IDENTIFICATION OF A VIRAL DETERMINANT OF VIRULENCE IN ROSS RIVER VIRUS THAT

MODULATES VIRAL REPLICATION AND FITNESS IN DISPARATE HOSTS

by

HENRI J JUPILLE

B.S., Colorado Mesa University, 2005

A thesis submitted to the

Faculty of the Graduate School of the

University of Colorado in partial fulfillment

of the requirements for the degree of

Doctor of Philosophy

Microbiology Program

2013

This thesis for the Doctor of Philosophy degree by

Henri J. Jupille

has been approved for the

Microbiology Program

by

Dave Barton, Chair

Thomas Morrison, Advisor

Kathryn Holmes

Caroline Kulesza

David Beckham

Date: 08/08/13

ii

Jupille, Henri J. (Ph.D., Microbiology)

Identification of a viral determinant of virulence in Ross River virus that modulates viral replication and fitness in disparate hosts.

Thesis directed by Assistant Professor Thomas Morrison.

ABSTRACT

Alphaviruses such as Ross River virus (RRV) and chikungunya virus cause debilitating and often chronic rheumatic disease in humans and are responsible for explosive epidemics which cause millions of cases of musculoskeletal inflammatory disease. Despite these outbreaks, the pathogenesis of these viruses is poorly understood. Our studies identified an RRV strain (DC5692) which, in contrast to the T48 strain, failed to cause musculoskeletal inflammation in mice. Using chimeric virus studies based on the DC5692 and T48 strains, we identified unique virulence determinants within both the nsP1 and PE2 coding regions responsible for attenuation.

Further study of these chimeric viruses showed that the specific mechanism of attenuation was different for the mutations in nsP1 and PE2, suggesting that they affect different aspects of the viral replication cycle. Through further study of the PE2 region, we identified a tyrosine (Y) to histidine (H) mutation at E2 position 18 (E2 Y18H) as a major attenuating mutation in mice. In vitro characterization studies showed that the E2

Y18H mutation caused a cell-type specific replication and fitness defect whereby a tyrosine conferred a large fitness advantage in mammalian cells, leading to enhanced viral replication in these cell types. In contrast, we showed a histidine at E2 position 18 iii

conferred a fitness and replication advantage in mosquito cells. Additional studies showed that the E2 Y18H mutation increased release of non-infectious virions from mammalian cells.

Taken together, these studies represent the first published reports of novel viral determinants of alphavirus-induced musculoskeletal disease located within the nsP1 and PE2 coding regions. Furthermore, the identification of a tyrosine or histidine residue at E2 position 18 in all alphaviruses in the Semliki Forest antigenic complex represents the first time a naturally occurring virulence determinant has been identified within the

N-flap domain of E2. Subsequent studies showing that the residue at position 18 affects viral fitness suggest that it may be acting as a “switching residue” during replication in disparate hosts. Finally, identification of a late stage specific replication defect in mammalian cells suggests an important and novel role for the N-flap domain of E2 during the assembly of alphavirus virions.

The form and content of this abstract are approved. I recommend its publication.

Approved: Thomas Morrison

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DEDICATION

I dedicate this work to my wife Marybeth, who has been an unwavering source of love and support throughout this entire process. Additionally, I would also like to dedicate this work to my parents who have supported me fully on my journey to this point in my life. I cannot imagine doing this without you all and am forever grateful for all the love and support you have given me over the years.

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ACKNOWLEDGMENTS

I would first like to thank the members of my committee for their many insightful questions and helpful advice throughout my research progression. I would also like to thank all past and present members of the Morrison Lab for all their assistance. I would like to extend special thanks to my mentor Dr. Morrison for allowing me the opportunity to join his lab, and for helping to train me to become a successful scientist. Without his patience and guidance, I would not be where I am today. I thank the many collaborators whose contributions allowed my research to move forward.

Specifically, I would like to thank Drs. John Aaskov and Michael Rossmann for providing the anti-E2 monoclonal antibody. I would also like to acknowledge the funding sources at the National Institutes of Health. Finally, I would like to acknowledge the students, faculty and staff of the Microbiology Department for all their guidance and support over the years.

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TABLE OF CONTENTS

CHAPTER

I. INTRODUCTION ...... 1

The Alphaviruses ...... 1

The Arthritogenic Alphaviruses...... 5

Chikungunya Virus...... 5

O’nyong-nyong Virus...... 7

Ross River Virus...... 8

Sindbis Virus...... 8

Mayaro Virus...... 9

Rheumatic Disease in Humans During Alphavirus Infection ...... 10

Alphavirus Pathogenesis ...... 13

Animal Models of Alphavirus-induced Rheumatic Disease ...... 16

Non-Human Primate Model of CHIKV Infection...... 16

Mouse Model of CHIKV Pathogenesis in IFN Deficient Mice ...... 17

Other Animal Models...... 17

Mouse Model of RRV- and CHIKV-induced Rheumatic Disease in Mice...... 18

Alphavirus Virions ...... 18

Alphavirus Genome...... 19

Alphavirus Gene Products and Functions ...... 23

Nonstructural Polyprotein Translation and Processing...... 23

nsP1...... 24

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nsP2...... 24

nsP3...... 25

nsP4...... 26

Structural Polyprotein Translation and Processing...... 27

Capsid...... 28

PE2...... 29

E1...... 31

6K...... 31

The Alphavirus Replication Cycle ...... 32

Alphavirus Entry and Fusion Process...... 32

Replication of Alphavirus RNA and Nonstructural Protein Processing...... 33

Alphavirus Structural Protein Translation and Virion Assembly...... 35

Alphavirus Determinants of Virulence ...... 38

Overview of Dissertation ...... 39

II. MATERIALS AND METHODS ...... 42

Viruses ...... 42

Construction of Virus cDNAs ...... 43

Site Directed Mutagenesis ...... 45

Cells ...... 46

RNA Infectivity Assays ...... 47

In Vitro Nonstructural Protein Processing ...... 47

Western Blots ...... 48

In Vitro Virus Replication ...... 48

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Competition Assays ...... 49

Protein Expression Kinetics Assay ...... 49

PFU Per Cell Assay ...... 50

Fusion-infection Assay ...... 50

Analysis of E2 Surface Expression ...... 51

Plaque Assays ...... 51

Mouse Experiments ...... 52

Quantification of Viral RNA ...... 53

Histological Analysis ...... 54

pH Stability Assay ...... 54

Statistical Analysis ...... 54

III. MUTATIONS IN NSP1 AND PE2 ARE CRITICAL DETERMINANTS OF ROSS RIVER VIRUS- INDUCED MUSCULOSKELETAL INFLAMMATORY DISEASE IN A MOUSE MODEL .. 56

Introduction ...... 56

Results ...... 58

RRV Strain DC5692 and Virus Derived from a Molecular Clone of RRV Strain DC5692 (RR87) Replicate like the T48 Strain in Vero Cells, but do not Cause Musculoskeletal Inflammatory Disease in Mice...... 58

A Chimeric Virus that Encodes the RRV DC5692 3` UTR in the Genetic Background of the T48 Strain (RR67) Caused Disease in Mice that was Indistinguishable from RRV T48 (RR64)- Induced Disease...... 62

Chimeric Viruses that Encode the RRV DC5692 nsP1 Coding Region in the T48 Strain Genetic Background are Attenuated In Vivo...... 64

Chimeric Viruses that Encode the RRV DC5692 PE2 Coding Region in the T48 Genetic Background are Attenuated In Vivo...... 66

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Attenuating Determinants in PE2, but not nsP1 Regulate Viral Tissue Titers in Mice...... 69

A Chimeric Virus that Encodes the RRV T48 Strain nsP1 and PE2 Coding Regions in the RRV DC5692 Genetic Background Caused Disease in Mice that was Indistinguishable from RRV T48 (RR64)- Induced Disease...... 73

Discussion ...... 74

IV. A TYROSINE TO HISTIDINE SUBSTITUTION AT POSITION 18 OF THE ROSS RIVER VIRUS E2 GLYCOPROTEIN IS A DETERMINANT OF VIRUS FITNESS IN DISPARATE HOSTS ...... 82

Introduction ...... 82

Results ...... 85

A Single Mutation in RRV, E2 Y18H, Results in Severe Attenuation in the Mouse Model...... 85

E2 Y18H Diminishes RRV Replication and Spread in Mice...... 87

E2 Y18H Affects RRV Replication in a Cell-Type Dependent Manner...... 88

The Amino Acid at RRV E2 Position 18 is a Determinant of Virus Fitness in Cells. .... 96

Additional Mutations at E2 Position 18 are Also Attenuating Mutations in Mice. .... 99

Discussion ...... 100

A Tyrosine at Position 18 is Advantageous in Mammalian Cells, Whereas a Histidine at Position 18 is Advantageous in Mosquito Cells...... 105

V. A TYROSINE TO HISTIDINE SUBSTITUTION AT RRV E2 POSITION 18 CAUSES A DEFECT IN THE LATE STAGE OF VIRUS REPLICATION IN MAMMALIAN CELLS, BUT NOT MOSQUITO CELLS ...... 108

Introduction ...... 108

Results ...... 109

Molecular Modeling of E2 position 18 within the glycoprotein spike complex...... 109

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The Amino Acid Residue at E2 Position 18 Does Not Impact pH Required for Viral Fusion...... 110

Low pH Treatment Enhances Entry of E2 Y18H Virus into BHK Cells...... 112

The E2 Y18H Mutation Does not Impact RRV Gene Expression or Protein Trafficking...... 114

A Tyrosine to Histidine Mutation at E2 Position 18 Causes Altered Cell to Cell Spread...... 115

A Tyrosine to Histidine Mutation at E2 Position 18 Causes a Late Stage Replication Defect in Mammalian Cells, but not Mosquito Cells...... 116

A Tyrosine to Histidine Mutation at E2 Position 18 Causes Release of Noninfectious Viral Particles from Mammalian Cells...... 119

Discussion ...... 121

VI. DISCUSSION AND FUTURE DIRECTIONS...... 125

Summary of Findings ...... 125

Amino Acid Substitutions Near the N-Terminus of E2 Can Affect Numerous Aspects of Viral Replication ...... 127

Conserved Cysteine Residues in the Nflap Domain Play Critical Roles During Alphavirus Assembly/Budding ...... 128

Mutations in Other Regions of E2 ...... 129

Alternating Replication of Alphaviruses in two hosts. Mammals vs. Mosquitos ...... 130

Genetic Pressure of Dual Hosts...... 130

Differences in Alphavirus Replication in Mammalian and Mosquito Cells...... 132

Location of E2 Position 18 Within the E1/E2 Dimer and Trimeric Spike ...... 135

Differences in Tyrosine or Histidine at Physiological pH ...... 137

Model for Role of E2 Position 18 During Replication in Mammalian Cells ...... 138

Proposed Role for E2 Position 18 During Replication in Mosquito Cells ...... 140

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Future Directions ...... 141

Mosquito Experiments...... 141

Mammalian Experiments...... 144

Summary of Findings ...... 146

REFERENCES ...... 149

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LIST OF TABLES

Table

1.1 Amino Acid Differences between RRV T48 (RR64) and RRV DC5692...... 61

4.1 Amino Acid Residue at Position 18 (or Equivalent) of the Alphavirus E2 Glycoprotein...... 90

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LIST OF FIGURES

Figure

1.1 Global Distribution of Alphaviruses and Recent Epidemics...... 6

1.2 Joint Manifestations of CHIKV Disease in Humans ...... 11

1.3 Mouse Model of RRV-Induced Musculoskeletal Disease...... 21

1.4 Alphavirus Virion Structure and Genome Organization ...... 22

1.5 Alphavirus Replication Cycle ...... 37

3.1 Multi-step Replication Analysis ...... 60

3.2 The RRV DC5692 Strain and Virus Derived from a Molecular Clone of the DC5692 Strain (RR87) are Attenuated in vivo...... 63

3.3 A Chimeric Virus that Encodes the RRV DC5692 3`UTR in the T48 Genetic Background (RR67) Caused Disease in Mice that was Indistinguishable from RRV-T48-induced Disease...... 65

3.4 Chimeric Viruses that Encode the RRV DC5692 nsP1 Coding Region in the T48 Strain Genetic Background are Attenuated in vivo...... 67

3.5 Chimeric Viruses that Encode the RRV DC5692 PE2 Coding Region in the T48 Genetic Background are Attenuated in vivo...... 70

3.6 A Small Plaque Phenotype on BHK-21 Cells Correlates with Attenuation in Vivo ...... 71

3.7 Attenuating Determinants in PE2, but not nsP1, Regulate Viral Tissue titers in Mice...... 72

3.8 Substitution of the DC5692 Strain nsP1 and PE2 Coding Regions with Those from the T48 Strain is Sufficient to Enhance the Virulence of the DC5692 Strain...... 75

4.1 RRV E2 Y18H is an Attenuating Mutation in a Mouse Model ...... 89

4.2 The E2 Y18H Mutation Reduces RRV Tissue Titers and Spread ...... 95

4.3 The E2 Y18H Mutation Affects RRV Replication in a Cell Type-Dependent Manner ...... 98

4.4 The E2 Y18H Mutation Affects RRV Replication in a Cell Type-Dependent Manner ...... 99 xiv

4.5 The Amino Acid at RRV Position 18 Toggles RRV Fitness in Disparate Host Cells ...... 101

4.6 A Tyrosine at E2 Position 18 is Required for Virulence in Mice and Enhanced Replication in Mammalian Cells ...... 102

5.1 Location of E2 Residue 18 Within the Virion ...... 111

5.2 pH of Fusion for RRV-T48 and E2 Y18H Mutant Virus in BHK and C2C12 Cells ...... 112

5.3 Acidic pH Treatment Increases Infectivity of E2 Y18H Mutant Virus ...... 115

5.4 The E2 Y18H Mutation does not Affect the Kinetics or Magnitude of RRV Structural Gene Expression ...... 116

5.5 The E2 Y18H Mutation does not Affect Trafficking of RRV Glycoproteins to the Plasma Membrane ...... 117

5.6 The E2 Y18H Mutation Affects the Rate of Cell to Cell Spread ...... 118

5.7 The E2 Y18H Mutation Impacts a Late Stage of the RRV Replication Cycle in Mammalian Cells ...... 120

5.8 The E2 Y18H Mutation Reduces the Infectivity of RRV in a Cell-type Dependent Manner. . 124

6.1 Proposed Model of RRV Replication in Mammalian Cells ...... 145

6.2 Proposed Model of RRV Replication in Mosquito Cells and Impact of E2 Y18H Mutation ... 146

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LIST OF ABBREVIATIONS

A: Alanine AA: Amino Acid BFV: Barmah Forest virus BHK: Baby Hamster Kidney CHIKV: Chikungunya virus CPV: Cytopathic Vacuole D: Aspartic Acid DALY: Disability Adjusted Life Year DPI: Days Post Infection EEEV: Eastern Equine Encephalitis virus ER: Endoplasmic Reticulum F: Phenylalanine GFP: Green Fluorescent Protein H: Histidine HPI: Hours Post Infection HS: Heparan Sulfate IFN: Interferon K: Lysine MAYV: Mayaro Virus MOI: multiplicity of infection NSAID: Non-steroidal anti-inflammatory drug NSP: nonstructural protein nt: nucleotide ONNV: o’nyong-nyong virus PBS: phosphate buffered saline PFU: Plaque Forming Unit RRV: Ross River virus SDS-PAGE: sodium dodecyl sulfate poly acrylamide gel electrophoresis SF: Semliki Forest SINV: Sindbis Virus TGN: Trans-Golgi Network VEEV: Venezuelan Equine Encephalitis virus WEEV: Western Equine Encephalitis virus WT: Wild Type Y: Tyrosine

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CHAPTER I

INTRODUCTION

The Alphaviruses

Alphaviruses (genus Alphavirus, family Togaviridae), are a group of enveloped, single-strand positive sense RNA viruses with 30 recognized species distributed worldwide [1]. A defining characteristic of several alphaviruses is the capacity to cause explosive epidemics of human disease, spreading the virus to new non-endemic regions, making them a critical global public health threat [2-13]. While the majority of alphavirus species have been isolated from terrestrial vertebrate and arthropod species, at least two alphavirus species have been identified which infect aquatic vertebrate hosts and arthropod vectors.

The Alphaviruses are maintained in nature via alternating transmission between various vertebrate hosts and invertebrate vector species. Among terrestrial alphaviruses, the Culex, and Aedes species mosquitoes act as the major vectors, while a specific vector has not been identified in transmission of aquatic alphaviruses. Isolation of aquatic alphaviruses from various species of sea louse however, support their role as invertebrate vectors [14].

In humans, alphavirus infection can result in either a severe, potentially fatal encephalitic disease, or an intense, debilitating, and often chronic arthritogenic disease.

Early studies attempted to associate disease with viral geographic distribution with some success. The New World alphaviruses such as Western Equine Encephalitis virus

1

(WEEV), Eastern Equine Encephalitis virus (EEEV), and Venezuelan Equine Encephalitis virus (VEEV) typically cause encephalitic disease, while the Old World alphaviruses such as chikungunya virus (CHIKV), Ross River virus (RRV) Sindbis virus (SINV), and o’nyong- nyong virus (ONNV) virus are associated with arthritogenic disease [15]. Recent studies have shown that using geographical distribution alone is not 100% accurate however, as

Mayaro virus (MAYV) causes arthritogenic disease despite being found only in the

Americas [16]. In aquatic vertebrate species, alphavirus infection has been shown in a laboratory setting to cause a sleeping sickness in Rainbow Trout, followed by development of lesions in the pancreas, heart, and muscle tissue [17]. Additionally, studies investigating Atlantic salmon populations showed that infection caused pancreatic disease in farmed salmon populations [18].

Terrestrial alphaviruses have been further classified from New or Old World into

7 distinct antigenic complexes determined by antibody cross-reactivity to viral antigens.

Recent genomic analyses of both terrestrial and aquatic alphaviruses showed that the alphavirus genus likely arose in an aquatic environment in the Southern Oceans before a transmission event led to a terrestrial replication cycle in Oceana [19]. The Old World alphaviruses then spread from Oceana to Africa and subsequently to the rest of the

Australasian region [19]. Phylogenetic analyses of multiple alphavirus species suggests that an Old World to New World transmission event likely introduced an ancestral alphavirus to the Americas, which has subsequently evolved to contain the viruses within the Western, Eastern, and Venezuelan Equine Encephalitis antigenic complexes

2

[20]. Interestingly, additional trans-oceanic transmission events are supported by phylogenetic analysis. Specifically, MAYV is found only in the New World, however infection in human populations results in a disease very similar to the arthritogenic alphaviruses. Upon phylogenetic analysis, MAYV was found to be more genetically similar to CHIKV and RRV, than any of the New World alphaviruses. Additional genetic analyses showed that the evolution of WEEV was the likely result of genomic recombination, with WEEV expressing nonstructural and capsid genes of an EEEV-like ancestor, combined with the E1 and E2 glycoprotein genes from a Sindbis (SINV)-like ancestor [20].

The arthritogenic alphaviruses are typically transmitted by various Aedes and

Culex species mosquitos which infect vertebrates during a blood meal. Infected vertebrates then spread the virus to naïve mosquitoes, again during a blood meal. Both

New and Old World alphaviruses have been isolated from numerous avian and mammalian species, suggesting that these viruses can productively infect a wide variety of host species [2, 21]. Despite the diversity of vertebrate hosts, many of the alphaviruses are thought to have major amplifying hosts in which the virus reaches the highest titer and may be asymptomatic. Studies with several alphaviruses have shown that the major amplifying host may vary based upon geographical location. In studies investigating RRV, it was shown that while marsupials serve as the best amplifying host for RRV, the marsupial populations are significantly smaller in regions near large cities

3

where seasonal human disease is common. This suggests that another mammalian species may be serving as the amplifying host in these areas [2]. In the case of SINV, it is thought that bird populations may function as the reservoir hosts in Northern Europe

[22], while CHIKV has traditionally been associated with transmission between non- human primates and mosquitoes [23]. During the 2004- 2011 CHIKV epidemic, the virus spread via a human-mosquito-human transmission cycle which allowed the virus to greatly expand its geographical range throughout the Indian Ocean Region [24] and beyond with the first reports of autochthonous transmission reported in Italy in 2009

[25] and France in 2010 [26].

In viruses which cause human disease, viral genetic determinants play critical roles in regulating the ability of a given virus to infect a given vector species [27-30].

Because productive viral replication in both vertebrate and invertebrate hosts is controlled by genetic determinants within the viral genome, mutations within the viral genome may impact viral fitness in one or more host species. Because of the dual host lifecycle, the alphavirus genome is under significant selective pressure, whereby mutations which enhance viral replication in one host, can potentially have a negative impact on replication in the alternate host. This pressure constrains alphavirus evolution to some extent, as the virus must balance replicative efficiency within two very different host systems.

4

The Arthritogenic Alphaviruses

The majority of alphavirus-induced rheumatic disease in human populations has been caused by CHIKV, RRV, MAYV, SINV, ONNV, and Barmah Forest virus (BFV). While these viruses have a broad geographic distribution, they have been shown to cause explosive epidemics which can spread the virus to new non-endemic regions (Figure

1.1). These epidemics have involved millions of cases of human disease, and had a significant impact on the populations affected. The disease burden of CHIKV in India during 2006 alone was estimated to be over 25,000 disability-adjusted life years (DALYs) from a suspected 1.39 million cases of CHIKV disease [31], highlighting the severity of disease and impact on local populations.

The globalization of our economies, along with increased international trade and travel, has only served to increase the public health threat posed by these viruses. As changing climates allow the vector species to increase their geographical distribution, some alphaviruses have the potential to spread globally as evidenced by small outbreaks of locally acquired CHIKV disease reported in Italy and France in recent years

[25, 26]. Additionally, imported cases of CHIKV have now been reported in at least 40 countries [32-36], highlighting the potential for CHIKV, and potentially other alphaviruses to become true global pathogens.

Chikungunya Virus. CHIKV was first isolated in 1952 during an outbreak of rheumatic disease in Tanganyika (what is present day Tanzania) [37]. Because of the symptoms of the disease described by patients, Robinson et al. characterized CHIKV

5

Figure 1.1 Global Distribution of Alphaviruses and Recent Epidemics. Approximate geographical location of disease outbreaks associated with arthritogenic alphaviruses. Shaded areas represent regions with endemic disease while dashed lines represent explosive epidemics of CHIKV and RRV. disease as “clinically indistinguishable from dengue” [37]. Because of the similarity of symptoms, there is the potential that CHIKV disease has been misdiagnosed as Dengue fever, perhaps as far back as the late 1700s [38]. Since 1952, sporadic epidemics have occurred, primarily affecting populations in Africa and Asia with the virus disappearing after each epidemic for approximately 7-8 years, or in some cases 2-3 decades [39-42].

In Africa, it is thought that CHIKV is maintained in a sylvatic cycle involving non-human primates while in Asia it is thought that humans serve as the primary host, and that outbreaks occur in more urban environments due to the urban habitat of the viral vector Aedes aegypti [43]. In 2004, epidemic CHIKV disease appeared in human

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populations on several islands in the Indian Ocean region, originating from an East

African strain [44]. As the epidemic continued and the virus spread, a single amino acid mutation A226V, arose in the E1 gene of some CHIKV strains which conferred the ability to productively infect and be transmitted by Aedes albopictus mosquitoes without compromising the virus’ ability to infect its original vector Aedes aegypti [29]. The broad geographical distribution of Aedes albopictus [45] enabled the virus to quickly spread throughout the Indian Ocean region, while the presence of Aedes aegypti enabled the virus to spread rapidly in urban environments within India resulting in approximately 6 million cases of human disease throughout the Indian Ocean region between 2004 and

2011 [46]. As discussed above, associated with this epidemic were the first reports of locally acquired CHIKV in Europe [25, 26], suggesting that there is the potential for another large scale explosive epidemic.

O’nyong-nyong Virus. ONNV is found in Africa, and has been responsible for several large outbreaks of human disease. The largest of these outbreaks occurred between 1959 and 1961 in Uganda and involved over 2 million cases of alphavirus- induced rheumatic disease [8, 47]. More recently, smaller epidemics have also been reported involving approximately 400 cases [8]. While ONNV is genetically distinct from other alphaviruses it is more closely related to CHIKV than to other members of the genus. Recent studies used monoclonal antibody inhibition assays to show that while

CHIKV-specific antibodies strongly neutralized ONNV, ONNV-specific monoclonal

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antibodies poorly inhibited CHIKV. These findings support the genetic studies showing that ONNV is a genetically distinct virus, closely related to CHIKV [48].

Ross River Virus. First isolated in 1959 from Aedes species mosquitoes [49] in

Townsville, Australia, RRV was not isolated from humans until 1972 [50]. It is currently the most common mosquito-borne human pathogen on the continent and is responsible for an estimated 5,000 cases of human arthritogenic disease within

Australia each year [51]. While RRV disease has been reported in all regions of the country activity often peaks during periods of high rainfall. In addition to this continuous disease burden, RRV was responsible for an explosive epidemic of alphavirus-induced rheumatic disease which occurred from 1979-1980 and involved more than 60,000 cases. During this epidemic, RRV-induced disease was reported in Australia, and on various islands throughout the region including Fiji, New Caledonia, the Cook Islands, and American Samoa [5, 12, 52, 53]. Additionally, travelers visiting Fiji in late 2003 through early 2004 contracted RRV disease suggesting that the virus may have remained endemic within the population of Fiji since its arrival during the 1979 epidemic [9].

Sindbis Virus. SINV was first isolated in 1952 from Culex species mosquitos in the village of Sindbis, near Cairo Egypt [54]. At the time of its isolation, no clinical disease signs in the local human population could be attributed to it, thus it was thought to be a virus without a disease. Only in 1963 was the first evidence of clinical disease associated with SINV infection in humans reported in South Africa [55]. SINV is one of the most widely distributed arboviruses throughout the Old World [56], and has been

8

responsible for causing epidemics in human populations in Northern Europe for decades. These epidemics are typically caused by one of three viruses which will be discussed in greater detail below.

The major SINV epidemics in human populations are largely due to a group of closely related SINV-like viruses: Ockelbo, Pogosta, and Karelian disease, which have caused thousands of cases of alphavirus-induced rheumatic disease in human populations in Northern Europe, specifically Sweden, Finland, and parts of Russia.

Further investigations into these outbreaks showed that infection primarily occurs in a temporal fashion, as these diseases are typically seen only in late summer or early fall, the timeframe when the local population enters the forests of the region to collect mushrooms and berries [22]. Additional studies have shown that local birds are likely the major amplifying host, and that the number of human disease cases can be correlated to these bird populations suggesting that these birds act as reservoir hosts for the virus [57]. These findings have subsequently been confirmed by studies which showed that in the case of Pogosta disease, epidemics typically occur every 7 years and that these epidemics are associated with large die-offs in bird populations [58].

Mayaro Virus. MAYV is unique among the arthritogenic alphaviruses for several reasons. It is the only arthritogenic alphavirus endemic in the New World and epidemics of MAYV disease are often small with between 30-100 cases of disease being reported each year. MAYV was first isolated in 1954 from workers in Trinidad who presented with characteristic arthritogenic symptoms [59]. Since these reports, isolates have been

9

recovered from numerous other countries in South America indicating that MAYV is fairly common in populations within the more rural regions of the continent [16].

