequence near the carboxyl terminus of the cytoplasmic domain of the cation-dependent mannose 6- phosphate receptor is necessary for the lysosomal enzyme sorting function. J Biol Chem 267:17110-17115. 348. Johnson KF, Kornfeld S. 1992. The cytoplasmic tail of the mannose 6- phosphate/insulin-like growth factor-II receptor has two signals for lysosomal enzyme sorting in the Golgi. J Cell Biol 119:249-257. 349. Miranda KC, Khromykh T, Christy P, Le TL, Gottardi CJ, Yap AS, Stow JL, Teasdale RD. 2001. A dileucine motif targets E-cadherin to the basolateral cell surface in Madin-Darby canine kidney and LLC-PK1 epithelial cells. J Biol Chem 276:22565-22572. 350. Matter K, Yamamoto EM, Mellman I. 1994. Structural requirements and sequence motifs for polarized sorting and endocytosis of LDL and Fc receptors in MDCK cells. J Cell Biol 126:991-1004. 351. Sandoval IV, Martinez-Arca S, Valdueza J, Palacios S, Holman GD. 2000. Distinct reading of different structural determinants modulates the dileucine- mediated transport steps of the lysosomal membrane protein LIMPII and the insulin-sensitive glucose transporter GLUT4. J Biol Chem 275:39874-39885. 352. Maxfield FR, McGraw TE. 2004. Endocytic recycling. Nat Rev Mol Cell Biol 5:121-132. 353. Wright PF, Ikizler MR, Gonzales RA, Carroll KN, Johnson JE, Werkhaven JA. 2005. Growth of respiratory syncytial virus in primary epithelial cells from the human respiratory tract. J Virol 79:8651-8654. 354. Schaap-Nutt A, Scull MA, Schmidt AC, Murphy BR, Pickles RJ. 2010. Growth restriction of an experimental live attenuated human parainfluenza virus type 2 vaccine in human ciliated airway epithelium in vitro parallels attenuation in African green monkeys. Vaccine 28:2788-2798. 355. Malkin E, Yogev R, Abughali N, Sliman J, Wang CK, Zuo F, Yang CF, Eickhoff M, Esser MT, Tang RS, Dubovsky F. 2013. Safety and immunogenicity of a live attenuated RSV vaccine in healthy RSV-seronegative children 5 to 24 months of age. PLoS One 8:e77104. 356. Diederich S, Dietzel E, Maisner A. 2009. Nipah virus fusion protein: influence of cleavage site mutations on the cleavability by cathepsin L, trypsin and furin. Virus Res 145:300-306. 357. Diederich S, Thiel L, Maisner A. 2008. Role of endocytosis and cathepsin- mediated activation in Nipah virus entry. Virology 375:391-400. 358. Brock SC, Goldenring JR, Crowe JE, Jr. 2003. Apical recycling systems regulate directional budding of respiratory syncytial virus from polarized epithelial cells. Proc Natl Acad Sci U S A 100:15143-15148.

