ABSTRACT SCHUCHMAN, RYAN MICHAEL. on New Host Cell

ABSTRACT

SCHUCHMAN, RYAN MICHAEL. On New Host Cell Proteins Involved in Alphavirus Replication (Under the direction of Dr. Dennis T. Brown).

These studies redefined the function of vacuolar-ATPase and identified new host cell proteins involved in alphavirus replication. Traditionally, the vacuolar-ATPase has been thought to induce conformational changes in the structural proteins by decreasing the pH in endomembrane compartments during infection through receptor-mediated endocytosis. The conformational changes in the structural proteins of the virus would allow for fusion of the virus particle and endomembrane compartment to release the viral genome into the cytoplasm.

However, using the heat-resistant strain of Sindbis virus, we have generated a bafilomycin- resistant mutant of Sindbis virus (BRSV). Using BRSV, and other mutants that were generated by reverse genetics, in combination with electron microscopy, quantitative polymerase chain reaction, and plaque assay, we showed that the vacuolar-ATPase helps facilitate direct entry at the plasma membrane, promotes synthesis of viral RNA, and functions in efficient assembly of mature virus particles.

Identifying host cell proteins that can be used as targets of antiviral therapeutic strategies is vastly important in controlling the impact of viruses on the human species. To that end, we have identified and validated sorting nexin 5 as host cell protein that has a critical function during the infection cycle of alphaviruses. The identification of host cell proteins in this study was made by purifying mature Sindbis virus particles from various cell backgrounds and interrogating the complement of proteins associated with the virus particles with liquid chromatography coupled with tandem mass spectrometry. Significantly, we also identified non- structural protein 2, the viral protease, as a component of the mature virus. The function of nsP2 outside of the role of the viral protease during replication is unknown, however it is possible that it may play some role in the initial phases of infection.

On New Host Cell Proteins Involved in Alphavirus Replication

by Ryan Michael Schuchman

A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Biochemistry

Raleigh, North Carolina 2018

APPROVED BY:

______Dennis T. Brown Cynthia L. Hemenway Committee Chair

______Raquel Hernandez Frank Scholle

DEDICATION

This work is dedicated to anyone who has ever dared to dream.

BIOGRAPHY

On 7 January 1988, Ryan Michael Schuchman was born at Cape Fear Valley Medical

Center in Fayetteville, North Carolina to Wendy and Michael. He would find himself graduating from Douglas Byrd Senior High School and Fayetteville State University for his undergraduate studies. Though he always loved the sciences, it wasn't until much introspection that he would decide that a doctoral degree in biochemistry was in his journey. He would eventually find himself completing his PhD at North Carolina State University under the guidance of Dr. Dennis

T. Brown.

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ACKNOWLEDGMENTS

Firstly, I want to thank Drs. Dennis Brown and Raquel Hernandez. I will never forget the generosity, advice, and friendship that you have extended to me through the last few years—it means the world to me. I also want to thank you for always allowing me to explore my curiosities. To the rest of my committee members, both present and past, Drs. Cindy Hemenway,

Stu Maxwell, and Frank Scholle, thank you for your guidance, encouragement, and advice through my education—I couldn’t have picked a better committee to advocate for me. Cindy, I also want to thank you for offering your beakers to me, but I'm glad that I never had to use them.

Next, I want to thank Michael—I know having stuck by my side almost every day of this adventure has been challenging. I am so very grateful for your patience, support, and especially for taking care of Watson and Newton when I couldn't be there because of late nights in lab.

I am incredibly indebted to my family; your encouragement, love, and support through the last 30 years have been paramount to my success. RueAnne, I'm going to need to sell a kidney on the black market to even come close to repaying you for all of the conversations, diversions, and late night car rides.

Drs. Finley Bryan, Faren Wolter, and Paul Swartz, you three will have to split the money that I get from my other kidney. The three of you have been instrumental in the maintenance of my sanity more times than I care to count.

To the best work wife a guy could ask for, Dr. Alex Breuer, I will always appreciate all of our conversations about science and life.

Next, in no particular order, I'd like to thank all of my dear friends that I met in Polk Hall:

Dmitry Grinevich, Dr. Davis Ferreira, Dr. Laura Herring, Dr. Annette Bodenheimer, Sophia

Yang, Matt Coco, Colin Woolard, Kate Emmerich, Hannah Conley, Kevin Blackburn, Amanda

Piper, Jonatan Isacsson, Gabby O’Neill, Dr. Tara Nash, Jigar Desai, Dipti Paudel, Dr. Joe

Maciag, Bryan Rogers, Kaylin Prestage, Dr. Melvin Thomas, Dr. Bob Grinshpon, and Dr. Brad

O’Dell. Thank you all for being part of my journey.

Lastly, I would be remiss if I did not mention my gratitude for the Cavanagh Lab: thank you all for your individual contributions to my education—my experiences with you are unforgettable.

TABLE OF CONTENTS

LIST OF FIGURES ...... vii

Chapter 1: Divergence from Cannon: an Additional Opportunity for Alphavirus Entry ... 1 1.1: Abstract ...... 2 1.2: Introduction:Alphaviruses, Their Superstructure, and Genomic Organization ...... 3 1.3: Alphavirus Structural Proteins ...... 5 1.4: The Study of Virus Entry ...... 8 1.5: A Brief Overview of Inhibitors used to Study Virus Entry ...... 9 1.6: Biological Complications in Studying Virus Entry ...... 11 1.7: Evidence for Receptor-Mediated Endocytosis ...... 13 1.8: Evidence for pH-Independent Entry...... 16 1.9: Summary ...... 20

Chapter 2: Role of the Vacuolar ATPase in the Alphavirus Replication Cycle ...... 21 2.1: Abstract ...... 22 2.2: Introduction ...... 22 2.3: Results ...... 26 2.4: Discussion ...... 44 2.5: Methods ...... 52 2.6: Acknowledgements ...... 56

Chapter 3: Comparative Characterization of the Sindbis Virus Proteome from Mammalian and Invertebrate Hosts Identifies nsP2 as a Component of the Virus Nucleocapsid and Sorting Nexin 5 as a Significant Host Factor for Alphavirus Replication ...... 57 3.1: Abstract ...... 58 3.2: Importance...... 59 3.3: Introduction ...... 60 3.4: Results ...... 66 3.5: Discussion and Conclusions ...... 80 3.6: Materials and Method ...... 88 3.7: Acknowledgements ...... 94

Chapter 4: Summary and Future Perspectives ...... 95

REFERENCES ...... 99

APPENDIX ...... 124

LIST OF FIGURES

Figure 1.1 An EM reconstruction of Sindbis Virus. In this reconstruction, the E1/E2 glycoprotein coat is seen in yellow, the lipid bilayer is seen in red, and the nucleocapsid core is seen in blue. Though not seen in this image, the E2 protein crosses the membrane and interacts with the capsid monomers. The capsid exhibits a T=4 triangulation number like the glycoprotein coat. Reproduced from Sharp, J., Nelson, S., Brown, D. and Tomer, K. 2006, Virology, 348, 216-223: Copyright 2006, with permission from Elsevier ...... 4

Figure 1.2 Schematic of E1:E2 interactions before E3 cleavage. (a) Model of the chikungunya virus heterodimer. The domains of the E1 protein are colored as follows: DI, red; DII, yellow; DIII, blue, and the fusion loop is colored orange The domains of the E2 protein are colored as follows: A, cyan; B, green; C, pink. The E3 protein is colored gray. Glycosylation sites are represented as balls and sticks and the residue bearing the glycosylation is indicated. Green sticks represent disulfide bonds. The large, black arrow represents the orientation of the viral membrane. The C-termini of the glycoproteins are depicted by a pink star (E2) and blue star (E1). The inset shows the relative positioning of the domains of the glycoproteins to each other. (b) A rotation of the glycoprotein heterodimer to show the E3 accessory protein. Reprinted by permission from Springer Nature: Nature, Glycoprotein organization of Chikungunya virus particles revealed by X-ray crystallography, Voss, J. et al., 2010 ...... 7

Figure 1.3 The deposition of the Sindbis virus genome at the plasma membrane. These images depict SVHR at 4°C (A) and 37°C (B). All panels have been treated with α-SVHR primary polyclonal antibodies and immune-gold labeled secondary antibodies. The full particles (A) can be seen empting their genomes (B) while the virus particle remains at the plasma membrane. The arrow indicates a putative apparatus used by the virus to deliver its genome and the scale bars represent a 50 nm measurement. Reproduced from Vancini, et. al., 2013 (68) ...... 18

Figure 2.1 Electron micrograph of Sindbis virus entry events on a BHK cell surface. Cells were infected with SVHR at an MOI of 1000 at room temperature for 15 min; virus particles were treated with anti-Sindbis structural protein polyclonal antibodies, then with immunogold-labeled secondary antibody and processed for electron microscopy. “F” indicates a full virus particle; “E” indicates an empty particle ...... 24

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Figure 2.2 The Effect of Metabolic Inhibitors on Alphavirus Entry. BHK cells were untreated (control), treated with cytochalasin-D (50µM), cyclohexamide (100µM), genestin (100µM), CCCP (40µM), monensin (50nM), or bafilomycin A1 (500nM) for 1 hour. After treatment, cells were infected with SVHR for 15 min at room temperature, fixed, processed, and treated with primary, secondary antibodies, and counted as described in the text. Error bars account for a 10% error and the percent inhibition for each treatment is shown ..... 28

Figure 2.3 The Effect of BAF on endosome acidification. Monolayers of BHK cells were either untreated (A), treated with 100nM BAF for 1 hour (B), or for 10 hours (C) before incubation with acridine orange and subsequent imaging. Cells with orange vesicles show acidic compartments (A) and cells lacking orange vesicles show endosome pH neutralization (B and C). Experiment was completed a minimum of three times...... 31

Figure 2.4 The effect of BAF on SVHR RNA production. BHK cells were pretreated with 100 nM BAF or tissue culture media at ambient temperature for 1 hour. Following this, cells were infected with the indicated virus (MOI=10 PFU/cell) in media with 100 nM BAF or without BAF at ambient temperature. Cells were then incubated at 37°C for the indicated amount of time in the presence or absence of BAF. RT-qPCR treatment groups are as follows: blue, SVHR without BAF; red, SVHR+100 nM BAF; green, BRSV without BAF; purple, BRSV+100 nM BAF. Experiment is an average of three biological replicates ...... 32

Figure 2.5 BAF time of addition assay. Identical BHK monolayers were treated with 100 nM BAF and infected with SVHR (MOI= 10 PFU/cell) at the indicated times and described orders. Infections took place and ambient temperature for 1 hour and then monolayers were transferred to 37°C for 10 hours. After incubation, cells were processed for RT-qPCR as described. Results presented are an average of 3 biological replicates, error bars represent the standard error of these replicates...... 33

Figure 2.6 RNA production in BRSV-derived mutants. BHK cells were treated with BAF as above, infected with the indicated virus, and assayed for RNA production by RT-qPCR at 10 hpi. The control treatment group (no BAF) is shown in blue and cells treated with 100 nM BAF are shown in red. Data presented are an average of of two biological replicates, error bars represent the standard error of these replicates ...... 35

Figure 2.7 The Effect of BAF on alphavirus glycoprotein expression. Monolayers of BHK cells were either untreated (A and C) or treated with 100nM BAF for 1 hour (B and D) before a 10 hour infection with SVHR (A and B) or BRSV (C and D). The negative control (E) is untreated and uninfected. All monolayers were fixed with methanol and incubated with anti-SVHR and Alexafluor secondary antibodies before microscopic examination ...... 37

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Figure 2.8 Mature virus production from BRSV-derived mutants. BHK cells were treated with 100 nM BAF for 1 hour. at room temperature, infected at an MOI of 10, and assayed for mature virus production by titration of the 20 hpi sample. The control treatment group (no BAF) is shown in blue and cells treated with 100 nM BAF are shown in red. Error bars represent the standard error of three biological ...... 38

Figure 2.9 The effect of BAF on mature virus production of SVHR or BRSV. BHK cells were either untreated or treated with 100 nm BAF at room temperature for 1 hour. Monolayers were then infected in presence or absence of BAF for 1 hour. Monolayers were incubated at 37°C for the indicated amount of time and the resulting supernatant was then titered. Treatment groups are as follows: blue, SVHR without BAF; red, SVHR+100 nM BAF; green, BRSV without BAF; purple, BRSV+100 nM BAF. Experiment is representative of two biological replicates ...... 41

Figure 2.10 The effect of BAF on viral assembly of SVHR or BRSV. Thin section electron micrographs of BHK cells infected with SVHR in the absence of BAF (A), BHK cells infected with BRSV in the absence of BAF (B), BHK cells infected with SVHR in the presence of 100 nM BAF (C), BHK cells infected with BRSV in the presence of 100 nM BAF. The arrows in A and B point to mature virus budding from the surface of cells. The arrow in C points to nucleocapsids bound to the surface of vesicular membranes. The arrow in D points to mature virus budding into the lumen of a vesicle. All infections were done at an MOI of 10 at room temperature for 1 hour. After infection, monolayers were moved to 37°C for approximately 18 hours. After incubation, monolayers were processed for electron microscopy as described. This experiment was performed at least 3 times and the images provided are representative observations ...... 42

Figure 2.11 Optimum RNA folds at 37ᵒC for SVHR ∆G=-21.18 (A), BRSV ∆G=-23.8 (B), and BRSV ∆G=-20.38 (C). These models of the E2 protein encompassing amino acid positions 15 and 18 suggest that the T15I and Y18H mutations may alter the secondary structure of this region of the RNA. These predicted conformational changes may recruit a different complement of host cell proteins that allow for efficient replication in the absence of an active V-ATPase. Fold models were made using Mfold (117) The blue circles indicate the sequences changed in the BRSV mutant ...... 49

Figure 2.12 Alignment of selected alphaviruses. This alignment of approximately 20-25 amino acids shows the region of the E2 protein containing the T15Y and Y18H mutations of BRSV (shown by arrows) in comparison with other alphaviruses. The tyrosine at position 18 is strictly conserved amongst the alphaviruses. Sequence of BRSV and SVHR are from this research. The other sequences are from reference 62 ...... 51

Figure 3.1 Sindbis virus (SINV) virion purification strategy and production flowchart. (A) Clarified viral preparations were purified over two potassium tartrate density gradients done in series using isopycnic ultracentrifugation and a final wash and pellet step. Negative controls (uninfected cell culture supernatant) were processed in parallel with the virus preparations. (B) Transmission electron micrographs were taken of the purified virions to confirm efficiency of this enrichment procedure. (C) An SDS-PAGE gel was stained with silver or with coomassie blue for each of the viral preparations. HepG2 preparations are presented here, and are representative of all viral preparations from all host cellular backgrounds ...... 65

Figure 3.2 Comparison of the Mammalian Viral Host Proteomes...... 69

Figure 3.3 Mosquito Phospholipid Scramblase Detection. (A) The NCBI BLAST alignment tool was used to align the protein sequence of phospholipid scramblase for Aedes albopictus and Homo sapiens. The “Query” is the Aedes albopictus sequence (Accession#: 604777565) and the “Sbjct” is the Homo sapiens sequence (Accession#: 10863877). Peptide sequences presented below the alignment are highlighted, red (mosquito) and yellow (human). (B) Is the MS2 spectra used to identify phospholipid scramblase in HEK293 and C7-10 cells ...... 71

Figure 3.4 Characterization of SINV nucleocapsid (NC) and glycoprotein fractions. Silver stain of whole virus and NC fractions after increasing treatments of Triton X-100. M, protein marker size (kDa.), lane 1: stained whole SINV, lane 2: whole virus treated with 4% Triton X-100 [NC fraction], lane 3: whole virus treated with 5% Triton X-100 [NC fraction], lane 4: Triton X-100 fractions containing glycoproteins. Note that capsid, E1 and E2 glycoproteins run slower (denoted by *) after treatment with Triton ...... 75