Additional studies have shown that habitation and employment in close proximity to the tropical forests in the region is often associated with an increased risk of infection [6, 16,

60]. While the numbers of human cases reported each year are relatively small it is possible that a number of MAYV infected patients are mis-diagnosed. Dengue virus causes a disease which is very similar to that of MAYV, thus the actual number of human cases of MAYV may be greater than are actually reported.

Rheumatic Disease in Humans During Alphavirus Infection

Alphavirus infection in human populations results in either encephalitic or musculoskeletal disease. Because the research presented in this dissertation addresses musculoskeletal disease only, the encephalitic disease will not be discussed in detail.

The disease process caused by the encephalitic alphaviruses has been thoroughly discussed in reviews by Stelle and Twenhafel [61], Davis et al. [62], and Zacks and

Passler [63].

Old World alphavirus infection in humans results in a musculoskeletal disease of varying severity. Several studies have shown that the proportion of symptomatic cases varies between viruses, with some like CHIKV causing symptoms in 80% of infections, while RRV causes symptoms in 25-50% [15]. During symptomatic infection by an arthritogenic alphavirus a well conserved series of events takes place which ultimately result in the manifestations of rheumatic disease. 10

Figure 1.2 Joint Manifestations of CHIKV Disease in Humans A. Acute arthritis involving wrist and interphalangeal joints in a patient with Chikungunya infection. B. Chronic arthritis involving wrists and metacarpophalangeal and interphalangeal joints in a patient with Chikungunya infection.1

Virus inoculation occurs via the bite of an infected mosquito, followed by primary replication near the site of inoculation. This primary infection produces a high titer serum viremia which can last between 5-7 days in both humans and animal models as assayed by detection of viral RNA [64, 65], and between 1-3 days in animal models as detected by plaque assay [65, 66]. During this viremic period, the virus spreads to additional tissues within the host, likely establishing productive infections within these tissues [2, 12, 35]. Development of clinical disease signs occurs shortly after this incubation period and in cases of less severe disease, manifest as fever, fatigue, headache, and nausea [67]. In cases of alphavirus-induced musculoskeletal disease,

1 Reprinted from Seminars in Arthritis and Rheumatism, Volume 41, Khasnis A. Schoen R. Clalbrese L., Emerging Viral Infection in Rheumatic Diseases, Pages 236-246, Copyright © 2011, with permission from Elsevier.

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clinical symptoms manifest as acute sudden onset high fever, maculopapular rash [7,

68], and intense often debilitating polyarthritis/arthralgia and myalgia/myositis. The intense pain and inflammation in the joints typically affects peripheral joints (ankles and wrists) and some larger joints (knees and shoulders) in a symmetrical fashion [6, 7, 37,

49]. A hallmark feature of alphavirus-induced rheumatic disease is the long-term and sometimes chronic nature of musculoskeletal symptoms.

Recovery from these musculoskeletal symptoms varies greatly between patients, and can range from weeks to years. While some studies have shown that many patients begin to recover after several weeks [67, 69], additional studies have shown that between 12-60% of patients will present with persistent arthralgia/arthritis 1-3 years after initial diagnosis [69-72]. Additional reports have suggested that alphavirus-induced musculoskeletal symptoms may persist for up to 8 years post-infection [73].

Several groups have examined cytokine levels in sera from patients diagnosed with CHIKV-induced chronic inflammatory disease, and found that IL-6 and Granulocyte

Macrophage Colony Stimulating factor were significantly up-regulated [69] in addition to the maintenance of a strong innate anti-viral response in association with IL-12 [74] at

18 months post infection. These studies have begun to elucidate the role of the host immune response during chronic infection and the etiology of chronic alphavirus-induce rheumatic disease, though additional studies will be required to fully understand this process.

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While the severity of the symptoms is variable between different alphaviruses, the anatomical locations of the symptoms appear consistent across all arthritogenic alphaviruses. In addition to these musculoskeletal symptoms, there have been reports of atypical outcomes such as neurological disease, hemorrhagic manifestations, myocarditis and deaths associated with CHIKV infection. These atypical outcomes were associated with age, and underlying medical conditions [75, 76].

Despite the large number of human cases of disease each year, and the significant impact of the disease on human populations, there are no specific anti-viral therapies available, and treatment is limited to analgesics, non-steroidal anti- inflammatory drugs (NSAIDs), and palliative care. Although there are currently no licensed vaccines available for prevention of these viruses, Baxter Healthcare

Corporation is currently in phase 3 clinical trials with a formalin treated, UV inactivated,

Vero cell-derived, aluminum hydroxide adjuvanted RRV vaccine. Additionally, the

National Institutes of Health and several biotech companies are investigating virus-like particle (NIH), live attenuated (Inviragen), or vaccine vector (Themis) based approaches for CHIKV. Currently, many of these vaccine candidates are undergoing pre-clinical evaluation or phase I clinical trials.

Alphavirus Pathogenesis

During alphavirus infection, the high viral loads in the serum early after infection are initially controlled by the Type I interferon (IFN) response. During the high titer viremic phase, it is thought that the virus spreads from the site of inoculation to other

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tissues within the host. As the infection continues, control via the Type-I IFN response is replaced by a strong neutralizing antibody response [77, 78]. While it is not known which cells are persistently infected during this phase, several groups have identified a broad range of cell types which can be productively infected. Labadie et al. showed that in a non-human primate model of CHIKV disease, macrophages served as the main cellular reservoirs during the late stages of infection [64]. Supporting these findings, Linn et al. showed that macrophage cell lines could be productively infected with RRV and were capable of producing virus for more than 50 days post-infection [79]. Studies from our group have shown that both CHIKV and RRV infect skeletal muscle as well as joint associated tissues [65]. Finally, studies by Ozden et al. showed that in human disease, skeletal muscle satellite cells were infected by CHIKV [80].

The rheumatic symptoms of patients are associated with a potent inflammatory response localized to joint and muscle tissues. This inflammatory response has been shown to be predominantly composed of macrophages in both CHIKV and RRV disease

[65, 80-82] and while several studies have implicated macrophages in driving the pathogenesis of these viruses, very few studies have investigated the molecular basis for this pathogenesis [83, 84]. Studies in our lab have shown that in a mouse model of RRV- induced inflammatory disease, both Arginase 1 and the Complement system play key roles in the regulation of this virus-mediated inflammatory disease [85, 86].

While the inflammatory response is gradually resolved in infected hosts, viral replication persists in various joint and muscle associated tissues for long periods of

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time after the onset of symptoms. In support of this hypothesis, unpublished studies in our lab have shown that CHIKV RNA persists in joint tissues for up to 84 days post infection in a mouse model of CHIKV infection and disease. Additionally, previous reports have shown the presence of RRV RNA in patient tissues 5 weeks after symptom onset [87], and CHIKV antigen in synovial tissues 18 months post symptom onset [74]. In a non-human primate model of CHIKV disease viral antigen and RNA were detectable for up to 90 days post infection [64]. This long term replication and the corresponding host inflammatory response to the virus, may help to explain the duration of the pathology associated with these infections. Though experimental evidence indicates that viral replication persists in these tissues, less well understood is how these viruses maintain persistence in the face of the strong innate and adaptive immune responses mounted by the host. Krejbich-Trotot et al. propose a model suggesting that CHIKV may evade the host immune response by hiding in apoptotic blebs, and maintaining infection in macrophage populations through repeated rounds of apoptosis and phagocytosis [88].

To combat this strong host innate immune response, alphaviruses have evolved mechanisms through which they can suppress the anti-viral state. Infection of type I IFN defective mice with both New and Old World alphaviruses is lethal, highlighting the role of the innate immune response in control of these viruses [78, 89]. In hosts with an intact innate immune response, the alphaviruses have evolved mechanisms which mediate shutoff of host-cell RNA transcription, preventing expression of host anti-viral proteins. The specific viral protein required for transcriptional shutoff differ based on

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the type of alphavirus. Studies have shown that the Old World alphaviruses accomplish this suppression through interactions of the viral nsP2 protein with host cell factors, while in the New World alphaviruses, this process is regulated by the viral capsid protein

[90, 91].

More recent studies have shown that arthritogenic alphaviruses may enhance viral replication in cells by co-opting the host cell autophagy pathway. While the specific mechanisms have not yet been identified, studies have shown that induction of autophagy delays the apoptotic death of the host cell during CHIKV infection [92]. Other studies with SFV have shown that this pathway may be regulated through expression of the viral glycoproteins [93], however additional studies will be required to determine the specific mechanisms required for this enhanced replication.

Animal Models of Alphavirus-induced Rheumatic Disease

Because of the persistent nature of alphavirus-induced rheumatic disease, and because of the epidemic potential of these viruses, additional research will be required to identify and characterize virus-host interactions, as well as determinants which contribute to disease severity and host specificity. To investigate these aims further, a number of animal models have been developed for use in studying various aspects of viral pathogenesis and the host response to infection.

Non-Human Primate Model of CHIKV Infection. Labadie et al. published a study in 2010 investigating CHIKV infection in non-human primates, specifically at sites of persistent infection. In this study adult cynomolgus macaques (Macaca fascicularis)

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were inoculated by various routes with between 101 and 108 PFU of virus and monitored for the development of disease. During the course of infection, the severity of disease observed in the animals was correlated with the infectious dose received. In animals inoculated with up to 106 pfu, fever and rash were the only observable disease signs, however joint effusion, subcutaneous edema, and death were observed in animals inoculated with 107 or 108 PFU of virus [64]. While these studies in non-human primates were the first to show disease signs similar to that of humans, the dose required to achieve this response is naturally implausible, and might result in an abnormal immune response to infection.

Mouse Model of CHIKV Pathogenesis in IFN Deficient Mice. Couderc et al. reported the development of a mouse model of CHIKV induced disease in a mouse model of infection and showed that the innate immune response, specifically the type I

IFN response, was critical to control of CHIKV infection. As expected, mice which were defective in Type I IFN signaling showed significantly higher viral loads in various tissues in addition to showing more severe disease signs than WT mice. While these studies show that the innate immune response is essential to control of CHIKV infection, neither the dose nor the route of inoculation required to achieve these phenotypes occur during normal infection conditions [78].

Other Animal Models. A recent publication by Teo et al. has done an excellent job summarizing a number of additional mouse models of alphavirus-induced rheumatic

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and neurological disease, examining mouse strain, routes of inoculation, as well as a brief summary of virus-induced disease [94].

Mouse Model of RRV- and CHIKV-induced Rheumatic Disease in Mice. Our lab has developed mouse models of both RRV- and CHIKV-induced rheumatic disease which recapitulate many aspects of the human disease [65, 82, 86]. In both models 3-4 week old C57BL/6 mice are inoculated with 103 pfu of virus in the left rear footpad. This dose and location were chosen because 103 pfu is similar to the inoculum delivered by an infected mosquito during a blood meal and the location allows for the tracking of viral spread from the site of inoculation to the draining lymph node, quadriceps muscle tissue, and subsequent spread to the contralateral side of the animal. After inoculation we can study the mice over time as they develop musculoskeletal disease similar to that observed in humans (Figure 1.3). In RRV infected mice, at approximately 15-20 days post infection the mice begin to resolve the inflammatory response, and the muscle tissue begins to regenerate. In CHIKV inoculated mice, virus-induced arthritis, tenosynovitis, and myositis is visible in tissue sections as late as 84 DPI recapitulating the persistent disease reported in human populations. Virus-induced pathology may also be present in

RRV-infected tissues at late times post-infection, however these studies have not yet been completed.

Alphavirus Virions

Alphavirus virions share many physical characteristics across species. Virions are spherical, enveloped particles of approximately 65-70 nm in size, with 240 heterodimers

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of the alphavirus glycoproteins E1 and E2 arranged to form 80 trimeric spike complexes in T=4 symmetry. This glycoprotein arrangement has been determined for several alphaviruses using cryo-EM techniques [95-101], and the crystal structure of the E1-E2 spike complex was recently solved for both SINV and CHIKV [102, 103]. The alphavirus glycoproteins are embedded in a lipid bilayer derived from the host cell, which in turn surrounds the viral nucleocapsid (Figure 1.4A). The nucleocapsid is comprised of 240 copies of the viral capsid protein, organized into a sphere of T=4 symmetry which surrounds a single copy of the alphavirus RNA genome [104].

Alphavirus Genome

In addition to sharing similar virion properties, all alphaviruses also possess a similar genomic organization. In general, the alphavirus genome is a positive-sense single strand of RNA between 11-12 kilo-bases in length. Additionally, it has been shown that this RNA possesses a 5`-7-methyl-guanosine cap, and a poly-A tail [105]. The genome contains 2 open reading frames (ORF) with the 5` 2/3 of the genome (ORF1), encoding 4 viral nonstructural proteins nsP1, nsP2, nsP3, and nsP4, which function in the replication of both negative and positive sense viral RNAs, while the 3` 1/3 (ORF2), encodes the viral structural proteins: capsid, PE2, 6K, and E1 (Figure 1.3B).

In addition to these coding regions, alphavirus genomes contain a number of important sequence elements in both the 5` and 3` untranslated regions (UTR) of the genome. Perhaps the most well studied of these sequence elements is a 51-nucleotide

(51 nt) conserved sequence element (CSE) which spans the 5`UTR and nsP1 coding

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sequence and is present in all members of the Alphavirus genus [106]. Analysis of this 51 nt CSE in SINV showed that this region is composed of a series of stem-loop structures which function in replication of both negative sense and positive sense RNA [107].

Additional studies have shown that this CSE regulates viral replication in a host-specific fashion. Mutations within this CSE resulted in a virus which was fully infectious in vertebrate cells, but significantly attenuated in invertebrate cells [106]. In addition to the 51 nt CSE, several groups have shown that other mutations within the 5` UTR can function as virulence determinants of disease in mouse models [89, 108].

The 3` UTRs of alphavirus genomes also encode a 19 nt CSE adjacent to the poly-A tail which is highly conserved among all alphaviruses and functions in RNA replication [109].

Other characteristic RNA elements in the 3` UTR include 40-60 nt repeat sequence elements, though these vary in sequence and length amongst different alphaviruses

[110]. Though the specific functions of these 3` repeat sequence elements remains unknown, recent studies have found that they may function in the deadenylation of viral

RNA [111].

Recent studies have shown that both the 5` and 3` UTRs of arthritogenic alphaviruses interact with various RNA-binding proteins in the cell to regulate transcription of viral RNA. The 5` UTR binds to heterogeneous nuclear ribonucleoprotein

A1, and this association facilitates transcription of the viral RNA [112]. Studies using

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Figure 1.3 Mouse Model of RRV-Induced Musculoskeletal Disease. Three to four week old C57BL/6J mice were inoculated with 103 pfu of RRV-T48 in the left read footpad and monitored daily for disease signs/progression. (A) At times indicated, mice were euthanized and perfused with 4% PFA. Five micron thick paraffin- embedded sections generated from quadriceps muscle were stained for H&E. Images are representative of mice at various times post-infection. (B) Five micron thick paraffin- embedded sections were generated from ankle/foot tissue and stained for H&E. (C) Mice were monitored daily for weight gain and development of musculoskeletal disease signs including loss of gripping ability, hind-limb weakness, and altered gait. 21

Figure 1.4 Alphavirus Virion Structure and Genome Organization A. Radially colored 3D reconstruction of VEEV showing the arrangement of the glycoprotein shell. E1 (green/E2 (blue) heterodimers surround the viral envelope (red), and nucleocapsid (yellow).2 B. Schematic organization of the alphavirus genome.

SINV have shown that HuR, a ubiquitously expressed RNA binding protein, recognizes unique U/A rich regions within the 3` UTR of SINV and regulates transcription of the viral

RNA. Additionally these studies showed that HuR also bound both RRV and CHIKV RNA, though these viruses do not possess the same U/A rich elements in their 3` UTR [113].

2 Reprinted from The EMBO Journal, Volume 30, Zhang et al., 4.4Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus, Pages 3854-3863, Copyright © 2011, under Creative Commons license.

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Together, these studies show that while specific sequence elements may vary between viruses, the interactions of host proteins with the 5` and 3` UTRs are critical for viral replication.

During the viral replication cycle, transcription of the negative sense replicative intermediate RNA gives rise to 2 RNA species as determined by density gradient centrifugation. The first of these species is a full length viral genome called the 49S RNA, which interacts with viral capsid protein to form nucleocapsids eventually giving rise to new virions, while the second called the 26S, or subgenomic RNA encodes only the structural genes of the viral genome. Recent studies have identified a unique RNA structure within the 26S transcript called the downstream loop, which allows translation of the 26S mRNA by an eIF2 independent manner, allowing the virus to replicate in the presence of activated protein kinase R. Critically, this structure was only required for replication in mammalian cells, while replication in insect cells was independent of this downstream loop structure [114].

Alphavirus Gene Products and Functions

Nonstructural Polyprotein Translation and Processing. As mentioned above, the 5` 2/3 of the alphavirus genome encodes the nonstructural proteins. Following infection and uncoating within the cytoplasm, translation of the viral RNA is initiated by the host cell. The nonstructural proteins are initially produced as the polyproteins P123, and P1234. In many arthritogenic alphaviruses, the majority of the polyprotein produced is P123 due to the presence of an opal termination codon at the 3` end of the 23

nsP3 sequence. During alphavirus infection, readthrough of this opal termination codon occurs approximately 10% of the time and allows for expression of small amounts of nsP4 [115]. Throughout the replication cycle, the nonstructural proteins function as various polyproteins and as four individual proteins generated by an ordered process of proteolytic cleavage.

nsP1. nsP1 functions in viral RNA replication and RNA capping [109]. Specifically, nsP1 encodes both guanine-7-methyltransferase and guanylyltransferase activities [116-

118]. Recently, enzymatically active nsP1 was purified and the kinetics of its role in the

RNA capping process were determined [119]. In addition to its role in the viral RNA replication and capping process, nsP1 is the sole membrane associated nonstructural protein and functions by anchoring viral replication complexes to endosomal membranes [120, 121]. Critically, this membrane association is required for the activation of its enzymatic functions during the capping process [122].

nsP2. The nsP2 protein has numerous roles during the alphavirus replication cycle. nsP2 functions as the viral nonstructural protease which is responsible for cleavage of the nonstructural polyprotein first in-cis at the nsP3/nsP4 junction, and subsequently in-trans at the nsP1/nsP2, and nsP2/nsP3 junction. The protease activity is located in the C-terminal domain of nsP2 and subsequent characterization studies have shown it to be a papain-like cysteine protease [123, 124]. In addition to the protease functionality, the N-terminal domain of nsP2 contains a nonfunctional methyltransferase-like domain. Several groups have investigated the function of this

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domain during infection. Mutational analysis showed that this domain was involved in the regulation of negative strand RNA replication and the development of cytopathic effect (CPE) during SINV infection [125]. This domain also plays a critical role during the capping of new viral genomes. Specifically, Vasiljeva et al. showed that the N-terminal region of nsP2 removes the γ,β-triphosphate bond at the 5` end of the RNA [126]. A recently published study by Akhrymuk et al. has identified a novel function for this domain which has addressed a curious phenotype regarding the cellular localization of nsP2. During Old World alphavirus infection, approximately 50% of mature nsP2 is localized to the nucleus of the cell [127] where it inhibits cellular transcription by inducing the rapid degradation of Rpb1, the catalytic subunit of the RNA polymerase II complex [128]. In addition to these functions, nsP2 possesses an N-terminal helicase domain critical for RNA duplex unwinding during the viral replication cycle [129].

nsP3. In contrast to the well characterized roles and functions of the other nonstructural proteins, significantly less is known about nsP3. It has been shown to be required for RNA synthesis, both in the individual state and in context of the polyprotein, and mutational analyses have shown that nsP3 plays a role in both viral pathogenicity in mice [130-132], and vector specificity [28]. Sequence alignment studies have shown that nsP3 is comprised of two major domains. The N-terminal domain contains a structurally conserved domain known as the macro domain and has been shown to bind ADP-ribose-1``-phosphate, which may allow nsP3 to recruit poly(ADP- ribosylated) cellular factors to the viral replication complex [133, 134]. Additional

25

studies have shown this domain to be important in neurovirulence and for viral replication in neurons [131]. In stark contrast to the well conserved N-terminus, the nsP3 C-terminal region is poorly conserved even among closely related alphavirus species. Despite the lack of sequence conservation, several studies have addressed potential roles for this domain in the alphavirus replication cycle. Neuvonen et al., intrigued by a conserved group of proline residues amongst alphavirus sequences, showed that nsP3 is involved in Src-homology 3 (SH3) domain recruitment of host cell amphiphysins, and that these amphiphysins were required for efficient viral RNA replication [135]. Additional studies have shown that the hyper-variable C-terminal domain of nsP3 mediates the formation of virus specific protein complexes. The specificity of nsP3 was shown by generating chimeric VEEV and SINV strains in which the hyper-variable domain of nsP3 had been exchanged. Using these chimeric viruses, Foy et al. showed that the protein complexes associated with VEEV or SINV replication were linked to the nsP3 sequence present. Interestingly, despite the different components in the VEEV and SINV replication complex, the authors showed that viral replication occurred regardless of which virus the nsP3 gene was originally from, suggesting that viral replication is only dependent on the presence of nsP3 [136].

nsP4. nsP4 is transcribed in low amounts during alphavirus infection as mentioned previously, and functions as the RNA-dependent RNA polymerase (RdRP).

The polymerase activity was identified due to the presence of a characteristic GDD motif in the c-terminal region of nsP4 which has been identified in numerous other viral RdRPs

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[137, 138]. Additional studies of the catalytic domain of nsP4 showed that it plays a key role in the maintenance and repair of the viral poly-A tail [139]. In contrast to the well conserved C-terminal catalytic core, the N-terminus of nsP4 is unique among viral

RdRPs. Alphaviruses possess a unique sequence which has been shown to be important in allowing nsP4 to bind to and interact with other nonstructural proteins throughout the replication process [140]. Recent studies utilizing purified functional nsP4 have confirmed the enzymatic roles of the protein within an in vitro system [141]. In addition to being expressed at lower levels because of the opal termination codon, the nsP4 protein is metabolically unstable. nsP4 degradation occurs based on the N-end rule pathway whereby the N-terminal residue can be correlated to protein stability. The N- terminal residue of nsP4 is a tyrosine which results in its rapid degradation [142].

Attempts to increase the amount of nsP4 present in an infected cell by removing the opal termination codon showed that excess nsP4 does not accumulate, but is rapidly degraded by these host pathways [143].

Structural Polyprotein Translation and Processing. Alphavirus structural protein translation occurs later in the replication cycle. The structural proteins are expressed as a polyprotein in similar fashion as the nonstructural proteins, and are translated from a positive sense subgenomic 26S mRNA produced in alphavirus infected cells. This RNA transcript contains the coding sequence for the viral capsid, E3, E2, 6k, and E1 proteins and is only expressed after sufficient amounts of negative sense viral RNA have been

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transcribed, and the nonstructural polyprotein has been processed into the mature individual nonstructural proteins.

Capsid. Capsid protein is cleaved from the nascent polyprotein by a serine protease domain located at the C-terminal end of the protein which acts in-cis [144].

Capsid protein then accumulates in the cytoplasm of the infected cell where it is able to interact with newly transcribed genomic RNA. In vitro models suggest that the recognition of the genomic encapsidation signal acts as a nucleation event whereby more capsid protein is incorporated, eventually leading to full encapsidation [145, 146].

In SINV, VEEV, WEEV, and EEEV this packaging signal is located in the nsP1 gene and consists of 4-6 stem-loop structures with conserved G nucleotides at the base of each loop. Viruses within the Semliki Forest antigenic complex differ in that the packaging signal is located in the nsP2 gene. Of note is the fact that capsid protein from SFV antigenic complex viruses are capable of interacting with packaging signals in either nsP1 or nsP2 [147]. In most alphaviruses, the presence of the packaging signal within the nonstructural genes ensures that only full length viral genomes are incorporated into nucleocapsids. Studies showed that Aura virus incorporated both 49S and 26S RNA suggesting that this particular virus has its packaging signal localized in the 26S sequence [148]. Upon completion of this encapsidation, the final nucleocapsid is comprised of 240 copies of capsid protein, organized into an icosahedral shell with T=4 symmetry surrounding a single molecule of viral RNA [95]. In addition to formation of nucleocapsids, during infection with New World alphaviruses capsid protein has been

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shown to function in host transcriptional shutoff instead of nsP2, as seen in the Old

World alphaviruses. Studies by Atasheva et al. have shown that not only are New World virus capsid proteins localized in the nucleus of infected cells [149], but that capsid protein interacts with host cell importin alpha/beta and CRM1 forming a complex that prevents normal nuclear transport from occurring in infected cells [150].

PE2. The alphavirus PE2 protein is a membrane-bound viral glycoprotein comprised of several distinct regions. The N-terminal region of PE2 encodes the viral E3 protein, which remains associated with E2 before being cleaved by the host protease furin late in the replication cycle. E3 has been shown to be critical to prevent premature conformational changes in the alphavirus spike complex during trafficking and processing in the secretory pathway [151]. Immediately downstream of the furin cleavage site is the E2 protein sequence. The E2 protein is comprised of 3 major domains A, B, and C, which possess distinct roles during the viral lifecycle, in addition to several smaller “arch domains” which are involved in conformational changes in the protein during exposure to low pH environments [103].

Structural studies of E2 show that domain A possesses many van der Waals interactions with both the E1 protein and neighboring E2 molecules within the trimeric spike complex suggesting, that E2 may have a role in stabilizing the trimeric spike complex [103]. Additional studies have shown that mutation of conserved cysteine residues in E2 domain A resulted in virus-specific assembly defects, suggesting that this region plays a critical role in the viral budding process [152].

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Domain B of the E2 protein functions as the major virus receptor recognition and binding region in addition to playing critical roles during the viral infection process.

Specifically, domain B is involved in binding to heparan sulfate (HS), a glycosaminoglycan present on many cell types [153], and that neutralizing antibodies specifically targeted residues within domain B [154]. Recent studies by Fields and Kielian have shown that in addition to receptor interactions, domain B is involved in the early steps of the viral fusion process during infection. Specifically, they showed that exposure to a low pH environment caused E2 domain B to lift away from the body of the spike, exposing the fusion peptide of E1 [155].

The C-terminal region of E2 encodes a transmembrane domain and cytoplasmic domain. The cytoplasmic domain is required for the viral budding process. Specifically, a hydrophobic stretch of amino acid (AA) residues in the cytoplasmic domain interact with a corresponding pocket on the surface of the viral nucleocapsid [156-158].

Additional domains in the E2 protein consist of domain C which is located between domain B and the cytoplasmic domain, and 2 “arch” regions which link domains A to B, and B to C. Specific functions for domain C have not been identified, though it has been shown to possess numerous interactions with E1 proteins both within and across heterodimers [103]. The arch domains are predicted to have multiple intra-dimer and intra-spike (across heterodimers) interactions with E1, in addition to intra-spike interactions with other E2 peptides. In addition to these interactions, recent structural studies identified 2 “acid sensitive regions” which have been shown to

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become disordered upon exposure to low pH, thus loosening the interactions which hold domain B in place [102].

E1. Like PE2, the alphavirus E1 protein is a membrane associated protein, and it functions as the viral fusion protein during infection. The protein is comprised of several domains: I, II, and III, with the fusion loop located near the middle of domain II [159].

Similar to PE2, E1 is involved in numerous van der Waals interactions with other E1 molecules. In addition, E1 is involved in lateral interactions which are critical for proper alphavirus budding [160].