178

359. Fulcher ML, Gabriel S, Burns KA, Yankaskas JR, Randell SH. 2005. Well- differentiated human airway epithelial cell cultures. Methods Mol Med 107:183- 206. 360. Juhasz K, Whitehead SS, Bui PT, Biggs JM, Crowe JE, Boulanger CA, Collins PL, Murphy BR. 1997. The temperature-sensitive (ts) phenotype of a cold-passaged (cp) live attenuated respiratory syncytial virus vaccine candidate, designated cpts530, results from a single amino acid substitution in the L protein. J Virol 71:5814-5819. 361. Collins PL, Hill MG, Camargo E, Grosfeld H, Chanock RM, Murphy BR. 1995. Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development. Proc Natl Acad Sci U S A 92:11563-11567. 362. Walsh EE, Hall CB, Schlesinger JJ, Brandriss MW, Hildreth S, Paradiso P. 1989. Comparison of antigenic sites of subtype-specific respiratory syncytial virus attachment proteins. J Gen Virol 70 ( Pt 11):2953-2961. 363. Walsh EE, Falsey AR, Sullender WM. 1998. Monoclonal antibody neutralization escape mutants of respiratory syncytial virus with unique alterations in the attachment (G) protein. J Gen Virol 79 ( Pt 3):479-487. 364. Mason RW, Green GD, Barrett AJ. 1985. Human liver cathepsin L. Biochem J 226:233-241. 365. Chauhan SS, Popescu NC, Ray D, Fleischmann R, Gottesman MM, Troen BR. 1993. Cloning, genomic organization, and chromosomal localization of human cathepsin L. J Biol Chem 268:1039-1045. 366. Gal S, Gottesman MM. 1988. Isolation and sequence of a cDNA for human pro- (cathepsin L). Biochem J 253:303-306. 367. Joseph LJ, Chang LC, Stamenkovich D, Sukhatme VP. 1988. Complete nucleotide and deduced amino acid sequences of human and murine preprocathepsin L. An abundant transcript induced by transformation of fibroblasts. J Clin Invest 81:1621-1629. 368. Ritonja A, Popovic T, Kotnik M, Machleidt W, Turk V. 1988. Amino acid sequences of the human kidney cathepsins H and L. FEBS Lett 228:341-345. 369. Ishidoh K, Kominami E. 1995. Procathepsin L degrades extracellular matrix proteins in the presence of glycosaminoglycans in vitro. Biochem Biophys Res Commun 217:624-631. 370. Nomura T, Fujisawa Y. 1997. Processing properties of recombinant human procathepsin L. Biochem Biophys Res Commun 230:143-146. 371. Salminen A, Gottesman MM. 1990. Inhibitor studies indicate that active cathepsin L is probably essential to its own processing in cultured fibroblasts. Biochem J 272:39-44. 372. Nishimura Y, Kawabata T, Furuno K, Kato K. 1989. Evidence that aspartic proteinase is involved in the proteolytic processing event of procathepsin L in lysosomes. Arch Biochem Biophys 271:400-406.

179

373. Roberts SR, Lichtenstein D, Ball LA, Wertz GW. 1994. The membrane- associated and secreted forms of the respiratory syncytial virus attachment glycoprotein G are synthesized from alternative initiation codons. J Virol 68:4538-4546. 374. Escribano-Romero E, Rawling J, Garcia-Barreno B, Melero JA. 2004. The soluble form of human respiratory syncytial virus attachment protein differs from the membrane-bound form in its oligomeric state but is still capable of binding to cell surface proteoglycans. J Virol 78:3524-3532. 375. Julenius K, Molgaard A, Gupta R, Brunak S. 2005. Prediction, conservation analysis, and structural characterization of mammalian mucin-type O- glycosylation sites. Glycobiology 15:153-164. 376. Puzer L, Cotrin SS, Alves MF, Egborge T, Araujo MS, Juliano MA, Juliano L, Bromme D, Carmona AK. 2004. Comparative substrate specificity analysis of recombinant human cathepsin V and cathepsin L. Arch Biochem Biophys 430:274-283. 377. Olson JK, Grose C. 1997. Endocytosis and recycling of varicella-zoster virus Fc receptor glycoprotein gE: internalization mediated by a YXXL motif in the cytoplasmic tail. J Virol 71:4042-4054. 378. Abdel Motal UM, Sentman CL, Zhou X, Robinson PJ, Dahmen J, Jondal M. 1995. Glycosylphosphatidylinositol-linked Db does not induce an influenza- specific cytotoxic T lymphocyte response or recycle membrane-bound peptides. Eur J Immunol 25:1121-1124. 379. Hedman K, Goldenthal KL, Rutherford AV, Pastan I, Willingham MC. 1987. Comparison of the intracellular pathways of transferrin recycling and vesicular stomatitis virus membrane glycoprotein exocytosis by ultrastructural double-label cytochemistry. J Histochem Cytochem 35:233-243. 380. Tirabassi RS, Enquist LW. 1998. Role of envelope protein gE endocytosis in the pseudorabies virus life cycle. J Virol 72:4571-4579. 381. Olson JK, Grose C. 1998. Complex formation facilitates endocytosis of the varicella-zoster virus gE:gI Fc receptor. J Virol 72:1542-1551. 382. Chen W, Feng Y, Chen D, Wandinger-Ness A. 1998. Rab11 is required for trans-golgi network-to-plasma membrane transport and a preferential target for GDP dissociation inhibitor. Mol Biol Cell 9:3241-3257. 383. Vogt C, Eickmann M, Diederich S, Moll M, Maisner A. 2005. Endocytosis of the Nipah virus glycoproteins. J Virol 79:3865-3872. 384. Moll M, Diederich S, Klenk HD, Czub M, Maisner A. 2004. Ubiquitous activation of the Nipah virus fusion protein does not require a basic amino acid at the cleavage site. J Virol 78:9705-9712. 385. Rowe RK, Suszko JW, Pekosz A. 2008. Roles for the recycling endosome, Rab8, and Rab11 in hantavirus release from epithelial cells. Virology 382:239- 249. 386. Beitia Ortiz de Zarate I, Cantero-Aguilar L, Longo M, Berlioz-Torrent C, Rozenberg F. 2007. Contribution of endocytic motifs in the cytoplasmic tail of