Figure 3.5 Western Blot Analysis of NC fractions of purified SVHR. (A) Western blot of whole purified SINV bound to anti-nsP2 Ab using the LI- CORE image-capturing system. M is the same marker as in Fig. 4; the 260, 70, and 38 kDa. markers do not fluoresce at the 700 and 800 nm. NIR wavelength used. (B) Western blot of same fraction as in panel A. Probed first with anti-C Ab (the yellow band) and second, with anti- whole SINV Ab, green bands. The arrows point to a lower C band which is the only band that fluoresces with the anti-C Ab and the upper arrow which indicates the upper green C band which only fluoresces with the anti-whole virus Ab. Thus, two capsid bands are resolved in this fraction. E2 is above C in green. *Note that E1 does not label with this Ab, while the capsid protein does ...... 76

Figure 3.6 Sindbis virus (SINV), Mayaro virus and CHIKV production from HEK293 cells. Cells lacking RBM3 or SNX5, or wild type cells were infected with wild type virus and infectious virus titers were determined by plaque assay. All three viruses assayed, SINV, MAYV and CHIKV replicated to high titers in wild-type HEK293. By contrast, the titers of SINV were significantly reduced >100 fold (p-0.03), ~10 fold for MAYV (p=0.003) and 40 fold for CHIKV (p=0.01) in the HEK293 ∆SNX5 with p values as determined by student’s t-test. For MAYV reduced viral replication was also observed in HEK293 ∆RBM3, however the effect was modest with a 5-fold reduction for both SINV and MAYV. CHIKV was more reduced in virus production than MAYV from HEK293 ∆SNX5 and HEK293 ∆RBM3 showing a relative inhibition of 40 and 36 fold, respectively ...... 79

CHAPTER 1

Divergence from Cannon: an Additional Opportunity for Alphavirus Entry

Ryan M. Schuchman, Dennis T. Brown, and Raquel Hernandez

North Carolina State University, Department of Molecular and Structural, Biochemistry, North

Carolina State University, Raleigh, NC 27695 USA

1.1 Abstract

The study of animal virus entry is a very difficult endeavor with many obscurities; thus the field of virology finds itself with vast chasms of disagreement on this topic. To further complicate this difficulty, characteristics specific to each virus system combine to make experimental interpretation difficult. For example, a virus species may be programmed to enter through differing pathways depending on the cell type and host species of cell that it infects (1).

Further, viruses can characteristically have high particle-to-PFU ratios that make observations by direct means difficult to interpret. These problems have plagued the field of virology for many years and will likely persist into the foreseeable future until methods and technologies advance to allow for increased resolution of these scientific impasses. This review will attempt to highlight the history, methodologies, and new developments within the field of alphavirus biology in the context of both receptor-mediated endocytosis and direct penetration hypotheses of virus penetration. Additionally, comparisons will be made between the proposed entry mechanisms of

Alphaviruses, and other enveloped viruses that may not follow orthodox pathways, to demonstrate the technical problems encountered by these studies.

Keywords: Alphaviruses and entry mechanisms

1.2 Introduction: Alphaviruses, Their Superstructure, and Genomic Organization

The prototypical Alphavirus (figure 1.1), Sindbis virus, has an icosahedral configuration and a triangulation number of 4 (2). The ~70nm virion is comprised of two nested protein shells with a host-derived membrane that resides between the two shells (2). The outer shell is composed of 240 copies each of the E1 and E2 glycoproteins which form heterodimeric E1/E2 complexes which further trimerize into the 80 glycoprotein spikes found on the surface of the virion (2, 3). The inner protein shell contains 240 copies of the capsid protein (C) and retains an identical icosahedral, T=4 configuration (2). Out of both of the structural glycoproteins, only E2 spans the membrane and interacts through hydrophobic interactions with the capsid protein to form a connection between the core of the protein and the surface components (4, 5). Within the capsid of the virion resides a single-stranded, ~11kb RNA molecule of positive polarity. From 5’ to 3’, this molecule contains genes for the nonstructural and structural proteins in the following order: nsP1, nsP2, nsP3, nsP4, capsid, E3, E2, 6K/TF, and E1 (6). This RNA molecule contains a

5’ methylguanosine cap and a 3’ poly-A tail and can function as infectious mRNA in the host cell (6).

Figure 1.1: An EM reconstruction of Sindbis Virus. In this reconstruction, the E1/E2 glycoprotein coat is seen in yellow, the lipid bilayer is seen in red, and the nucleocapsid core is seen in blue. Though not seen in this image, the E2 protein crosses the membrane and interacts with the capsid monomers. The capsid exhibits a T=4 triangulation number like the glycoprotein coat. Reproduced from Sharp, J., Nelson, S., Brown, D. and Tomer, K. 2006, Virology, 348, 216-223: Copyright 2006, with permission from Elsevier.

1.3 Alphavirus Structural Proteins

Alphaviruses have three main proteins that contribute to the structure of the mature virion: E1, E2, and capsid. Recently, nsP2, the viral protease has been found associated with mature infectious particles and may also participate in a structural capacity (manuscript in review). Here, the current knowledge pertaining to the structure and/or function of the structural proteins will be discussed. E1 and E2 are both glycoproteins that interact on the surface of the virion to facilitate entry into the host cell (2, 3, 7). Evidence has been put forth that implicates E2 in receptor recognition and E1 as the fusogenic element involved in the fusion event between the virus and host endosomal membrane (7).

E1 (47.3 kDa) is considered a Class II virus membrane fusion protein and exhibits a β- dominated secondary structure that forms an ectodomain of elongated tertiary structure containing three domains (figure 1.2). The X-ray crystal structure of the E1 ectodomain of

Semliki Forest Virus (SFV) has been characterized (8); SFV-E1 bears 50% sequence identity and

68% similarity to that of Sindbis virus E1. Domain I (d1) is a β-sandwich consisting of 8 β strands (10 total β strands in the domain) that encodes high-mannose, N-linked glycosylation sites at residues 139 and 245, though these specific glycosylation sites are not conserved across the alphaviruses, or may be absent (8-11). E1 Domain II (dII) is comprised of 13 β strands that form a finger-like projection, an α helix, and 3 one-turn 3/10 helices (8). DII is not a continuous domain in itself, but is formed by 2 “excursions” of the polypeptide that interact to form the two opposing sides of the DII module (8). DII also contains a notable functional feature: the putative fusion loop. The fusion loop exists at the tip of the elongated E1 structure and, as would be expected, contains ~20 hydrophobic residues (12). This putative fusion loop is proposed to facilitate fusion by its insertion into the membrane of the host endosomal membrane during the

5 fusion (7) event of the virion and endosomal membrane, as described by the receptor-mediated endocytosis hypothesis. Domain III (dIII) connects the ectodomain to the transmembrane domain that anchors E1 to the viral membrane. This domain contains 8 β strands that are organized into a fold that is characteristic of the immunoglobulin super family (8). Immediately after the DIII domain is the C-terminal transmembrane region of approximately 24 residues that anchors E1 to the envelope of the virion. This region is not represented in the crystal but has been modeled into the CryoEM density of Sindbis virus (7). Depending on the alphavirus, there may be a short E1 tail that extends past the membrane (8).

Preliminary structures of the E2 (46.9 kDa) protein have been presented (figure 1.2), however, the accuracy of these structures falls under question (13, 14). Uncertainties in one study (13) regarding the biological relevance of the structure arise because the structure was characterized in the absence of phospholipids and because the E1 and E2 proteins were grown recombinantly, connected with a linker without the normal proteolytic processing or the membrane anchors. The resulting crystal structure showed all cysteine residues participating in disulfide bridges (8). However, an analysis of a whole infectious virus preparation (particle/PFU ratio ~1) (15) by LC-MS/MS showed that two disulfide bonds (C49-C114 and C259-C271) are available for chemical reduction and are therefore not present, as proposed by a crystal structure

(8) suggesting that preparations of recombinant virus proteins used for crystallization may adopt various conformations.

Figure 1.2: Schematic of E1:E2 interactions before E3 cleavage. (a) Model of the chikungunya virus heterodimer. The domains of the E1 protein are colored as follows: DI, red; DII, yellow; DIII, blue, and the fusion loop is colored orange The domains of the E2 protein are colored as follows: A, cyan; B, green; C, pink. The E3 protein is colored gray. Glycosylation sites are represented as balls and sticks and the residue bearing the glycosylation is indicated. Green sticks represent disulfide bonds. The large, black arrow represents the orientation of the viral membrane. The C-termini of the glycoproteins are depicted by a pink star (E2) and blue star (E1). The inset shows the relative positioning of the domains of the glycoproteins to each other. (b) A rotation of the glycoprotein heterodimer to show the E3 accessory protein. Reprinted by permission from Springer Nature: Nature, Glycoprotein organization of Chikungunya virus particles revealed by X-ray crystallography, Voss, J. et al., 2010.

The capsid protein (29.4 kDa) forms a T=4 shell around the genomic RNA of the alphaviruses (16). Lee, et al. characterized the structure of the Sindbis capsid protein using X-ray crystallography (4). In this study, a hydrophobic pocket was observed that was occupied by segments of proximal capsid proteins in the crystal structure (4). It was suggested after aligning the amino acid sequences of capsid and E2 that this hydrophobic pocket could accommodate the carboxy-terminal tail of the E2 protein (4). This hypothesis has been supported by other studies

(2, 5). It is interesting to note that in a study presented by Sokolosky et al. affinities between the capsid protein and genomic viral RNA are different: capsid-RNA interactions in the cytoplasm during early infection are specific while those between capsid and RNA in the mature virus are not (17). In this study, capsid proteins bearing mutations that disrupt interactions with RNA highlight the importance of capsid-protein interactions in early infection. Disruption of these interactions contributes to an increase of RNA stability during infection, and an increase in intracellular immunity of the RNA to cell (17). Additionally, this study found that the wild type capsid protein assists in promoting the translation of viral RNA (17).

1.4 The Study of Virus Entry

Many methodologies have been used in the attempt to study virus entry and many are performed in combination with metabolic inhibitors. To correctly resolve which aspect of the virus replication cycle is being affected in the experiment, it is important that care be taken to directly measure the specific process in question. For instance, if a metabolic pathway (e.g., receptor-mediated endocytosis) is being investigated as a route of entry, the metric of the experiment should be a direct measurement of virus endocytosis, not of virus RNA synthesis, protein synthesis, viral glycoprotein expression on the surface of the cell, nor mature virus

8 production. At best, assay of the biochemical events that are downstream of the actual event in question can be broadly resolved into early or late events in the virus replication cycle. In addition, any experiment that is conducted where observations are made downstream of the event in question should have proper controls in place eliminating secondary effects of any treatment.

For instance, if RNA synthesis is the measure of an experiment with a metabolic inhibitor it should be shown that the inhibitor does not interfere with the function of the viral replication complex itself or any other aspect of RNA synthesis. Unfortunately, the literature contains studies that, while valuable, may not completely describe the specific events being investigated because of these two experimental flaws.

1.5 A Brief Overview of Inhibitors used to Study Viral Entry

Chemical inhibitors have been a staple of studying entry pathways of viruses for decades

(18-21). This section will describe the function of a few inhibitors of the pathways that viruses may use to enter cells and some of the metabolic inhibitors used in the pursuit of this investigation. These inhibitors range from non-specific buffers, like ammonium chloride, that neutralize the low pH of endomembrane compartments (19) to more specific compounds like bafilomycin, Dynasore, PitStop, cytochalasin-D, and monensin. Others exist however, only a few inhibitors will be discussed here (19, 22-24) as these inhibitors have been used to determine the involvement and mechanisms of endocytosis during the process of alphavirus infection (19-21,

25).

One traditional method to study virus infection by endocytosis has been to neutralize the endosomes using different agents. Bafilomycin (BAF) is a specific inhibitor of the vacuolar

ATPase, (V-ATPase) the universal proton pump of eukaryotes (24, 26). In the cell V-ATPase is

9 localized to the plasma membrane and endomembrane compartments and its’ main function is to maintain the pH homeostasis of the cell (27). The V-ATPase is composed of approximately 20 subunits which form 2 domains (reviewed in (28)). The cytosolic V1 domain is responsible for

ATP hydrolysis while the membrane bound VO domain performs proton translocation.

Bafilomycin acts on the V-ATPase by antagonizing the c subunit of the enzyme’s Vo domain

(29, 30). This antagonism prevents the translocation of protons through the respective membrane thereby allowing disruption of the low pH. Ammonium chloride is a frequently used compound in the field of virology and is classified as a lysosomotropic weak base because of its capability of infiltrating the endomembrane compartments of the cell and neutralizing them (19). Similar to

BAF, the rational for using ammonium chloride is to buffer the pH of the endocytic compartments to neutrality. However, the nonspecific nature of ammonium chloride has been shown to have secondary effects during Sindbis virus infection that prevented the processing of viral non-structural proteins having a direct effect on RNA synthesis (21).

Endocytosis is a complex process that includes pinocytosis, and phagocytosis functionalities (reviewed in (31)). These two sub-pathways of endocytosis have differing functions; pinocytosis facilitates the intake of small extracellular components and water, while phagocytosis allows for the intake of larger materials. Clathrin-dependent and clathrin- independent modes of activity further divide the ways in which pinocytosis can occur (32).

Because of the many different types of endocytosis that can occur and the differing proteins that facilitate them, there are numerous inhibitors that can be used to delineate which type of endocytosis is used by viruses to gain entry into the cell (18, 22, 23).

The formation of endocytic pits is required for uptake of extracellular materials. The structure of the pits formed by clathrin is through the repeated association of clathrin molecules

10 that deform the cytoplasmic surface of the plasma membrane (PM). These invaginations are subsequently sealed and released into the cytoplasm. The function of clathrin can be inhibited by the treatment of cells with hypertonic sucrose (33) or by depleting the cells of potassium (34).

More specific inhibitors of the function of clathrin-coated pits include Dynasore and Pitstop.

These two inhibitors inhibit different aspects of clathrin-dependent endocytosis: Dynasore inhibits the activity of dynamin, which forms rings between the PM and the neck of the clathrin- coated pits thereby inhibiting the GTPase activity of the complex and thus prevents the release of the clathrin-coated vesicles (22). Pitstop interacts with the N-terminus of clathrin, which prevents the association of endocytic cargo with the clathrin scaffold (23). Despite secondary effects that the compounds used to study virus entry may have in the infection process, the compounds remain useful and may even provide unexpected insights into the infection as will be discussed.

1.6 Biological Complications in Studying Virus Entry

As stated previously, the infectivity of the virus sample (as measured by some form of titration) and the cell lines being used can have profound impact on the outcome of an experiment and its interpretation. A study performed by Alfson and colleagues demonstrated that the infectivity of Ebola virus can influence the course of infection in macaques (35). In this study, virus from two distinct stocks that had differing infectivities (8,400 particles/PFU and 511 particles/PFU) were used and animals were infected with 100, 1, or 0.001 PFU/macaque (35).

All animals died from the infection with the exception of animals infected with 0.001 PFU from the stock of virus with the higher relative infectivity (511 particles/PFU) (35). Because the 0.001

PFU infection from the stock of virus with an infectivity of 8,400 particles/PFU resulted in death while the same infection done with an infectivity of 511 particles/PFU did not, it was concluded

11 that the particles that are traditionally classified as defective retain some sort of biological significance as there is an increase in animal mortality despite the fact that defective particles are unable to produce cytopathic effects in tissue culture (35). Differing biological activities between particles of influenza virus that are classically defined as infective or defective have also been observed and may be conditional. Many of these observations have been summarized in a review by Brooke (36).