During virus entry, the major role of E1 is to facilitate fusion of the viral envelope with the membrane of the host endosome, releasing the nucleocapsid into the cytoplasm of the host cell. This process involves several low-pH induced conformational changes in the protein culminating in the insertion of the fusion loop peptide into the host endosomal membrane, followed by fusion of the membranes. The specific changes and fusion mechanism required for this process have previously been well characterized by Kielian et al. [161].

6K. Similar to nsP3, the functions of the viral 6K protein have been less well characterized compared to the other structural proteins. Studies have shown that 6K is required for efficient virion budding, and that deletion or mutation of the 6K protein resulted in significantly lower virus production from infected cells in association with an increase in the number of multi-cored particles present [162, 163]. It is interesting to note that while 6K is required for efficient production of new virions and fusion activity,

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examination of extensively washed viral particles devoid of 6K showed no observable differences as compared to normally derived virus [164]. It has also been shown that 6K is incorporated into new virions, but that it is a minor component of the virion [164].

Several studies have attempted to identify the specific role of 6K on viral replication, and have shown that it is responsible for the formation of ion channels and the permeability of host-cell membranes, and that this activity often leads to rapid cell death, suggesting that this protein could be playing a role similar to other “viroporin”- like proteins [165, 166].

The Alphavirus Replication Cycle

Alphavirus Entry and Fusion Process. Alphavirus infection occurs in a multi-step process. Upon delivery by the bite of an infected mosquito, the virus first binds to an unknown cell surface receptor via interactions in domain B of the E2 protein. While the specific receptor in humans is unknown, experimental studies have shown that a broad range of cellular proteins can function as alphavirus receptors including: heparan sulfate, integrins, prohibitin, laminin, and DC-SIGN/L-SIGN [167-171]. Upon engagement of the receptor, the virion is endocytosed via a clathrin dependent mechanism [172].

The endosome is then targeted for either recycling or degradation, which results in its acidification. When the pH in these early endosomes reaches a critical threshold of 6.3 to 5.8, conformational changes are induced in the viral glycoproteins. Specifically, exposure to the low pH environment causes domain B of the E2 protein to loosen its interactions with the fusion loop of E1, allowing it to insert into the endosomal

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membrane [155]. In addition, numerous other E1/E2 interactions are weakened, resulting in the rotation of E2 towards the outside of the spike, allowing the E1 proteins to assemble into a homo-trimer [173-175]. A ring of between 5 and 6 E1 homo-trimers is required for fusion of the viral envelope with the endosomal membrane [176]. This fusion event results in release of the viral nucleocapsid into the host cell where it is rapidly disassembled [177]. While the specific mechanism of capsid disassembly has not been defined, Wengler et al. have shown that in SINV, sequence elements surrounding capsid position 106 function as a ribosomal binding site, suggesting a model whereby host ribosomes disassemble the incoming nucleocapsid [178]. This disassembly of the viral nucleocapsid releases the viral RNA into the cytoplasm where host cell translation machinery begins synthesizing the viral nonstructural polyproteins (Figure 1.5).

Replication of Alphavirus RNA and Nonstructural Protein Processing. Synthesis of the nonstructural polyprotein P1234 is followed by the cleavage of the nsP3/nsP4 bond in-cis by the nsP2 protease domain, generating P123 and free nsP4. This process repeats itself until high enough levels of P123 are present that the nsP2 protease domain of P123 acts in –trans, cleaving the nsP1/nsP2 bond of another P123 molecule giving rise to free nsP1, P23, and nsP4. Only after cleavage of the nsP1/nsP2 bond can the nsP2 protease domain of P23 act in –trans to cleave the nsP2/nsP3 bond, producing individual nonstructural proteins [179, 180]. This nonstructural protein processing is regulated temporally and acts to regulate negative strand and positive strand synthesis of the viral RNA [180].

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Soon after synthesis, P123 and nsP4 begin synthesizing negative sense viral RNA for use as a template during the genomic replication cycle. Cleavage of nsP1 from the

P123 polyprotein results in a nsP1-P23-nsP4 complex which functions in both positive and negative sense RNA replication, until cleavage of the nsP2/nsP3 bond results in a nsP1-nsP2-nsP3-nsP4 complex which is capable only of positive sense RNA synthesis

[181].

Numerous studies have shown that alphavirus RNA replication occurs in structures termed “type I cytopathic vacuoles” (CPV-I) which contain numerous membranous invaginations (spherules) connected to the membrane through narrow necks (Figure 1.5). These spherules are localized to either the plasma membrane [182], or on endosomal membranes [120]. Viral nsP replication complexes are localized to these CPVs through the membrane binding domain of nsP1, and are thought to offer a semi-protected site for replication of viral RNA. A similar mechanism has been characterized during replication of flock house virus, a model system for replication of positive sense single strand viral RNA, which showing that this mechanism of viral genomic replication is well conserved across many positive sense RNA viruses [183].

Once sufficient viral negative sense RNA has been synthesized and sufficiently high levels of the individual nsPs are available, production of new positive sense viral

RNA is initiated. This produces two major RNA species. The first of these is the full length viral genome, which is promptly capped by nsP1, and which then begins to accumulate in the cytoplasm where it will interact with capsid protein to form new

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nucleocapsids. In addition to full length genomes, transcription of a second RNA transcript is initiated at the 26S subgenomic promoter of the negative sense RNA. This

26S subgenomic RNA encodes the viral structural genes and is translated by the host cell, initiating structural protein processing (Figure 1.5).

Alphavirus Structural Protein Translation and Virion Assembly. Capsid cleavage from the structural polyprotein results in the exposure of an ER translocation signal which mediates translation of the PE2 protein into the lumen of the ER [184]. Near the c-terminus of PE2 is a stop transfer signal which serves to anchor E2 into the membrane of the ER. Following the membrane anchor sequence a second ER translocation signal directs the translation of the viral 6K protein into the ER lumen. A final ER translocation signal immediately following the C-terminus of 6K directs translation of E1 into the ER lumen. A final stop transfer signal is located just upstream of the c-terminus of E1 which serves to anchor E1 into the ER membrane.

The polyprotein is rapidly processed during translation by the host-cell signalase protease, resulting in individual PE2, 6K and E1 proteins. Folding of both PE2 and E1 begins shortly after translation of the proteins into the ER, and requires various folding enzymes, chaperones, and disulfide bonds [185-188]. It has also been shown that both

PE2 and E1 are rapidly glycosylated at all potential N-linked glycosylation sites [189].

After being properly folded and glycosylated PE2 and E1 have been shown to associate as heterodimers and to maintain this association throughout the secretory pathway [190]. Once associated into their heterodimeric conformation, both PE2 and E1

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proteins are palmitoylated at several conserved cysteine residues located in the cytoplasmic region of PE2 and near the transmembrane domain of E1 [191, 192].

Following palmitoylation, PE2/E1 heterodimers are trafficked through the Golgi apparatus where they oligomerize into a trimeric spike composed of 3 PE2/E1 heterodimers. In the Golgi, the host-cell protease furin cleaves the E3/E2 bond in PE2, generating the fully mature E1/E2 heterodimer [193]. Although the E3/E2 bond has been cleaved, E3 remains closely associated with the E1/E2 heterodimer, preventing its premature triggering while in the acidic environment of the cellular secretory pathway

[151].

Once the glycoproteins have been trafficked through the trans-Golgi network

(TGN), they are delivered into a “Type II Cytopathic Vacuole” (CPV-II), a structure derived from the TGN. These CPV-IIs are spindle shaped compartments comprised of long tubular structures which contain large numbers of trimeric spikes arranged in a hexagonal lattice with the cytoplasmic domains facing the outer wall of the structure

[194, 195]. The long tubular structures within the CPV-IIs are then proposed to fuse with the plasma membrane, delivering large quantities of trimeric spikes arranged into a hexagonal lattice to the cell surface. Once the glycoprotein complexes have arrived at the plasma membrane, hydrophobic residues in the cytoplasmic tail of E2 interact with a corresponding hydrophobic pocket of capsid protein [157]. This interaction is thought to provide the free energy required for viral assembly and budding, propelling the

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Figure 1.5 Alphavirus Replication Cycle Alphaviruses enter target cells through receptor mediated endocytosis, and subsequent acidification of the endosomal environment. Exposure to the acidic pH causes conformational rearrangements of the E1 and E2 proteins which expose the fusion loop of the E1 peptide. Insertion of the fusion loop into the endosomal membrane results in further conformational changes leading to fusion between the viral and endosomal membranes releasing the nucleocapsid into the cytoplasm. Disassembly of the nucleocapsid exposes the viral genome to host cell translational machinery and initiates translation of viral nonstructural proteins. nsP1 helps form CPV-I RNA replication complexes on endosomal membranes. Transcription of negative strand replication intermediates and complete processing of nonstructural proteins initiates transcription of full length 49S viral genomic RNA and 26S subgenomic structural RNA. Subgenomic RNA is translated to produce Capsid, PE2, 6K, and E1 proteins. Capsid is cleaved and accumulates in the cytoplasm, interacting with viral genomic RNA forming nucleocapsids. The remaining structural proteins are translated into the lumen of the ER, and processed further, generating E1/PE2 heterodimers. These dimers are trafficked to the trans-Golgi network and cleaved into E1/E2 dimers by Furin. Glycoproteins are then trafficked to CPV-IIs where they oligomerize into trimeric spikes before finally being delivered to the plasma membrane. Once at the plasma membrane, nucleocapsids interact with the cytoplasmic tails of E2, and initiate budding, and release of new virions. 37

nucleocapsid through the plasma membrane, completing its envelopment with the full complement of 80 trimeric spikes arranged with T=4 symmetry (Figure 1.5).

Alphavirus Determinants of Virulence

In addition to studies investigating the host immune response to viral infection, another critical aspect of alphavirus research is identification of viral determinants of virulence. Through investigation of viral genetic mutations that regulate virus-induced disease, it becomes possible to define the molecular mechanisms which the virus utilizes to cause disease within the host. Because of the error-prone nature of viral RdRPs during infection, alphaviruses do not exist as a static population of homogenous particles with identical genomes, but as a constantly evolving group of closely related, but genetically distinct quasi-species. While these strains may all bear the same name, slight genetic differences can significantly impact virulence by modulating key aspects of the viral replication cycle.

Studies of SINV for example, identified a single glycine to histidine mutation at

E2 position 55 which altered neurovirulence in a mouse model of disease in addition to regulating viral attachment to neuroblastoma cells. Further studies suggest that the enhanced neurovirulence was likely due to enhanced replication within neuronal tissues

[196-198]. Additional studies of alphaviruses such as SINV and VEEV have shown that mutations within the E2 glycoprotein can significantly impact viral protein receptor targeting, conferring heparan sulfate binding activity and attenuating disease in animal models [167, 199]. Studies in SFV showed that a single threonine to isoleucine

38

substitution at position 12 in domain A of the E2 protein can have a significant effect on viral infection by decreasing the pH threshold for fusion [200]. In addition, studies investigating the mechanisms of attenuation of the CHIKV 181/25 vaccine strain showed that this E2 T12I substitution, in addition to a glycine to arginine substitution at E2 position 82, were responsible for attenuation of the virus in vertebrate hosts [201]. Such studies are critical to enhancing our understanding of the alphavirus lifecycle, and are crucial in the development of successful vaccines and novel specific anti-viral therapies.

A second key element in the study of alphavirus virulence determinants is that of viral adaptation. Because of their rapid infection cycle, and because of the error-prone

RdRP nsP4, alphaviruses are capable of quickly accumulating mutations that enhance replication in a particular host. As mosquito transmitted arboviruses, alphaviruses are under extreme evolutionary pressure to maintain their ability to replicate efficiently in both invertebrate and vertebrate cells. Repeated passage of alphaviruses exclusively in either host causes the virus to adapt to that host, enhancing virulence and replication within that specific host. Conversely, this adaptation and enhanced virulence can result in the virus becoming less fit in the reciprocal host [202-204].

Overview of Dissertation

Despite the critical importance of these global pathogens, at the time this work was begun very little was known about viral genetic determinants of alphavirus-induced rheumatic disease. While many studies have been undertaken prior to the work presented here, these experiments focused almost exclusively on determinants critical 39

for neurovirulence of Old World alphaviruses. While neurovirulence of these viruses has been extensively studied in mice, it is not the disease caused by Old World alphaviruses in human populations. The studies presented herein were aimed at identifying viral genetic determinants of virulence in alphavirus-induced rheumatic disease. These studies were initiated by the identification of a strain of RRV (DC5692), which in contrast to the prototypic T48 strain, was attenuated in a mouse model of alphavirus-induced musculoskeletal disease. Based on this finding, I generated a panel of chimeric viruses which contained portions of the DC5692 genome in the T48 genetic background. These chimeric viruses were then tested in the mouse model for ability to establish disease.

These studies showed that amino acid substitutions in the nsP1 coding region, and the

PE2 coding region were independently capable of attenuating RRV-induced disease in mice. These findings were confirmed through gain of virulence studies whereby replacing the nsP1 and PE2 coding region of the mouse avirulent strain with the T48 sequence produced a virus capable of causing WT levels of disease in mice.

Further studies focused on the amino acid mutations within the PE2 region identified by the chimeric virus studies, and showed that a single tyrosine to histidine mutation at position 18 of the E2 glycoprotein was responsible for attenuation in mice.

In vitro replication assays showed that a tyrosine at E2 position 18 resulted in a significant fitness advantage for the virus in mammalian cells and led to enhanced viral titers, while a histidine at position 18 conferred a fitness advantage in mosquito cells with increased virus yield. Subsequently, studies were undertaken to define the

40

molecular mechanisms responsible for the attenuation of the mutant virus in mammalian cells and simultaneous enhancement of the mutant virus in mosquito cells.

Utilizing numerous characterization assays, I showed that the E2 Y18H mutation resulted in a significant increase in the particle to pfu ratio of virus derived from mammalian cells, suggesting that the attenuation is due to a budding defect where non- infectious virions are released from infected cells.

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CHAPTER II

MATERIALS AND METHODS

Viruses

The T48 strain of RRV was isolated from Aedes vigilax mosquitos in Queensland,

Australia. Prior to cDNA cloning, the virus was passaged 10 times in suckling mouse brain followed by two passages on Vero cells [205, 206]. RRV strain DC5692 was isolated in 1995 from Aedes camptorhynchus mosquitos in the Peel region of Western Australia

[207]. The virus was passaged 1 time in C6/36 cells, 1 time in Vero cells and 1 time in

BHK-21 cells prior to cDNA cloning [66]. Viral stocks were generated from full-length wild-type and mutant virus cDNAs as previously described [66]. Briefly, plasmids encoding virus cDNAs were linearized by digestion with Sac I (NEB). 5`-capped full-length

RNA transcripts were generated in vitro using SP6-specific mMessage mMachine transcription kits (Ambion). Full-length transcripts were electroporated into BHK-21 cells

(ATCC CCL-10) using a Gene Pulser electroporator (Bio-Rad). Culture supernatants were harvested at 24 h after electroporation, centrifuged for 20 min at 3,000 rpm, aliquoted and stored at -80°C. Stocks were titrated by plaque assay on BHK-21 cells. For purified virus stocks, virus particles were banded on a 60%-20% discontinuous sucrose gradient by centrifugation at 24,000 rpm in a Beckman SW-24 rotor. Banded virus was collected and centrifuged through 20% sucrose at 24,000 rpm in a Beckman SW-24 rotor. Virus pellets were then resuspended, aliquoted and stored at -80°C.

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Construction of Virus cDNAs

Plasmids encoding full-length and chimeric viral cDNAs are designated by the prefix "p". Infectious virus derived from the cDNA clone does not contain the prefix "p".

Plasmid pRR64 (provided by Richard Kuhn, Purdue University) encodes the full-length cDNA of the T48 strain of RRV. To generate a chimeric virus cDNA that encodes the RRV

DC5692 3’UTR in the T48 genetic background, a shuttle vector (pRR65) was created by religation of pRR64 that had been digested with Afl II and Psh AI followed by a klenow fill in reaction. This resulted in a plasmid with a unique Hind III site at RRV position

11329, which is at the junction of the E1 gene and the 3’UTR and a unique Sac I site at the 3’ end of the RRV poly A tail. The 3’UTR of RRV DC5692 was PCR amplified with a 3’ primer that engineered a Sac I restriction site downstream of the poly A tail. The RRV

DC5692 3’UTR was inserted into the Hind III-Sac I site of pRR65 to create pRR66. The

Xma I-Sac I fragment of pRR66 was inserted into the Xma I-Sac I site of pRR64 to generate pRR67 which encodes a chimeric virus genome composed of the RRV DC5692

3’UTR in the T48 genetic background.

To generate pRR77, the RRV DC5692 Eag I-XbaI fragment was inserted into the

Eag I-Xba I site of pRR64. To generate plasmids pRR76 and pRR79, the RRV DC5692 BssH

II-Eag I fragment was inserted into the BssH II-Eag I site of pRR77 and pRR64, respectively. To generate pRR94 and pRR95, pRR64 and pRR79 were digested with Eco

RI and the ~8300 bp fragment was gel isolated and religated to generate pRR90 and pRR91, respectively. This eliminated nucleotides 6407-11884 of the RRV sequence and

43

generated plasmids with a unique Sap I site just 20 bp upstream of the junction of the nsP1 and nsP2 coding regions. Next, the BssH II-Sap I fragments were swapped between pRR90 and pRR91 to create pRR92, which encodes RRV DC5692 strain sequence from

BssH II-Sap I and T48 strain sequence from Sap I-Eag I, and pRR93, which encodes T48 strain sequence from BssH II-Sap I and RRV DC5692 strain sequence from Sap I-Eag I.

Finally, the BssH II-Eag I fragment of pRR92 and pRR93 was inserted in the BssH II-Eag I site of pRR64 to create pRR94 and pRR95, respectively.

To generate plasmid pRR73, the RRV DC5692 Xma I-Hind III fragment was inserted into the Xma I-Hind III site of pRR65 to generate pRR71. The Hind III-Sac I fragment of pRR71was inserted into the Hind III-Sac I site of pRR64 to generate pRR72.

Finally, the RRV DC5692 Rsr II-Xma I fragment was cloned into the Rsr II-Xma I site of pRR72 to generate pRR73. Plasmid pRR78 was generated by inserting the RRV DC5692

Xba I-Rsr II fragment into the Xba I-Rsr II site of pRR64. To generate plasmids pRR100, pRR101, and pRR102, pRR64 and pRR73 were digested with Age I, to remove the Age I-

Age I fragment, and then religated at the Age I site to create pRR96 and pRR97, respectively. The RRV DC5692 Age I-Rsr II fragment was inserted into the Age I-Rsr II site of pRR96 and pRR97 to yield pRR98 and pRR99, respectively. Next, the pRR64 Age I-Age

I fragment was inserted into the Age I site of pRR98 and pRR99 to generate pRR100 and pRR101, respectively. The RRV DC5692 Age I-Age I fragment was inserted into pRR96 to yield pRR102. Comparison of the RR64 and DC5692 genome sequences revealed only 2 synonymous nucleotide differences within the first 200 nucleotides of the nsP1 coding

44

sequence of these two RRV strains. Therefore, pRR87 encodes the entire coding region of RRV DC5692 except for the first 193 nucleotides (nucleotides 272–11329). To generate pRR87, the RRV DC5692 BssH II-Xba I fragment was inserted into pRR72 to create pRR82. Next, the RRV DC5692 Rsr II-Xma I fragment was inserted into pRR78 to create pRR85. Finally, The Xba I-Xma I fragment of pRR85 was inserted into pRR82 to create pRR87. To generate plasmid pRR106, pRR87 was digested with Eco RI and the

~8300 bp fragment was gel isolated and religated to generate pRR104. This eliminated nucleotides 6407-11884 of the RRV sequence and generated a plasmid with a unique

Sap I site just 20 bp upstream of the junction of the nsP1 and nsP2 coding regions. Next, the BssH II-Sap I fragment from pRR64 was cloned into pRR104 to create pRR105, which encodes RRV T48 strain sequence from BssH II-Sap I and DC5692 strain sequence from

Sap I-Eag I. The BssH II-Xba I fragment of pRR105 was inserted in the BssH II-Eag I site of pRR87 to create pRR106. To create plasmid pRR108 and pRR109, the Age I-Xma I fragment from pRR102 was inserted into the Rsr II-Xma I site of pRR87 to create pRR107. Finally, the Xba I-Xma I fragment of pRR107 was inserted into the Xba I-Xma I site of pRR87 or pR106 to create pRR108 and pRR109, respectively.

Site Directed Mutagenesis

Single amino acid substitutions (E3 R59G, E2 Y18H, E2 I67M, E2 H94R, E2 R251K,

E2 H256Q, and E2 E302V) were generated by site-directed mutagenesis of plasmid pRR64, which encodes the RRV-T48 genome, using the QuikChange II XL Site-Directed

Mutagenesis Kit (Agilent). The mutagenized Xba 1-Rsr II fragment was subcloned back 45

into pRR64. Clones for each mutant were verified by sequencing. To verify that the mutations were present in virus stocks, virion RNA was isolated, reverse transcribed, cloned into pCR2-TOPO and a portion of the E2 coding region was sequenced. For competition studies, a synonymous mutation was introduced into the RRV-T48 genome in plasmid pRR64 that eliminated the endogenous Rsr II restriction site at position 9573.

The Xba I (6340)/Xma I (10693) fragment from this mutagenized plasmid was sequenced, digested, and ligated into the same sites in pRR64 and pRR64 E2 Y18H to generate plasmids pRR64ΔRsrII and pRR64 E2 Y18H ΔRsrII.

Cells

BHK-21 cells (ATCC CCL-10) were grown in α-minimal essential medium (Gibco) supplemented with 10% bovine calf serum (Hyclone), 10% tryptose phosphate broth, penicillin and streptomycin, and 0.29 mg/ml L-glutamine. C2C12 murine muscle cells

(ATCC CRL-1772) were grown in high glucose Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% fetal bovine serum (Lonza), penicillin and streptomycin, 0.29 mg/ml L-glutamine, and 110 mg/L sodium pyruvate. Normal primary human synovial fibroblasts were obtained commercially (Asterand) and grown in DMEM/F12 medium

(Gibco) supplemented with 10% fetal bovine serum (Lonza), penicillin and streptomycin, and 0.29 mg/ml L-glutamine. Aedes albopictus clone C6/36 mosquito cells (ATCC CRL-

1660) were grown in minimum essential media with Earle’s Salts (Gibco) supplemented with 5% fetal bovine serum (Lonza), non-essential amino acids (Gibco), penicillin and streptomycin, and 0.29 mg/ml L-glutamine. 46

RNA Infectivity Assays

BHK-21 cells were plated at 5.0 × 104 cells/well in 12-well dishes. Cells were transfected with 100 ng, 10 ng, 1 ng, and 0.1 ng of 5′-capped full-length virus RNA transcripts using Lipofectamine 2000 according to the manufacturer’s instructions

(Invitrogen). At 1.5 hours post-transfection, growth media was removed and cell monolayers were overlaid with 0.5% immunodiffusion agarose. Forty hours post transfection, cells were stained with neutral red, plaques were enumerated, and the

PFU per microgram of RNA was determined.

In Vitro Nonstructural Protein Processing

5′-capped full-length RNA transcripts were generated in vitro using SP6-specific mMessage mMachine in vitro transcription kits (Ambion). 0.64 μg of each viral RNA was added to a 100-μl reticulocyte lysate reaction supplemented with [35S]-methionine

[208]. Reaction mixtures were also supplemented with KCl to a final concentration of

0.05 M, and then incubated at 30°C for 40 min, at which point the chase was initiated by adding unlabeled methionine to a final concentration of 1 mM and cycloheximide at a final concentration of 0.6 mg/ml. Samples were then incubated at 37°C for the duration of the chase. Five-microliter samples were removed at 0, 20, 40, 60, and 80 min into the chase and placed into 20 μl of gel loading buffer. Samples were heat denatured at 95°C for 5 min and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) (10% polyacrylamide). Gels were analyzed on a phosphorimager.

47

Western Blots

Cell lysates were collected at 6, 9, 12, 18, and 24 hpi and mixed 1:1 in 2x Laemmli buffer and boiled for 5 minutes. Lysates were separated by SDS-PAGE and proteins were transferred to polyvinylidene fluoride membranes (Bio-Rad). Membranes were blocked in 5% milk dissolved in PBS/0.1 % Tween (PBS-T) and blocked membranes were probed with RRV immune ascitic fluid (ATCC VR-1246AF) at 1:1000 in 5% milk dissolved in PBS-T.

Secondary donkey anti-mouse-HRP conjugated antibody (GE Healthcare) was used for detection at 1:2500 in 5% milk dissolved in PBS-T. Membrane images were obtained using a Chemi-Doc XRS+ system (Bio-Rad) and band intensities were quantified using

ImageLab software (Bio-Rad).

In Vitro Virus Replication

Triplicate wells were inoculated with virus at a multiplicity of infection (moi) of either 0.01, or 5. Viruses were adsorbed to cells for 1 hour at 30°C or 37°C. Wells were then washed three times with 1 ml of room-temperature phosphate-buffered saline

(PBS). One ml of growth medium was then added to each well, and cells were incubated at 37°C (C2C12, Vero, Primary human fibroblasts) or 30°C (C6/36). For cumulative growth analysis, 100 µl samples of culture supernatants were removed at various times post-infection and an equal volume of fresh growth medium was added to maintain a constant volume within each well. Samples were stored at -80°C for analysis by plaque assays on BHK-21 cells.

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Competition Assays

C6/36 or C2C12 cells were plated in 24-well dishes. Growth media was removed and triplicate wells were inoculated at an moi of 0.01 (C6/36) or 1 (C2C12) with a 1:1,

1:10, or 10:1 ratio of either RRV-T48: RRV-T48 E2 Y18H-ΔRsrII or RRV-T48-ΔRsrII: RRV-

T48 E2 Y18H. Viruses were adsorbed to cells for 1 hour. Wells were then washed three times with 1 ml of room-temperature PBS. One ml of growth media was then added to each well. Culture supernatants were collected at 24 hours post-inoculation and stored at -80°C for analysis. Viral titers in the supernatants were quantified by plaque assays and used to infect additional C6/36 cells or C2C12 cells at an moi of 0.01 and 1, respectively. Total RNA was extracted from supernatants using a PureLink RNA-mini kit

(Life Technologies) and cDNA was generated using Superscript III Reverse Transcriptase

(Life Technologies). RRV genomic DNA was amplified via PCR using primers designed to flank the endogenous Rsr II restriction site (Fwd: E2 9194- 5`-

CACTACCAGTACTGACAAGACC-3`. Rev: 6K 9869 5`-CCACAGATATGCCATAGTCTCAGC-3`).

PCR reactions were purified using a PCR cleanup kit (Qiagen), and digested with Rsr II

(NEB). Digested PCR products were run on a 1.8% agarose gel, stained with ethidium bromide (Sigma), and imaged using a Chemi-Doc XRS+ (Bio-Rad). Relative band intensities were quantified using Image Lab 4.0 software (Bio-Rad).

Protein Expression Kinetics Assay

C6/36 or C2C12 cells were plated in 24-well dishes and inoculated at an moi of 5 with either RRV-T48 or RRV-T48 E2 Y18H. At 6, 9, 12, 18 and 24 hpi, cell lysates were

49

collected via harvest of cell culture supernatants followed by one wash in room temperature PBS. After removal of PBS wash, 100 µl ice-cold RIPA buffer with protease inhibitors (Roche) was added to wells. Wells were then scraped and lysates stored at -

80°C until analysis. SDS-PAGE and western blotting were performed as described above.