180

herpes simplex virus type 1 glycoprotein B to virus replication and cell-cell fusion. J Virol 81:13889-13903. 387. Krzyzaniak MA, Mach M, Britt WJ. 2009. HCMV-encoded glycoprotein M (UL100) interacts with Rab11 effector protein FIP4. Traffic 10:1439-1457. 388. Bruce EA, Digard P, Stuart AD. 2010. The Rab11 pathway is required for influenza A virus budding and filament formation. J Virol 84:5848-5859. 389. Zenner HL, Yoshimura S, Barr FA, Crump CM. 2011. Analysis of Rab GTPase-activating proteins indicates that Rab1a/b and Rab43 are important for herpes simplex virus 1 secondary envelopment. J Virol 85:8012-8021. 390. Nakatsu Y, Ma X, Seki F, Suzuki T, Iwasaki M, Yanagi Y, Komase K, Takeda M. 2013. Intracellular transport of the measles virus ribonucleoprotein complex is mediated by Rab11A-positive recycling endosomes and drives virus release from the apical membrane of polarized epithelial cells. J Virol 87:4683- 4693. 391. Diederich S, Sauerhering L, Weis M, Altmeppen H, Schaschke N, Reinheckel T, Erbar S, Maisner A. 2012. Activation of the Nipah virus fusion protein in MDCK cells is mediated by cathepsin B within the endosome-recycling compartment. J Virol 86:3736-3745. 392. Diederich S, Moll M, Klenk HD, Maisner A. 2005. The nipah virus fusion protein is cleaved within the endosomal compartment. J Biol Chem 280:29899- 29903. 393. Trento A, Galiano M, Videla C, Carballal G, Garcia-Barreno B, Melero JA, Palomo C. 2003. Major changes in the G protein of human respiratory syncytial virus isolates introduced by a duplication of 60 nucleotides. J Gen Virol 84:3115- 3120. 394. de-Paris F, Beck C, de Souza Nunes L, Machado AB, Paiva RM, da Silva Menezes D, Pires MR, dos Santos RP, de Souza Kuchenbecker R, Barth AL. 2014. Evaluation of respiratory syncytial virus group A and B genotypes among nosocomial and community-acquired pediatric infections in Southern Brazil. Virol J 11:36. 395. Choudhary ML, Anand SP, Wadhwa BS, Chadha MS. 2013. Genetic variability of human respiratory syncytial virus in Pune, Western India. Infect Genet Evol 20:369-377. 396. Visser A, Delport S, Venter M. 2008. Molecular epidemiological analysis of a nosocomial outbreak of respiratory syncytial virus associated pneumonia in a kangaroo mother care unit in South Africa. J Med Virol 80:724-732. 397. Zhang ZY, Du LN, Chen X, Zhao Y, Liu EM, Yang XQ, Zhao XD. 2010. Genetic variability of respiratory syncytial viruses (RSV) prevalent in Southwestern China from 2006 to 2009: emergence of subgroup B and A RSV as dominant strains. J Clin Microbiol 48:1201-1207. 398. Pretorius MA, van Niekerk S, Tempia S, Moyes J, Cohen C, Madhi SA, Venter M, Group SS. 2013. Replacement and positive evolution of subtype A and B respiratory syncytial virus G-protein genotypes from 1997-2012 in South Africa. J Infect Dis 208 Suppl 3:S227-237. 181