Despite accepted observations that other enveloped viral species can enter a cell by direct methods at the plasma membrane (37-40), there is still quite a large amount of opposition to any model suggesting that the alphaviruses are capable of similar entry routes that lack pH dependence. A study that underscores the complexity of virus entry was performed by Whitbeck and colleagues using vaccinia virus (1). This virus contained a beta-galactosidase reporter gene cloned into the viral genome and its expression was assayed during infection in the presence or absence of BAF (1). In this experiment transcription and translation of the reporter virus genome is required for the expression of the beta-galactosidase gene (β-gal), and the BAF used in the experiment should have no direct effect on enzyme activity. This study however, demonstrated that differences in beta-galactosidase activity were detected in different cells infected with vaccinia and treated with BAF (1). This situation shows that BAF is not directly contributing to the decrease in activity of beta-galactosidase implicating other host factors in the expression of

β-gal. Using this reporter assay in several cell types, the authors were able to conclude that vaccinia virus is capable of entry by pH-dependent and pH-independent routes. HIV, herpesvirus, coronavirus, and influenza A have also been shown to possess the ability to enter cells using a pH-independent fusion mechanism (37, 38, 40-42).

In contrast to the alphavirus structural proteins described above that are used for entry by fusion, influenza, coronavirus, herpes virus, and HIV are known to utilize proteins with a vastly different structure. Class II fusion proteins are only known to be used by the arboviruses. While the fusion machinery between the viruses discussed appears to be different, these particular cases highlight the ability for enveloped viruses to enter cells by means that do not absolutely require endosomal acidification.

1.7 Evidence for Receptor-Mediated Endocytosis

The model of alphavirus entry by endocytosis states that upon receptor mediated ingestion of the virus and subsequent delivery into endosomal compartments, the virus takes advantage of the decreasing pH to facilitate conformational changes in the glycoproteins of the virus (43). The conformational rearrangements then expose a hydrophobic loop of the E1 protein, containing approximately 20 amino acids, proposed to be critical for membrane fusion upon insertion into the endosome membrane (12).

Countless studies have been conducted that seek to demonstrate the ability of alphaviruses to enter cells by receptor-mediated endocytosis (14, 19, 20, 44-46). In pursuit of this line of study, experiments by Smit and colleagues began to characterize the biophysical properties of membrane fusion between Sindbis virus particles and liposomes that were composed of a mixture of cholesterol and sphingolipids (47). This study questioned what specific pH exposure was required by the virus to render the particle fusion-competent with the liposomes. It was determined that this value was a pH of 5.0 with some fusion occurring at a pH as high as 6.0. In this experiment it was also determined that Sindbis virus was capable of fusing in this pH range but required artificial membranes containing large amounts of cholesterol

13 despite the lack of proteins or other potential virus receptors in the liposomes (47). This large cholesterol requirement was explained by the suggestion that the large quantities of cholesterol used could promote membrane curvature that would provide favorable conditions for virus- liposome fusion (48). However, there are two inconsistencies with the liposome model 1) the large amount of cholesterol required for fusion and 2) the mechanism of fusion of a small fusion domain with a protein free lipid bilayer. It is known that membrane lipid composition affects curvature (48) however insect hosts of alphaviruses are cholesterol auxotrophs and infections in these cells cannot utilize high levels of cholesterol.(49). Additionally, although alphavirus and influenza encode different classes of fusion proteins, one mechanistic quandary remains. The membrane-fusion mechanism of entry for influenza has been suggested to require that the fusion protein penetrates the plasma membrane to at least the interface between the inner and outer leaflets a distance occupied by ~ 20Å (50). The pitch of the α-helical fusion domain being 1.4Å requires ~ 14 amino acids to reach the bilayer interface (8). Thus the attributes afforded by the fusion proteins of the alphaviruses would seem insufficient, in this case, for fusion to occur as only 10 amino acids constitute the fusion loop and would not reach such a depth to maintain productive interactions to induce fusion. Indeed, fusion from without and within still occur in the alphaviruses (51, 52) despite the lack of depth reached by the fusion loop. This observation can be explained by interaction between the viral fusion proteins and various proteins on the PM or endosomal membranes which could promote the merging of the viral and host membranes resulting in fusion (1).

The biological properties of virus infection were also investigated using BAF, which inhibits the action of V-ATPase. The pH decrease in endomembrane compartments is accomplished by the action of the V-ATPase during endomembrane maturation and so

14 perturbing its function was of interest to those studying virus entry by endocytosis. One such study by Pérez and Carrasco questioned the role of the V-ATPase in entry for enveloped viruses like Semliki Forest virus, influenza virus, vaccinia virus, herpes simplex virus-1, and Sendai virus; non-enveloped viruses like polio virus and adenovirus were also represented (53). By observing events downstream of entry, like RNA and protein synthesis after treatment with BAF, the authors concluded that inactivation of the V-ATPase, and therefore inhibition of fusion after endocytosis, was responsible for the differences seen between treated and untreated experiments

(53). Further, it was concluded that BAF had an effect on all enveloped viruses tested with the exception of Sendai virus. Similar to Sendai virus, all non-enveloped viruses were unaffected in their ability to infect cells after treatment with BAF (53). However, another explanation of this observation may be that replication of Sendai virus may be less dependent on the activity of the

V-ATPase, in view that virus entry was not assayed directly. A more recent study (54) has confirmed V-ATPase requirement as a host factor involved in dengue virus maturation and has been observed with other flaviviruses and influenza virus (55-57). Further, a study by Duan and colleagues demonstrated that the V-ATPase interacts directly with the prM protein of dengue virus and that this interaction is required for proper secretion of dengue virus from the cell (58).

The observation that dengue virus requires the V-ATPase for secretion was made by disrupting the interaction between prM and the V-ATPase. V-ATPase has been found to be involved in glycoprotein transport in the past (26) and could be involved in dengue maturation. However, the study by Duan evaluated the successful inhibition of entry by BAF treatment with immunofluorescence to assay for the production of glycoproteins on the surface of the cell (58).

As stated, protein translation occurs downstream of entry and conclusions should be drawn with caution.

In addition to the V-ATPase, various other host cell proteins have been implicated in viral infection by receptor-mediated endocytosis and, like the V-ATPase, are vastly associated with the membrane of endosomal compartments. Rab5 and Rab7 have also been implicated in the replication of alphaviruses, however there appear to be differences in the necessity of these proteins by membrane-containing viruses (59). These proteins contribute to viral infection by controlling the trafficking of endomembrane compartments as their association with these compartments is thought to be linked to endomembrane maturation (60-63). A more recent study by Stiles and colleagues has shown by siRNA screening that TSPAN9 is a host cell protein, whose function is largely unknown, that may be involved with entry by receptor mediated endocytosis by increasing the ability of alphaviruses to fuse with the membrane of the endosome

(64).

1.8 Evidence for pH-Independent Entry

The idea that the alphaviruses are capable of pH-independent entry began with the observations that Sindbis virus-mediated cell fusion takes place as a two-step event requiring the virus to be exposed first to low pH and then returned to neutrality (51, 65). This situation does not occur in the physiology of cells as the pH of the endomembrane compartments changes unidirectionally from neutrality to around a pH of 4 (44). Also, many of the studies in the formative years of alphavirus biology assayed biochemical events downstream of entry as the reporter (19, 53). This was because of inherent difficulties in directly observing and capturing the entry of viruses that are of both a known particle-to-PFU ratio and one that optimally approaches unity. The fact that alphaviruses do not produce empty particles because of their maturational properties lends them the advantage over other viruses in that they inherently have a lower

16 particle-to-PFU ratio. However, they could still exhibit higher particle-to-PFU ratios because of defects in the virus particle after release from the cell. The complication of the increased particle- to-PFU ratio has been overcome by the use of the heat-resistant strain of Sindbis virus (SVHR) that was originally isolated by Burge and Pfefferkorn (66, 67) which expresses high structural stability.

Electron microscopy (EM) continues to be an invaluable tool for studying the superstructure of cells and viruses and has been used extensively in the study of infection for many families of virus. However, infectivities of viruses, as discussed, greatly complicate direct observations by electron microscopy, as it is known that exposing the cell to excess ligands can induce endocytosis. Direct observations of virus infections at the entry stage can obscure evidence by other means because of potential artifacts induced by exposing the cell to large numbers of noninfectious virus particles. An EM study by Vancini, et al. observed for the first time, in a direct fashion, an alphavirus particle depositing its genome into the host cell (figure

1.3) in a manner that is not by receptor-mediated endocytosis (16, 68). This study, aside from the direct nature of observation, has the advantage over others that it utilizes SVHR which correctly prepared produces nearly 100% infectious virus particles so all cellular responses are representative of response to a single, infectious particle. In this study, an apparatus for the transfer of the genome from virus to cell was also observed (68). The observation of this apparatus agrees with experiments by Paredes et al. that demonstrate that a protrusion forms at the fivefold axis of purified infectious virus after exposure to low pH (16). Because exposure to low pH did not result in complete disassembly of the glycoprotein lattice, conformational changes induced by exposure to low pH may be indicative of similar changes that occur after interaction with the receptor(s) on the surface of the host cell (16).

Figure 1.3: The deposition of the Sindbis virus genome at the plasma membrane. These images depict SVHR at 4°C (A) and 37°C (B). All panels have been treated with α-SVHR primary polyclonal antibodies and immune-gold labeled secondary antibodies. The full particles (A) can be seen empting their genomes (B) while the virus particle remains at the plasma membrane. The arrow indicates a putative apparatus used by the virus to deliver its genome and the scale bars represent a 50 nm measurement. Reproduced from Vancini, et. al., 2013 (68).

An early study in 1991 by Edwards investigated the role of low pH in Sindbis virus infection, by genetic means, in Chinese hamster ovary cells (69). Despite being defective in endosomal acidification, these cells were still susceptible to Sindbis virus infection. At the time of this study, the genetic cause of the inability of these cells to acidify endosomes at non- permissive temperatures was unknown, but it was speculated that inactivation of the V-ATPase could be a cause. Stemming from this study, the involvement of the V-ATPase in alphavirus infection was recently investigated in the infection of SVHR using a combination of BAF treatment and a GFP reporter gene encoded by the virus under the control of an independent promotor (25). Despite treatment with BAF, infection was still observed. Importantly, a condition tested in this study examined the effect of BAF treatment on infection after transfecting the virus into the host cells. This particular experiment did not yield a productive infection when BAF was present throughout (pretreatment, during transfection, and after transfection). Because of this, the V-ATPase is implicated in viral processes that pertain to events in viral replication that are independent of entry. Potential viral processes that are dependent on the function of the V-ATPase are currently under investigation our laboratory. In these as yet unpublished experiments, we generated a mutant Sindbis virus that was resistant to

BAF (BRSV). Importantly, in this study, the mutations that accounted for the mutant phenotype were not in the viral replication proteins. When the RNA replication of wild type and mutant virus was compared with BAF treatment, BRSV was shown to overcome blockages in replication. Analysis of SVHR viral replication during a time of addition assay showed that BAF inhibits viral replication when cells are treated 5 minutes before infection, a time point which does not allow for endosomal pH neutralization before virus entry can occur (by endocytosis, for example). Further, a time point with BAF treatment 30 minutes after infection resulted in a

19 similar decrease in RNA production, a time in which entry should have been complete (70).

These experiments demonstrate that the effect of BAF is due to an inhibited V-ATPase and not because of neutralization of pH, nor because of off-target effects of the drug on the viral replication proteins.

1.9 Summary

The field of virus entry has been fraught with many challenges that have led to very intensive questioning. During the course of studying the entry characteristics of the viruses presented in this review, it is apparent that nature is rarely simple and well-defined: many viruses that were thought to enter cells by only one mechanism have been shown to do so by several.

Despite the fact that some viruses may exhibit a preferential mode of entry, it is imperative that all potential routes are characterized. Aside from academic pursuits, this knowledge is valuable for applied science, particularly the development of antiviral therapeutics directed toward entry.

Simply put, the design of therapeutics for only one route of entry may not prevail if other avenues can establish infection. For these reasons the entry of alphaviruses has been re-examined and a paradigm shift in what was once thought to be canon in virology has been shown to not be absolute.

CHAPTER 2

Role of the Vacuolar ATPase in the Alphavirus Replication Cycle

Ryan M. Schuchman1, Ricardo Vancini1, Amanda Piper1, Denitra Breuer1, Mariana Ribeiro1,

Davis Ferreira1,2, Joseph Magliocca1, Veronica Emmerich1, Raquel Hernandez1, and Dennis T.

Brown1*

1Department of Molecular and Structural Biochemistry, North Carolina State University,

Raleigh, NC, USA

2 Institute of Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil

Heliyon, 2018, Volume 4, Issue 7, e00701.

* Corresponding author. Mailing address: Department of Molecular and Structural Biochemistry,

North Carolina State University, Raleigh, NC 27695. Phone: (919) 515-5765. Fax: (919) 515-

2047. E-mail: [email protected].

2.1 Abstract

We have shown that Alphaviruses can enter cells by direct penetration at the plasma membrane (R. Vancini, G. Wang, D. Ferreira, R. Hernandez, and D. Brown, J Virol, 87:4352-

4359, 2013). Direct penetration removes the requirement for receptor-mediated endocytosis exposure to low pH and membrane fusion in the process of RNA entry. Endosomal pH as well as the pH of the cell cytoplasm is maintained by the activity of the vacuolar ATPase (V-ATPase).

Bafilomycin is a specific inhibitor of V-ATPase. To characterize the roll of the V-ATPase in viral replication we generated a Bafilomycin A1(BAF) resistant mutant of Sindbis virus (BRSV).

BRSV produced mature virus and virus RNA in greater amounts than parent virus in BAF- treated cells. Sequence analysis revealed mutations in the E2 glycoprotein, T15I/Y18H, were responsible for the phenotype. These results show that a functional V-ATPase is required for efficient virus RNA synthesis and virus maturation in Alphavirus infection.

2.2 Introduction

Sindbis virus is the prototype Alphavirus in the Togaviridae family. These viruses consist of a positive-polarity, single-stranded RNA that is housed in an internally situated nucleocapsid that is in turn surrounded by an outer protein shell. A membrane, whose lipid composition is host-dependent, lies between the nucleocapsid and outer protein shell (2, 3). The outer protein envelope is composed of trimers of the E1 and E2 protein heterodimers (2, 3). The proteins of the outer shell provide the tools by which the virus attaches to the cell surface (E2) and a mechanism that facilitates the entry of the viral genome into the cell (E1) (7). We have previously shown that this entry event takes place at the cell surface by direct penetration in a

time and temperature dependent process with an activation energy (Ea) of ca. 27 Kcal/mol (71).

We have also demonstrated that viral entry does not require endocytosis, acidification, or membrane fusion, as suggested by others (16, 18, 68, 72). Past studies may have incorrectly classified entry as the pH-sensitive step due to the use of indirect reporters that addressed events downstream of entry, i.e. transcription or translation (19, 20). The model suggesting that receptor-mediated endocytosis is the mechanism for virus entry was challenged by the development of an electron microscopy protocol that directly visualized the entry process (16,

68). This protocol uses immunogold-labeling to facilitate the identification of empty virus particles on the surface of the cell that would have otherwise been overlooked after entry took place (Figure 2.1).

Once the genome has entered the host cell, the viral polycistronic RNA is translated into

4 non-structural proteins, nsP1-4. nsP1 functions as a methyltransferase to add a methylguanosine cap to newly synthesized viral RNA (73-76). nsP2 is the protease that cleaves the nonstructural polypeptide and also is the viral helicase (77-81). nsP3 is an accessory in the synthesis of the negative RNA strand (82, 83); finally, nsP4 is the RNA-dependent RNA polymerase (84-86). The structural proteins are translated from a subgenomic viral 26S RNA.