PFU Per Cell Assay

C2C12 or C6/36 cells were plated in 48-well dishes and allowed to adhere for 6 or 12 hours respectively. Growth media was removed and cells were infected at an moi of 5 with either RRV-T48-double promoter (dp) GFP or RRV-T48 E2 Y18H-dpGFP. Viruses were adsorbed to cells for 1 hour. Wells were washed 5 times with 200 µl room temperature PBS. Following removal of terminal wash, 400 µl of normal growth media was added to each well. At 18 hpi, culture supernatants were collected for titer on BHK-

21 cells, and cells were collected via scraping (C6/36) or trypsin (C2C12) and analyzed for % GFP positive cells using a FACScalibur (Beckton Dickenson) and FlowJo analysis software (Tree Star). Total number of infected cells was calculated and titer data was used to determine the pfus released per infected cell.

Fusion-infection Assay

Fusion of viruses with the plasma membrane was assayed using a variation of a well-established protocol utilized to investigate fusion of Sindbis virus and Semliki Forest virus [209-211]. C2C12 cells were plated in 12-well dishes and allowed to adhere for 18 hours. Cells were washed twice with ice cold RMed (RPMI without sodium bicarbonate +

2% BSA + 10 mM HEPES). RRV-T48-dpGFP and RRV-T48 Y18H-dpGFP viruses were

50

diluted in ice cold RMed at an moi of 5 and allowed to adsorb to cells for 90 minutes on ice. Cells were then treated with buffered ice cold pH media (pH < 6.0: RMed + 20mM

Sodium Succinate, pH ~6.0: RMed + 10mM Sodium Succinate + 10mM MES, pH > 6.0: RMed

+ 20mM MES) and shifted to 37°C for 1 minute and returned to ice. Buffered fusion media was removed and replaced with normal growth media containing 20 mM NH4Cl to prevent secondary infection and cells were returned to 37°C. At 18 hpi, cells were collected, fixed overnight in 1% PFA, and then analyzed for % GFP positive cells using a

FACScalibur (Becton Dickson) and FlowJo analysis software (Tree Star).

Analysis of E2 Surface Expression

C2C12 cells were plated in 12-well dishes and mock-inoculated or inoculated at an moi of 5 with either RRV-T48 or RRV-T48 E2 Y18H. At 6 and18 hpi, cells were collected and incubated for 1 hour on ice with D7 anti-RRV E2 mouse monoclonal antibody [212], a kind gift from Drs. John Aaskov (Queensland University of Technology) and Michael Rossmann (Purdue University). Bound D7 was visualized by incubating cells with Phycoerythrin (PE)-conjugated goat-anti-mouse secondary antibody for 1 hour.

After staining, cells were fixed overnight with 1% PFA and analyzed for E2 surface expression using a FACScalibur (Becton Dickson) and FlowJo analysis software (Tree

Star).

Plaque Assays

BHK-21 cells or C2C12 cells were seeded into 6-well dishes. Growth medium was removed and cell monolayers were inoculated with serial 10-fold dilutions of virus-

51

containing samples in virus diluent (1x PBS + 1% BSC, + 1x Mg2+/Ca2+). Samples were adsorbed for 1 hour at 37°C, followed by overlay with 0.5% immunodiffusion agarose

(MP Biomedicals) in medium for 38-40 hours. Plaques were visualized by neutral red staining (Sigma). Plaque sizes were measured using a 10x Scale Lupe (Peak) and plaque numbers were enumerated to determine the number of BHK or C2C12 plaque forming units (pfu) per ml of culture supernatant and mouse serum, or BHK-pfu per gram of tissue.

Mouse Experiments

C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, Maine) and bred in house. Animal husbandry and experiments were performed in accordance with all University of Colorado School of Medicine Institutional Animal Care and Use

Committee guidelines. Although RRV is classified as a biosafety level 2 pathogen, due to its exotic nature all mouse studies were performed in a biosafety level 3 laboratory.

Three to four week old mice were used for all studies. Mice were inoculated in the left rear footpad with 103 or 105 pfu of virus in diluent (PBS/1% bovine calf serum) in a 10 µl volume. Mock-infected animals received diluent alone. Mice were monitored for disease signs and weighed at 24 hour intervals. Disease scores were determined by assessing grip strength, hind limb weakness, and altered gait as previously described using the following system: 1= very mild deficit in hind paw gripping ability of injected foot only; 2= very mild deficit in bilateral hind paw gripping ability; 3= bilateral loss of gripping ability, mild bilateral hind limb paresis; altered gait not readily observable; 4=

52

Bilateral loss of gripping ability, moderate bilateral hind limb paresis, observable altered gait; difficulty righting self; 5= bilateral loss of gripping ability, severe bilateral hind limb paresis, altered gait, unable to right self; 6= moribund [66, 85]. To determine viral titers in tissues, mice were euthanized by thoracotomy, blood was collected, and mice were perfused by intracardial injection of 1x PBS. The right and left ankles and right and left quadriceps muscles were removed by dissection and weighed. Tissues were homogenized in 1x PBS supplemented with 1% bovine calf serum, Ca2+, and Mg2+ with a

MagNA Lyzer (Roche) and stored at -80°C. The amounts of virus present in tissue homogenates were quantified by plaque assays on BHK-21 cells.

Quantification of Viral RNA

RRV RNA in mouse tissues or virus stocks was quantified as previously described

[85]. Briefly, a sequence-tagged (small caps) RRV-specific RT primer (4415-5'- ggcagtatcgtgaattcgatgcAACACTCCCGTCGACAACAGA-3') was used for reverse transcription and a tag sequence-specific reverse primer (5'-

GGCAGTATCGTGAATTCGATGC-3') was used with a RRV sequence-specific forward primer (RRV: 4346 5'- CCGTGGCGGGTATTATCAAT-3') and internal Taqman probe (RRV:

4375 5'-ATTAAGAGTGTAGCCATCC-3') during qPCR to enhance specificity. To create a standard curve, 10-fold dilutions from 108 to 100 copies of RRV genomic RNA, synthesized in vitro, was spiked into RNA from BHK-21 cells and reverse transcription and qPCR were performed in an identical manner. Absolute quantification was performed using a Light Cycler 480 (Roche). RRV RNA from virus stocks was extracted by

53

heating 5µl culture supernatant with 500ng of RRV-specific RT primer at 94°C for 5 minutes, followed by 70°C for 5 minutes before continuing as described above.

Histological Analysis

At the times indicated, mice were sacrificed by exsanguination, and perfused by intracardial injection of 4% paraformaldehyde, pH 7.3. Tissues were embedded in paraffin and 5-µm sections were prepared. To assess histopathological changes such as tissue inflammation and damage, sections were stained with hematoxylin and eosin

(H&E) and evaluated by light microscopy. pH Stability Assay

Stability of virions after pH treatment was determined by incubating 5*104 pfu of either RRV-T48 or RRV-T48-E2 Y18H virus were incubated at 4°C in media with pH adjusted values of 7.0, 6.5, 6.0, 5.5, and 5.0 for 30 minutes. Samples were then neutralized to a pH of 7.0 and samples were tittered in triplicate via plaque assay on

BHK-21 cells.

Statistical Analysis

Data were analyzed with Prism 5 software (GraphPad Software). Disease scores, percent weight gain, mouse tissue titers, and in vitro growth curve data were evaluated for statistically significant differences by two-way analysis of variance (ANOVA) followed by Bonferroni’s post-test. The pfu/cell experiments were evaluated using a two-tailed t- test with Welch’s Correction. C6/36 cell competition assays were evaluated using one- way ANOVA followed by Tukey’s Multiple Comparison Test while the C2C12 cell

54

competition assays were evaluated using two-tailed t-tests. A P value < 0.05 was considered statistically significant. All differences not specifically indicated to be significant were not significant (P > 0.05).

55

CHAPTER III

MUTATIONS IN NSP1 AND PE2 ARE CRITICAL DETERMINANTS OF ROSS RIVER VIRUS-

INDUCED MUSCULOSKELETAL INFLAMMATORY DISEASE IN A MOUSE MODEL3

Introduction

RRV is a positive-sense, single-stranded RNA virus in the Alphavirus genus of the family Togaviridae [20]. RRV is among a group of related mosquito-transmitted alphaviruses, which includes CHIKV, MAYV, ONNV, and others, that typically cause a debilitating musculoskeletal inflammatory disease in humans. These viruses are an emerging disease threat due to their ability to cause explosive epidemics. Past epidemics include a 1979-to-1980 epidemic of RRV disease in the South Pacific that involved more than 60,000 patients[2] and a 1959-to-1962 epidemic of ONNV in Africa that involved at least 2 million infections [3]. Since 2004, CHIKV has caused major epidemics in multiple countries in the Indian Ocean region with disease estimates on the order of 1–6 million cases[4].

Clinical manifestations following infection with arthritis/myositis-associated alphaviruses develop following an incubation period ranging from 2 to 12 days [2, 11].

The disease is most commonly characterized by fever, intense pain in the peripheral

3 Portions of this chapter are reprinted from Virology, Volume 410, Jupille et al., Mutations in nsP1 and PE2 are critical determinants of Ross River virus-induced musculoskeletal inflammatory disease in a mouse model, Pages 216-227, Copyright © 2011, with permission from Elsevier

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joints, myalgia, and an impaired ability to ambulate [2, 213, 214]. A number of studies indicate that musculoskeletal pain lasts for months to years in a subset of persons infected with RRV or CHIKV, however, the cause of these long lasting symptoms is unclear

[2, 70, 215-218]. There are currently no licensed antivirals or vaccines for any of the arthritis/myositis-associated alphaviruses. Treatment is limited to supportive care with analgesics and anti-inflammatory drugs [2, 11].

To study the pathogenesis of arthritis/myositis-associated alphaviruses, we have developed a mouse model of RRV-induced disease, based on subcutaneous inoculation of 1000 pfu of the T48 strain of RRV into the footpad of 3–4 week-old C57BL/6 mice, that recapitulates many aspects of the human disease [65, 81, 86]. Studies with the RRV mouse model demonstrated that, following a high titer serum viremia, bone/joint-associated tissues and skeletal muscle tissue are the primary targets of RRV replication [65]. RRV replication at these sites results in a severe inflammatory response with abundant tissue damage leading to deficits in grip strength and an altered gait. This model recapitulates many aspects of human RRV and CHIKV infection, including i) high titer serum viremia in people infected with RRV or CHIKV [24, 74], ii) the detection of RRV RNA and antigen in human joint tissue[87, 219], iii) the detection of CHIKV antigen in quadriceps muscle biopsies [80], iv) the presence of mononuclear inflammatory infiltrates in joints and muscle tissue of RRV or CHIKV infected patients [74, 80, 219, 220], and v) the disease symptoms experienced by infected patients. Thus, understanding the host and viral

57

factors that contribute to pathogenesis in this mouse model may aid the development of therapies and vaccines to treat or prevent human disease.

A number of studies have identified genomic regions, genes, or specific molecular determinants that contribute to alphavirus neurovirulence [89, 198, 199, 221-233].

However, no studies have identified viral determinants important for alphavirus-induced musculoskeletal inflammatory disease. Utilizing our RRV disease model, we report here the identification of a murine-attenuated RRV strain (DC5692). To identify genetic determinants associated with virulence, chimeric viruses composed of various portions of the mouse virulent T48 strain and the DC5692 strain were constructed and analyzed in vitro and in vivo. These studies identified critical, yet distinct, roles for determinants encoded in the nonstructural protein 1 (nsP1) and the PE2 coding regions in the pathogenesis of RRV-induced musculoskeletal inflammatory disease.

Results

RRV Strain DC5692 and Virus Derived from a Molecular Clone of RRV Strain

DC5692 (RR87) Replicate like the T48 Strain in Vero Cells, but do not Cause

Musculoskeletal Inflammatory Disease in Mice. We have developed a mouse model of

RRV-induced rheumatic disease that has been utilized to identify host factors that promote inflammation and damage of musculoskeletal tissues following RRV infection

[81, 83, 86 2006, 234]. In contrast, the viral genetic determinants of Alphavirus-induced musculoskeletal inflammatory disease are unknown. In an effort to identify such determinants, we first sought to identify RRV strains that were defective for the

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induction of inflammatory disease in vivo, but which replicated like the virulent RR64

(virus derived from the molecular clone of the T48 strain) in vitro. This strategy was adopted to eliminate viruses that may be attenuated in vivo due to intrinsic replication differences in simple mammalian cell culture systems. Screening of multiple RRV isolates resulted in the identification of one candidate strain (RRV DC5692), which replicated with similar kinetics to RR64-derived virus in Vero cells (Figure 3.1), but unlike RR64,

DC5692 did not cause overt disease signs in infected mice (Figure 3.2A). At 10 dpi, RR64- infected mice had severe inflammation and tissue damage in musculoskeletal tissues such as the quadriceps muscles (Figure 3.2B). Similar to control mice, mice inoculated with 103 pfu of RRV DC5692 did not have evidence of inflammation or damage of quadriceps muscle tissue at 10 dpi (Figure 3.2B). The fact that the DC5692 strain showed similar replication to the T48 strain in Vero cells, but a defect in the induction of virus- induced disease, suggested that this virus might be a useful tool for identifying critical viral determinants of T48 strain-induced musculoskeletal inflammatory disease.

Therefore, as a first step in this process, we sought to create a full length molecular clone of DC5692 with the goal of using this clone to create chimeric viruses with RR64 to map viral virulence determinants.

To establish this system, the complete genome sequence, except the 5′ UTR, of

RRV strain DC5692 was determined (Genbank HM234643) and compared to the

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sequence of RR64. This analysis revealed a total of 356 nucleotide differences (96.8% identity) within the coding region of the genomes (nucleotides 79-11,330). As shown in

Table 1.1, 47 of the 356 nucleotide changes resulted in an amino acid difference.

Figure 3.1 Multi-step Replication Analysis A. Schematic representations of the genomes of the T48 strain molecular clone RR64 (white), RRV strain DC5692 (grey), and the DC5692 molecular clone RR87. B. Vero cells were inoculated with 0.01 pfu/cell of RR64, RRV strain DC5692, or RR87. At the times indicated, 100 μl of supernatant was harvested and virus titers were determined by plaque assay on BHK-21 cell monolayers. The dashed line indicates the limit of detection. No statistically significant differences were detected.

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Table 1.1 Amino Acid Differences between RRV T48 (RR64) and RRV DC5692.

nsP1 nsP2 nsP3 nsP4 S79C R11K I192V T7I A112S A31T S330P V29A L224I E116D T331M T89A C416F M140T V362L V99A S424N T175A V412I A359V L463I S406T K427T E399D R464K A431T V438I A435del. V459I V461A K514N K494E I510V S518P Capsid E3 E2 E1 K73Q R59G Y18H S120L N84K I67M V304A P89S H94R L413M R251K H255Q E302V

Due to the existence of only 2 synonymous nucleotide differences within the first

200 nucleotides of the nsP1 coding sequence of these two RRV strains, a molecular clone that encodes nucleotides 272-11329 of RRV strain DC5692 (pRR87; Figure 3.1A) was constructed as described in Materials and Methods. Infectious virus derived from the molecular clone of strain DC5692 (RR87) replicated like the biological isolate and

RR64 in Vero cells (Figure 3.1B). Furthermore, inoculation of mice with 103 pfu of RR87 did not result in detectable disease signs or evidence of inflammation of quadriceps muscle tissue (Figure 3.2B).

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A Chimeric Virus that Encodes the RRV DC5692 3` UTR in the Genetic

Background of the T48 Strain (RR67) Caused Disease in Mice that was

Indistinguishable from RRV T48 (RR64)- Induced Disease. Sequence analysis revealed a number of differences in the nucleotide sequences of the RRV DC5692 3′ UTR compared to the 3′ UTR of the T48 strain (Figure 3.3A). These differences include a 79 nucleotide deletion within the DC5692 3′ UTR, which eliminates most of repeat element 2, a 34 bp deletion, which eliminates a large region of repeat element 3, an 11 nucleotide deletion, and numerous other nucleotide changes. Thus, the RRV DC5692 strain 3′ UTR (not including the poly A tail) is 398 nucleotides in length with 2 intact repeat elements (1 and 4) whereas the 3′ UTR of the T48 strain is 523 nucleotides in length and encodes 4 repeat elements (1–4). To test whether the 3′ UTR of the T48 strain contributed to virulence in the mouse model, a chimeric virus encoding the RRV DC5692 strain 3′ UTR in the T48 strain genetic background (RR67) was generated (Figure 3.3B). Inoculation of

103 pfu of either RR64 or RR67 resulted in disease signs (Figure 3.3C) and inflammation of quadriceps muscles (Figure 3.3D) that were indistinguishable. These data, together with the data that indicated that the 5′ and 3′ UTRs of the T48 strain were not sufficient to enhance the virulence of the DC5692 strain (Figure 3.2), suggest that the 3′ UTRs of these RRV strains do not encode major virulence determinants in this mouse model of inflammatory rheumatic disease.

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Figure 3.2 The RRV DC5692 Strain and Virus Derived from a Molecular Clone of the DC5692 Strain (RR87) are Attenuated in vivo. A. Twenty-four-day-old C57BL/6J mice were inoculated with PBS (n = 3), or 103 pfu of RR64 (n = 4), RRV strain DC5692 (n = 5), or the molecular clone of DC5692, RR87 (n = 5) by injection in the left rear footpad. Mice were scored for the development of disease signs including loss of gripping ability, hind-limb weakness, and altered gait. Each data point represents the arithmetic mean ± standard deviation (SD). Data was evaluated for statistically significant differences by ANOVA (P < 0.0001) followed by Bonferroni's Multiple Comparison Test. B. At 10 dpi, mice were sacrificed and perfused with 4% paraformaldehyde. Five micron-thick paraffin-embedded sections generated from quadriceps muscles were stained with H&E. Images are representative of three mice per group.

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Chimeric Viruses that Encode the RRV DC5692 nsP1 Coding Region in the T48

Strain Genetic Background are Attenuated In Vivo. To test whether important virulence determinants of T48 strain-induced musculoskeletal inflammatory disease are encoded in the nonstructural region of the genome, a panel of chimeric virus cDNAs was constructed (Figure 3.4). Full-length RNAs encoding the parental and chimeric virus genomes formed plaques with similar efficiency after transfection into BHK-21 cells

(data not shown), whereas all of the chimeric viruses that encode the nsP1 coding region from the DC5692 strain had a small plaque phenotype on BHK-21 cells compared to RR64 (data not shown). Similar to virus derived from the DC5692 molecular clone,

RR87, inoculation of mice with RR76, which encodes nsP1, nsP2, nsP3, and the N- terminal portion of nsP4 from RRV DC5692 strain in the T48 strain genetic background resulted in severely diminished inflammation of quadriceps muscle tissue and disease signs compared to RR64 (Figures 3.4A and 3.4B; P ≤ 0.05). To further map the determinants underlying this phenotype, we compared chimeras encoding either nsP1- nsP2 (RR79) or nsP3 and the N-terminal portion of nsP4 (RR77) from RRV DC5692 in the

T48 genetic background. As shown in Figure 3.4A and 3.4B, while RR77 elicited disease signs identical to the virulent RR64 virus, RR79 was significantly attenuated (P≤ 0.01).

Additional chimeras encoding either nsP1 or nsP2 from the RRV DC5692 strain in the

T48 strain genetic background (RR94 and RR95, respectively) demonstrated that the presence of the nsP1, but not the nsP2, coding region of the DC5692 strain resulted in

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Figure 3.3 A Chimeric Virus that Encodes the RRV DC5692 3`UTR in the T48 Genetic Background (RR67) Caused Disease in Mice that was Indistinguishable from RRV- T48-induced Disease. A. Alignment of the 3′ UTR of the RRV strains DC5692 and T48. The underlined TAA (first row) is the E1 stop codon. The italicized and underlined sequences are the repeat elements present in the T48 3′ UTR. B. Schematic representations of the T48 derived (white) and DC5692 derived (grey) sequence within the genomes of RR64 and RR67. C. Twenty-four-day-old C57BL/6J mice were inoculated with 103 pfu of RR64 (n = 4), RRV strain DC5692 (n = 4), or RR67 (n = 4) by injection in the left rear footpad. Mice were scored for the development of disease signs including loss of gripping ability, hind-limb weakness, and altered gait. Each data point represents the arithmetic mean ± SD. No statistically significant differences were detected. D. At 10 dpi, mice were sacrificed and perfused with 4% paraformaldehyde. Five micron-thick paraffin-embedded sections generated from quadriceps muscles were stained with H&E. Images are representative of four mice per group. 65

diminished inflammation in skeletal muscle tissues and significantly less severe disease signs (Figures 3.4A and 3.4B; P≤0.001). To investigate whether the attenuated phenotype of RR94 was due to inefficient processing of the chimeric P12 protein, we compared nonstructural protein processing kinetics of the parental viruses, RR64 and

RR87, to RR94. No differences in nonstructural protein processing were observed in vitro (data not shown). Taken together, these data suggest that a determinant(s) in nsP1 plays a critical role in the pathogenesis of the musculoskeletal inflammatory disease caused by the mouse virulent T48 strain.

Chimeric Viruses that Encode the RRV DC5692 PE2 Coding Region in the T48

Genetic Background are Attenuated In Vivo. While the data presented above demonstrated that determinants within nsP1 are required for the induction of inflammatory disease by the T48 strain, we also sought to assess the relative contribution of the viral structural genes to disease. Therefore, a second panel of chimeric viruses was constructed to test whether important virulence determinants of

T48 strain-induced inflammatory disease are encoded in the structural region of the genome (Figure 3.5). Full-length RNAs encoding these chimeric virus genomes formed plaques with similar efficiency compared to the T48 strain after transfection into BHK-21 cells (Figure 3.6B). Interestingly, similar to the chimeric viruses encoding nsP1 from the

DC5692 strain, all of the chimeric viruses that encode PE2 from the DC5692 strain had a small plaque phenotype on BHK-21 cells compared to the T48 strain (Figure 3.6A).

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Figure 3.4 Chimeric Viruses that Encode the RRV DC5692 nsP1 Coding Region in the T48 Strain Genetic Background are Attenuated in vivo. A. Twenty-four-day-old C57BL/6J mice were inoculated with 103 pfu of RR64, RR76, RR79, RR77, RR94, or RR95 by injection in the left rear footpad. At 10 dpi, mice were sacrificed and perfused with 4% paraformaldehyde. Five micron-thick paraffin- embedded sections generated from the quadriceps muscle were stained with H&E. Images are representative of three mice per group. B. Twenty-four-day-old C57BL/6J mice were inoculated with 103 pfu of RR64 (n = 19), RR76 (n = 9), RR77 (n = 11), RR79 (n = 9), RR94 (n = 7), or RR95 (n = 3) by injection in the left rear footpad. Mice were scored for the development of disease signs including loss of gripping ability, hind-limb weakness, and altered gait. Each data point represents the arithmetic mean ± standard error of the mean (SEM). Data are combined from four independent experiments and were evaluated for statistically significant differences by ANOVA (P < 0.0001) followed by Bonferroni's Multiple Comparison Test. 67

Inoculation of mice with RR73, which encodes the very C-terminus of E2, 6K, and E1 from the RRV DC5692 strain in the T48 strain genetic background resulted in musculoskeletal inflammation and disease signs indistinguishable from those observed following inoculation of RR64 (Figure 3.5). In contrast, inoculation of mice with a chimera that encodes the C-terminal domain of nsP4, capsid, E3, and the majority of E2 from RRV DC5692 (RR78) or a chimera that encodes E3, E2, 6K, and E1 from RRV DC5692

(RR101) resulted in an attenuated phenotype (Figures 3.5A and 3.5B; P ≤ 0.01). Taken together, these findings suggested that an important determinant(s) was located in the

PE2 coding region. To confirm these finding, two additional chimeras, RR102 and RR100, were constructed. Inoculation of mice with RR102, which encodes portions of RRV

DC5692 nsP4 and capsid in the T48 strain genetic background, resulted in musculoskeletal inflammation and disease signs indistinguishable from T48-inoculated mice (Figures 3.5A and 3.5B). Inoculation of mice with RR100, which encodes RRV

DC5692 E3 and the majority of E2 in the genetic background of the T48 strain resulted in an attenuated phenotype (Figures 3.5A and 3.5B; P≤0.05). Thus, all chimeras that encode the PE2 coding region of the RRV DC5692 strain in the T48 strain genetic background (RR78, RR101, and RR100) were attenuated when inoculated into mice.

These findings suggest that in addition to nsP1, determinant(s) in the PE2 coding region also plays a critical role in the pathogenesis of RRV-induced musculoskeletal inflammatory disease.

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Attenuating Determinants in PE2, but not nsP1 Regulate Viral Tissue Titers in

Mice. To investigate whether the attenuation of RR94 and RR100 chimeric viruses in vivo was associated with differences in viral loads in tissues, mice were inoculated with

T48, RR87, RR94, or RR100 and the amounts of infectious virus present in serum, joint, and skeletal muscle tissues at 1, 3, and 5 dpi were quantified by plaque assays (Figure

3.7). The viral loads in tissues of RR87-inoculated mice were significantly lower compared to T48-inoculated mice at all time points examined (Figure 3.7). Interestingly, the amounts of infectious virus in tissues of RR94-inoculated mice were similar to the amounts in tissues of T48-inoculated mice, suggesting that a determinant(s) in RRV nsP1 regulates activation of host inflammatory responses in musculoskeletal tissues independent of effects on viral titers in these tissues (Figure 3.7). In contrast, the pattern of tissue titers detected in mice inoculated with RR100 was very similar to that detected in mice inoculated with RR87, with the exception of serum titers at 3 dpi

(Figure 3.7).

At 1 dpi, RR87 and RR100 titers were dramatically lower than T48 and RR94 titers in serum, ankle joints, and quadriceps muscles. Whereas in T48 and RR94 tissues peak viral titers occurred by 1 dpi, RR87 and RR100 tissue titers reached a lower peak by

3 dpi and then returned to lower levels by 5 dpi. These findings indicate that the attenuating determinant(s) in the RRV DC5692 PE2 coding region negatively impact efficient viral replication in tissues.

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Figure 3.5 Chimeric Viruses that Encode the RRV DC5692 PE2 Coding Region in the T48 Genetic Background are Attenuated in vivo. A. Twenty-four-day-old C57BL/6J mice were inoculated with 103 pfu of RR64, RR73, RR78, RR101, RR102, or RR100 by injection in the left rear footpad. At 10 dpi, mice were sacrificed and perfused with 4% paraformaldehyde. Five micron-thick paraffin- embedded sections generated from quadriceps muscles were stained with H&E. Images are representative of three mice per group. B. Twenty-four-day-old C57BL/6J mice were inoculated with 103 pfu of RR64 (n = 10), RR73 (n = 7), RR78 (n = 4), RR101 (n = 3), RR102 (n = 3), or RR100 (n = 7) by injection in the left rear footpad. Mice were scored for the development of disease signs including loss of gripping ability, hind-limb weakness, and altered gait. Each data point represents the arithmetic mean ± SEM. Data are combined from three independent experiments and were evaluated for statistically significant differences by ANOVA (P < 0.0001) followed by Bonferroni's Multiple Comparison Test.