399. Tran DN, Pham TM, Ha MT, Tran TT, Dang TK, Yoshida LM, Okitsu S, Hayakawa S, Mizuguchi M, Ushijima H. 2013. Molecular epidemiology and disease severity of human respiratory syncytial virus in Vietnam. PLoS One 8:e45436. 400. Auksornkitti V, Kamprasert N, Thongkomplew S, Suwannakarn K, Theamboonlers A, Samransamruajkij R, Poovorawan Y. 2014. Molecular characterization of human respiratory syncytial virus, 2010-2011: identification of genotype ON1 and a new subgroup B genotype in Thailand. Arch Virol 159:499- 507. 401. Tabatabai J, Prifert C, Pfeil J, Grulich-Henn J, Schnitzler P. 2014. Novel respiratory syncytial virus (RSV) genotype ON1 predominates in Germany during winter season 2012-13. PLoS One 9:e109191. 402. Pierangeli A, Trotta D, Scagnolari C, Ferreri ML, Nicolai A, Midulla F, Marinelli K, Antonelli G, Bagnarelli P. 2014. Rapid spread of the novel respiratory syncytial virus A ON1 genotype, central Italy, 2011 to 2013. Euro Surveill 19. 403. Weis M, Maisner A. 2015. Nipah virus fusion protein: Importance of the cytoplasmic tail for endosomal trafficking and bioactivity. Eur J Cell Biol 94:316- 322. 404. Wu XS, McNeil BD, Xu JH, Fan JM, Xue L, Melicoff E, Adachi R, Bai L, Wu LG. 2010. Ca2+ and calmodulin initiate all forms of endocytosis during depolarization at a nerve terminal (vol 12, pg 1003, 2009). Nature Neuroscience 13:649-649. 405. MacDonald PE, Eliasson L, Rorsman P. 2005. Calcium increases endocytotic vesicle size and accelerates membrane fission in insulin-secreting INS-1 cells. J Cell Sci 118:5911-5920. 406. Grant BD, Donaldson JG. 2009. Pathways and mechanisms of endocytic recycling. Nature Reviews Molecular Cell Biology 10:597-608. 407. Reiche J, Schweiger B. 2009. Genetic variability of group A human respiratory syncytial virus strains circulating in Germany from 1998 to 2007. J Clin Microbiol 47:1800-1810. 408. Rebuffo-Scheer C, Bose M, He J, Khaja S, Ulatowski M, Beck ET, Fan J, Kumar S, Nelson MI, Henrickson KJ. 2011. Whole genome sequencing and evolutionary analysis of human respiratory syncytial virus A and B from Milwaukee, WI 1998-2010. PLoS One 6:e25468. 409. Eshaghi A, Duvvuri VR, Lai R, Nadarajah JT, Li A, Patel SN, Low DE, Gubbay JB. 2012. Genetic variability of human respiratory syncytial virus A strains circulating in Ontario: a novel genotype with a 72 nucleotide G gene duplication. PLoS One 7:e32807.

182