Figure 2.1: Electron micrograph of Sindbis virus entry events on a BHK cell surface. Cells were infected with SVHR at an MOI of 1000 at room temperature for 15 min; virus particles were treated with anti-Sindbis structural protein polyclonal antibodies, then with immunogold-labeled secondary antibody and processed for electron microscopy. “F” indicates a full virus particle; “E” indicates an empty particle.

While Sindbis virus is an Alphavirus, it belongs to a broader group of arthropod-borne viruses, the arboviruses. The arboviruses are mainly represented in nature by three families

(Flaviviridae, Togaviridae, and Nairoviridae [formerly Bunyaviridae]) and representative species include chikungunya virus, Mayaro virus,and (alphavirus), West Nile virus, dengue virus, yellow fever virus, Zika virus, (flavivirus), and Crimean-Congo Hemorrhagic Fever virus

(Nairoviridae) (87). A vaccine exists for yellow fever virus, but effective treatments or prophylactics for the others are still not available (88). By fully characterizing the replication cycle of these viruses, using Sindbis virus as the accepted model system for Alphaviruses, new therapeutic targets and strategies may be identified and developed.

SVHR (Sindbis virus heat-resistant) is a strain of Sindbis virus that is structurally stable and has a particle-to-PFU ratio that approaches unity when properly prepared (89). This characteristic ensures that nearly every virus particle is infectious making a morphological study of entry possible.

We have investigated the role that V-ATPase plays in the Sindbis virus replication cycle by producing a virus mutant of SVHR, resistant to the effects of bafilomycin A1 (BAF). BAF is a specific inhibitor of the Vacuolar ATPase (V-ATPase), known to be involved in controlling the pH of the organelles in the secretory pathway (24, 90). Other known functions of the V-ATPase range from control of pH homeostasis to vesicular trafficking within the cell, whose functions are dependent upon an isoform of the V-ATPase. For instance, the a2 isoform is responsible for early endosome acidification, and the a3 isoform targets the Trans Golgi in the secretory pathway, other isoforms and combinations exist (91-93). It has been shown previously that interruption of the secretory pathway by BAF results in an accumulation of Golgi-derived vesicles (94).

The goal of this study is to identify what the effect of an inhibited V-ATPase has on alphavirus replication by examining entry, RNA production, virus maturation, and egress of

SVHR compared to a BAF-resistant Sindbis virus mutant (BRSV).

2.3 Results

2.3.1 Effect of Metabolic Inhibitors on Alphavirus entry

The effect of metabolic inhibitors on the entry of SVHR in Baby Hamster Kidney cells

(BHK) was examined using the indicated concentration of each inhibitor shown in figure 2.2.

After cells were treated for an hour with the respective inhibitor, the cells were infected at a multiplicity of 1,000 PFU/cell for 15 minutes at ambient temperature, fixed, processed, incubated with a primary anti-SVHR polyclonal antibody, and then incubated with immunogold beads- conjugated with secondary antibody. This high MOI is not for synchronization, but rather needed to ensure the observation of a sufficient number of virus particles on the surface of the cell. For each treatment, the number of full and empty particles out of a total of 100 was determined. In all cases, particles less than 60% of the full-sized diameter were not included in the dataset to ensure that the core of the virus particle would be in the plane of the thin section. That the appearance of empty particles is not an artifact of thin sectioning for the electron microscope is underscored by the fact that the appearance of empty particles is time and temperature dependent and follows

Arrhenius kinetics. If control values are set as 100% and all the treatments are set as % of control

Cytochalasin-D, cyclohexamide, and genistein were found to reduce the number of empty particles to approximately 80% of control value. Carbonyl cyanide-m-chlorophenylhydrazone

(CCCP), monensin, and BAF were found to reduce the number of empty particles to

26 approximately 55%, 60% and 65%, of control level respectively. The most significant inhibition of entry was seen in treatments with ionophores (CCCP and monensin). This indicates a requirement for membrane potential. A lesser effect was seen with BAF treatment, which also has a secondary effect on membrane potential. Endocytosis of virions or the presence of virus in endosomes was not seen in this study as was the case in our previous studies involving direct observation (4,9).

Figure 2.2: The Effect of Metabolic Inhibitors on Alphavirus Entry. BHK cells were untreated (control), treated with cytochalasin-D (50µM), cyclohexamide (100µM), genestin (100µM), CCCP (40µM), monensin (50nM), or bafilomycin A1 (500nM) for 1 hour. After treatment, cells were infected with SVHR for 15 min at room temperature, fixed, processed, and treated with primary, secondary antibodies, and counted as described in the text. Error bars account for a 10% error and the percent inhibition for each treatment is shown.

2.3.2 Generation of a BAF-Resistant Virus.

Twenty-two serial passages of SVHR in BHK cells treated with 100nM BAF produced a mutant that was resistant to BAF (25). The mutant virus was deemed BAF-resistant after the titer rose above 108 PFU/mL, non-adapted virus grown in the presence of BAF produced a titer of

4x106 PFU/mL. When the resulting mutant was sequenced, we discovered three mutations in the structural proteins: one in E1 (L222F) and two in E2 (T15I, Y18H). There were no mutations found in the replication (nonstructural) proteins.

2.3.3 Effect of BAF on alphavirus RNA Replication

Studies on the effect of BAF on alphavirus replication using radiolabeled RNA have been reported, but those experiments were only carried out to approximately 3.5 to 4 hours post infection (hpi) (19). We monitored viral RNA replication of SVHR and BRSV in the presence and absence of BAF using RT-qPCR over the course of 10 hours. A concentration of 100nM

BAF and a treatment time of 1 hour were seen to be sufficient to nearly neutralize all acidic compartments. This was determined by incubating cell monolayers with acridine orange (5

µg/mL), which only labels acidic compartments (95). No labeling was seen in BAF treated cells

(Figure 2.3) and this effect persisted past 24 hours after treatment (data not shown) (25). In the absence of BAF treatment, assay of viral RNA synthesis of SVHR and BRSV infected cells showed that both virus infections approached a maximum RNA copy number of approximately

108 molecules per approximately 9.5x105 cells (Figure 2.4). For SVHR, RNA production in the presence of BAF lagged behind production of SVHR RNA in the absence of drug by approximately 2 orders of magnitude at 3 hpi but slowly increased production over 10 hours.

During the same time course, BRSV in the absence of drug produced amounts of RNA nearly equivalent to SVHR in the absence of drug. In the presence of BAF, on average BRSV produced approximately 1.7x104, 1.2x106, 4.1x106, and 4.0x106 copies of RNA per approximately 9.5x105 cells at 3, 5, 7, and 10 hpi, respectively. These results indicate that previous assays conducted by others may have been prematurely terminated at a time point where a low level of RNA was produced; resulting in the conclusion that entry had not occurred. In this particular assay, it became difficult to measure RNA production beyond 10 hours because monolayers lost their integrity when cells lysis and detachment began, possibly skewing the measurement. To separate the apparent inhibition of RNA production from that of virus entry, a time of addition assay

(Figure 2.5) was conducted in which BAF was added to monolayers at time increments of 1 hour, 5 minutes prior to infection, or 15 minutes, and 30 minutes post infection. All infections were allowed to proceed to 10 hours before monolayers were processed for RT-qPCR. As anticipated, there was a significant reduction in RNA replication for the 1 hour pretreatment

(reduction to approximately 1.5% of untreated control). RNA production was inhibited for the

BAF treatment 5 minutes prior to infection (reduced to approximately 1.3% of untreated control), as well as the treatments at 15 minutes after infection (reduced to approximately 4% of untreated control) and 30 minutes after infection (reduced to approximately 10.5% of untreated control).

Figure 2.3: The Effect of BAF on endosome acidification. Monolayers of BHK cells were either untreated (A), treated with 100nM BAF for 1 hour (B), or for 10 hours (C) before incubation with acridine orange and subsequent imaging. Cells with orange vesicles show acidic compartments (A) and cells lacking orange vesicles show endosome pH neutralization (B and C). Experiment was completed a minimum of three times.

Figure 2.4: The effect of BAF on SVHR RNA production. BHK cells were pretreated with 100 nM BAF or tissue culture media at ambient temperature for 1 hour. Following this, cells were infected with the indicated virus (MOI=10 PFU/cell) in media with 100 nM BAF or without BAF at ambient temperature. Cells were then incubated at 37°C for the indicated amount of time in the presence or absence of BAF. RT-qPCR treatment groups are as follows: blue, SVHR without BAF; red, SVHR+100 nM BAF; green, BRSV without BAF; purple, BRSV+100 nM BAF. Experiment is an average of three biological replicates.

Figure 2.5: BAF time of addition assay. Identical BHK monolayers were treated with 100 nM BAF and infected with SVHR (MOI= 10 PFU/cell) at the indicated times and described orders. Infections took place and ambient temperature for 1 hour and then monolayers were transferred to 37°C for 10 hours. After incubation, cells were processed for RT-qPCR as described. Results presented are an average of 3 biological replicates, error bars represent the standard error of these replicates.

2.3.4 T15I/Y18H Mutations are Responsible for Bypass in BAF-Induced Blockages of RNA Synthesis.

To determine which of the three mutations make the greatest contribution to the BRSV phenotype we made single mutant strains of SVHR (E2:T15I, E2:Y18H, E1:L222F) and double mutant strains of SVHR (E2:T15I/Y18H, E2:T15I/E1:L222F, E2:Y18H/E1:L222F) that contain permutations of the single mutations. The mutants were then tested with regard to RNA synthesis

(Figure 2.6). In the absence of BAF treatment, SVHR and all of the BRSV single- and double- mutants exhibited RNA production of similar amounts, an average of 5.0x108 copies. (Figure

2.6). However, with BAF treatment, E2:T15I had a reduction of RNA production to an average of 1.7x106 copies, and E2:Y18H and E1:L222F had a decrease to 1.9x106 and 1.4x106 copies, respectively. Similarly, E2:T15I/E1:L222F and E2:Y18H/E1:L222F had a reduction of RNA production to 2.6x106 and 6.7x105 copies, respectively. By contrast, E2:T15I/Y18H only had

RNA production reduced to 2.4x107 copies with BAF treatment. SVHR in the presence of BAF produced an average RNA copy number of 1.2x107. The RNA levels of BAF-treated mutants examined in this experiment compare to the BAF-treated BRSV at 10 hpi. (Figure 2.6). These mutants indicate that viral RNA encoding a threonine to an isoleucine at the 15th position and a tyrosine to a histidine at the 18th position of the E2 gene is sufficient to confer the partial ability to bypass BAF-induced blockages of RNA replication.

Figure 2.6: RNA production in BRSV-derived mutants. BHK cells were treated with BAF as above, infected with the indicated virus, and assayed for RNA production by RT-qPCR at 10 hpi. The control treatment group (no BAF) is shown in blue and cells treated with 100 nM BAF are shown in red. Data presented are an average of of two biological replicates, error bars represent the standard error of these replicates.

2.3.5 T15I/Y18H Mutations are Responsible for the Ability to Bypass Blockages of BAF in Virus Egress.

To determine which mutations have the greatest contribution to the BRSV phenotype with regard to virus egress (Figure 2.8), the single mutant strains of SVHR (E2:T15I, E2:Y18H,

E1:L222F) and double mutant strains of SVHR (E2:T15I/E2:Y18H, E2:T15I/E1:L222F,

E2:Y18H/E1:L222F) that contain permutations of the single mutations described above were again used (those presented in Figure 2.6). In the absence of BAF, all viruses produced mature virus with an average titer of about 2.1x109 PFU/mL. BAF treatment caused a dramatic reduction in titer to approximately 9.1x105 PFU/mL for E2:T15I, E2:Y18H, E1:L222F,

E2:T15I/E1:L222F, and E2:Y18H/E1:L222F. The E2:T15I/E2:Y18H mutant produced a titer that was approximately 9.5x107 PFU/mL in the presence of drug. BRSV, in the presence of BAF produced an average titer of approximately 7.3x107 PFU/mL (Figure 2.8). This result indicates that the E2:T15I/E2:Y18H mutations confer the partial ability to bypass BAF-induced blockages in viral egress. The effect of the BRSV mutations are exemplified in the immunofluorescence assay performed for SVHR and BRSV in the presence and absence of BAF (Figure 2.7). In this experiment, BRSV has infected fewer BAF-treated cells than either SVHR or BRSV has infected in cells not treated with BAF. This confirms that BRSV has a greater ability to infect cells that do not have acidified endomembrane compartments.

Figure 2.7: The Effect of BAF on alphavirus glycoprotein expression. Monolayers of BHK cells were either untreated (A and C) or treated with 100nM BAF for 1 hour (B and D) before a 10 hour infection with SVHR (A and B) or BRSV (C and D). The negative control (E) is untreated and uninfected. All monolayers were fixed with methanol and incubated with anti-SVHR and Alexafluor secondary antibodies before microscopic examination.

Figure 2.8: Mature virus production from BRSV-derived mutants. BHK cells were treated with 100 nM BAF for 1 hour. at room temperature, infected at an MOI of 10, and assayed for mature virus production by titration of the 20 hpi sample. The control treatment group (no BAF) is shown in blue and cells treated with 100 nM BAF are shown in red. Error bars represent the standard error of three biological replicates.

2.3.6 Effect of BAF on Virus Assembly and Egress

To characterize what effects an inhibited V-ATPase had on egress of the viruses in question, we determined the titer of the progeny viruses on BHK monolayers after SVHR or

BRSV was released from control or BAF-treated cells during a 20 hr. time course (Figure 2.9).

The titers of BRSV under both treatments and BAF treated-SVHR were all within about one order of magnitude from the SVHR control at 3 hpi. Greater disparity between the treatment groups began around 5 hpi. BRSV in the absence of BAF at 20 hpi was produced at titers comparable to the untreated SVHR control > 1010 PFU/mL. With BAF treatment, both SVHR and BRSV had a reduction of approximately 4 and 2 orders of magnitude, respectively, by 20 hpi compared to the untreated groups. The titers of virus produced at the end of 20 h were 4x106

PFU/mL for SVHR and 6x108 PFU/mL for BRSV in the presence of drug. We confirmed that at the 10 hpi time point BAF treatment did not produce excessive empty virus particles in any of the treatment groups by comparing the RNA levels to the virus titer in PFU/mL (data not shown). Thus, BAF treatments did not contribute to an altered particle to PFU ratio. The genome-to-PFU ratios calculated were approximately between 10 and 20 for all treatment groups.

To determine the ultimate fate of maturing virus particles, observations of monolayers 20 hpi, of SVHR and BRSV under the same experimental conditions as described for the egress experiment, were made using electron microscopy (Figure 2.10). BRSV and SVHR are seen to bud normally from the cell surface in the absence of BAF (Figure 2.10 A, B). However, SVHR and BRSV nucleocapsids accumulate at the surface of vesicular bodies when infected in the presence of BAF (Figure 2.10 C, D). In BAF-treated cells that have been infected with BRSV, the virus is able to overcome the block in maturation and is enveloped in the membrane of the

39 internal vesicles. Release of mature virions occurs as cells succumb to the rigors of infection, drug treatment, and lysis.

Figure 2.9: The effect of BAF on mature virus production of SVHR or BRSV. BHK cells were either untreated or treated with 100 nm BAF at room temperature for 1 hour. Monolayers were then infected in presence or absence of BAF for 1 hour. Monolayers were incubated at 37°C for the indicated amount of time and the resulting supernatant was then titered. Treatment groups are as follows: blue, SVHR without BAF; red, SVHR+100 nM BAF; green, BRSV without BAF; purple, BRSV+100 nM BAF. Experiment is representative of two biological replicates.