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Figure 3.6 A Small Plaque Phenotype on BHK-21 Cells Correlates with Attenuation in Vivo (A) BHK-21 cells were inoculated with serial 10-fold dilutions of parental and chimeric viruses. Following a one hour adsorption at 37°C, cell monolayers were overlaid with 0.5% immunodiffusion agarose, stained with neutral red, and plaque sizes were measured. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 as determined by the Kruskal-Wallis test followed by Dunn’s Multiple Comparison Test. (B) BHK-21 cells were transfected with 100 ng, 10 ng, 1 ng, and 0.1 ng of 5`-capped full-length virus RNA transcripts. At 1.5 hours-post-transfection, growth media was removed, and cell monolayers were overlaid with 0.5% immunodiffusion agarose. Forty hour post-transfection, cells were stained with neutral red, plaques were enumerated, and the PFU per microgram of RNA was determined. No statistically significant differences were observed as determined by the Kruskal-Wallis test followed by Dunn’s Multiple Comparison Test.

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Figure 3.7 Attenuating Determinants in PE2, but not nsP1, Regulate Viral Tissue titers in Mice. Twenty-four-day-old C57BL/6J mice were inoculated with 103 pfu of RR64 (filled circles), RR87 (open circles), RR94 (filled squares), or RR100 (open squares) by injection in the left rear footpad. At 1, 3, and 5 dpi mice (n = 3–6) were sacrificed, blood was collected via cardiac puncture, and mice were perfused by intracardial injection with 1X PBS. Tissues were dissected, weighed, and homogenized and the amount of infectious virus present was quantified by plaque assay on BHK-21 cells. Dashed lines indicate the limit of detection. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 as determined by ANOVA. 72

A Chimeric Virus that Encodes the RRV T48 Strain nsP1 and PE2 Coding Regions in the RRV DC5692 Genetic Background Caused Disease in Mice that was

Indistinguishable from RRV T48 (RR64)- Induced Disease. We next constructed a panel of chimeric viruses to test whether the nsP1 and PE2 coding regions derived from the

T48 strain were sufficient to convert the DC5692 strain, which is highly attenuated in mice (Figure 3.2), into a virus that causes musculoskeletal inflammatory disease (Figure

3.8). Each of these chimeric virus genomes had similar RNA infectivity compared to the

T48 strain (data not shown). Inoculation of mice with RR106, which encodes the T48 nsP1 coding region in the DC5692 strain genetic background (the reciprocal virus to

RR94), resulted in severely diminished inflammation of quadriceps muscle tissue and disease signs compared to T48 (Figures 3.8A and 3.8B; P ≤ 0.01). Similarly, inoculation of mice with RR108, which encodes the T48 PE2 coding region in the DC5692 strain genetic background (the reciprocal virus to RR100), resulted in an attenuated disease phenotype (Figures 3.8A and 3.8B; P≤0.05). These results indicated that neither the T48 nsP1 nor PE2 coding regions alone were sufficient to restore virulence of the DC5692 strain to that of the T48 strain. Inoculation of mice with RR109, which encodes both the

T48 nsP1 and PE2 coding regions in the DC5692 genetic background, resulted in inflammation of musculoskeletal tissue and disease signs indistinguishable from T48- inoculated mice (Figures 3.8A and 3.8B). Interestingly, although neither the T48 nsP1

(RR106) nor the PE2 coding regions alone (RR108) were sufficient to reverse the small plaque phenotype of the DC5692 strain (RR87) on BHK-21 cells, RR109 formed plaques

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on BHK-21 cells that were indistinguishable from those formed by the T48 strain

(Figure 3.6A). These findings indicate that both the T48 nsP1 and PE2 coding regions together are necessary and sufficient to convert strain DC5692 into a virus that caused musculoskeletal inflammatory disease in mice indistinguishable from that caused by the

T48 strain.

Discussion

A large number of studies, utilizing mouse models of SINV, SFV, EEEV, or VEEV neuropathogenesis, have shed light on the genetic determinants of Alphavirus neurovirulence. In contrast, no studies have identified viral genetic determinants that promote Alphavirus-induced rheumatic disease. We identified a strain of RRV (RRV strain DC5692) that replicates like the mouse virulent T48 strain in Vero cells but fails to initiate musculoskeletal inflammation following inoculation into mice. These findings suggested that RRV strain DC5692 could be used to identify RRV genetic determinants critical for the development of disease in the mouse model. Thus, we generated a molecular clone of RRV strain DC5692 and a panel of T48-DC5692 chimeric viruses and used them to identify specific genes associated with the musculoskeletal inflammatory disease.

The 3′ UTRs of Alphaviruses encode a number of sequence elements. These elements include a 19 nucleotide sequence adjacent to the poly A tail that is highly conserved among alphaviruses and functions in RNA replication [109]. In addition,

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Figure 3.8 Substitution of the DC5692 Strain nsP1 and PE2 Coding Regions with Those from the T48 Strain is Sufficient to Enhance the Virulence of the DC5692 Strain. A. Twenty-four-day-old C57BL/6J mice were inoculated with 103 pfu of RR64, RR106, RR108, or RR109 by injection in the left rear footpad. At 10 dpi, mice were sacrificed and perfused with 4% paraformaldehyde. Five micron-thick paraffin-embedded sections generated from quadriceps muscles were stained with H&E. Images are representative of three mice per group. B. Twenty-four-day-old C57BL/6J mice were inoculated with 103 pfu of RR64 (n = 10), RR106 (n = 5), RR108 (n = 7), or RR109 (n = 9) by injection in the left rear footpad. Mice were scored for the development of disease signs including loss of gripping ability, hind-limb weakness, and altered gait. Each data point represents the arithmetic mean ± SEM. Data are combined from three independent experiments and were evaluated for statistically significant differences by ANOVA (P < 0.0001) followed by Bonferroni's Multiple Comparison Test.

Alphavirus 3′ UTRs contain 40-60 nucleotide repeat sequence elements that vary in sequence and length among different alphaviruses [110]. The biologic function of the repeat sequence elements is unclear, although recent evidence suggests these elements may regulate viral RNA deadenylation [111]. Alignment of the DC5692 and T48 genome sequences revealed a number of differences in the 3′ UTRs of these two viruses.

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Extensive variability in 3′ UTRs among RRV isolates has been previously reported [235], however, whether these differences impact the development of disease is not known.

To test whether the differences in the 3′ UTRs contributed to pathogenesis in the mouse model, we generated a chimeric virus that encodes the DC5692 3′ UTR in the T48 backbone (RR67). Inoculation of mice with RR67 resulted in disease indistinguishable from RR64-inoculated mice, suggesting that the 3′ UTR of DC5692 is not sufficient to attenuate T48 in this model of musculoskeletal inflammatory disease. Furthermore,

RR87, which is phenotypically indistinguishable in vitro and in vivo from the DC5692 biological isolate, encodes the complete DC5692 coding region bracketed by the 5′ and

3′ UTRs of T48. Our experiments indicated that replacing the 3′ UTR of DC5692 with the

T48 3′ UTR did not result in a gain of virulence.

To investigate whether genes within the nonstructural coding region of the genome contributed to pathogenesis in the mouse model, a series of chimeric viruses was constructed and tested in vivo for their ability to cause disease. From these experiments, we discovered that substitution of the T48 nsP1 with that from DC5692, which differ by six amino acids, generated a virus (RR94) that failed to cause disease in mice. In contrast to RR94, chimeric viruses encoding DC5692 nsP2, nsP3, or various portions of nsP4 in the T48 genetic background caused disease like T48. Interestingly,

RR94 exhibited no defect in viral replication or specific infectivity in BHK-21 cells (Figure

3.6) and viral loads of RR94 in serum and musculoskeletal tissues were indistinguishable from those of T48-inoculated mice from 1-5 dpi, suggesting that a determinant(s) in

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nsP1 modulates the host inflammatory response independent of effects on RRV tissue titers during the initiation phase of the disease in mice.

Alphavirus nsP1 is a replicase protein that functions in viral RNA replication and viral RNA capping [109]. In studies of temperature sensitive (ts) mutants of SFV and

SINV, only three ts mutations have been mapped to nsP1 and each of these mutants is specifically defective in minus-strand synthesis [236, 237]. In addition, nsP1 encodes guanine-7-methyltransferase and guanylyltransferase activities which are essential for capping and cap methylation of viral genomic and subgenomic RNAs [116-118, 238]. The structure of nsP1 is unknown and deletion studies have failed to define specific enzymatic subdomains. However, specific mutations that affect the binding of enzymatic substrates have been located in the first 310 amino acids of nsP1 [236]. For example, two conserved acidic residues, D62/D88 in RRV nsP1, are essential for S- adenosyl-L-methionine binding and residues S23 and V302 may function in GTP binding

[236].

Previous studies have identified determinants within nsP1 critical for Alphavirus neurovirulence. In all alphaviruses, nsP1 contains 1-3 cysteine residues at positions 418-

420 (SFV numbering) and the nsP1 protein of Semliki Forest virus (SFV) and Sindbis virus

(SINV) is palmitoylated at these conserved cysteines [239, 240]. Mutation of the SFV nsP1 palmitoylation site (CCC to AAA at position 418 to 420) generated a virus that was avirulent in mice [240]. Interestingly, the mouse attenuated RRV strain DC5692 encodes a phenylalanine instead of a cysteine at RRV nsP1 position 416 (C416F). However, in

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contrast to the T48-nsP15692 chimera (RR94), the SFV mutant had dramatically altered growth in BHK-21 cells and dramatically reduced tissue titers in mice at all time points examined [240], suggesting there is a different mechanism of attenuation for RR94. In other studies, a single amino acid change within the nsP1/nsP2 cleavage domain of the neurovirluent SINV strain AR86 (nsP1 T538I), which alters nonstructural protein processing and synthesis of the alphavirus subgenomic RNA, resulted in loss of virulence in mice [208, 232, 241]. Despite these differences, the mutant AR86 replicated as well as the wild-type virus in BHK-21 cells and in the brains of mice following an intracranial inoculation [241]. However, the mutant virus exhibited a restricted pattern of spread within brain tissues, suggesting that the nsP1 mutation regulated the efficiency of viral spread or viral clearance. Furthermore, AR86 with the attenuating T538I nsP1 mutation induced a more robust type I interferon response in both tissue culture and mice [242].

Interestingly, when an analogous mutation was introduced into the nsP1/nsP2 cleavage domain of T48 nsP1 (nsP1 A532V), the mutant RRV was also a more potent inducer of type I interferon in tissue culture, suggesting that determinants within this region of nsP1 regulate alphavirus-induced interferon responses [242]. Compared to T48, the

DC5692 strain does not encode any amino acid changes in the nsP1/nsP2 cleavage domain, suggesting that the kinetics of nonstructural protein processing will not be altered, however, this remains to be determined. In addition, it may be of interest to test whether the amino acid changes present in the DC5692 nsP1 also impact interferon responses.

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Our studies suggest that nsP1 plays a critical role in the initiation of musculoskeletal inflammation and are the first to directly implicate a nonstructural protein in the pathogenesis of Alphavirus-induced inflammatory rheumatic disease. The divergent in vivo phenotypes of T48 and RR94, highly inflammatory vs. non- inflammatory, respectively, suggest that a specific determinant(s) within T48 nsP1 may promote the host inflammatory response, independent of viral titers at the normal sites of inflammation. Comparison of the T48 and DC5692 nsP1 sequences revealed a total of

36 nucleotide changes. Six of these changes resulted in an amino acid coding change

(S79S, A112S, L224I, C416F, S424N, and L463I). Future experiments will assess whether one or a combination of these amino acid changes is responsible for the attenuated phenotype.

In this study, we also investigate whether genes within the structural region of the genome contributed to pathogenesis in the mouse model. Our experiments identified a critical role for a determinant(s) in the PE2 gene, as substitution of the T48

PE2 gene with that from DC5692, which differs by 7 amino acids, resulted in an attenuated phenotype. In contrast to the RR94 chimera, tissue titers in mice inoculated with RR100 (which encodes the DC5692 PE2 gene in the T48 background), were dramatically altered compared to RR64-inoculated mice. These findings support a model in which determinants within PE2 and nsP1 regulate distinct steps within the pathogenic sequence that leads to musculoskeletal inflammation and disease.

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The mechanism(s) by which the DC5692 PE2 protein leads to attenuation will require further study. PE2 and the mature E2 form of the protein have a number of roles in the viral life cycle, including roles in virus attachment and entry, virion assembly, and budding. In addition, the mature E2 glycoprotein is the major target of neutralizing antibodies. A number of studies have implicated determinants in the E2 glycoprotein in

Alphavirus neurovirulence. For example, alternating intracranial passage of a laboratory- adapted SINV in neonatal and weanling mice led to the selection of a SINV strain with increased neurovirulence [243]. A glutamine to histidine at E2 position 55, which enhances binding and entry into neurons as well as virus-induced apoptosis of neurons, was identified as the primary determinant associated with the neurovirulent phenotype

[196, 197]. It will be of interest to test whether the amino acid changes present in the

DC5692 E2 protein regulate RRV binding and entry of specific cell types associated with disease, such as connective tissue fibroblasts, osteoblasts, myofibers, and/or macrophages. In other studies, serial passaging of SINV or VEEV in BHK-21 cells resulted in attenuating mutations associated with adaptation to use of heparan sulfate as a receptor by the acquisition of positively charged amino acid residues in E2 [199, 244].

Based on the low passage history and absence of amino acid changes to positively charged residues, it is unlikely that the DC5692 PE2 protein confers enhanced binding to heparan sulfate, but this remains to be formally tested.

RRV has been isolated from all mainland states and territories of Australia and the island territory of Tasmania. RRV is also endemic in Papua New Guinea. Genetic

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types predominate in geographic regions [245, 246], particularly eastern vs. western

Australia, and this may be due to non-migratory vertebrate hosts allowing for microevolution of geographically separated isolates. The T48 strain was isolated in

Queensland in eastern Australia from Aedes vigilax, whereas the DC5692 strain was isolated in the far southwestern region of Western Australia from Aedes camptorynchus.

It would be interesting to investigate whether the genetic types found in these regions are associated with distinct disease outcomes in humans.

In summary, utilizing pathogenic and apathogenic strains of RRV and a chimeric virus approach, we have identified critical, yet distinct, roles for nsP1 and PE2 in the pathogenesis of RRV-induced rheumatic disease in mice. These are the first studies to identify viral gene regions that promote alphavirus-induced rheumatic disease. Future studies will investigate the specific determinants within nsP1 and PE2 that impact disease outcomes and the mechanism(s) by which these determinants mediate pathogenesis.

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CHAPTER IV

A TYROSINE TO HISTIDINE SUBSTITUTION AT POSITION 18 OF THE ROSS RIVER VIRUS

E2 GLYCOPROTEIN IS A DETERMINANT OF VIRUS FITNESS IN DISPARATE HOSTS4

Introduction

Arthritogenic alphaviruses (genus Alphavirus, family Togaviridae), including RRV,

CHIKV, ONNV, and MAYV virus, are a group of mosquito-transmitted viruses with positive-sense, single-stranded RNA genomes that cause musculoskeletal inflammatory diseases in humans [15]. In addition to causing endemic disease in Australia, Africa, Asia, and South America, these viruses are capable of causing explosive epidemics. Previous outbreaks include a 1959-1962 epidemic of o’nyong-nyong fever in Africa involving at least 2 million cases [247], and a 1979-1980 epidemic of RRV disease in Australia and islands in the South Pacific which involved more than 60,000 cases [2]. Since 2004,

CHIKV has caused a series of epidemics in the Indian Ocean region resulting in millions of cases of severe, debilitating, and often persistent, arthralgia [4]. Furthermore, autochthonous transmission resulting in the first CHIKV disease outbreaks in Europe and the Pacific region occurred in Italy in 2009 [25], in France in 2010 [26], and in New

4 Portions of this chapter are with permission from, Jupille el at., A Tyrosine-to-Histidine Switch at Position 18 of the Ross River Virus E2 Glycoprotein Is a Determinant of Virus Fitness in Disparate Hosts, Journal of Virology, Volume 87, Pages 5970-5984, Copyright ©2013, with permission from American Society for Microbiology.

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Caledonia in 2011 [248]. These examples illustrate the ability of these viruses to re- emerge and to spread to new geographical regions.

Clinical manifestations which follow infection with an arthritogenic alphavirus develop after an incubation period of between 2 to 12 days. Human disease is most commonly characterized by fever, maculopapular rash, intense pain in the peripheral joints, myalgia, and difficulty ambulating [2, 43]. A multitude of studies have indicated that musculoskeletal pain persists for months to years in a subset of patients infected with RRV or CHIKV, however the underlying cause of these persistent symptoms remains unclear [15, 249]. No specific therapies or licensed vaccines are currently available. Treatment is limited to supportive care with analgesics and anti-inflammatory drugs [15, 249].

To study the pathogenesis of arthritogenic alphaviruses, we utilize a previously described mouse model of RRV-induced disease based on subcutaneous inoculation of the T48 strain of RRV into the footpad of 3-4 week old C57BL/6 mice [65, 81, 250].

Studies using this model have shown that following a high titer serum viremia, bone/joint-associated tissues and skeletal muscle tissue are the primary sites of RRV replication [65]. Viral replication in these tissues leads to severe inflammation with significant tissue damage and associated deficits in grip strength and altered gait.

Human disease associated with infection by an arthritogenic alphavirus shows a similar progression with: i) high titer serum viremia [24, 74], ii) detection of virus RNA and/or antigen in musculoskeletal tissues [87, 219], iii) the presence of mononuclear

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inflammatory infiltrates in musculoskeletal tissues [74, 80, 219, 220] and iv) difficulty ambulating and performing routine tasks. These similarities suggest that understanding host and viral factors which contribute to disease in this mouse model will increase our understanding of the pathogenesis of these viruses and may aid in the development of therapies and vaccines to treat or prevent human disease.

Previously, we reported that substitution of the PE2 coding region of the RRV-

T48 strain with that from the attenuated DC5692 strain resulted in an attenuated disease phenotype in mice [66]. Here, we find that a single tyrosine (Y) to histidine (H) substitution at position 18 of the RRV-T48 E2 glycoprotein (E2 Y18H) was sufficient to generate a virus that caused dramatically less severe musculoskeletal disease in the mouse model. The attenuated phenotype of RRV-T48 E2 Y18H was associated with reduced viral loads in tissues and less efficient virus spread. Consistent with these data,

RRV-T48 E2 Y18H replicated less well in murine and human cells in vitro. In contrast, the

RRV-T48 E2 Y18H mutant virus replicated more efficiently than RRV-T48 in C6/36 mosquito cells. Competition studies confirmed that RRV-T48 E2 Y18H had a fitness advantage in mosquito cells and a fitness disadvantage in mammalian cells.

Interestingly, all sequenced Ross River viruses encode either a Y or H at E2 position 18 and this holds true for other alphaviruses in the Semliki Forest antigenic complex. These findings suggest that a tyrosine- histidine switch at E2 position 18 functions as a critical regulator of RRV fitness in invertebrate and vertebrate cells.

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Results

A Single Mutation in RRV, E2 Y18H, Results in Severe Attenuation in the Mouse

Model. In previous studies, substitution of the PE2 coding region of the mouse virulent

T48 strain of RRV with that from the attenuated DC5692 strain generated a RRV that was severely attenuated in a mouse model of alphavirus-induced musculoskeletal inflammatory disease [66]. Furthermore, substitution of the PE2 coding region of the attenuated DC5692 strain with that from the virulent T48 strain, along with substitution of the nsP1 coding region, was sufficient to restore full virulence to the DC5692 strain in the mouse model [66]. These findings indicated that the PE2 coding region encodes critical virulence determinants of RRV-T48-induced rheumatic disease. Sequence comparison between the PE2 regions of the DC5692 and T48 strains showed that there were nucleotide differences in this region which resulted in seven amino acid changes

(E3: R59G; E2: Y18H, I67M, H94R, R251K, H255Q, and E302V) [66]. To investigate the role of specific amino acid changes in attenuation, we introduced each DC5692 coding change individually into the infectious clone of the RRV-T48 strain and tested the ability of virus derived from these clones to cause disease in the mouse model. Inoculation of a cohort of mice (n = 3 mice per group) with 103 BHK pfu of each virus revealed that RRV

E3 R59G, E2 I67M, E2 H94R, E2 R251K, E2 H256Q, or E2 E302V caused disease signs similar to the virulent RRV-T48 strain (Figures 4.1A and 4.1B, P > 0.05). In contrast, mice inoculated with E2 Y18H developed significantly milder disease signs (Figures 4.1A and

4.1B, P < 0.001), similar to our previously published findings with a chimeric virus

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encoding the complete DC5692 PE2 coding region in the T48 strain genetic background

[66]. Consistent with these data, the severity of inflammatory tissue pathology in quadriceps muscle tissue was similar for RRV-T48, E3 R59G, E2 I67M, E2 H94R, E2

R251K, E2, H256Q, and E2 E302V whereas it was markedly less severe in sections derived from mice inoculated with E2 Y18H (Figure 4.1C). To confirm these results, additional mice were inoculated with RRV-T48 (n = 10) or RRV-T48 E2 Y18H (n = 11) and monitored for disease development. As shown in Figures 4.1D and 4.1E, mice inoculated with E2 Y18H had significantly less severe disease signs. In addition, mice inoculated with 105 BHK pfu of RRV-T48 E2 Y18H still exhibited mild disease signs (Figures 4.1D and

4.1E). Taken together, these findings indicate that a single amino acid change from the

T48 strain-encoded tyrosine to the DC5692 strain encoded histidine at position 18 in the

RRV-T48 E2 glycoprotein results in dramatic attenuation of the virus in this mouse model of inflammatory musculoskeletal disease.

We next analyzed available sequence data to determine the amino acid residues present at E2 position 18 of various Ross River virus isolates (Table 4.1). Interestingly, all sequenced Ross River viruses encode either a Y or H at E2 position 18, suggesting a critical role for these two amino acids at this position during the RRV replication cycle.

Based on serological cross-reactivity, viruses in the Alphavirus genus are classified in seven antigenic complexes [19]. All E2 sequences from members of the Semliki Forest

(SF) antigenic complex, which includes RRV, CHIKV, Mayaro virus, o'nyong nyong virus, and others, possess either a Y or H at E2 position 18 (Table 4.1), further supporting an

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important role for these two amino acids at E2 position 18 for this group of medically important alphaviruses.

E2 Y18H Diminishes RRV Replication and Spread in Mice. To investigate the extent to which the reduced disease severity observed in mice inoculated with RRV-T48

E2 Y18H was associated with effects on viral replication and spread, mice were inoculated in the left rear footpad with 103 BHK-pfu of RRV-T48 or RRV-T48 E2 Y18H and the amounts of infectious virus present in tissues at 1, 3, 5, and 7 days post-inoculation

(dpi) were quantified by plaque assays. At 1 dpi, the titer of RRV-T48 E2 Y18H was significantly lower in the left ankle (14.6-fold decrease, P = 0.0004) (Figure 4.2A), a site near the site of inoculation. The titers of RRV-T48 E2 Y18H in the left ankle remained significantly lower at 3, 5, and 7 dpi (P = 0.0045, 0.0021, and 0.0004, respectively). More dramatic differences in virus titers were detected in tissues distal to the site of inoculation. At 1 dpi, the titer of RRV-T48 E2 Y18H was significantly lower in the right ankle (29.5-fold decrease, P = 0.0004) (Figure 4.2B), the left quadriceps (77.4-fold decrease, P = 0.0008) (Figure 4.2C), and the right quadriceps (115-fold decrease, P =

0.0004) (Figure 4.2D) compared to the titers of RRV-T48. Again, viral loads of RRV-T48

E2 Y18H in these tissues remained significantly lower at 3, 5, and 7 dpi (Figures 4.2B-D).

Consistent with these findings, infection with RRV-T48 E2 Y18H produced a significantly lower serum viremia at 1 dpi (59.6-fold decrease, P = 0.0013) and became undetectable in the serum more rapidly than RRV-T48 (Figure 4.2E). To confirm these data, we utilized qRT-PCR to quantify RRV-T48 and RRV-T48 E2 Y18H RNA levels in ankle tissues and

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found that RRV-T48 E2 Y18H inoculated mice had reduced RRV RNA levels at 1 and 3 dpi in both the left and right ankles (data not shown). These data indicate that the E2 Y18H mutation severely reduced RRV-T48 replication and spread in the mouse model, thus, providing a likely explanation for the reduced disease severity.

E2 Y18H Affects RRV Replication in a Cell-Type Dependent Manner. We next investigated the extent to which the E2 Y18H mutant virus exhibited differences in replication in cell culture. To mimic a site of infection in vivo, virus growth analyses were performed in C2C12 murine muscle cells. At an moi of 0.01, we detected significantly reduced titers of RRV-T48 E2 Y18H in culture supernatants by 12 hours post-inoculation

(hpi) (P < 0.05) (Figure 4.3A). At 18 and 24 hpi, titers of RRV-T48 E2 Y18H were reduced

17.8 (P < 0.05) and 80-fold (P < 0.001), respectively, compared to titers of RRV-T48. At an moi of 5 (Figure 4.3B), we also detected significantly reduced titers of RRV-T48 E2

Y18H in culture supernatants at 18 and 24 hpi, although the differences were less dramatic than those detected when C2C12 cells were inoculated with an moi of 0.01

[15-fold (P < 0.001) and 11.3-fold (P < 0.001) at 18 and 24 hpi, respectively]. Similar differences in virus yields were detected in murine L929 fibroblasts and RAW 264.7 macrophages (Figure 4.4).

To investigate whether the E2 Y18H mutation also affected replication in human cells, virus growth analyses were performed in primary human synovial fibroblasts.

Similar to the murine cells, at an moi of 0.01 we detected significantly reduced titers of

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Figure 4.1 RRV E2 Y18H is an Attenuating Mutation in a Mouse Model Three- to four-week old C57BL/6J mice were inoculated with PBS or 103 pfu of RRV- T48, E3 R59G, E2 Y18H, E2 I67M, E2 H94R, E2 R251K, E2 H256Q, or E2 E302V in the left rear footpad (n = 3 per group). At 24-hour intervals, mice were (A) assessed for weight gain and (B) scored for the development of disease signs including loss of gripping ability, hind-limb weakness, and altered gait. Each data point represents the arithmetic mean +/- SD. Data were evaluated for statistically significant differences by two-way ANOVA followed by Bonferroni’s test. *** P < 0.001. (C) At 10 dpi, mice were sacrificed, and perfused with 4% paraformaldehyde. Five-micron thick paraffin-embedded sections generated from the quadriceps muscle were stained with H & E. Images are representative of 3 mice per group. (D-E) Cumulative % weight gain and disease scores for PBS-inoculated mice (n = 3), mice inoculated with 103 pfu RRV T48 (n=10), 103 pfu RRV-T48 E2 Y18H (n=14), or mice inoculated with 105 pfu E2 Y18H (n=3). Data are combined from 3-4 independent experiments. Each data point represents the arithmetic mean +/- SD. Data were evaluated for statistically significant differences by two-way ANOVA followed by Bonferroni’s test. ** P < 0.01, *** P < 0.001. 89

Table 4.1 Amino Acid Residue at Position 18 (or Equivalent) of the Alphavirus E2 Glycoprotein.

Antigenic Cell culture Amino Acid Accession Complex Virus Strain Source Location Year history at PYXX?C Number

Grallina Ross River Virus 2975 cyanoleuca (bird) Australia 1965 6x SMB, 1x Vero Y ACV67004.1

Microeca Australia Ross River Virus 2982 fascinans (bird) (northeast) 1965 6x SMB, 1x Vero Y ACV66994.1

Poephila Australia Ross River Virus 3078 personata (bird) (northeast) 1965 6x SMB, 1x Vero Y ACV66996.1

Macropus agilis Australia Ross River Virus 8961 (wallaby) (northeast) 1965 6x SMB, 1x Vero H ACV66998.1

Macropus agilis Australia Ross River Virus 9057 (wallaby) (northeast) 1968 6x SMB, 1x Vero H ACV67000.1

Aedes Australia 1x C6/36, 1x Ross River Virus DC5692 camptorynchus (western) 1995 Vero, 1x BHK-21 H ADJ78349.1

Culex Australia Ross River Virus K1503 annulirostris (Northwest) 1984 2x C6/36, 1x Vero H AAB00026.1

Semliki Forest virus Semliki Forest Mixed Aedes and New South Ross River Virus NB1053 Culex sp. Whales 1989 1x C6/36, 3x Vero H AAB00050.1

New South NP_740684. Ross River Virus NB5092 Aedes vigilax Whales 1969 1x SMB X 1

Australia C6/36 cells Ross River Virus PW11 Homo sapiens (Northwest) 2009 (previous paper) H AEC49766.1

Australia C6/36 cells Ross River Virus PW14 Homo sapiens (Northwest) 2009 (previous paper) H AEC49788.1

Australia C6/36 cells Ross River Virus PW7 Homo sapiens (Southwest) 2010 (previous paper) H AEC49747.1

PMID: Australia 19759236 Ross River Virus QML1 Homo sapiens (northeast) 2004 1x C6/36 H Table S2

Australia C6/36 cells Ross River Virus SN39 Homo sapiens (Ease Coast) 2009 (previous paper) H AEC49809.1

Australia C6/36 cells Ross River Virus SN85 Homo sapiens (Eastern) 2009 (previous paper) H AEC49829.1

SW2027 Aedes Australia Ross River Virus 6 camptorynchus (Southwest) 1991 1x C6/36, 1x Vero H AAB00011.1

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Antigenic Cell culture Amino Acid Accession Complex Virus Strain Source Location Year history at PYXX?C Number

Aedes Australia Ross River Virus SW2191 camptorynchus (Southwest) 1988 4x C6/36 H AAB00003.1

Aedes Australia Ross River Virus SW876 camptorynchus (Southwest) 1987 2x C6/36, 2x Vero H AAB00002.1

Australia 6x SMB, 1x Ross River Virus T48 Aedes vigilax (Northern) 1959 C6/36, 1x Vero Y ACV67002.1

Australia (Northern 1x C6/36, 3x BHK- Ross River Virus V993 Aedes vigilax Territory) 1986 21 H AAB00025.1

Culex Australia Ross River Virus WK20 annulirostris (Northwest) 1977 ?x SMB, 1x Vero H AAB00024.1

Unknown 2005 Homo sapiens PMID: - Chikungunya Virus 06-027 (CS Fluid) 16700631 2006 1x C6/36 H CAJ90488.1

India 2003 Chikungunya Virus 653496 Homo sapiens (Nagpur) ? Unknown H AAR84279.1

Angola Unknown, 1x Chikungunya Virus M2022 Unknown Angola 1962 C6/36 H ADG95956.1

ArD p-5 (From PMID: Chikungunya Virus 93229 Aedes dalzieli Senegal 1993 2041028) H ADG95948.1

Central DakAr B Anopheles Africa 5x SMB, 2x Vero, Chikungunya Virus 16878 (Ceilia) funestus (Bououi) 1984 1x C6/36 H ADG95881.1

Chikungunya Virus DRDE-07 Homo sapiens ECS African 2007 Unknown H ACA81773.1

Gibbs Chikungunya Virus 63-263 Homo sapiens India 1963 Unknown H ADG95936.1

India IND-06- Unknown (PMID: (Maharashtr Chikungunya Virus MH2 17554030) a) 2006 1x C6/36 H ABN04192.1

LR2006- Chikungunya Virus OPY1 Homo sapiens La Reunion 2006 1x Vero H ABD95938.1

3x SMB, 1x Vero, Chikungunya Virus PM2951 Aedes aegypti Senegal 1966 1x C6/36 H ADG95883.1

Ross Low- 16x SMB, 1x Chikungunya Virus passage Homo sapiens Tanzania 1953 Vero, 1x C6/36 H ADG95932.1

Chikungunya Virus RSU1 Homo sapiens Indonesia 1985 2x Vero, 1x C6/36 H ADG95906.1

P176, 3x SMB, 1x Chikungunya Virus S27 Homo sapiens Tanzania 1953 BHK-21 H AAO33341.1

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Antigenic Cell culture Amino Acid Accession Complex Virus Strain Source Location Year history at PYXX?C Number

Chikungunya Virus SL-CK1 Homo sapiens Sri Lanka 2007 1x Vero, 1x C6/36 H ADG95913.1

Sri Lanka (Japanese Chikungunya Virus SL10571 Homo sapiens patient) 2006 ?x Vero H BAH97933.1

Chikungunya Virus SL15649 Homo sapiens Sri Lanka 2006 ?x Vero H ACZ72971.1

21x SMB, 1x Chikungunya Virus TH35 Homo sapiens Thailand 1958 C6/36 H ADG95930.1

Onyong'nyong Homo sapiens Virus Gulu and Anopheles Uganda 1959 Unknown H P22056.1

Onyong'nyong Virus SG650 Homo sapiens Uganda 1996 1x Vero H AAC97205.1

IBH1096 Igbo Ora 4 Homo sapiens Nigeria 1966 Unknown H AAC97207.1

TRVL Trinidad and Mayaro virus 4675 Homo sapeins Tobego 1954 7x SMB, 1x BHK Y AAO33335.1

Homo sapiens French Mayaro virus MAYLC (Lab tech) Guiana 1998 1x Vero cells Y AAY45742.1

Culex tritaenioryhnchu Getah virus HB0234 s Giles China 2002 BHK or C6/36 Y ABV68937.1

LEIV 16275 Getah virus Mag Aedes sp. Russia 2000 Unknown Y ABR23661.1

LEIV Getah virus 17741 Culex sp. Mongolia 2000 Unknown Y ABR23663.1

South Getah virus Korea Porcine South Kora 2004 Unknown Y AAU85260.1

Armigeres Getah virus YN0540 subalbatus China 2005 ?x BHK Y ABV68939.1

Adult female P13/SM2/BHK21- Sagiyama virus Original mosquitos Japan 1956 1 Y AAO33337.1

Bebaru virus Original Culex sp. Malaysia 1956 Unknown Y AEJ36225.1

Una virus Original Psorophora ferox Brazil 1959 Suckling mice Y AEJ36237.1

Semliki Forest waiting for 1942 virus A7 reference Uganda ? Y CAD90834.1

Semliki Forest A774 (A7 waiting for 1942 virus strain) reference Uganda ? 5x MBA13 cells Y CAA55002.1

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Antigenic Cell culture Amino Acid Accession Complex Virus Strain Source Location Year history at PYXX?C Number

Semliki Forest Aedes 10x SMB from virus L10 Abnormalis Uganda 1952 original isolate Y AAM64227.1

Culex Semliki Forest tritaenioryhnchu virus Vietnam s Vietnam 1971 ?x SMB Y ACB12688.1

Barmah Forest Culex 1x SMB, 1x Vero, NP_054024. virus BH2193 annulirostris Australia 1974 1x C6/36, 1x BHK W 1

Western Equine Horse Brain Ps, Encephalitis Virus 71V1658 Equine Brain USA 1971 1x SM F ACT75286.1

Western Equine 85- Encephalitis Virus 452NM Culex tarsalis USA 1985 2x Suckling Mice F ACT75290.1

Western Equine Encephalitis Virus Imperial Culex tarsalis USA 2005 2x Vero F ACT75278.1

Western Equine McMilla 2x Mice, 1x SMB, Encephalitis Virus n Homo sapiens Canada 1941 2x Vero F ACN87273.1

Western Equine Montana 1x Duck Embryo Encephalitis Virus -64 Equine Brain USA 1967 cells F ACT75282.1

Buteo jamaicensis Highlands J virus 585-01 (Hawk) USA 2001 1x Vero H ACO59902.1

Strix varia Georgia Highlands J virus 744-01 (barred owl) (USA) 2001 Unknown H ACZ34298.1

WEEV Highlands J virus B-230 Equine brain Florida (USA) 1964 SMB H ACT32135.1

Colorado unpassaged (no Fort Morgan virus CM4-146 Oeciacus vicarius (USA) 1974 information) R ACT68009.1

Sindbis virus 631310 Culex sp. India 1963 Unknown T ACU25461.1

Sindbis virus 95M116 Aedes cinereus Sweeden 1995 Vero cells T ACU25469.1

B322/23 Sindbis virus /24 Motacilla alba India 1953 10x mice T ACU25463.1

Girdwoo ~195 Sindbis virus d Culex univittatus Egypt 5 Unknown T AAA86134.1

Unkn NP_740675. Sindbis virus hrsp Unknown Unknown own ?X CEFs T 1

Culex 1966 tritaenioryhnchu - Sindbis virus MRE-16 s Malaysia 1969 AP61 or C6/36 T AAC59319.1

Unkn Sindbis virus R2215 Unknown Unknown own Unknown T Unknown

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Antigenic Cell culture Amino Acid Accession Complex Virus Strain Source Location Year history at PYXX?C Number

SA80- Saude Sindbis virus 294 Culex univittatus Arabia 1980 Unknown T ACU25467.1

Sindbis virus SAAR86 Culex sp. South Africa 1954 unknown T Unknown

Culex Sindbis virus SW6562 annulirostris Australia 1990 Vero cells T AAM10630.1

C6/36 cells, BHK Sindbis virus XJ-160 Anopheles sp. China 1990 cells T AAC83379.1

Edsybn Ockelbo virus 82-5 Culiseta sp. Sweeden 1982 1x Vero T AAA96973.1

Eastern Equine FL93- Encephalitis virus 1637 Culex erraticus Florida (USA) 1993 1x Vero D ADB08661.1

Eastern Equine Phoca vitulina Massacheus Encephalitis virus MA06 (seal) etts, (USA) 2006 1x Vero D ACY66806.1

EEEV Eastern Equine ME7713 Massacheus 1x mosquito, 1x Encephalitis virus 2 Mosquito etts, (USA) 1977 C6/36 D AAC53758.1

Eastern Equine Culiseta New Jersey 6x unknown, 1x Encephalitis virus NJ/60 melanura (USA) 1959 suckling mice D ABQ63086.1

Eastern Equine Connecticut Encephalitis virus Williams Equine Brain (USA) 1990 1x Vero D AAC53760.1

Venezuelan equine encephalitis virus 243937 Horse Venezuela 1992 2x Suckling Mice R AAC71998.2

Venezuelan equine 4x Suckling Mice, encephalitis virus Fe-37c Culex sp. Florida (USA) 1963 6x Vero R P36330.1

Venezuelan equine Syrian hampster, encephalitis virus MX01-22 Syrian hamster. Mexico 2001 1x Vero R AAW30006.1

VEEV

Venezuelan equine encephalitis virus SH3 Homo sapiens Venezuela 1993 1x Vero R AAC71999.2

Venezuelan equine Trinidad Trinidad and 1x Guinea Pig, 6x encephalitis virus Donkey Donkey Tobego 1943 Vero R AAC19322.1

Venezuelan equine encephalitis virus ZPC738 Hamster Venezuela 1997 none R AAD27803.1

Dead Horse Middelburg virus 857 Spleen Zimbabwe 1993 3x Vero, 2x BHK Y ABP73666.1

Original Unknown (serial Ndumu virus isolate Aedes sp. South Africa 1959 pasasge in SMB) Y AEJ36231.1

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Figure 4.2 The E2 Y18H Mutation Reduces RRV Tissue Titers and Spread Three- to four-week old C57BL/6J mice were inoculated with 103 pfu of RRV T48 (closed circles) or E2 Y18H mutant virus (open squares) by injection in the left rear footpad. At 1, 3, 5, and 7 dpi, mice (n = 4-7) were sacrificed, blood was collected via cardiac puncture, and mice were perfused via intracardial injection with 1X PBS. Tissues were dissected, weighed, homogenized, and the amounts of infectious virus present was quantified via plaque assays. Dashed lines indicate the limit of detection. Data were evaluated for statistically significant differences by two-way ANOVA followed by Bonferroni’s test. * P < 0.05, ** P < 0.01, *** P < 0.001.

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RRV-T48 E2 Y18H in culture supernatants at 18 and 24 hpi [82-fold decrease (P < 0.05) and 36-fold decrease (P < 0.001), respectively] (Figure 4.3C). At an moi of 5, the titers of

RRV-T48 E2 Y18H in culture supernatants of the primary human synovial cells were reduced approximately 3-fold at 18 and 24 hpi, however, these differences were not statistically significant (Figure 4.3D). In contrast to these findings, when Aedes albopictus C6/36 cells were inoculated at an moi of 0.01 (Figure 4.3E), we detected significantly higher titers of RRV-T48 E2 Y18H in culture supernatants at 12 and 18 hpi

[13-fold (P < 0.01) and 7.7-fold (P < 0.001) increase, respectively]. When C6/36 cells were inoculated at an moi of 5 (Figure 4.3F), no significant differences were detected in viral titers. Because of the temperature difference between the C2C12 and C6/36 cell growth analyses, we next investigated replication in C2C12 cells at 30°C to determine if

RRV-T48 E2 Y18H conferred a temperature sensitive phenotype. At an moi of 0.01, we observed that RRV-T48 E2 Y18H replicated to significantly lower titers than RRV-T48 at both 37°C and 30°C (Figure 4.3G). Further characterization studies of the RRV-T48 E2

Y18H virus indicated that the mutation did not affect virion stability at 37° (data not shown). Taken together, these data suggest that a tyrosine at RRV-T48 E2 position 18 provides a replication advantage in murine muscle cells whereas a histidine at RRV-T48 position 18 provides a replication advantage in C6/36 mosquito cells.

The Amino Acid at RRV E2 Position 18 is a Determinant of Virus Fitness in Cells.

To confirm that a Y or H at RRV E2 position 18 enhances RRV fitness in mammalian and mosquito cells, respectively, we utilized a competition assay [204, 251]. A synonymous

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mutation (9569-5`-gccggtCcgcc-3`) → (9569- 5`-gccggtAcgcc-3`) was introduced into the

RRV-T48 and RRV-T48 E2 Y18H genomes which ablated the endogenous Rsr II restriction site (ΔRsrII). C6/36 cells were inoculated with 1:1, 1:10, or 10:1 ratios of either RRV-T48:

E2 Y18HΔRsrII, or RRV-T48ΔRsrII: E2 Y18H at an moi of 0.01 and virus-derived from these infections was serially passaged for a total of 5-10 passages. RNA was isolated from culture supernatants, the region containing the Rsr II restriction site was amplified, and the relative abundance of each competitor was quantified by measuring the intensity of Rsr II-digested DNA fragments as described in the Materials and Methods.

As shown in Figures 4.5A and 4.5B, after 5 passages we detected increased amounts of

RRV-T48 E2 Y18H in supernatants derived from C6/36 cell cultures which had been inoculated at a 1:1 ratio. Importantly, similar results were obtained in parallel competitions when either RRV-T48 (Figure 4.5A) or RRV-T48 Y18H (Figure 4.5B) carried the Rsr II marker. Consistent with these results, RRV-T48 Y18H remained the predominant genotype detected in culture supernatants from cells that were inoculated with a 1:10 ratio of RRV-T48: RRV-T48 Y18H (Figures 4.5C and 4.5D). After 5 passages we detected a shift in favor of RRV-T48 E2 Y18H when cells were inoculated with a 10:1 ratio of RRV-T48: RRV-T48 Y18H (Figures 4.5E and 4.5F). To further verify this, the 10:1 ratio samples were passaged an additional 5 times. By passage 10, RRV-T48 E2 Y18H was significantly more abundant than RRV-T48 (P < 0.001) (Figures 4.5E and 4.5F).

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Figure 4.3 The E2 Y18H Mutation Affects RRV Replication in a Cell Type-Dependent Manner C2C12 murine muscle cells (A and B), primary human synovial fibroblasts (C and D), or C6/36 mosquito cells (E and F) were inoculated with RRV T48 or RRV-T48 E2 Y18H at an moi of 0.01 (A, C, and E) or 5 (B, D, and F). At 0 (input), 1, 6, 12, 18, and 24 hpi the amounts of infectious virus present in culture supernatants were quantified by plaque assays. Each data point represents the arithmetic mean +/- SD. Data were evaluated for statistically significant differences by two-way ANOVA followed by Bonferroni’s test. * P < 0.05, ** P < 0.01, *** P < 0.001. (G) C2C12 murine muscle cells were inoculated with RRV-T48 or RRV-T48 E2 Y18H at an moi of 0.01 and incubated at 37°C or 30°C. At 0 (input), 1, 12, and 24 hpi the amounts of infectious virus present in culture supernatants were quantified by plaque assays. Each data point represents the arithmetic mean +/- SD. Data were evaluated for statistically significant differences by two-way ANOVA followed by Bonferroni’s test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Taken together, these data suggest that a histidine at E2 position 18 of RRV provides a fitness advantage in mosquito cells. In stark contrast, similar competition experiments demonstrated that a tyrosine at RRV E2 position 18 provided a strong fitness advantage in C2C12 murine muscle cells by three passages (Figures 4.5G and 4.5H). 98

Figure 4.4 The E2 Y18H Mutation Affects RRV Replication in a Cell Type-Dependent Manner L929 murine fibroblasts (A), or RAW murine macrophages (B), were inoculated with RRV T48 or RRV-T48 E2 Y18H at an moi of 0.01. At 0 (input), 1, 6, 12, 18, and 24 hpi the amounts of infectious virus present in culture supernatants were quantified by plaque assays. Each data point represents the arithmetic mean +/- SD. Data were evaluated for statistically significant differences by two-way ANOVA followed by Bonferroni’s test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Additional Mutations at E2 Position 18 are Also Attenuating Mutations in Mice.

To investigate the effect of additional amino acid changes at E2 position 18, we introduced additional mutations: Y18A, Y18D, and Y18F at E2 position 18 into the RRV-

T48 strain and tested the ability of virus derived from these clones to cause disease in the mouse model. Inoculation of a cohort of mice (n = 3- 4 mice per group) with 103 BHK pfu of RRV-T48, E2 Y18H, E2 Y18F, or E2 Y18D revealed that only RRV-T48 inoculated mice developed significant disease, while all other E2 position 18 mutants were significantly attenuated (Figure 4.6A and B). Further studies investigating in vitro

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replication kinetics showed that similar to E2 Y18H, all other E2 position 18 mutations resulted in reduced viral replication in mammalian cells (Figure 4.6C).

Discussion

Few studies have identified viral virulence determinants that contribute to alphavirus-induced rheumatic disease. Using chimeric viruses, we previously reported that substitution of the PE2 coding region of the T48 strain of RRV with that from the attenuated DC5692 strain resulted in an attenuated disease phenotype in a mouse model of alphavirus-induced musculoskeletal inflammatory disease [66]. In gain of virulence studies, we found that substitution of both the nsP1 and the PE2 coding regions of the DC5692 strain with those from the virulent T48 strain was required for a gain of virulence [66]. In this study, we identified a single amino acid substitution, E2

Y18H, which functioned as the major attenuating mutation within the PE2 coding region. Further, we showed that attenuation in the mouse model due to the E2 Y18H mutation was associated with reduced RRV-T48 loads in tissues and reduced RRV-T48 spread, suggesting that the mutation affected the RRV-T48 replication cycle.

Consistent with these data RRV-T48 E2 Y18H replicated less efficiently than RRV-

T48 in a variety of murine and human cells. In contrast to the effects of this mutation in mammalian systems, RRV-T48 E2 Y18H replicated more efficiently in C6/36 mosquito cells. Competition studies in both mammalian and mosquito cells confirmed that a

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Figure 4.5 The Amino Acid at RRV Position 18 Toggles RRV Fitness in Disparate Host Cells C6/36 cells were inoculated at an moi of 0.01 with a 1:1 ratio (A and B), a 1:10 ratio (C and D), or a 10:1 ratio (E and F) of either T48:E2 Y18HΔRsrII (A, C, and E) or T48ΔRsrII:E2 Y18H (B, D, and F). At 24 hpi, culture supernatants were collected and titered on BHK-21 cells. New C6/36 cells were then inoculated at an moi of 0.01. This process was repeated a total of 5-10 times. At the passages shown, total RNA was isolated from culture supernatants and a portion of the E2 gene was amplified by RT- PCR. PCR amplicons were digested with Rsr II and band intensities of undigested and digested products were quantified to determine the relative ratios of each virus genome. Data were evaluated for statistically significant differences by two-way ANOVA followed by Bonferroni’s test. ** P < 0.01, *** P < 0.001. C2C12 cells were infected at an moi of 1 with a 1:1, 1:10, or 10:1 ratio of T48:E2 Y18HΔRsrII (G) or T48ΔRsrII:E2 Y18H (H). At 24 hpi, culture supernatants were collected and titered on BHK-21 cells. New C2C12 cells were then inoculated at an moi of 1. This process was repeated a total of three times and the relative amounts of each genotype present in culture supernatants were quantified as described above. Data were analyzed for statistically significant differences by a two-tailed t-test.

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Figure 4.6 A Tyrosine at E2 Position 18 is Required for Virulence in Mice and Enhanced Replication in Mammalian Cells Three- to four-week old C57BL/6J mice were inoculated with PBS or 103 pfu of RRV- T48, E2 Y18H, E2 Y18D, or E2 Y18F in the left rear footpad (n = 3-4 per group). At 24- hour intervals, mice were (A) assessed for weight gain and (B) scored for the development of disease signs including loss of gripping ability, hind-limb weakness, and altered gait. Each data point represents the arithmetic mean +/- SD. Data were evaluated for statistically significant differences by two-way ANOVA followed by Bonferroni’s test. *** P < 0.001. (C) C2C12 cells were infected at MOI 0.01 with RRV- T48, E2 Y18H, E2 Y18A, or E2 Y18F. At 0 (input), 1, 6, 12, and 24 hpi the amounts of infectious virus present in culture supernatants were quantified by plaque assays. Each data point represents the arithmetic mean +/- SD.

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tyrosine at RRV E2 position 18 is advantageous in mammalian cells whereas a histidine at RRV E2 position 18 is advantageous in mosquito cells. Thus, our studies suggest that the amino acid at RRV E2 position 18 toggles the fitness of RRV in disparate hosts.

To identify attenuating mutations encoded in the PE2 region, we introduced each of the attenuated RRV DC5692 strain PE2 amino acid coding changes, 7 in total, individually into the genetic backbone of the virulent RRV T48 strain. This panel of viruses was then tested in our mouse model of RRV-induced musculoskeletal inflammatory disease, a model that recapitulates many aspects of the human disease caused by arthritogenic alphaviruses [15, 65, 66]. These studies revealed that a single amino acid change, E2 Y18H, was sufficient to recapitulate the attenuated phenotype of a chimeric virus that encoded the entire DC5692 PE2 coding region in the genetic backbone of the T48 strain [66]. In comparison to RRV-T48, the E2 Y18H mutant virus was attenuated by all measurements of disease severity: body weight changes, musculoskeletal disease signs such as altered gait and diminished grip strength, and histopathological changes in skeletal muscle tissues (Figure 4.1). While E2 Y18H was identified as the major attenuating mutation, other mutations in this region (such as E2

R251K and E2 H256Q) altered body weight changes compared to RRV-T48, suggesting that these mutations may also make minor contributions to virulence. To investigate the mechanisms of attenuation, we measured the amounts of infectious virus present in tissues near and distal to the site of inoculation at time points throughout the acute stage. These experiments demonstrated that the attenuated disease phenotype of RRV-

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T48 E2 Y18H was associated with significantly reduced viral loads in tissues, and less efficient spread of the virus to tissues distal to the site of inoculation (Figure 4.2), suggesting that the E2 Y18H mutation altered a critical aspect of the RRV replication cycle.

Recently, the structure of the Sindbis virus and CHIKV E2 proteins were solved

[102, 103], allowing for the accurate placement of RRV E2 position 18 within the context of the alphavirus E1-E2 heterodimer (Figure 5.1). Based on the published structures, E2 position 18 is localized on the underside of E2, near the fusion loop of the E1 protein in what Voss et al. term the “N-flap” domain, which is composed of E2 amino acid residues

16-30 [103]. While a variety of studies have identified virulence determinants within the

E2 glycoprotein of various alphaviruses, many of these determinants are located near the distal-most “petal” region of E2 in domain B or the outer surface of domain A which have been implicated in host-cell receptor binding, antibody neutralization, and the acquisition of mutations that increase E2 and virus binding to glycosaminoglycans [196,

198, 199, 201, 222, 225, 230, 252-254]. For example, an amino acid substitution in the

E2 protein of CHIKV, G82R was identified as a major attenuating determinant in the

181/clone 25 vaccine strain [201]. CHIKV E2 position 82 is located on the outer surface of E2 domain A and a glycine to arginine mutation at this position has been postulated to increase interactions with glycosaminoglycans, which was identified as a possible attenuating mechanism for the CHIKV 181/ clone 25 strain [255]. Based on the localization of position 18 within the E2 glycoprotein, it is unlikely that a tyrosine to

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histidine mutation at this position alters the interaction of RRV with cell surface glycosaminoglycans. This conclusion is supported by our own experiments that failed to detect enhanced susceptibility of RRV-T48 E2 Y18H plaque formation to preincubation with heparin (data not shown). Furthermore, a single amino acid change on the exposed petal region of RRV E2, N218R, has been shown to confer heparan sulfate binding to

RRV [153, 256]. In addition to the R82G mutation, E2 T12I was also identified as a major attenuating mutation of the CHIKV 181/clone 25 vaccine strain. Interestingly, the identical mutation in Semliki Forest virus reduced the pH required to trigger E1 fusion activity by regulating the dissociation of the E1/E2 dimer [200]. Although this position is near E2 position 18, the crystal structure of the CHIKV E2 glycoprotein suggests that position 12 and position 18 are in distinct domains and, as discussed below, we did not observe any effect of the RRV E2 Y18H mutation on the pH of RRV entry. Thus, to our knowledge, this study is the first to identify an important alphavirus virulence determinant in the N-flap domain of the E2 glycoprotein.

A Tyrosine at Position 18 is Advantageous in Mammalian Cells, Whereas a

Histidine at Position 18 is Advantageous in Mosquito Cells. Our studies in mice indicated that a histidine at E2 position 18 diminished RRV replication in tissues. To investigate if the RRV-T48 E2 Y18H mutation affected RRV replication in cultured cells, we performed virus growth analyses in a variety of murine cell lines and primary human synovial cells. These experiments showed that the E2 Y18H mutant virus replicated to significantly lower titers compared to RRV-T48 (Figure 4.3). Furthermore, these studies

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indicated that the effects of the E2 Y18H mutation on replication capacity were not limited to murine tissues and cells. As an arbovirus, RRV is maintained in nature by alternating cycles of replication in vertebrates and mosquitoes. Strikingly, RRV-T48 E2

Y18H replicated more efficiently than RRV-T48 in C6/36 mosquito cells, suggesting that a histidine at E2 position 18 enhances RRV fitness in mosquito cells. In both mammalian and mosquito cells, our results indicated that the effects of the E2 Y18H mutation on yields of infectious virus were moi-dependent, suggesting that the E2 Y18H mutation has subtle effects in a single replication cycle that are amplified after several cycles of replication. In addition, our experiments showed that the E2 Y18H mutant virus replicated less efficiently than RRV-T48 in C2C12 cells regardless of temperature, suggesting that the advantage of the E2 Y18H mutant virus in C6/36 cells was not due to the lower incubation temperature. In competition experiments in C2C12 cells, we detected a rapid shift in favor of the virus containing a tyrosine at E2 position 18 (Figure

4.4). In contrast, competition experiments in C6/36 cells demonstrated that a histidine at E2 position 18 is advantageous for the virus, even when starting at 10-fold lower levels. Taken together, these experiments suggest that the amino acid at RRV E2 position 18 toggles the fitness of RRV in mammalian and mosquito cells. Important future studies will investigate the extent to which a histidine at E2 position 18 provides a fitness advantage in mosquitoes.