Figure 2.10: The effect of BAF on viral assembly of SVHR or BRSV. Thin section electron micrographs of BHK cells infected with SVHR in the absence of BAF (A), BHK cells infected with BRSV in the absence of BAF (B), BHK cells infected with SVHR in the presence of 100 nM BAF (C), BHK cells infected with BRSV in the presence of 100 nM BAF. The arrows in A and B point to mature virus budding from the surface of cells. The arrow in C points to nucleocapsids bound to the surface of vesicular membranes. The arrow in D points to mature virus budding into the lumen of a vesicle. All infections were done at an MOI of 10 at room temperature for 1 hour. After infection, monolayers were moved to 37°C for approximately 18 hours. After incubation, monolayers were processed for electron microscopy as described. This experiment was performed at least 3 times and the images provided are representative observations.

2.4 Discussion

The goal of this study was to characterize the involvement of V-ATPase in the replication cycle of Sindbis virus. This was accomplished by observing the effects of various inhibitors on virus entry using electron microscopy and by producing a BAF-resistant mutant virus. The phenotypes of the two viruses were determined in the context of RNA replication and mature virus production in the presence and absence of BAF. Early and late stages in the viral replication cycle were monitored using RT-qPCR, virus titration by plaque assay, and electron microscopy.

Our selection of metabolic inhibitors for the EM studies (Figure 2.2) is represented by an inhibitor of endocytosis (cytochalasin-D) (45), a eukaryotic translation inhibitor (cyclohexamide)

(96), a caveolae-mediated endocytosis inhibitor (genistein) (97), two ionophores (CCCP and monensin) (98, 99), and a V-ATPase inhibitor (BAF) (24). The observation that empty virus particles were produced found bound to the cell plasma membrane using our EM protocol after treatment with cytochalasin-D is direct evidence that the Alphavirus RNA can enter the cells by pathways not requiring receptor-mediated endocytosis. These observations confirm our previous observations that virus entry can occur at the cell surface (68) and that the V-ATPase has some effect on the replication process independent of endosome pH or virus penetration. As proposed by others, V-ATPase could participate in other aspects of the virus replication cycle. For instance, V-ATPase has been shown to reduce egress of Japanese encephalitis virus (55), dengue virus (58), vesicular stomatitis virus (55), and herpes simplex virus (55).

One mechanism by which viral RNA could be introduced into a host cell is by physical translocation. It was considered possible that an enzymatic component of the cell, like a ribosome in close proximity to the cytoplasmic side of the plasma membrane, could attach to

43 viral RNA and facilitate its entry into the cell in an event concurrent with viral protein translation. This event would be mechanistically similar to the transcription-dependent entry event of bacteriophage T7 (100-102). This does not seem to be the case because cyclohexamide treatment, which inhibits protein synthesis, only reduced the number of empty virus particles by

20% during the 15-minute infection period (Figure 2.2) while reducing virus production ten-fold

(68).

The observation that RNA entry follows Arrhenius kinetics with a low activation energy of 27Kcal/mol suggests that there is an enzymatic component involved with genome entry and that genome entry is not physically forced, as is the case with T4 bacteriophage (103, 104). The treatment of the monolayers with ionophores shows a more dramatic reduction in virus entry (a reduction of approximately 40% for CCCP and monensin) compared to the other treatments.

Taken together with the observation that the kinetics of entry follows Arrhenius kinetics, and is inhibited by ionophores, these data suggest the entry event relies partly on an unidentified protein component and a gradient of ions across the cell membrane, an observation that has been made previously (105).

The generation of a BAF resistant mutant (BRSV) raises interesting questions to consider for the mechanism of infection and replication of the Alphaviruses. Certainly, there are countless studies that investigate the roles of membrane fusion and endosomal pH in the receptor mediated endocytosis of Sindbis virus (19, 43, 52, 59, 64, 106-110); these observations do not preclude the possibility that the virus can enter through other routes, such as direct penetration (2, 16, 25, 68,

111). Previous studies have set out to characterize the intra- and intermolecular architecture of the Alphavirus structural proteins and because of these, questions have arisen regarding the functional role of the specific mutations found in BRSV (7, 10, 14, 112). It has been suggested,

44 because of these previous studies, that the mutations that have arisen in the analogous BRSV mutant, SFV fus-1, merely act to destabilize the interactions of the fusion proteins, thereby increasing the pH at which fusion occurs (7, 10, 14, 112). This situation can certainly be true in the context of the receptor-mediated endocytosis model, but considering the direct, morphological evidence provided by this study, and studies referenced in this work, we believe these criticisms to be peripheral and are not evidence that direct penetration at the plasma membrane is precluded. In addition, our immunofluorescence experiment (Figure 2.7) with

SVHR and BRSV shows that BRSV mutations do not cause fusion to occur at neutrality. If this were the case, one would expect the entire BAF-treated monolayer to be infected with BRSV.

This is supported by the fact that alphavirus infection has been shown to be a process that eventually allows for ions to leak through the PM (113-115).

It is also important to point out that selection of an SFV mutant requiring lower than normal pH for fusion also produced the mutant T15I. The studies that attempted to characterize the intermolecular contacts between the proteins of glycoprotein spikes involve the fitting of recombinant-expressed fused ectodomains of the envelope proteins that are then fitted into densities acquired from cryo-electron microscopy reconstructions of whole virus particles (7).

Structural studies such as these may reveal sound information about intramolecular contacts; but because of the lack of the viral membrane, the native super-structure of the glycoprotein coat may be compromised. Furthermore, Whitehurst, et al. have shown that a disulfide bridge proposed by the crystal structure of the E1 protein does not exist in the intact virion (15). For these reasons, it may be possible that the BRSV mutations are not involved in protein-protein interactions within the glycoprotein spike and therefore do not destabilize the interactions with

E1.

The phenotype of BRSV supports the hypothesis that the Alphaviruses can enter cells by direct penetration because infection took place in the absence of endosome acidification but with reduced levels of RNA replication (68). A nonfunctional V-ATPase did not inhibit the synthesis of viral RNA for BRSV, but suppressed RNA synthesis for SVHR (Figure 2.4). Similarly, a pretreatment of monolayers with BAF suppresses chikungunya virus RNA replication after entry occurs (data not presented). A functional V-ATPase also appears to be involved in viral maturation and egress. After determining the amount of mature virus produced in the presence and absence of BAF, we observed there was a large difference (~2.5 orders of magnitude) in the amount of mature virus that was released from the SVHR and BRSV-infected cells (Figure 2.9).

The fact that RNA replication was inhibited by BAF addition after the monolayers were inoculated with virus shows that the suppression of RNA synthesis seen in this study is not due to an interruption in virus entry (Figure 2.5). It has been demonstrated by others that at least half of all virus has entered the cell by 10-15 minutes after addition of virus to cell monolayers and entry has reached near completion by 30 minutes (70). Studies involving direct observation by electron microscopy revealed that 50% of attached particles released their RNA in 15 min. at room temperature (68). Additionally, reduction in RNA replication with the presence of BAF only 5 minutes before infection, a time where pH neutralization has not taken place (assayed by acridine orange, data not shown), indicates that a functional ATPase, not low pH, is required for the efficient initiation of RNA synthesis.

We turned to electron microscopy to determine any differences in viral maturation because of the relatively large amounts of BRSV RNA produced together with a deficit of mature virus particles (Figures 2.9 and 2.10). The budding of BRSV virus particles into the lumen of the vesicles of BAF-treated cells demonstrates that these mutations allow the virus to

46 bypass the normal maturational pathway. The virus is likely released as the cells break up during an extended incubation period. The BRSV mutations confer a property to the virus such that when the primary pathway of maturation—specifically, the secretory pathway of the cell—is inhibited, a secondary pathway can be utilized. In agreement with prior studies (26, 55, 58), this inhibition of the secretory pathway by BAF accounts for the lag in Sindbis virus production, seen in Figure 2.9. It appears that both of the mutations in E2 (T15I/Y18H) provide a significant contribution to the ability of the virus to resist the RNA-suppressing consequence of an inhibited

V-ATPase as well as the maturational obstructions (Figures 2.6 and 2.8). The E2:T15I mutant seems to have a relatively high efficiency of virus maturation compared to the other mutants, with the exception of the E2:T15I/E2:Y18H double mutant.

Because the E2:T15I/E2:Y18H mutant retains a total RNA producing activity greater than the other mutants and because both of these mutations lie in the E2 structural protein, we suggest that a change in secondary structure in the E2 gene RNA sequence may provide a cis- acting element that facilitates RNA synthesis in the altered cytoplasmic environment (Figure

2.11) (116). Additionally, the altered sequence of E2 protein, once translated, may similarly recruit additional or different host factors involved in viral maturation in the absence of a functional V-ATPase. A previously published article provides evidence that the V-ATPase is involved in the egress of dengue virus (58). In our experiments, SVHR entry has clearly occurred in the presence of BAF, but the BRSV mutations are required for a productive infection. Finally, serially passaging BRSV in the absence of BAF did not result in the restoration of the SVHR sequence (data not shown). This indicates that the mutations in BRSV have no negative effect on virus replication in the absence of drug. The single mutant E2:T15I is particularly interesting. A previous study in which Semliki Forest virus was mutated (fus-1) to require a lower than normal

47 pH in order to fuse artificial liposomes also resulted in this same amino acid change which aligns to the same position in the alphavirus genome (Figure 2.12). It was also shown that fus-1 infection was more sensitive to NH4Cl concentration, indicating a more acidic environment was required for infection (112). In our hands, this single mutant grew in the presence of BAF, indicating that it is infecting cells at a higher pH than would be expected in light of the fus-1 study. This observation, in addition to others (51), separates the events surrounding virus entry into host cells from those related to in vitro fusion of virus with liposomes (44). Both the fusogenic mutant, designated fus-1, and our BAF-resistant mutant have a threonine-to-isoleucine mutation at a conserved residue, E2-15. The fus-1 mutant was shown to revert back to the wild type sequence during infections in the presence of ammonium chloride. Our findings indicate that there is no selective pressure to favor the SVHR wild type sequence in the absence of BAF and suggests that the mechanism of action of ammonium chloride inhibition is quite different than that of BAF.

Figure 2.11: Optimum RNA folds at 37ᵒC for SVHR ∆G=-21.18 (A), BRSV ∆G=-23.8 (B), and BRSV ∆G=-20.38 (C). These models of the E2 protein encompassing amino acid positions 15 and 18 suggest that the T15I and Y18H mutations may alter the secondary structure of this region of the RNA. These predicted conformational changes may recruit a different complement of host cell proteins that allow for efficient replication in the absence of an active V-ATPase. Fold models were made using Mfold (117) The blue circles indicate the sequences changed in the BRSV mutant.

Finally, the mutations E2:T15I/Y18H give no apparent selective advantage to the virus except in the unnatural situation of a nonfunctional V-ATPase. Published sequences of

Alphaviruses indicate that the combination of amino acid changes in BRSV does not exist in examined variants of natural virus populations.

Figure 2.12: Alignment of selected alphaviruses. This alignment of approximately 20-25 amino acids shows the region of the E2 protein containing the T15Y and Y18H mutations of BRSV (shown by arrows) in comparison with other alphaviruses. The tyrosine at position 18 is strictly conserved amongst the alphaviruses. Sequence of BRSV and SVHR are from this research. The other sequences are from reference 62.

2.5 Methods

2.5.1 Tissue culture

BHK-21 cells were cultured in minimal essential medium (MEM) supplemented with

10% fetal bovine serum, 5% tryptose phosphate broth, 2 mM glutamine, 50 µg/mL gentamicin, and 10 mM HEPES (pH 7.4). Cells were maintained at 37°C with 5% CO2.

2.5.2 Drug treatment, infections, acridine orange staining, and electron microscopy

BHK cells were cultured in 6-well plates until ~90% confluent. For infections in the presence of BAF, monolayers were incubated with complete MEM containing 100 nM BAF for

1 hour at RT. Virus was diluted in MEM in the presence or absence of 100 nM BAF to give a final MOI of approximately 10 for BRSV and SVHR. Cells were infected for 1 hour at RT

(25°C). Inoculum was removed after infection and replaced with MEM either containing or lacking 100 nM BAF (Tocris). Monolayers were incubated for indicated times at 37°C. Live cell monolayers were stained with acridine orange (Figure 2.3) as described previously to verify the activity of BAF (95). BHK cell monolayers were processed as described previously (68).

2.5.3 Immunofluorescence Assay

BHK monolayers were infected as above. After 10 hours, the monolayers were fixed with

100% ice-cold methanol. The monolayers were then incubated with anti-SVHR (1:1000) in PBS supplemented with 0.1% BSA for 1 hour at ambient temperature. The monolayers were then

52 carefully washed with PBS supplemented with 0.1% BSA three times. The monolayers were then incubated with and Alexafluor-conjugated secondary antibody for one hour and then the monolayers were washed as before. The monolayers were then left in PBS for observation under

UV light.

2.5.4 RT-qPCR

All samples for RT-qPCR were extracted from monolayers of 6-well plates after appropriate treatment. Extraction was accomplished with Trizol (Thermo-Fisher) using manufacturer’s instructions. Following extraction, RNA concentrations were normalized to total

RNA concentration. cDNA of each sample was made using random hexamer primers from the

Maxima First Stand cDNA Synthesis Kit (Thermo) following the instructions of the manufacturer. cDNA was then either stored at -80°C or used for the Real Time PCR. RT-qPCR on all samples was done on an AB 7500 Fast Dx instrument (Applied Biosystems) with the following primers and TaqMan probe:

SVHR Sense: 5’-TGTGTACACCATCTTAGC-3’

SVHR Antisense: 5’-CAAAGGTATGCACAACTG-3’

SVHR Probe: 5’-FAM-AGTGCCTGACGCCATA-MGB-NFQ-3’

Samples were prepared in TaqMan Fast Advanced Mastermix (Thermo) following the instructions of the manufacturer and the following 40-round cycle was used for amplification:

95°C, 3 sec; 60°C, 30 sec. Results were analyzed using the 7500 Fast System v1.4.0 software

(Applied Biosystems). All RNA replication experiments were analyzed against a standard curve made from the purified Toto1101 infectious clone for SVHR.

2.5.5 Titration by plaque assay

Viral titrations were done on BHK cells in 6-well plates that were ~95% confluent. 250

µL of virus from each 10-fold serial dilution was added to the monolayers and allowed to adsorb for 1 hour at RT. Following infection, the supernatant was replaced with 2 mL of 1X MEM containing 1% agarose. The assays were allowed to incubate for approximately 18 h at 37°C.

Following this incubation, a 2 mL overlay of 1X PBS-D, 1% agarose, and 0.06% phenol red was added to the wells and allowed to incubate for 4 hour at 37°C protected from light. Visible plaques were subsequently counted.

2.5.6 Entry assay

Cells were treated with the indicated drug for 1 hour. The monolayers were infected for

15 min at an MOI of 1000 PFU/cell at room temperature, and processed as described (16, 68).

2.5.7 Generation of BRSV mutant and Site-Directed Mutagenesis

Twenty-two serial passages of SVHR in BHK cells treated with 100nM BAF produced a mutant that was resistant to BAF. The BHK cells were pretreated with BAF for 1 hour at room temperature and then infected with SVHR. After the 1 hour viral adsorption at room temperature, the flask was transferred to 37°C for ~24 hours after which the supernatant was harvested. BHK cells were infected with BRSV and the RNA extracted using Trizol (see RT-qPCR above). The resulting RNA was deep sequenced and the resulting contigs. compared to the Toto1101 sequence (accession J02363). A build of the resulting sequence data in CLC workbench compared to Sindbis virus strains in the database generated a table of variants differing from the

54 reference strain. Only sequence data which was not from a Sindbis variant or resulted in the conservation of the known amino acid sequence was considered a mutation. Three mutations in the structural proteins were thus identified: one in E1 (L222F) and two in E2 (T15I, Y18H).

There were no mutations found in the replication (nonstructural) proteins. These mutations were further studied as single and multiple virus templates.