The Alphavirus genus is composed of a diverse group of viruses. Based on serological cross-reactivity, viruses in the Alphavirus genus are classified in seven

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antigenic complexes, and these classifications are largely supported by recent phylogenetic analyses based on complete genomic sequences [19]. We analyzed available sequence data to determine the amino acid residues present at E2 position 18 of alphaviruses from each of the antigenic complexes (Table 4.1). Interestingly, all E2 sequences from members of the Semliki Forest (SF) antigenic complex, which includes

Ross River virus, possess either a tyrosine or histidine at position 18, suggesting a critical role for these two amino acids at this position during the virus replication cycle. Perhaps more interesting is the passage history of the various isolates, where we observed that viruses which possess a tyrosine at E2 position 18 tended to have a more extensive passage history in mammalian cells, while viruses which possess a histidine at position

18 tended to have a more insect heavy passage history. However, this does not appear to hold true for CHIKV which invariably encodes a histidine at E2 position 18. Further studies will be required to determine if the host passage history affects the amino acid residue at E2 position 18.

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CHAPTER V

A TYROSINE TO HISTIDINE SUBSTITUTION AT RRV E2 POSITION 18 CAUSES A DEFECT IN

THE LATE STAGE OF VIRUS REPLICATION IN MAMMALIAN CELLS, BUT NOT MOSQUITO

CELLS5

Introduction

We previously showed that a tyrosine to histidine mutation at position 18 of the

RRV E2 glycoprotein functioned as a major attenuating mutation in a mouse model of

RRV-induced musculoskeletal disease and that this attenuation was associated with reduced viral tissue titers and reduced spread in mice [66]. Additionally, we showed that a tyrosine at E2 position 18 conferred a significant fitness advantage during replication in mammalian cells, while a histidine at this position conferred a fitness advantage in mosquito cells. These studies suggest that the E2 Y18H mutation affects viral replication in a cell type dependent manner. To identify the specific mechanism of viral attenuation we used several well characterized experimental systems to investigate multiple aspects of viral replication including: the pH required for viral entry into host cells, kinetics of structural protein expression and trafficking of viral glycoproteins, as well as budding

5 Portions of this chapter are with permission from, Jupille el at., A Tyrosine-to-Histidine Switch at Position 18 of the Ross River Virus E2 Glycoprotein Is a Determinant of Virus Fitness in Disparate Hosts, Journal of Virology, Volume 87, Pages 5970-5984, Copyright ©2013, with permission from American Society for Microbiology.

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assays to determine both the number of infectious virions produced per infected cell and the total number of viral particles released per infected cell.

During the course of these investigations we showed that while E2 Y18H does not alter the pH of virus entry, it does seem to play a role in pH stability as a histidine at

E2 position 18 led to enhanced viral infectivity following low pH treatment and neutralization. During infection of mammalian cells we showed that the E2 Y18H mutation led to a significant decrease in the number of PFUs released per infected cell, suggesting that in mammalian cells this mutation causes a late stage replication defect.

In contrast, a H at E2 position 18 confers a slight, but statistically significant increase in

PFUs released from mosquito cells. Finally, our studies show that in mammalian cells, the E2 Y18H mutation results in a significant increase in the genome-to-pfu ratio, suggesting that a large number of E2 Y18H virions released from mammalian cells are non-infectious.

Results

Molecular Modeling of E2 position 18 within the glycoprotein spike complex.

Recently the atomic structures of both CHIKV and SINV E2 proteins were solved. Based on these structures, E2 position 18 is located on the underside of E2, near the fusion loop of the E1 protein in what Voss et al. term the “N-flap” region of domain A, which is composed of E2 amino acid residues 16-30 (Figure 5.1A) [102, 103]. In the CHIKV structure, a histidine at E2 position 18 is predicted to interact with E1 residues 228-230

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(Figure 5.1B) within the E1/E2 heterodimer and residues 142-146 in arch1 of the E2 molecule present in the neighboring heterodimer (Figure 5.1 B-D).

The Amino Acid Residue at E2 Position 18 Does Not Impact pH Required for

Viral Fusion. Due to the proximity of E2 position 18 to the fusion loop of E1 and the unique pKa properties of histidine [257], we tested whether the E2 Y18H mutation affected the pH of RRV entry. Previous studies in Semliki Forest virus identified a virus which was less sensitive to the acidic pH of the endosome during infection, requiring a more acidic environment to activate the fusion activity of the glycoproteins [258].

Additional studies of this SFV strain showed that a single threonine to isoleucine mutation at E2 position 12 was responsible for this pH sensitivity defect [200]. Because of the proximity of this residue to E2 position 18, we used previously described fusion assays [209-211] to determine what effect the amino acid residue at E2 position 18 was playing on the pH required for viral entry into mammalian cells. RRV-T48-dpGFP or RRV-

E2 Y18H-dpGFP virus were adsorbed at 4°C on BHK or C2C12 cells at moi 0.01 or 5 respectively. After 90 minutes at 4°C, the inocula were removed and cells were treated with pH adjusted fusion media at pH: 7.0, 6.2, 6.1, 6.0, 5.9, 5.8, or 5.6. Cells were shifted to 37°C for 1 minute to trigger fusion of pH activated virus, and returned to 4°C. Fusion media were removed, and growth media were added to each well. Growth media were supplemented with 20 mM NH4Cl to prevent secondary infection, and cells were incubated at 37°C for 18 hours. Cells were then harvested and analyzed for GFP expression by flow cytometry.

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Figure 5.1 Location of E2 Residue 18 Within the Virion A. Surface view of the CHIKV E1 (green) and E2 heterodimer showing the location and structural orientation of E2 domain A (red), E2 domain B (magenta), E2 domain C (maroon), E2 arches (tan), and E2 position 18 (blue). B. Expanded view of E2 position 18 (blue) and potential interacting residues in E1 (green). C. Stereo-3D diagram of E2 Histidine 18 (blue) and potential interacting residues from both E1 (green) and E2 (yellow). D. Stero-3D diagram of E2 Tyrosine 18 (blue) and potential interacting residues from both E1 (green) and E2 (yellow). Images modeled based on Protein Data Bank code 3N42 (A and B) and 2XFC (C and D) and displayed using PyMol (http://www.pymol.org) 111

Analysis of %GFP positive cells demonstrated that the amino acid at E2 position 18 did not alter the pH required for RRV fusion in mammalian cells (Figure 5.2). Importantly, these experiments show that RRV fusion and entry requires a pH between 6.0- 5.6, a finding consistent with other alphaviruses within the SFV antigenic complex [259, 260].

Low pH Treatment Enhances Entry of E2 Y18H Virus into BHK Cells. Although the amino acid residue at E2 position 18 did not play a role in the pH of fusion, because of the unique pKa of histidine we hypothesized that the residue at E2 position 18 could alter another aspect of viral replication.

Figure 5.2 pH of Fusion for RRV-T48 and E2 Y18H Mutant Virus in BHK and C2C12 Cells GFP expressing RRV-T48 or E2 Y18H mutant virus was adsorbed to the surface of C2C12 (A), or BHK (B) cells for 90 minutes. Fusion at the plasma membrane was triggered by treatment with pH adjusted media, then incubated at 37°C for 18 hours in normal growth media supplemented with 20mM NH4Cl to prevent secondary infection. At 18HPI, cells were harvested, fixed in 2% PFA, and analyzed for GFP Expression by flow cytometry.

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In addition to the well characterized irreversible conformational changes that occur when the alphavirus glycoproteins are exposed to acidic pH during membrane fusion and entry, several studies have shown that these glycoproteins can undergo reversible conformational changes when exposed to an acidic pH for 30 minutes before being neutralized. This treatment allows the viral spike proteins to undergo fusion triggering under the acidic conditions, followed by an “untriggering” event upon neutralization

[261, 262]. These studies further showed that the glycoprotein conformation assumed after the neutralization step is different from that of untreated virus, suggesting that the viral glycoproteins can refold into a “hybrid” conformation which maintains their fusogenic ability [262, 263].

To investigate the role of tyrosine or histidine at position 18 on the stability of virions at different pHs, we incubated 5*104 PFU of RRV-T48 or E2 Y18H mutant virus across a range of pH values from 8.0 to 5.0 for 30 minutes at 4°C. The virus samples were then neutralized and titered on BHK cells. Surprisingly treatment of RRV E2 Y18H virus at low pH enhanced viral entry into BHK cells. Specifically, we showed that treatment of E2 Y18H virus at pH 5.0 resulted in a ~52-fold increase in titer compared to

E2 Y18H virus treated at pH 7.0 (Figure 5.3). In addition, treatment of both RRV-T48 and

E2 Y18H at pH 5.0 resulted in a significantly higher titer of E2 Y18H mutant virus than

RRV-T48 (~230-fold, P < 0.001). These findings suggest that E2 position 18 plays a critical role in regulating spike stability within the virion. In addition, these studies suggest that

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E2 position 18 may be essential for the formation of the “hybrid” conformation reported by Meyer and Waarts.

The E2 Y18H Mutation Does not Impact RRV Gene Expression or Protein

Trafficking. To investigate if the E2 Y18H mutation affects RRV gene expression, C2C12 or C6/36 cells were infected with RRV-T48 or RRV-T48 E2 Y18H (moi = 5) and levels of capsid protein at various times post-inoculation were analyzed by western blots (Figure

5.4A and 5.4B). Despite significant differences in the yields from these two viruses in

C2C12 cells, but not C6/36 cells infected at this moi (Figure 4.3B and 4.3F), quantification of capsid protein expression in C2C12 and C6/36 cells showed no significant differences between RRV-T48 or RRV-T48 E2 Y18H at any time point (Figure

5.4A and 5.4B). Additionally, we utilized flow cytometry and immunofluorescence to quantify expression of E2 on the surface of infected C2C12 cells. As seen in Figure 5.5B, the amounts of E2 protein expressed on the surface of C2C12 cells infected with RRV-

T48 or E2 Y18H mutant virus were similar at both 6 and 18 hpi as quantified by mean fluorescence intensity. Similar staining intensity was also observed when RRV E2 surface expression was analyzed at 18 hpi via immunofluorescence in C2C12 cells infected with

RRV-T48 or E2 Y18H mutant virus at an moi of 1 (Figure 5.5C). Taken together, these results suggest that the E2 Y18H mutation did not alter expression of E2, or glycoprotein trafficking to the plasma membrane.

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Figure 5.3 Acidic pH Treatment Increases Infectivity of E2 Y18H Mutant Virus 5*104 pfu of RRV-T48 or E2 Y18H were treated at indicated pH levels for 30 minutes at 4°C before being neutralized to pH ~7.0. Infectious virus was titered by plaque assay on BHK cells. Data were evaluated for statistically significant differences by two-way ANOVA followed by Bonferroni’s test. *** P < 0.001.

A Tyrosine to Histidine Mutation at E2 Position 18 Causes Altered Cell to Cell

Spread. We next investigated whether the E2 Y18H mutation affected the ability of the virus to spread from cell to cell. C2C12 cells were inoculated at an moi of 0.01 with either RRV-T48-dpGFP, or E2 Y18H-dpGFP mutant virus (n=5) and fixed at 18 or 24 hpi.

Observation of formation of foci of infected cells suggested that RRV-T48-dpGFP infection formed dense foci with a high percentage of adjacent cells infected. In contrast, E2 Y18H-dpGFP forms more diffuse foci with greater distance between infected cells (Figure 5.6A) While not quantitative, these finding suggest that the E2

Y18H mutation reduces viral spread within BHK cell monolayers.

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Figure 5.4 The E2 Y18H Mutation does not Affect the Kinetics or Magnitude of RRV Structural Gene Expression C2C12 murine muscle cells, or C6/36 mosquito cells were inoculated at an moi of 5. A. At 6, 9, 12, 18, and 24 hpi capsid protein expression was analyzed by western blots. B. Relative capsid band intensities (n=3) were quantified using ImageLab 4.0 software. No statistically significant differences were detected between genotypes.

A Tyrosine to Histidine Mutation at E2 Position 18 Causes a Late Stage

Replication Defect in Mammalian Cells, but not Mosquito Cells. To determine if the E2

Y18H mutation affects a late stage of the RRV replication cycle, we inoculated C2C12 or

C6/36 mosquito cells with recombinant viruses that express GFP (RRV-T48-dpGFP or

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Figure 5.5 The E2 Y18H Mutation does not Affect Trafficking of RRV Glycoproteins to the Plasma Membrane C2C12 cells were inoculated at an moi of 5. At 6 and 18 hpi, cells were harvested and stained for E2 surface expression with an anti-E2 monoclonal antibody followed by a PE- conjugated secondary antibody. A. A representative histogram displaying the anti-E2- specific fluorescence intensity of mock-, RRV T48-, and E2 Y18H mutant-infected cells at 18 hpi. B. The mean fluorescence intensity (MFI) of E2 staining at 6 and 18 hpi was determined by flow cytometry (n=3). Each bar represents the arithmetic mean +/- SD. C. C2C12 cells were inoculated at an moi of 1 with either RRV-T48 or E2 Y18H mutant virus. At 18HPI, cells were fixed and surface stained for the presence of E2 with an anti-E2 monoclonal antibody followed by an alexafluor 568λ-conjugated secondary antibody.

RRV-T48 E2 Y18H-dpGFP) and quantified the yield of infectious virus per GFP-positive cell. When C2C12 cells were infected with RRV-T48-dpGFP or RRV-T48 E2 Y18H-dpGFP, we detected a significant (P < 0.0001), but less than 2-fold difference in the percent of

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Figure 5.6 The E2 Y18H Mutation Affects the Rate of Cell to Cell Spread C2C12 cells were inoculated at an moi of 0.01 with RRV-T48-dpGFP or E2 Y18H- dpGFP and incubated at 37°C for 18 or 24 hpi. At 24HPI, cells were fixed in 2% PFA, and GFP foci were imaged using MetaMorph Software. A. Representative images of foci of cells infected with the GFP expressing viruses at 24 HPI. B. The average number of GFP positive cells/focus was enumerated for 10 foci at 18 and 24 HPI.

GFP-positive cells (Figure 5.7A) despite a 41-fold difference in pfus ( P < 0.0001) in culture supernatants (Figure 5.7C). Based on these data, we calculated that E2 Y18H infected C2C12 cells released approximately 40 pfu/cell, while RRV-T48 infected C2C12 cells released approximately 1200 pfu/cell (Figure 5.7E), a 24-fold difference (P <

0.0001). In C6/36 cells, no significant differences in the percent of GFP positive cells were detected by flow cytometry (Figure 5.7B) or fluorescent microscopy (data not shown). The yield of infectious virus present in culture supernatants (Figure 5.7D, P =

0.0044) and the pfus released per infected cell (Figure 5.7F, P = 0.0004) were significantly higher for RRV-T48 E2 Y18H compared to RRV-T48, however, the magnitude of these effects were quite small. Taken together, our analyses of RRV gene expression

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and virus production suggest that the E2 Y18H mutation affects a late stage of the RRV replication cycle in mammalian cells but not insect cells

A Tyrosine to Histidine Mutation at E2 Position 18 Causes Release of

Noninfectious Viral Particles from Mammalian Cells. To determine the extent to which the differences in production of infectious virus from C2C12 cells were associated with differences in the total number of particles produced, we used qRT-PCR to quantify the number of RRV RNA copies present in clarified cell culture supernatants [152]. C2C12 cells infected with either RRV-T48 or the E2 Y18H mutant virus produced similar copy numbers of RRV genomes/ml of culture supernatant (Figure 5.8A) despite the significantly reduced pfu/ml detected in the supernatants of cells infected with the E2

Y18H mutant virus (Figure 5.8B, P < 0.0001). Previous western blot and qRT-PCR analyses of sucrose gradient-purified virus stocks of RRV-T48 and RRV-T48 E2 Y18H grown in BHK-21 cells indicated that the E2 Y18H mutation had an approximate 2-fold effect on particle-to-PFU ratios (data not shown). To investigate whether the E2 Y18H mutation conferred cell-type-dependent effects on the particle-to-PFU ratio, RRV-T48 and RRV-T48 E2 Y18H were grown in BHK-21 cells or C2C12 cells and the amounts of

RRV genomes and infectious virus present in culture supernatants were quantified by qRT-PCR and plaque assays, respectively (Figure 5.8C-D). In addition, a portion of the culture supernatants was utilized for sucrose-gradient purification of viruses which were then subjected to similar analyses (Figure 5.8F-G).

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Figure 5.7 The E2 Y18H Mutation Impacts a Late Stage of the RRV Replication Cycle in Mammalian Cells C2C12 murine muscle cells or C6/36 mosquito cells were inoculated with GFP- expressing RRV-T48 or E2 Y18H at an moi of 5. (A and B) At 18 hpi, the percent of GFP- positive cells was quantified by flow cytometry. Each data point represents an independent culture of cells. Data were analyzed for statistically significant differences by two-tailed t-tests with Welch’s correction. (C and D) At 18 hpi, the amounts of infectious virus present in culture supernatants were quantified by plaque assays. Each data point represents an independent culture of cells. Data were analyzed for statistically significant differences by two-tailed t-tests with Welch’s correction. (E and F) Yield of infectious virus released per infected cell was calculated based on the viral titer of the culture supernatants and the % GFP positive cells. Each data point represents an independent culture of cells. Data were analyzed for statistically significant differences by two-tailed student’s t-test with Welch’s Correction. 120

As shown in Figure 5.8C, the amount of infectious RRV-T48 E2 Y18H virus compared to infectious RRV-T48 virus in cell culture supernatants was reduced in both cells lines, although the magnitude of the difference was greater in C2C12 cells (72-fold) as compared to BHK-21 cells (6.7-fold). In contrast, the amounts of RRV-T48 and E2 Y18H

RNA present in cell culture supernatants were not significantly different in cell culture supernatants of infected BHK-21 cells or C2C12 cells (Figure 5.8D). Based on these data, the genome-to-PFU ratio of the E2 Y18H mutant virus derived from C2C12 cells was increased 82-fold compared to C2C12-derived RRV-T48 (Figure 5.8E). In contrast, the difference in the genome-to-PFU ratios of RRV-T48 and the E2 Y18H mutant virus grown in BHK-21 cells was smaller in magnitude (6.1-fold) (Figure 5.8E). Similar results were detected with sucrose gradient-purified virus (Figures 5.8F-G), although the magnitude of the differences in genome-to-PFU ratios between RRV-T48 virus and the E2 Y18H mutant virus were reduced in both BHK-21 cells and C2C12 cells (2.4-fold and 15-fold, respectively). Taken together, these results indicate that the E2 Y18H mutation had a cell-type dependent effect on production of infectious virus from cells causing fewer infectious virions to be released from infected cells. These data suggest that the E2 Y18H mutation may affect the assembly or stability of fully infectious virions.

Discussion

The E2 glycoprotein has a variety of functions during viral entry and exit [264].

We previously showed that the E2 Y18H mutation caused a significant replication defect in mammalian cells. This defect was not associated with effects on the pH of RRV entry

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(Figure 5.2), RRV gene expression kinetics (Figure 5.4), or expression of E2 on the cell surface (Figure 5.5). Instead, we found that the number of pfus released per RRV-T48 E2

Y18H-infected C2C12 cell was significantly reduced in comparison to RRV-T48-infected

C2C12 cells (Figure 5.7). In contrast, in mosquito cells the same analyses yielded the opposite result, with RRV-T48 E2 Y18H-infected cells yielding slightly more pfus per cell, further supporting that the E2 Y18H mutation has cell type-dependent effects and suggesting that the E2 Y18H mutation affects a late stage of the RRV replication cycle in mammalian cells. Interestingly, quantification of RRV RNA in clarified cell culture supernatants, as an indicator of virion particle production [152], revealed that the E2

Y18H mutation did not affect particle release into the culture supernatants (Figure. 5.8

A-B). Instead, our analyses indicated that the E2 Y18H mutation reduced the infectivity of RRV particles in a cell-type-dependent manner (Figure 5.8 C-H). In the crystal structure of the CHIKV E2 glycoprotein, Voss et al. identified that E2 position 18 is involved in E2-E1 contacts within E1-E2 heterodimers that form the trimeric spikes

(Figure 5.1), as well as E2-E2 intra-spike contacts across E1-E2 heterodimers [103]. These data, together with our findings, suggest that the amino acid at E2 position 18 may alter lateral interactions that are known to be critical for assembly of the trimeric spikes

[160]. Interestingly, Li et al. previously described similar results to our findings, where an alanine (A) to valine (V) mutation at position 251 of the Sindbis virus E2 glycoprotein, which arose during repeated passages of the virus in clone C7-10 Aedes albopictus mosquito cells, resulted in a virus which was defective for production of infectious virus

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from avian but not mosquito cells [265]. However, in contrast to our findings, the

Sindbis virus A251V mutation seemed to prevent the release of extracellular virions in a cell-type-dependent manner [265]. More recently, Snyder et al. reported that mutation of conserved cysteines at positions 19 and 22 within the N-flap domain of the RRV-T48

E2 glycoprotein resulted in assembly defects and altered particle morphology [152], supporting the idea that the N-flap domain of E2 functions during the late stages of the

RRV replication cycle, likely in assembly of infectious particles.

In addition to these findings our data also suggest that E2 Y18H affects viral pH stability, whereby treatment of E2 Y18H virus at a low pH followed by neutralization significantly increased its infectivity as compared to RRV T48. Several studies utilizing other alphaviruses have shown that viral glycoproteins can undergo partial triggering if exposed to low pH outside the context of infection.

These studies further showed that upon neutralization of the viral sample, the glycoproteins are capable of refolding into a hybrid conformation while maintaining their fusogenic ability [262, 263]. It is possible that this “hybrid conformation” phenotype could play a role in determining host-type specific replication differences, although additional experiments will be required to determine the precise processes regulated by the amino acid at position 18 and how those processes differ in vertebrate and invertebrate hosts.

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Figure 5.8 The E2 Y18H Mutation Reduces the Infectivity of RRV in a Cell-type Dependent Manner. C2C12 murine muscle cells were inoculated with GFP-expressing RRV-T48 or E2 Y18H at an moi of 5. (A) RRV RNA copies and (B) PFU present in clarified C2C12 culture supernatants were quantified by qRT-PCR and plaque assays respectively. Each data point represents an individual cell culture. Data were analyzed for statistically significant differences by two-tailed t-tests with Welch’s correction. (C-H) RRV-T48 and E2 Y18H were grown in C2C12 cells and BHK-21 cells. Clarified cell culture supernatants were analyzed directly (C-E), or virions were purified through a 60%/20% discontinuous sucrose gradient prior to analysis (F-H). (C and F) PFU and (D and G) RRV RNA copies were quantified by plaque assays and qRT-PCR respectively. (E and H) The genome-to- PFU ratios were calculated based on the number of genomes/ml present in culture supernatant or gradient-purified virus stocks. 124

CHAPTER VI

DISCUSSION AND FUTURE DIRECTIONS

Summary of Findings

In the studies presented, we utilized pathogenic and apathogenic strains of RRV in a chimeric virus approach to identify novel determinants of virulence within the nsP1 and PE2 coding regions. Additionally we showed that the mutations in nsP1 and PE2 resulted in distinct phenotypes, and impacted different steps during disease development. Specifically, we showed that the determinant within the PE2 coding region was associated with reduced viral loads in infected tissues as well as impaired ability to spread in mice. While the specific molecular mechanism of attenuation in the nsP1 mutant has not been fully characterized, studies in our laboratory suggest that the attenuation may be due to enhanced sensitivity to the Type I IFN response in mice.

Current efforts to identify the specific amino acid mutations required for the attenuated phenotype and preliminary results suggest that two mutations act as the major determinants for this phenotype. Several other groups have also identified mutations within nsP1 that alter virulence. While some studies characterized the effect of temperature sensitive mutations [236, 237], other studies investigated mutations which impact neurovirulence [239, 240]. Recently, an attenuating mutation at position 538 of nsP1 was shown to impact neurovirulence in mice by increasing induction of the Type I

IFN response, suggesting that nsP1 may play a critical role in host immune suppression

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[242]. In contrast, our studies represent the first report of a nonstructural protein functioning as a determinant of virulence in alphavirus-induced musculoskeletal disease.

Utilizing site-directed mutagenesis, we introduced each of the DC5692 PE2 coding changes into the T48 genetic background and identified a single Y to H mutation at E2 position 18 which functioned as the major attenuating mutation in the PE2 coding region. Additional studies showed that the reduced viral loads and failure to spread originally identified in the chimeric studies could be mapped to this mutation.

Examination of numerous alphavirus PE2 sequences (Table 4.1) showed that all viruses within the Semliki Forest antigenic complex, either a Y or H was present at E2 position

18, indicating that these amino acids may be critical to the viral lifecycle. In vitro replication assays further showed that a Y at E2 position 18 conferred a significant replication advantage in mammalian cells, while a H at this position conferred a slight replication advantage in mosquito cells, suggesting that this position may have a unique role in viral fitness. Competition studies in both mammalian and mosquito cells showed this hypothesis to be correct, with a Y at E2 position 18 resulting in a large fitness advantage in mammalian cells, while a H caused a fitness advantage in mosquito cells.

Because of this unique role for E2 position 18 in the viral replication cycle in both mammalian and mosquito hosts, we next investigated which molecular mechanism(s) were impacted by the E2 Y18H mutant virus in mammalian cells. We showed that the mutation did not affect pH of entry, the kinetics of viral gene expression, or the trafficking of E2 to the plasma membrane. Studies investigating viral particles released

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per infected cell however showed that the E2 Y18H mutation caused a significant reduction in the amount of infectious virus released per infected mammalian cell. In contrast, we showed that a H at position 18 conferred a slight increase in PFU release from infected mosquito cells. Further analysis showed that in mammalian cells, the E2

Y18H mutation increased the release of noninfectious particles as determined by genome-to-pfu and protein-to-pfu assays. Taken together these studies represent the first published reports of a virulence determinant within the Nflap domain of PE2 which impacts viral fitness and replication in a cell-type dependent fashion.

Amino Acid Substitutions Near the N-Terminus of E2 Can Affect Numerous Aspects of

Viral Replication

While our studies represent the first report of naturally occurring virulence determinants within the Nflap domain of E2, numerous groups have investigated the effects of induced mutations near the N-terminus of the E2 protein and showed that they function in multiple aspects of the viral replication cycle. Kielian et al. utilized a non-specific mutagenesis approach to generate the SFV strain fus-1, an attenuated strain in which a threonine to isoleucine mutation at E2 position 12 caused the virus to undergo fusion at a significantly lower pH (pH: <5.5), than the WT SFV strain (pH: ~6.2)

[200, 258]. This alteration in pH sensitivity led to a decrease in the rate of viral penetration from endosomal compartments to the cytoplasm of infected cells, resulting in reduced levels of viral replication during infection [200, 258]. Interestingly, studies investigating the molecular mechanisms behind the attenuation of the CHIKV vaccine

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strain 181/25 identified two mutations which were required for attenuation in mice, one of which was E2 T12I [201]. Combined with the studies of fus-1, these data suggest that E2 position 12 is involved with critical E1/E2 interactions during infection.