Construction of the BRSV single and double mutants was accomplished with PfuTurbo

(Agilent Technologies) using the manufacturer’s instructions and the following primers on a

Toto1101 template:

E2_T15I_FWD: 5’-CCCCTACTTGGGCATATGCTCGTACTGCC-3’

E2_T15I_REV: 5’- GGCAGTACGAGCATATGCCCAAGTAGGGG-3’

E2_Y18H_FWD: 5’- TTGGGCACATGCTCGCACTGCCACCATACTG-3’

E2_Y18H_REV 5’- CAGTATGGTGGCAGTGCGAGCATGTGCCCAA-3’

E2_T15I/Y18H_FWD: 5’- GCCCCTACTTGGGCATATGCTCGCACTGCCACCAT-3’

E2_T15I/Y18H_REV: 5’-ATGGTGGCAGTGCGAGCATATGCCCAAGTAGGGGC-3’

E1_L222F_FWD: 5’- AGCACAGACATTAGGCTATTCAAGCCTTCCGCC-3’

E1_L222F_REV: 5’- GGCGGAAGGCTTGAATAGCCTAATGTCTGTGCT-3’

2.6 Acknowledgements

DTB would like to thank Elmer Pfefferkorn (Dartmouth Medical School) for introducing him to Sindbis virus in 1967. It has been a long and wonderful relationship.

CHAPTER 3

Comparative Characterization of the Sindbis Virus Proteome from Mammalian and Invertebrate Hosts Identifies nsP2 as a Component of the Virus Nucleocapsid and Sorting Nexin 5 as a Significant Host Factor for Alphavirus Replication

Ryan Schuchman2, Andy Kilianski1, Amanda Piper2, Ricardo Vancini2, José M.C. Ribeiro3,

Thomas R. Sprague4, Farooq Nasar4, Gabrielle Boyd5, Raquel Hernandez*2, Trevor Glaros*1

Journal of Virology, 2018, JVI-0694

1US Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD, USA

2Department of Molecular and Structural Biochemistry, North Carolina State University,

Raleigh, NC, USA

3National Institute of Allergy and Infectious Diseases, Laboratory of Malaria and Vector

Research, Rockville, MD, USA

4U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, USA

5Excet, Inc. 6225 Brandon Ave, Suite 360, Springfield, VA 22150, USA

*Corresponding Authors [email protected] (TG)

[email protected] (RH)

Running head: Host proteins involved in Sindbis virus replication

3.1 Abstract

Recent advances in mass spectrometry methods and instrumentation now allow for more accurate identification of proteins in low abundance. This technology was applied to Sindbis virus, the prototypical alphavirus to investigate the viral proteome. To determine if host proteins are specifically packaged into alphavirus virions, Sindbis virus (SINV) was grown in multiple host cells representing vertebrate and mosquito hosts and total protein content of purified virions was determined. This analysis identified host factors not previously associated with alphavirus entry, replication, or egress. One host protein, sorting nexin 5 (SNX5), was shown to be critical for the replication of three different alphaviruses, Sindbis, Mayaro and Chikungunya virus. The most significant finding was that in addition to the host proteins, SINV non-structural protein 2

(nsP2) was detected within virions grown in all host cells examined. The protein and RNA- interacting capabilities of nsP2 coupled with its presence in the virion support a role for nsP2 during packaging and/or entry of progeny virus. This function has not been identified for this protein. Taken together, this strategy identified at least one host factor integrally involved in alphavirus replication. Identification of other host proteins provides insight into alphavirus-host interactions during viral replication in both vertebrate and invertebrate hosts. This method of virus proteome analysis may also be useful for the identification of protein candidates for host- based therapeutics.

3.2 Importance

Pathogenic Alphaviruses, such as Chikungunya and Mayaro virus, continue to plague public health in developing and developed countries alike. Alphaviruses belong to a group of viruses vectored in nature by hematophagous (blood-feeding) insects and are termed Arboviruses

(arthropod-borne viruses). This group of viruses contains many human pathogens such as dengue fever, West Nile and Yellow fever viruses. With few exceptions there are no vaccines or prophylactics for these agents leaving one third of the world population at risk of infection.

Identifying effective antivirals has been a long term goal for combating these diseases not only because of the lack of vaccines but also because they are effective during an ongoing epidemic.

Mass spectrometry-based analysis of the Sindbis virus proteome can be effective in identifying host genes involved in virus replication and novel functions for virus proteins. Identification of these factors is invaluable for the prophylaxis of this group of viruses.

Keywords: Alphavirus; Sindbis virus; Viral Proteomics; Proteomics; Mass Spectrometry

3.3 Introduction

Epizootics of re-emerging and novel pathogenic viruses have increased in recent years, causing infections of susceptible human populations in some of the poorest countries in the world and imposing constant risks on public health. With increasing weather events, travel, and sexual transmission, an even larger population may be at risk. The Alphavirus genus (Family

Togaviridae) includes emerging and well characterized vector-borne diseases including Eastern,

Western, and Venezuelan Equine Encephalitis Viruses (EEEV, WEEV, VEEV), Chikungunya virus (CHIKV) Ross River (RRV), and Mayaro virus (MAYV). Sindbis virus (SINV), the prototype of this group is nonpathogenic (117). These pathogens are vectored in nature from zoonotic reservoirs, through mosquitoes or ticks to humans, and cycle from vertebrate to invertebrate hosts (arboviruses) (118). Upon emergence, these viruses spill over into the human population and cause severe disease. Viruses are obligate parasites of their hosts and require the host’s biosynthetic machinery to propagate all virus infections, necessitating prophylactic treatments that do not harm the host. With few exceptions, all arbovirus diseases lack effective vaccines and antivirals (119). Host-based therapeutics are thought to be alternative avenues, but identifying targets for this approach requires a detailed understanding of how these viruses interact with their host throughout the replication cycle (120, 121). Identification of host cofactors to virus enzymes or specific host factors involved in viral replication and packaging is challenging. The research presented herein focused on the identification of host proteins within

SINV virus particles. This was done in an effort to identify host factors associated with virus replication and address any functional relevance these specific proteins might have in viral propagation. In an effort to determine if mass spectrometry could 1) identify host proteins in the virus particle that could participate in subsequent infections or 2) identify host proteins trapped

60 in the particle during the process of maturation, virus was grown in three different vertebrate and one invertebrate host cell line.

Because of this parasitic characteristic it was originally thought necessary to specifically target virus factors for antiviral and vaccine development. However, this approach has not always been successful, so methods to target host factors required for viral propagation have been sought. Certain DNA and RNA viruses have long been known to package host factors into virus encoded progeny, to participate in subsequent infections. It is well established that retroviruses package host lysyl-tRNA synthetase (LysRS), by contact with Gag, which targets tRNA to prime transcription to DNA upon infection of a new host (122). Host factors have also been previously observed in virus particles such as influenza (123) vesicular stomatitis virus

(124) vaccinia (125) and herpes simplex virus (126) [reviewed in (127)].

Infection by SINV begins with attachment of E2 protein to specific cell receptor(s) (6).

The formation of this complex is proposed to induce conformational changes within the virus that produce a pore within the membrane and facilitates the release of the positive sense, single- stranded viral RNA directly into the cell (68, 128-130). In the cytoplasm, the RNA is then translated into four non-structural proteins, nsP1, nsP2, nsP3, and nsP4 (131). Together, these proteins, in association with unknown host proteins, comprise the replication complex which is found associated with the plasma membrane in vertebrate cells (132). Transcription of viral RNA produces polycistronic full length and a subgenomic 26S RNAs. In both vertebrate and invertebrate cells the non-structural proteins are translated from full length RNA while the structural proteins are translated from the subgenomic 26S RNA. The 26S RNA encodes the proteins in the order Capsid (C) PE2 (processed to E3 and E2), 6K/TF (transframe), and E1, assembled in the endoplasmic reticulum (ER) and processed by host signalase and furin proteins

(133). The matured virus from all host species contains the structural proteins C, E1 and E2. 6K is not found associated with Sindbis virions but LC-MS/MS analysis of the 6K chimera- frameshift TF protein have identified the latter in Sindbis particles (134). In vertebrate hosts, the glycoproteins mature via the exocytic pathway and are delivered to the plasma membrane.

Concurrently, the capsid protomers are pre-assembled into viral nucleocapsids with full length

RNA found within the cytoplasm and are transported to the plasma membrane, where they acquire their glycoprotein-modified outer shell (6). The virus fully matures as E1 and E2 modified plasma membrane surrounds the nucleocapsid (NC) core and is extruded into the surrounding environment (3, 135) in a process that excludes the preponderance of host plasma membrane proteins (136). In invertebrate cells the virus takes an alternate path to maturation.

Translation and replication complexes are housed in cytoplasmic endosomes termed “virus factories” where all assembly mechanics occur. Fully formed virions are then secreted into the media by fusion of the endosomes with the cell plasma membrane (137, 138). In both hosts, the final virion is composed of each of the structural proteins, C, E1 and E2, in a 1:1:1 stoichiometric ratio producing a highly stable and symmetrical virus (139). The mature structure contains an outer protein shell composed of the glycoproteins E1 and E2 separated by a host-derived membrane from the inner NC core. The lipid composition of the membrane will vary between vertebrate or invertebrate hosts especially with respect to cholesterol, which is not synthesized by the class Insecta (49, 140). In nature, arboviruses are maintained in a complex cycle involving infections between the virus vertebrate reservoir, the insect vector and the human host and can replicate in many host backgrounds (89). For this reason, we used cell lines of various tissue types and host backgrounds to characterize the compliment of host cell-derived proteins. To determine the host protein content of the model Alphavirus particle, SINV, a mass spectrometry-

62 based approach was utilized to identify host proteins within virions that might not be resolvable using other methods (141). Additionally, mass spectrometry can allow for the identification of proteins that are specifically associated with the virus structure or alternatively have become trapped or assembled into the structure during the process of maturation.

In this study, highly purified SINV preparations derived mosquito and mammalian cells were analyzed using LC-MS/MS to determine if previously undetected host proteins were packaged into virus particles. More than 124 unique host proteins were discovered in virus replicated in the human cell backgrounds of HEK293 (human embryonic kidney cells) or HepG2

(human hepatic cancer) cells and in hamster BHK21 (baby hamster kidney) cells more than 38 host proteins were identified. When the host protein profiles between mammalian species were cross compared, eight proteins were found in common. SINV was also prepared in a mosquito

C7-10 Aedes albopictus cell line, an Aedine cell line which exhibits both lytic and persistent infections (142). Although more than 50 host proteins were identified in the mosquito background, phospholipid scramblase was the most abundant and interestingly was the only protein found in both the invertebrate and vertebrate backgrounds. A detailed mapping of the viral proteome revealed a nearly 75% coverage of SINV’s structural polyprotein, which contains peptides detected for capsid, E3, E2, and E1, and TF but not 6K. An unexpected and intriguing finding was that peptides from SINV’s non-structural polyprotein were also detected, but specifically only for nsP2, the viral protease, which was not previously detected (143-145).

In order to determine a related function of these co-identified proteins during SINV replication we attempted to generate stable homozygous knockouts of each of the eight identified host proteins, in HEK293 cells. We were able to obtain homozygous knockouts for sorting nexin

5 (SNX5) and RNA binding protein 3 (RBM3), others were lethal. Using these knockout cell

63 lines, we observed a significant suppression in Sindbis virus production when compared to wild- type HEK293 cells. To confirm this observation, we also performed the same analysis using

Mayaro and Chikungunya virus. Viral replication was also inhibited for both these viruses. To the best of our knowledge, this is the first report of a sorting nexin, SNX5, playing a critical role in alphavirus replication. Collectively, this LC-MS/MS approach has expanded the tools that can be used for understanding the mechanics of alphavirus replication while also identifying novel host targets with therapeutic potential.

Figure 3.1: Sindbis virus (SINV) virion purification strategy and production flowchart. (A) Clarified viral preparations were purified over two potassium tartrate density gradients done in series using isopycnic ultracentrifugation and a final wash and pellet step. Negative controls (uninfected cell culture supernatant) were processed in parallel with the virus preparations. (B) Transmission electron micrographs were taken of the purified virions to confirm efficiency of this enrichment procedure. (C) An SDS-PAGE gel was stained with silver or with coomassie blue for each of the viral preparations. HepG2 preparations are presented here, and are representative of all viral preparations from all host cellular backgrounds.

3.4 Results

3.4.1 Determination of Viral Purity

In order to effectively analyze the virus associated host protein content, ultra-pure preparations of virions were prepared. Host background contamination from cellular vesicles or debris not specifically incorporated needs to be eliminated by the virus purification procedure prior to mass spectrometry analysis. The high particle/PFU ratio of SVHR, its structural stability and known exclusion of host proteins from the virus modified membrane make this an ideal experimental model. First, virus was grown and supernatant collected from infected and non- infected cells. The supernatant was harvested from a single cycle infection prior to cell lysis; crude viral preparations were clarified by low speed centrifugation to remove large complexes and particulates. Negative control fractions were run through the same process as virus- containing fractions. The clarified material was then run through two serial potassium tartrate density gradients using isopycnic ultracentrifugation. Virus from the second purification was then pelleted through PBS and the pellet was washed as the final purification step. To determine if this purification scheme (Figure. 3.1A) resulted in visually pure preparations of virus particles free of contaminating organelles or membrane fragments, fractions were visualized using transmission electron microscopy by negative stain (Figure 3.1B). The electron micrographs of the three-step purified SINV preparations visually show high purity virus preparations, with little evidence of cell debris or other cell-associated membrane-bound vesicles. To determine if there was any other protein contamination present in the preparations, each purified prep was resolved on an SDS-PAGE gel and stained with silver or coomassie blue as described above. Similar to the transmission electron micrographs, the SDS-PAGE analysis also confirmed that there were

66 no major protein impurities in the preparations (Figure 3.1C). The primary bands in the viral preparations were from the virus structural proteins E1, E2, and capsid.

3.4.2 Host and Virus Protein Identification and Functional Analysis of Purified SINV Virions

Following the confirmation of the purity of the virus preparations, SINV and negative control preparations were analyzed, in parallel, using a FASP LC-MS/MS method. Viral preparations and negative controls from BHK21 (hamster, Cricetulus griseus), C7-10 (mosquito,

Aedes albopictus), HEK293 (human), and HepG2 (human) cell host backgrounds were analyzed to determine if any host proteins could be detected. Each sample was analyzed in technical triplicate on a virgin C18 HPLC column to ensure that carry over from sample to sample would not result in false host protein identifications. LC-MS/MS spectral data was searched using the

SEQUEST algorithm in Proteome Discoverer 1.4 (Thermo Fisher) to identify peptides and align them to known host proteins from each background. Each purified virus preparation yielded unique host protein identifications with only a limited number of host proteins being identified in the negative controls. Keratin was often identified in the negative control and was not considered for functional interpretation because it is shed by cells in culture or introduced during sample processing. In the human backgrounds 124 host proteins were identified; 91 in HepG2 cells and

58 in HEK293 cells. Using the PANTHER classification to characterize these protein identifications, a significant portion of the proteins fell into three major functional classes; binding, catalytic activity, and receptor activity.

Compared to the human proteins found associated with SINV, an additional 38 host proteins were identified from the BHK21 hamster background and 51 proteins from the C7-10

67 mosquito background. It is of note that mammalian and insect cells do not always express mammalian protein orthologs. Both hamster and human genomes and proteomes are well characterized; however, the same level of characterization does not exist in the mosquito host

Aedes albopictus as the genome sequence was very recently published (146). To analyze the raw data obtained from the C7-10 cell background, a curated draft proteome from Aedes albopictus was utilized (see methods). Many of the proteins identified in the C7-10 background classify in similar functional categories and classes as the proteins identified in the mammalian backgrounds. Interestingly, for the mosquito background, phospholipid scramblase was overwhelmingly represented when compared to the other mosquito host proteins identified.

Scramblase was detected as 8 unique peptide sequences a total of 57 times (PSMs) for total sequence coverage of 30%.

Figure 3.2: Comparison of the Mammalian Viral Host Proteomes.

3.4.3 Coinciding Host Protein Signatures between Human, Hamster, and Mosquito Cell Backgrounds

When comparing human virus preparations (HEK293 vs. HepG2) only to each other, we observed a 34% overlap or identified 20 proteins, respectively (Figure 3.2). Given that the limiting factor which dictates detection by LC-MS/MS is dynamic range and instrument sensitivity, the host proteins that were commonly detected between each preparation were also in the highest abundance (indicated by the total number of PSMs, ∑#PSMs) within these virus particles. In addition to comparing preparations within the same species, we also compared the host proteins identified across species. Using this strategy we identified 8 protein signatures that were conserved between hamster and human (Figure 3.2). Cellular nucleic acid binding protein 6

(CNBP6), RNA binding protein 3 (RBM3), myosin (MYO), and claudin (CLDN) were present in virus particles grown in all backgrounds. Interestingly, the exact peptide sequence was identified for CNBP6, RBM3, and sorting nexin 5 (SNX5) from all vertebrate derived samples. When we compared the mammalian backgrounds to the mosquito background we found that only phospholipid scramblase was conserved. Also of note, we detected that the scramblase peptides overlapped even though only 51% of the amino acid sequence is conserved between mosquito and human proteins (Figure 3.3).

Figure 3.3: Mosquito Phospholipid Scramblase Detection. (A) The NCBI BLAST alignment tool was used to align the protein sequence of phospholipid scramblase for Aedes albopictus and Homo sapiens. The “Query” is the Aedes albopictus sequence (Accession#: 604777565) and the “Sbjct” is the Homo sapiens sequence (Accession#: 10863877). Peptide sequences presented below the alignment are highlighted, red (mosquito) and yellow (human). (B) Is the MS2 spectra used to identify phospholipid scramblase in HEK293 and C7-10 cells.

3.4.4 Viral Structural and Non-structural Proteins Identified in Purified SINV Virions

In addition to the detailed analysis of the host proteome contained within the SINV virion, the viral proteome within SINV virions was also examined. Raw data gathered from each

SINV preparation regardless of host background was pooled together to generate a comprehensive sequence map of SINV structural and non-structural polyprotein coverage. For

SINV’s structural polyprotein, 91 unique peptide sequences were detected a total of 10,833 times, resulting in a sequence coverage total of 74%. Peptides from every processed structural protein were detected except 6K. However, we did detect a single peptide (RLPGEGR) corresponding to TF protein, which is reported to co-purify with NC (147). In this study, we also searched our raw data against SINV non-structural polyprotein. From this analysis, we only detected peptides from nsP2, the virus protease. No peptides from any other non-structural protein were detected. For nsP2, we detected 20 unique peptide sequences a total of 121 times.

When compared to the structural polyprotein, peptides corresponding to nsP2 were detected with a frequency of roughly 1%. To confirm that nsP2 is not co-purifying with other structural proteins via protein-protein interactions, we performed an additional high salt wash in 1M KCl on viral pellets prepared in the BHK21 background. LC-MS/MS analysis of these preparations still resulted in high sequence coverage of nsP2 and no other non-structural proteins.

To identify if the nsP2 protein was associated with the SINV outer shell or inner core, we attempted to fractionate the inner core from the outer shell containing the glycoproteins. NC cores were fractionated by treating purified virus particles with 4% or 5% Triton X-100 for 1 hr. at 37ᵒC to remove the glycoprotein shell and the fractions were separated by ultracentrifugation.

Silver stain of 4-12% Bis-Tris gradient gels of the purified fractions (Figure 3.4) demonstrate

72 that the NC fractions shown in lanes 2 and 3 are not completely stripped of glycoproteins showing visible amounts of E1 and E2 on the gel after treatment with 5% Triton. From these silver stained fractions it could be concluded that the Triton X-100 treatment was not sufficiently stringent to eliminate all of the outer glycoprotein shell from the cores. Additionally, it was clear that treatment of the fractions with Triton appeared to shift the mobility of C, E1 and E2 proteins as seen by the appearance of slower migrating proteins in the 4% Triton X-100 fraction (Figure

3.4, lane 2) compared to the 5% Triton X-100 fraction (Figure 3.4, lane 3) and the observed E1 and E2 proteins in the glycoprotein fraction seen in figure 3.4, lane 4. Additionally the doublet C band in figure 3.4 has unlabeled C protein bands similar to those seen in Figure 3.5B, lane 1 described below.

Thus, none of the extractions utilizing treatment of the purified virus with 4% Triton X-

100 produced fractions pure enough to unambiguously assign the location of nsP2 to a particular sub fraction of the virus. However, to definitively identify the virus protein species which were seen in the extracted fractions, western blots were done using polyclonal antibodies against expressed capsid protein only (147) (raised in rabbits, a gift from S. Mukhopadhyay) or a combination of anti-C Ab and anti-SVHR whole virus (raised in rabbits) and is shown in Figure

3.5, B. This analysis showed that the anti-capsid Ab only recognized the bottom band of the

~30kDa. doublet while both C bands were recognized by the anti-whole SVHR Ab. The anti-

SVHR C/E2 antibody recognized only E2 at roughly 50kDa. These observations indicate that after Triton treatment, the doublet band running at ~30kDa. contains 2 forms of C protein. The lower band only binds to the polyclonal anti-C Ab while the whole SINV Ab virus reacted with both bands of C. The specific lot of anti-whole virus Ab used does not recognize E2, while E1 and C are recognized (data not shown). To positively identify nsP2 protein association with

73 purified SINV, a second blot was made and probed with anti-nsP2 antibody (a gift of R. Hardy) shown in Figure 3.5, A. Thus we verified that nsP2 was associated with purified virus but could not definitively identify the location in the particle.

Figure 3.4: Characterization of SINV nucleocapsid (NC) and glycoprotein fractions. Silver stain of whole virus and NC fractions after increasing treatments of Triton X-100. M, protein marker size (kDa.), lane 1: stained whole SINV, lane 2: whole virus treated with 4% Triton X-100 [NC fraction], lane 3: whole virus treated with 5% Triton X-100 [NC fraction], lane 4: Triton X- 100 fractions containing glycoproteins. Note that capsid, E1 and E2 glycoproteins run slower (denoted by *) after treatment with Triton.

Figure 3.5: Western Blot Analysis of NC fractions of purified SVHR. (A) Western blot of whole purified SINV bound to anti- nsP2 Ab using the LI- CORE image-capturing system. M is the same marker as in Fig. 4; the 260, 70, and 38 kDa. markers do not fluoresce at the 700 and 800 nm. NIR wavelength used. (B) Western blot of same fraction as in panel A. Probed first with anti-C Ab (the yellow band) and second, with anti- whole SINV Ab, green bands. The arrows point to a lower C band which is the only band that fluoresces with the anti-C Ab and the upper arrow which indicates the upper green C band which only fluoresces with the anti-whole virus Ab. Thus, two capsid bands are resolved in this fraction. E2 is above C in green. *Note that E1 does not label with this Ab, while the capsid protein does.

3.4.5 Virus-associated Mammalian Host Protein Requirement for Virus Replication

A Stable knockouts of each of the conserved host proteins were attempted in human

HEK293 cells. We were able to obtain homozygous knockouts for sorting nexin 5 (SNX5) and

RNA binding protein 3 (RBM3). To establish that this observation is not specific just to Sindbis virus, but rather to alphaviruses, we repeated these studies using the ‘new-world’ Mayaro, a pathogenic virus (27) and CHIKV which produces acute febrile polyarthralgia (148). Using these knockout cell lines, we observed a significant suppression in Sindbis, Mayaro and Chikungunya virus production by standard plaque assay when the cells were grown in the SNX5, or RBM3 knockout cell lines when compared to wild-type HEK293 (Figure 3.6). Because the virus produced from these knockout cell lines compared to virus produced from wild type HEK293 cells was infectious as determined by plaque assay on BHK cells, we conclude that these proteins are not absolutely required for infectivity. While SNX5 is important in the efficient maturation of infectious alphavirus intracellular trafficking of virus was not affected because there was not a significant amount of intracellular virus found in the SNX5 negative cells (data not shown). All three viruses assayed, SINV, MAYV and CHIKV replicated to high titers in wild-type HEK293, with titers of 5 x 109, 1 x 108 , and 4.2 x 107 PFU/mL, respectively. By contrast, the titers of

SINV were significantly reduced >100 fold (p-0.03), ~10 fold for MAYV (p=0.003) and 40 fold for CHIKV (p=0.01) in the HEK293 ∆SNX5 (p value determined by student’s T-test). For

MAYV reduced viral replication was also observed in HEK293 ∆RBM3, however the effect was modest with a 5-fold reduction for both SINV and MAYV. CHIKV was more reduced in virus production than MAYV from HEK293 ∆SNX5 and HEK293 ∆RBM3 showing a relative inhibition of 40 and 36 fold, respectively. Of the two proteins identified, the data demonstrates

77 that the deletion of SNX5 is much more deleterious to alphavirus replication than RBM3, thus

RBM functions are not further discussed.

Figure 3.6: Sindbis virus (SINV), Mayaro virus and CHIKV production from HEK293 cells. Cells lacking RBM3 or SNX5, or wild type cells were infected with wild type virus and infectious virus titers were determined by plaque assay. All three viruses assayed, SINV, MAYV and CHIKV replicated to high titers in wild-type HEK293. By contrast, the titers of SINV were significantly reduced >100 fold (p-0.03), ~10 fold for MAYV (p=0.003) and 40 fold for CHIKV (p=0.01) in the HEK293 ∆SNX5 with p values as determined by student’s t-test. For MAYV reduced viral replication was also observed in HEK293 ∆RBM3, -fold reduction for both SINV and MAYV. CHIKV was more reduced in virus production than MAYV from HEK293 ∆SNX5 and HEK293 ∆RBM3 showing a relative inhibition of 40 and 36 fold, respectively.

3.5 Discussion and Conclusions

The lack of a significant advancement in the development of effective antivirals, vaccines, and investigative tools for the study of pathogenic viruses remains a major gap in the ability to protect susceptible populations from diseases caused by emerging and re-emerging pathogens. Basic understanding of virus-host biology remains central to the development of new approaches, therefore; novel techniques must be harnessed to increase this basic scientific knowledge. Here, LC-MS/MS was utilized to determine if the prototypical alphavirus, Sindbis virus, specifically packaged host proteins into its virus particles. A two-stage purification process for SINV virions, followed by FASP digestion procedure and mass spectrometry analysis, revealed that a variety of host proteins were incorporated into SINV particles from each host background. Central to this approach was ensuring that the viral preparations were free of virion- associated impurities and cellular debris. Transmission electron microscopy and a silver-stained

SDS-PAGE gel revealed that our approach yielded high purity viral preparations that were not contaminated with other cellular debris, membrane fragments, or any significant protein contamination (Figure 3.1, B&C). The observation that 6K was not found associated with the

SINV particles is expected since 6K is not virion-associated in SVHR (149). The Aedes albopictus mosquito proteome is not well annotated so processing of mass spectrometry data from the C7-10 background required our group to generate a manually curated mosquito proteome. Despite this limitation, the functional role of these proteins being associated with

SINV replication will aid in experimental validation of their proteomic annotation. In the mammalian backgrounds, the identification of a core group of coinciding host proteins creates strong leads for further antiviral potential (Figure 3.3). These proteins should serve as the first set

80 of host proteins interrogated for their functional relevance during the SINV replication cycle and will be discussed first.

In this study, sorting nexin 5 (SNX5) was found to be associated with virus particles when SINV was grown in two mammalian cell lines HEK293 and BHK21. This host protein has cellular functions which are germane to the replication pathway of SINV because virus replication and maturation is integrally associated with the endo and exocytic host pathways. The sorting nexins (SNX) are a family of cytoplasmic proteins associated with a larger protein complex known as the retromer complex (150). This complex plays a vital role in endosomal protein sorting and this machinery is conserved across all eukaryotes (151). This complex is involved in endosome to Golgi trafficking and also in endosome to plasma membrane sorting and signaling. These membrane movement events are required to recycle receptors and other plasma membrane components during normal cellular processes. Members of this family contain a phox (PX) domain (152), which is a phosphoinositide binding domain (PIP) (153). Specific

SNX5 functions include endosomal sorting via the phosphoinositide-signaling pathway and micropinocytosis (154). However, while SNX5 was found only in two of the three mammalian cell lines tested, SNX5 is known to be expressed in HepG2 cells, the third mammalian line (155).

Although no SNX5 was found associated with virions produced from HepG2 cells, this may be a result of the level of expression of SNX5 by the HepG2 cells as well as the dynamic range of the

LC-MS/MS analysis. This may also explain why SNX5 was not found associated to virions from

C7-10 cells because SNX orthologs are also expressed in the Class Insecta (156). An alternative explanation for the lack of detection of SNX5 from insect cells is that alphaviruses mature via secretion from internal vesicles and not through the budding at the plasma membrane (137).

To investigate the requirement of this protein by SINV during replication, stable HEK293

SNX5 knockout cell lines were constructed. By replicating SINV in these cell lines we were able to show that SNX5 is essential for the growth of Sindbis, Chikungunya, and Mayaro viruses. In the absence of SNX5, replication of virus was found to be significantly inhibited by infectious virus titration by plaque assay of progeny virus. SNX5 has been shown to be critical in the replication of other intracellular pathogens as well, which is relevant to this analysis. Currently, a scenario is beginning to emerge in which the retromer complex may be involved in the restriction of pathogens to the host cell by its’ functional exploitation or repression (157). The best documented pathogen is Chlamydia, a common human bacterial disease. Recently, a pair of studies have elucidated a role for SNX5 as a restriction factor for pathogenesis. The intracellular bacteria Chlamydia require membrane structures to replicate and produce infectious progeny within host cells. Both studies determined that SNX5 can negatively impact the membrane rearrangements required by Chlamydia to replicate efficiently (158, 159). To counter this restrictive effect, Chlamydia sequesters SNX5 in replicative bodies and prevents its action on membrane rearrangements, allowing replication. It is known that membrane spherules are involved in plus strand RNA replication of alphaviruses (160, 161). It has also been shown that membrane rearrangements and composition are required for assembly of the alphaviruses during virus maturation or budding (136). Additionally, SNX5 was recently been found to co-localize with Ebolavirus during the entry phase in an internalization process via micropinocytosis (162).

Collectively these observations raise the question of whether membrane reorganization occurs during alphavirus penetration of the host membrane or budding at the plasma membrane by an

SNX mediated process. Although we have shown that SNX5 is essential for alphavirus replication, the exact effect on replication or the mechanism of inhibition is unknown. Other

82 viruses, such as human papilloma virus (HPV) and human immunodeficiency virus (HIV) are known to commandeer the retromer complex for efficient infection via specific mechanisms involving virus entry into the cell (122, 127). Evidence also implicates the retromer complex in a role during hepatitis C virus (HCV) infection as a method to recruit host proteins to the replication complex on a phophoinositol-4-phosphate rich membrane (163, 164). It is possible that SNX5 might be hijacked by SINV proteins directly or indirectly during particle penetration, early steps of the replication cycle or conversely during particle assembly to induce events necessary for infectious particles to be formed efficiently. Research to uncover the exact role of

SNX5 during SINV replication is warranted and could further uncover potential host candidates for anti-viral therapeutics.

While SNX5 has been implicated in the replication of infectious alphaviruses, the other proteins identified which were found in common (Figure 3.2) in the viral proteome by the reported method have less-clear potential roles in SINV replication. We did not detect phospholipid scramblase in two (both mammalian) of the four cell lines investigated and was the only host protein which vertebrate and invertebrate derived SINV particles displayed in common.