While E2 position 18 is in a different sub-domain of E2 than position 12, the similar attenuation of the E2 Y18H mutant in mammalian cells initially led us to hypothesize that a H at E2 position 18 could also cause a shift in pH sensitivity of RRV. As shown in Figure 5.2 however, we did not see any correlation between the amino acid residue at E2 position 18 and the pH required for fusion induced at the plasma membrane. These findings showed that position 18 and the Nflap domain play a critical role in the alphavirus replication cycle.

Conserved Cysteine Residues in the Nflap Domain Play Critical Roles During Alphavirus

Assembly/Budding

Snyder et al. recently investigated the role of conserved cysteine residues during alphavirus infection and replication [152]. They identified several cysteine residues which were conserved across all alphaviruses arranged in a characteristic disulfide isomerase motif. Mutation of these conserved residues in both RRV and SINV resulted in several different defects in the late stages of viral replication. Of particular interest was the finding that mutating the cysteine residue at RRV E2 position 19 resulted in a PE2 processing defect which resulted in PE2 being incorporated into progeny virions. In contrast, mutagenesis of the cysteine residue at SINV E2 position 16 (the homologous site to RRV E2 position 19) to a serine residue, was shown to be lethal with no release of

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infectious virus from infected cells. Additionally, mutagenesis of cysteines at RRV E2 position 22 and SINV E2 position 19 to serine, resulted in defects in PE2 processing as well as altered virion morphology such as multi-cored particles and particles of various size.

Although these studies involve residues which are physically close to E2 position

18, the attenuated phenotypes of these viruses are significantly different from that observed in the E2 Y18H mutant virus, suggesting that the mechanisms of virus attenuation are different. Based on the conserved nature of the cysteine residues mutated by Snyder et al., and because of their potential role in disulfide bond formation and protein folding, it is not unexpected that mutation of these conserved cysteine residues would significantly disrupt normal virion assembly. In contrast, either tyrosine or histidine has been reported in every SFV antigenic complex sequence investigated to date. Taken together with the data from Voss et al., suggesting that E2 position 18 may engage in both E1/E2 and E2/E2 interactions [103], the presence of either Y or H at E2 position 18 suggest that the mechanism of attenuation is more subtle than the disruption caused by mutating highly conserved residues known to be critical for protein folding.

Mutations in Other Regions of E2

Virulence determinants have been identified within the E2 glycoproteins of various alphaviruses. Many of these determinants are located near the distal-most

“petal” region of E2 in domain B or the outer surface of domain A which have been

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implicated in host-cell receptor binding, antibody neutralization, and enhanced binding of E2 to cell surface glycosaminoglycans [196, 198, 199, 201, 222, 225, 230, 252-254].

Studies involving domain C have confirmed its critical role in efficient virus assembly and budding. By studying the mutations in the cytoplasmic domain of E2 several groups have identified a series of highly conserved hydrophobic amino acids which interact with viral nucleocapsids during budding [156-158, 266, 267]. It is important to note however, that these mutations prevent viral budding and therefore have not been isolated during natural infections.

Alternating Replication of Alphaviruses in two hosts. Mammals vs. Mosquitos

Genetic Pressure of Dual Hosts. Alphaviruses must replicate alternately in vertebrate and invertebrate hosts during their transmission cycle. In consequence these viruses must walk an “evolutionary tightrope”, maintaining replicative efficiency in two very different hosts in the face of unique selective pressures placed on the virus by each host. Mutations that enhance viral replication in vertebrate cells often result in reduced viral replication in invertebrate cells and vice versa. Investigations of the mutational rate of alphaviruses, during repeated virus passage exclusively in either vertebrate or invertebrate cells showed evolution of adaptive mutations that enhanced viral replication in that particular host. As expected, these adaptive mutations often significantly attenuated the virus in the other host. Additionally, alternating passage in invertebrate and vertebrate cells was required to maintain efficient replication of RRV in both cell types [202-204, 251, 268-270]. Taken together, these studies illustrate how the

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alternation of vertebrate and invertebrate hosts during the alphavirus lifecycle places significant evolutionary pressures on the viruses.

While the “evolutionary tightrope” limits the ability of alphaviruses to acquire large numbers of adaptive mutations, several groups have identified mutations in E1 and E2 that enhance viral replicative capacity in one host while not impacting the ability of the virus to replicate in in the reciprocal host. During the 2004-2011 CHIKV epidemic, a single alanine to valine mutation in E1 at position 226 was identified which allowed

CHIKV to undergo a vector shift, productively infecting Aedes albopictus mosquitos in addition to Aedes aegypti [44, 271]. Importantly, this mutation did not alter virulence in humans, and may have contributed to the rapid spread of CHIKV across the Indian

Ocean region. Second, a threonine to alanine mutation at E1 position 98 further enhanced the ability of CHIKV to infect Aedes albopictus mosquitos while not impacting replication or virulence in Aedes aegypti or Homo sapiens [30]. Finally, a leucine to glutamine mutation at E2 position 210 was shown to cause a significant increase in dissemination in Aedes albopictus, without altering fitness in either Aedes aegypti, or vertebrate cell lines [30].

We first became interested in the differences between viral replication in mammalian vs. mosquito cells based on our in vitro replication kinetics assays which showed that the E2 Y18H mutation led to attenuation in mammalian cells while enhancing replication in mosquito cells. These differences became more apparent during our fitness assays, which showed that a tyrosine at E2 position 18 conferred a

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fitness advantage in mammalian cells, while a histidine at this same position conferred a fitness advantage in mosquito cells. Together, the data suggests that the amino acid at position 18 might act as a “switching” residue, where a histidine is selected for during replication in mosquitos, while in mammalian cells, a tyrosine is selected for. While many of the studies investigating mutations in structural genes have shown attenuation in one or both host species, our findings represent the first report of a mutation which toggles viral fitness for one host or the other.

Differences in Alphavirus Replication in Mammalian and Mosquito Cells.

During alphavirus replication in mammalian and mosquito cells many aspects of the replication cycle are shared, however there are several key differences which must be addressed in order to determine what specific effects the E2 Y18H mutation has on viral replication in each host.

The first potential difference involves the temporal regulation of PE2 cleavage into E2/E3. During infection of mammalian cells, PE2 cleavage occurs in the trans-Golgi network and is mediated by the host protease furin [193]. During replication in mosquito cells however, viral glycoprotein processing kinetics are significantly different.

Specifically, PE2 is cleaved shortly after translation while the glycoproteins are localized in the lumen of the ER [191]. It is not known which specific host-cell protease is responsible for the ER-localized PE2 cleavage in mosquito cells, however preliminary studies of alphavirus replication in Drosophila cells identified the insect protease d-Furin as being the key enzyme responsible for this cleavage [272]. This difference is critical

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because of studies which showed that the presence of E3 during glycoprotein trafficking is required to prevent premature triggering of the viral spike proteins [193], and further that E3/E2 interactions are maintained after furin cleavage, but only under acidic conditions [151]. Because cleavage of PE2 in mammalian cells occurs in the trans-Golgi, the acidic pH of this compartment ensures that E3 remains associated with the spike protein and is only released upon exposure to neutral pH. Processing of PE2 in the ER of mosquito cells however, leads to a potential problem during replication, as the pH of the

ER has been shown to be neutral [273]. Processing of PE2 at neutral pH may allow E3 to be released from the spike protein immediately following cleavage, allowing the mature

E1/E2 heterodimers to be exposed to the acidic pH of the secretory pathway during processing.

A second major difference between the alphavirus replication cycle in mammalian vs. mosquito cells is the mechanism of virion release from infected cells.

Virion production in mammalian cells occurs when viral nucleocapsids interact with the cytoplasmic tail of viral E2 glycoproteins located at the plasma membrane Virions bud through the plasma membrane acquiring all of its membrane bound glycoproteins as it buds. In mosquito cells however, alphavirus glycoproteins are embedded in the lipid bilayers of large intracellular cytoplasmic vacuoles. Viral nucleocapsids interact with the cytoplasmic tails of the glycoproteins as they do in mammalian cells, however instead of budding through the plasma membrane, the maturing virions appear to bud into the large vacuolar space [274]. Mature virions accumulate in these large vacuoles and

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eventually these vacuoles begin to fuse together before fusing with the plasma membrane where they release numerous new virions [275, 276]. A third major difference is that in mosquito cells alphaviruses establish persistent infections, while in mammalian cells alphavirus infection is cytolytic [277, 278].

A fourth difference between alphavirus replication in mammalian vs. mosquito cells is cholesterol dependence. Alphavirus infection of mammalian cells is cholesterol- dependent and cholesterol is required for efficient virus assembly/budding [259, 279,

280]. Because mosquitos are cholesterol auxotrophs, they must procure any cholesterol they require via blood meals. This lifestyle can place evolutionary pressure on the virus, selecting for mutations which reduce the cholesterol dependence of the virus.

Mutations at E1 position 226 are associated with a cholesterol independent phenotype

[172, 259, 279-281]. This finding is especially crucial given the role of the E1 A226V mutation in CHIKV during the 2004-2011 epidemic [29, 279]. Although these mutations can be selected for during natural transmission, it is unlikely to explain the results of our in vitro studies because there is a significant amount of cholesterol in the cell culture medium which would prevent cholesterol depletion within the membranes of either mammalian or mosquito cells.

A final key distinction between mammalian and mosquito cells are the types of carbohydrate which can be generated during glycosylation. Both PE2 and E1 undergo N- linked glycosylation at several asparagine residues. While this process occurs in both mammalian and mosquito cells, the specific type of glycan added can vary greatly

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depending on the host. During replication in mosquito cells, all glycosylation sites receive pauci-mannose or high-mannose sugars, while in mammalian cells, viral glycoproteins display a mix of complex and high-mannose carbohydrates. The exact ratio is determined by the accessibility of the site to the various glycosyl transferases within the Golgi [282]. Though this pathway has been shown to play a critical role in alphavirus replication, the E2 Y18H mutation does not affect any of the RRV glycosylation sites, thus we hypothesize that this is not the cause of E2 Y18H attenuation in mammalian cells.

Finally, there is a large temperature differential between mammalian and mosquito cells. While mammalian cells require a constant temperature of approximately

37°C, mosquito cells require between 28°C and 30°C. There are many mutations in various alphavirus genes which confer a temperature-sensitive phenotype [138, 143,

237]. Our studies with E2 Y18H showed that infection of mammalian cells at either 37°C or 29°C resulted in similar attenuation, suggesting that the mutation did not confer a temperature-sensitive phenotype.

Location of E2 Position 18 Within the E1/E2 Dimer and Trimeric Spike

Because of our findings that the E2 Y18H mutation increased the particle-to-pfu ratio of virions released from mammalian cells, we wanted to investigate the role of E2 position 18 in greater detail. Based on the recently published crystal structures of the

CHIKV heterodimer and trimeric spike, we showed that the side chain of Histidine at position 18 is pointing outward from the body of the E2 protein towards the Arch

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domain from the neighboring E2 molecule in the pre-fusion complex [103]. Interestingly,

Voss et al. propose a number of van der Waals interactions between E2 position 18 and residues 228-230 of E1 within the dimer, as well as E2 residues 142-146 in the Arch region of the neighboring E2 molecule suggesting that the amino acid residue at E2 position 18 could be playing a role in maintaining critical E1/E2 or E2/E2 interactions.

[103].

We next used the molecular modeling software PyMol to mutate the histidine residue at position 18 in the published CHIKV structure to a tyrosine while maintaining the orientation of the side chain [283]. As seen in Figure 5.1, a tyrosine at E2 position 18 could significantly impact the proposed van der Waals interactions between E2 position

18 and E1 228-230 or E2 144-148. This hypothesis is based on the presence of the hydroxyl group in tyrosine being in closer proximity to these interacting residues than the 5-member ring found in histidine. This suggests that a tyrosine at E2 position 18 could serve to either strengthen or weaken the proposed van deer Waals interactions, which in turn could affect the ability of the viral glycoproteins to undergo pH-induced conformational changes. More specifically, the strength of these interactions may be influenced by the charged nature of both tyrosine or histidine residues at E2 position 18.

Mutation of this position to alanine, aspartic acid, or phenylalanine resulted in virus attenuation both in vitro and in vivo. Together, these findings indicate that the nature of the interactions between the residue at E2 position 18 and its purported binding

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partners in E1 and E2 may be dependent on the presence of positively charged, aromatic amino acid residues

Differences in Tyrosine or Histidine at Physiological pH

While our analysis of the CHIKV E2 crystal structure suggested that the amino acid residue at position 18 could be impacting E1/E2 or E2/E2 interactions, it is possible that the dramatically different pKa of both tyrosine and histidine may be the driving force behind these changes. While tyrosine has a pKa greater than 10, histidine has a pKa of 6.04, although within a protein, the pKa of histidine can shift to ~6.4. This

“physiological” pKa means that minor differences in pH which occur in the cell can alter the positive charge associated with histidine residues. During the alphavirus replication cycle, the viral glycoproteins are exposed to a multitude of different pH levels during processing and trafficking. These different pH levels have been shown to impact the protonation state of histidine residues and that these changes are critical for various roles during the viral replication cycle [284].

Our data suggest that the protonation state of histidine at E2 position 18 is affecting viral budding. While the pH within the ER is fairly neutral (~7.1), the pH within the Golgi is approximately 6.5 [273]. The trans-Golgi network and secretory vesicles have an even lower pH of approximately 6.2 [285] and 5.5 [286] respectively. These localized pH differences, coupled with the difference in pKa between tyrosine and histidine suggest that the protonation kinetics of E2 position 18 are likely different for viruses with a tyrosine or histidine at E2 position 18. The charge of a tyrosine residue

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would not change from initial translation of the protein throughout the secretory pathway, while a histidine residue would gain a positive charge once the glycoproteins arrive in the medial-, or trans-Golgi, where the acidic pH leads to protonation of the histidine. This difference could be impacting the interactions between E2 position 18 and the other amino acids in E1 and E2 proposed by Voss et al. [103]. This protonated state of histidine persists until the glycoproteins have been delivered to the plasma membrane. Once exposed to the extra-cellular environment and its neutral pH, a histidine at position 18 may lose its positive charge potentially altering its binding affinity with its interacting residues. This altered binding affinity would reduce the stability of the glycoproteins, allowing the spike to be permanently triggered under less stringent conditions. This hypothesis is supported by our studies which show that low pH treatment (triggering), followed by neutralization (un-triggering) restores some of the infectivity to histidine containing virus, while not impacting infectivity of tyrosine containing virus. These findings together led us to propose the following models for the role of tyrosine and histidine in both mammalian and mosquito cells.

Model for Role of E2 Position 18 During Replication in Mammalian Cells

Because of our data suggesting that a histidine at E2 position 18 is associated with release of non-infectious virions (Figure 5.8), we hypothesize that in mammalian cells a histidine residue at E2 position 18 results in a less stable and thus more sensitive conformation of the alphavirus spike complex. Because of the PE2 processing kinetics in mammalian cells, and the association of E3 with the E1/E2 heterodimers, it is unlikely

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that the residue at E2 position 18 would cause the spike proteins to be triggered during processing. Once the heterodimers are delivered to the plasma membrane however, a histidine at E2 position 18 may result in reduced E1/E2, or E2/E2 interactions, thereby rendering the spike complex more sensitive to triggering under improper (higher pH) conditions. Once triggered, the virions are considered non-infectious. This model is supported by our experimental findings in mammalian cells.

Studies in our lab and others have shown that the conformational changes of the alphavirus glycoproteins may be somewhat reversible. Exposure of virions to acidic pH for 30 minutes induced triggering of the virus spikes as expected. Subsequent neutralization of these samples however, showed that the spikes underwent a partial

“untriggering”, restoring fusogenic activity to the virions [262]. Additional studies using conformation dependent monoclonal antibodies further showed that the conformations in the “untriggered” virions was a “hybrid” conformation, distinct from both pre-and post-fusion conformations [262, 263]. Using this method with RRV containing either a tyrosine or histidine at E2 position 18, we showed that we could restore infectivity to

RRV containing a histidine at E2 position 18 through this triggering and un-triggering reaction (Figure 5.3). Thus, we propose that in mammalian cells a histidine at E2 position 18 causes the spike complex to more easily undergo conformational changes which render the virion non-infectious. Forced triggering under acidic conditions followed by neutralization, restores some of this lost infectivity by refolding the spike complexes into an infectious “hybrid” conformation. In contrast to this, a tyrosine at E2

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position 18 results in greater spike complex stability preventing pre-mature triggering, and maintaining infectivity of the virions. Because of this enhanced stability, we do not see a “recovery” of infectious tyrosine containing virus after low pH treatment and neutralization. This hypothesis may also explain our experimental findings in mosquito cells where a histidine at E2 position 18 appears to have a fitness and replication advantage compared to a tyrosine at position 18.

Proposed Role for E2 Position 18 During Replication in Mosquito Cells

As discussed previously, a major difference in the processing of alphavirus glycoproteins in mosquito cells vs. mammalian cells is the compartment in which PE2 cleavage occurs. In mammalian cells, this step occurs late in the replication cycle where

PE2 is in the acidic trans-Golgi compartment. This effectively ensures that E3 will remain associated with the E1/E2 heterodimer, preventing their premature triggering under the acidic conditions of the secretory pathway [151]. In mosquito cells, PE2 cleavage occurs much earlier, while PE2 is still within the ER [191]. Because PE2 processing occurs in the

ER of mosquito cells, the neutral pH may allow E3 to be released from the E1/E2 heterodimer. This dissociation results in exposure of the mature heterodimer to the acidic pH of the secretory pathway during glycoprotein processing. This exposure to acidic pH would induce spike triggering in virus with either tyrosine or histidine at E2 position 18. Once new viral particles have budded into the large cytoplasmic vacuoles and have been released to the extra-cellular environment, the neutral pH induces refolding of the triggered spikes into the “hybrid” conformation, restoring infectivity to

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histidine containing virus, while not impacting tyrosine containing virus. This would confer a slight growth advantage to virus with a histidine at E2 position 18 consistent with our experimental results investigating viral replication and fitness in mosquito cells.

Future Directions

Mosquito Experiments. Several experiments will be required to test our hypothesis that early PE2 cleavage leads to enhanced infectivity of histidine containing virus. The most direct method of investigating this is the use of a PE2 cleavage deficient virus. Several groups have shown that E3 cleavage is required for virus entry and infectious of host cells. In particular, Sjoberg et al. used a furin resistant strain of SFV which remained sensitive to proteinase K treatment in vitro, to show that the presence of E3 in virions suppressed the pH-induced triggering of the E1/E2 heterodimer [151]. To investigate the role of early PE2 cleavage in mosquito cells on infectivity, it would first be necessary to make the furin-resistance mutation in both RRV-T48 and E2 Y18H genetic backgrounds. This mutation would force incorporation of PE2 into new virions, preventing the low pH induced triggering proposed in our model. Once PE2-containing virions have been recovered from infected cells, E3 cleavage would be performed in vitro using proteinase K. The now fully mature virions would then be used to infect mosquito and mammalian cells to determine if maintenance of E3 throughout the secretory pathway suppresses the enhanced infectivity of E2 Y18H virus derived from mosquito cells.

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A second strategy to examine the effect of the E2 Y18H mutation in mosquito cells is to examine the fitness of a tyrosine or histidine at E2 position 18 under conditions which prevent the acidification of the secretory pathway. Numerous have used ammonium chloride, bafilomycin, or chloroquine to prevent acidification of endosomes. Utilizing our genetically marked viruses, competition assays could be performed to investigate the effect of either a Y or H at position 18 in the presence or absence of lysomotropic drugs. Because of the alphavirus entry process, it would be necessary to infect the cells prior to drug treatment, essentially resulting in a single round of replication. If our mosquito model is correct, we would expect that progeny virion with the E2 Y18H mutation would possess a growth advantage in mosquito cells in the absence of the lysomotropic drugs, but that addition of the drugs would result in virus with the same phenotype as mammalian cell-derived virus.

Another critical experiment is investigation of the role of the E2 Y18H mutation on RRV replication in whole mosquitos. Because of previous studies showing that mutations in CHIKV E2 caused an increase in the ability of the virus to establish disseminated infection in Aedes albopictus [30], it would be interesting to see if the E2

Y18H mutation confers a similar enhanced replication or spread compared to the RRV-

T48 strain in this context. Mosquito populations would be infected with either RRV-T48 or E2 Y18H mutant virus through an infected blood meal, and at various times post- infection mosquitoes would be collected and viral tissue titers could be quantified to determine the growth phenotype of both strains in a tissue dependent manner. It would

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also be possible to adapt these assays to compare the rates of persistent infection within the various tissues, in addition to quantifying the amount of virus produced from persistently infected cells. Tissue tropism and persistent infection could also be monitored through the use of fluorescence microscopy. While we cannot say what impact the E2 Y18H mutation will have on replication in whole mosquitoes, our experimental data suggests that the mutation will lead to enhanced viral replication within various tissues, and will lead to enhanced dissemination of the virus within the mosquito.

A final experiment to test the role of the E2 Y18H mutation as a “switching residue” would be to allow infected mosquitos to infect naïve mice with either RRV-T48 or E2 Y18H mutant virus. After infection, the mice would be monitored for development of disease signs, and at various times post-infection, tissues could be collected and total viral RNA could be isolated. This viral RNA could then be sequenced, and disease severity in each animal could be correlated to the amino acid residue at E2 position 18.

Because of the error prone nature of the viral RdRP, alphaviruses are able to quickly accumulate adaptive mutations to enhance replication in a particular host. Based on our switching hypothesis, we would expect that some of the mice inoculated with the E2

Y18H mutant virus would develop disease signs, and that sequencing viral isolates from tissues within these animals would identify a histidine to tyrosine reversion at E2 position 18, restoring the virus to its mouse virulent form.

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Mammalian Experiments. In order to test our hypothesis that a histidine at E2 position 18 results in increased sensitivity of the virus spike to pH-induced conformational changes there are several experiments which could be performed. The first set of experiments is the mutation of all potential E2 position 18 interacting residues. E2 position 18 is thought to engage in van der Waals interactions with residues

228-230 in E1 within the heterodimer, and residues 142-146 in E2 within the trimeric spike. After mutating each of these residues in both RRV-T48 and E2 Y18H, the resultant viruses would be tested in our animal model, and assayed for their ability to cause disease. If any of these second site mutations attenuate the virus to a similar degree as

E2 Y18H it could imply that the mutated residue and E2 position 18 are responsible for maintaining their van der Waals interactions throughout the lifecycle of the virus.

Another key experiment that will be required to investigate the role of Y or H at

E2 position 18 is to examine the stability of the “hybrid” conformation assumed by the virions after acidic pH treatment and subsequent neutralization. In this assay, samples of both RRV-T48 and E2 Y18H mutant virus would be treated at low pH for 30 minutes followed by neutralization and incubation at 4°C for between 0 and 24 hours prior to plaquing on BHK cells. If the hybrid conformation is unstable, we would expect to see a greater restoration of infectivity in samples titered shortly after neutralization and less infectivity as time passes between neutralization and titering. It would also be interesting to examine mosquito cell-derived RRV-T48 and E2 Y18H and see if in these

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Figure 6.1 Proposed Model of RRV Replication in Mammalian Cells Maintenance of E3 association with E1/E2 heterodimers prevents premature triggering of alphavirus spike complexes. Exposure to neutral pH allows E3 to dissociate from heterodimers, priming the spike complex for conformational changes leading to membrane fusion and virus entry. In virus containing histidine at E2 position 18, this shift to neutral pH results in deprotonation of His 18, leading to weakened E1/E2 and E2/E2 interactions leading to abnormal spike morphology. A tyrosine at this position is not affected by the shift to neutral pH and maintains normal spike morphology. cases the infectivity of E2 Y18H mutant virus was enhanced by low pH treatment. Based on our hypothesis, we predict that we would not see any increase in infectivity in either virus after low-pH induced fusion and subsequent neutralization.

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Figure 6.2 Proposed Model of RRV Replication in Mosquito Cells and Impact of E2 Y18H Mutation Early PE2 cleavage during RRV replication in mosquito cells causes E3 dissociation while the glycoprotein complex is still in the ER. Acidic pH of the secretory pathway induces partial triggering of mature RRV trimeric spikes. Upon budding to the neutral extracellular space, spikes refold into a “hybrid” conformation, enhancing infectivity of virus with histidine at E2 position 18, while not affecting virus with a tyrosine at this position.

Summary of Findings

The studies presented here identified novel viral genetic determinants of virulence within both nsP1 and PE2 which attenuate RRV-induced disease in a mouse model. Additional studies showed that these determinants were individually sufficient to attenuate RRV-induced disease in mice, however differences in viral titers in infected tissues suggest that the specific mechanisms of attenuation are likely different for each region. 146

Further studies of the determinant within the PE2 coding region showed that a single Y to H substitution at E2 position 18 functioned as a major virulence determinant within this region. Studies of the E2 Y18H mutation showed that while a Y at E2 position

18 led to enhanced viral replication in mammalian cells, a H at the same position led to enhanced replication in mosquito cells. Competition experiments showed that the AA residues at E2 position 18 also regulated viral fitness in mammalian and mosquito cells.

Finally, studies were undertaken to identify the specific molecular mechanism affected by E2 position 18 during replication. Studies of viral entry, gene expression, and structural protein trafficking showed no difference between RRV-T48 or E2 Y18H genotypes suggesting that the attenuation in mammalian cells was occurring at a step downstream of glycoprotein trafficking. Studies investigating pfu release per infected cell showed a significant decar5ease in the number of pfus released from mammalian cells infected with E2 Y18H mutant virus. In contrast, the E2 Y18H mutation resulted in a slight increase in the number of pfus released from mosquito cells suggesting that the residue at this position impacts RRV replication in a host-type dependent manner.

Additionally, studies investigating the impact of the E2 Y18H mutation on particle-to-pfu and genome-to-pfu ratios showed that in mammalian cells, similar numbers of E2 Y18H and RRV-T48 particles were being released into the culture supernatant. Combined with infectious titer data, these studies suggested that the E2 Y18H mutation renders a large portion of released virus non-infectious.

147

By using molecular modeling, the specific interactions between E2 position 18 and E1 positions 228-230, and E2 positions 142-146 were visualized using both a tyrosine and histidine. Combined with our experimental data, these findings led us to hypothesize that a histidine at E2 position 18 resulted in a virus which was more sensitive too pH-induced conformational changes, while a tyrosine had no impact on pH-sensitivity. The infectivity of the histidine containing virus was able to be restored following acidic pH treatment and neutralization of virus, inducing refolding into a

“hybrid” conformation. In mammalian cells, the presence of E3 with the E1/E2 heterodimers prevents this triggering, leading to non-infectious virus release in mammalian cells. In mosquito cells however, we hypothesize that he PE2 processing kinetics allow the E1/E2 heterodimers to be triggered by the acidic pH of the secretory pathway, and are then neutralized inducing formation of the “hybrid” conformation under natural infection conditions.

Together, these studies have increased our understanding of viral genetic determinants of alphavirus-induced musculoskeletal disease in addition to identifying novel roles for the E2 protein during the RRV replication cycle. The presence of either Y or H at E2 position 18 in all members of the Semliki Forest antigenic complex suggests that the knowledge gained during the course of these studies may be applicable to other globally important viruses within this complex.

148

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