For the co-identified proteins to provide meaningful information on the SINV replication pathway a putative functional role for the virion associated proteins must first be identified.

Some hypothetical functions of these proteins during SINV replication are suggested below and are amenable to testing. Phospholipid scramblase functions to passively reorganize phosphatidyl inositols (PIPs) interchangeably from the outer and inner leaflet of the cell membranes. During lytic death induced by infection of vertebrates by alphaviruses, membranes are key targets for destruction upon the initiation of cell death by apoptosis and necrosis (165, 166). It has become

83 increasingly documented that PIPs are involved in these processes (167). Thus, PIPs may accumulate during the latter part of the infection as cell lysis approaches. The mosquito C7-10 cells line is the invertebrate cell line which expresses the most SINV induced lysis of the Aedes derived linages (168). Because this process is passive, there is no clear signaling network between scramblase and SNX5. Additionally, phospholipid scramblase 1 has been found to interact with influenza A suppressing nuclear import and thereby inhibiting virus replication

(169).

It is of special interest that junctional adhesion molecule C (JAMC), claudin (CLDN), and myosin are all components of the epithelial cells tight junctions (TJ) that provide a physical barrier between cells (170). These cell structures have been shown to be important for hepatitis C

(HEP C) viral entry, replication, dissemination, and exit from the cell (163, 170). Desmoplakin is a component of the desmosome, a cell structure that is also involved in cell to cell adhesion.

How this protein relates to SINV replication is also not clear. Cellular nucleic acid binding protein 6 (CNBP6) and RNA binding protein 3 (RBM3) both have the ability to bind single stranded RNA, the species of RNA encoding the SINV genome (171). In fact, RBM3 has already been shown to be associated with the SINV particle (172). Possible functions for these host proteins are to participate in virus translation and/or also participate as components of the RNA replication complex (173, 174). These RNA-binding proteins could protect viral RNA from host cell antiviral sensors while also promoting translation of viral polyproteins upon progeny virus entry into the host cell cytoplasm (174, 175). The heat-shock proteins HSP70/71 are important throughout viral replication cycles, although it remains unclear if these molecules play a role by incorporation into alphavirus and SINV virions (176, 177). HSP70 is known to be involved in the assembly of other RNA-containing viruses such as cucumber necrosis virus (178), and

84 hepatitis C virus (164) and are physically associated with lentivirus particles (179). Another protein chaperone, BIP (HSPA5), belonging to the HSP70 superfamily has been shown to be required for the assembly of SINV (180, 181). The identification of multiple host factors within

SINV particles using this method opens avenues for continued exploration into how these sequestered proteins influence the production of infectious virions or some part of the replication cycle. To definitively identify a role of these proteins further analysis is required.

Of major significance is the identification of a SINV non-structural protein, nsP2 associated with the virus particles, an unpredicted and fascinating finding. Alphavirus non- structural proteins are important for virus replication, but have not been found encapsidated into the virus particle. In this study, LC-MS/MS data lead to the discovery that nsP2 is incorporated into SINV virus particles when grown in each of the different host cells tested. It was considered that nsP2 could be co-purifying with SINV as a non-specific contaminant but its’ association with the virion was confirmed by performing additional high salt washes of the virus preparations to eliminate non-specific binding. To determine whether the nsP2 protein was associated in the outer glycoprotein shell or was held in the nucleocapsid core, the inner and outer shells of purified virus were prepared as described above, dissociated from one another with Triton X-100 and the fractions were run on a 4-12% Bis-Tris SDS PAGE gradient gel. To definitively identify the proteins, western blots were performed on the nucleocapsid enriched fraction using anti-whole SINV Ab and anti-capsid Ab. Capsid proteins and E2 were identified in the NC fraction. nsP2 was found incorporated into whole purified virus although a specific association with the outer shell or inner core could not be established.

This finding is supported by evidence that nsP2 plays a direct or indirect role in RNA encapsidation in Venezuelan Equine Encephalitis virus (VEEV) another alphavirus (182). This

85 discovery is significant because nsP2 is a multifunctional enzyme previously thought only to function in RNA replication as the viral protease and triphosphatase (NTPase) (183). As the viral protease, its’ functions range from cis and trans cleavage of viral non-structural polyproteins to innate immune evasion early in viral replication (184-189). While all proposed enzymatic roles for nsP2 have not been verified nor all the identified roles fully understood, one role of this protein is as a transcription factor i.e. a structural role. The presence of nsP2 in the SINV suggests that this protein may have an additional structural role within the SINV virion or may be required upon virus entry because it was identified in virions produced in all host backgrounds. The incorporation of an enzyme in a structural role is not novel among viruses.

Thymidylate synthase and dihydrofolate reductase are DNA synthesis and replication enzymes encoded by the phage T4. However, they are also essential structural components of the phage baseplate and thus essential elements for phage infectivity (190). Conversely, by incorporating nsP2 into progeny virions, SINV may use its enzymatic and immune antagonist roles to facilitate the infection at the earliest stages of viral entry, before translation of the plus-stranded viral RNA occurs. It may be involved in recruitment of capsid protein to the RNA nucleation site thereby participating in NC assembly. Alternatively nsP2 associated with the genomic RNA could establish the translation complex which initiates production of the non-structural proteins and then switches to replicate viral RNA. Any or all of these functions are possible and will be investigated. It is feasible that prophylactic targeting of viral nsP2, which has no other known substrates (131), would serve to disrupt the infection. Functional and structural roles for nsP2 both in cis and trans imply that the protein can assume multiple conformations and may not be amenable to rational design of functional inhibitors for non-enzymatic structures. However,

86 pharmacological targeting of nsP2 is an excellent candidate for an anti-viral therapeutic and would fulfill the first tenant of a good therapeutic, virus specificity.

Overall, this study has produced significant results that contribute to the understanding of alphavirus-host biology while identifying SNX5 as a potential target for antiviral therapy. LC-

MS/MS analysis of highly pure viral particles determined that SINV specifically incorporates host proteins into progeny virus particles. These proteins have a variety of functions that can be investigated for a role in SINV replication, with a core group of proteins conserved in multiple host backgrounds serving as a logical starting point. Finally, the novel discovery of SINV nsP2 incorporation into virus particles suggests a specific interaction and a possible role for nsP2, during replication or assembly. By interrogating SINV particles for members of the host background proteome, a better understanding of the virus-host interactions necessary for alphavirus entry, replication, protein processing, and particle assembly prior to budding can be established.

3.6 Materials and Methods

3.6.1 Cells, Virus Growth, Preparation, and Purification

The heat resistant SINV (SVHR) strain used in this study was provided to Dr. Dennis

Brown (NC State University). This strain was isolated by Burge and Pfefferkorn in 1966 by collecting virus that was resistant to heating to 54ᵒC (67). The choice of strain is important because this strain produces high titers (1010 pfu/mL) of highly infectious and physically stable virus. Chikungunya virus West African strain 37997 was grown and harvested as described in

(191). Mayaro virus strain CH was utilized for the study and was kindly provided by Dr. Scott

Weaver at UTMB. Propagation of HepG2 cells (human), and C7-10 cells (mosquito) was as described previously (192). Influenza A/PR/8/34 and the MDCK cells used to propagate and titer the virus were a kind gift of S. Laster and propagated as detailed in (193). All cells were obtained from American Tissue Culture Collection (Vero: CCL-81, HEK293: #CRL-1573, HepG2: #HB-

8065, BHK21: #C-13) except for the C7-10 (Aedes albopictus) cells which were obtained from internal collections (194). Culture medium from each uninfected cell culture was harvested and served as a negative control. Virus and negative controls were harvested from 10 T-75 flasks

(Corning) which produces enough virus to form a visible band in a 30 mL potassium tartrate gradient. Cells were infected at an MOI of 10 PFU/mL, allowed to replicate for a single cycle and harvested at 18 hours post infection to ensure that no cell lysis took place. The supernatants were clarified by low speed centrifugation. Twenty mL of the resulting virus was loaded onto a

15-35% linear potassium tartrate gradient and twice purified by isopycnic ultracentrifugation

(Beckman SW-28 rotor, 18 hours at 24,000 RPM). The resulting band of purified virus was collected and washed twice by pelleting the virus in 1X PBS. A sample of the purified

88 population was then visualized by transmission electron microscopy to check for contaminating cellular organelles or membrane fragments as previously described (71). Additionally, to check for co-purified protein contaminants, each preparation was resolved on a 4-12% Bis-Tris SDS-

PAGE gradient gel (Invitrogen) as described previously (195) and stained with silver in the method of Wray (196) or with coomassie blue. Visualized bands (silver stain only) were excised and an in-gel digestion was performed as described previously (197) prior to LC-MS/MS analysis for protein identification.

3.6.2 Sindbis virus particle analysis for nsP2

Gradient purified SINV was washed with 1M KCl to remove non-specifically bound proteins Purified SINV particles were then separated into glycoprotein and nucleocapsid fractions by treatment with 4% or 5% Triton X-100 for 1 hour at 37ᵒC and pelleted for 45 min through PBS at 45,000 RPM in a Beckman XL 90 centrifuge (198). The supernatant

(glycoprotein fraction) was saved and the resulting pellet (nucleocapsid fraction) was washed twice by pelleting through PBS in the same manner. The nucleocapsid fraction was then subjected to LC-MS/MS analysis, SDS-PAGE, or fluorography. Location of the nsP2 protein within the outer or inner shells of the particle was done using staining of 4-12% Bis-Tris gradient gels and western blot of the glycoprotein and NC fractions of the particle. Virus particles (~5µg per lane) were prepared in SDS-PAGE sample buffer supplemented with 5% beta- mercaptoethanol and boiled for approximately 10 minutes. The samples were run on an SDS-

PAGE Bis-Tris 12% gradient gel and transferred to a PVDF membrane. The membrane was blocked with 5% Carnation nonfat dry milk in PBS for 1 hour at ambient temperature. The membrane was then probed for one hour with a rabbit antibody raised against nsP2 (a gift from

R. Hardy) or anti C protein (a gift from S. Mukhopadhyay) diluted in PBS supplemented with

5% dry milk and 0.1% TWEEN 20 at ambient temperature. The blot was washed 3 times for 5 minutes each with PBS supplemented with 0.1% TWEEN 20 at ambient temperature. Finally, the membrane was probed with an anti-rabbit secondary antibody conjugated with IRDye© 800CW near infrared dye diluted in PBS supplemented with 5% dry milk and 0.1% TWEEN 20, and

0.02% SDS at ambient temperature. The blot was subsequently washed 3 times for 5 minutes each with PBS supplemented with 0.1% TWEEN 20 at ambient temperature, washed once with water, dried, and imaged according to LI-COR Odyssey CLx instructions.

3.6.3 Protein Extraction and Digestion

Viral preparations and their respective negative controls were processed for LC-MS/MS analysis using the filter-aided sample preparation (FASP) method as previously described (199).

Briefly, following purification the total protein concentration was determined and all preparations were normalized using sterile PBS to 0.5 µg/µL, aliquoted in 10µg fractions, and stored at -80ᵒC. A total of 10µg of total protein (20µL) was mixed 1:1 with MPER (Thermo

Fisher) supplemented with 50 mM dithiothreitol (DTT) and heated to 95ᵒC for 10 minutes. Once cooled to room temperature, the samples were mixed with 200µL of UA (8M urea, 100mM Tris-

HCl pH 8.5), placed over a 30kDa filter spin column (Millipore- Ultracell YM-30), and centrifuged at 14,000G for 30 minutes at room temperature to collect all proteins on the filter membrane. Denatured and reduced proteins were then alkylated by adding 100µL of IAA solution (0.05M iodoacetamide in UA) to each filter and incubating at room temperature in the dark for 20 minutes. Following alkylation, the samples were centrifuged at 14,000G for 20 minutes to remove the alkylation solution. Next, each sample was washed three times with

100µL of UA and then three times with 100µL of 100mM triethylammonium bicarbonate

(TEAB). Centrifugation at 14,000G for 30 minutes was used to remove each wash including the final wash. To digest the captured protein, 100µl of a trypsin digestion solution (10 µg/mL in

100mM TEAB) was placed on each membrane and incubated in a sealed tube overnight at 37ᵒC with shaking. After incubation, the peptides were collected for LC-MS/MS analysis in a clean tube by centrifuging each tube for 30 minutes at 14,000G. The membrane was washed by centrifugation one time with 50µL 100mM TEAB and once with 50µL of 0.5M NaCl. All washes were collected and pooled with the final peptide eluate. Each sample was then acidified using 10% trifluoroacetic acid until the final pH was roughly 2-3. Prior to mass spectrometric analysis, each sample was desalted using C18 desalting columns (Thermo Fisher) according to the manufacturer’s directions.

3.6.4 Liquid Chromatography Mass Spectrometry Analysis

Tryptic peptides were analyzed (technical triplicates) on an Orbitrap ELITE mass spectrometer coupled with the Easy-nLC II liquid chromatography pump system. Dried peptides were reconstituted in 3% acetonitrile/0.1% formic acid and resolved on virgin Picofrit 15cm X

75µm ID HPLC column packed with 5µm BioBasic C18 particles 300Å (New Objective) using a

130 minute multistep gradient [0-5 min: 5-10%B, 6-110 min: 10-35%B, and 111-130 min: 35-

95%B]. For the gradient, the A buffer is 3% acetonitrile/0.1% formic acid and the B buffer is

95% acetonitrile/0.1% formic acid. Orbitrap MS1 scans were performed at a resolution of

120,000 at 400 m/z, with a scan range of 110-2000 m/z. The top 20 precursors were selected for

MS2 data-dependent fragmentation. An MS2 spectrum was acquired using the iontrap scanning in normal mode (Top 20 Method). The minimum signal required to trigger a data-dependent scan

91 was 5000. Collision induced dissociation (CID) was used to generate MS2 spectra with the following settings: normalized collision energy 35%, default charge state 2, isolation width 2 m/z, and activation time 10 ms. AGC target was set to 1 x 106 for MS and 5 x104 for MS/MS with a maximum accumulation time of 200 ms. Dynamic exclusion was set for 60 seconds for up to 500 targets with a 5 ppm mass window. A lock mass of 445.120025 was used for internal calibration to improve mass accuracy.

3.6.5 Mass Spectrometry Data Processing

Spectra data were processed using Proteome Discoverer 1.4 with the SEQUEST search algorithm against a Sindbis virus polyprotein database (Uniprot ID: P03317) merged with either

Homo sapiens (RefSeq Tax ID: 9606) or Cricetulus griseus (RefSeq Tax ID: 10029). The Aedes albopictus-deducted proteome (146) data was downloaded from VectorBase (200). Additional peptides were downloaded from the NCBI TSA database. The non-redundant proteomes were organized on excel spreadsheets (201) and annotated as described previously (202). The resulting

FASTA file was merged with the SINV polyprotein and used to search against the SINV mosquito preparations. Dynamic modifications were set for carbamidomethylation of cysteine

[+57.02 Da], oxidation of methionine [+15.99 Da], and N-terminal acetylation [+42.011].

MS/MS spectra were searched with a precursor mass tolerance of 10 ppm and a fragment mass tolerance of 0.6 Da. Trypsin was specified as the protease with a maximum number of missed cleavages set to 2. A false discovery rate was calculated using PERCOLATOR and was set at

<1% to score high confidence peptide identifications. Grouping and functional analysis was performed using the PANTHER classification system for the human background only using each protein id’s accession number (203).

3.6.6 Phenotypic Studies with Stable Knockout HEK293 Cells

HEK293 knockout (KO) cell lines were purchased from Applies Biological Materials Inc.

(Richmond, BC, Canada). SNX5 and RBM3 KO was confirmed by Sanger sequencing. Cell lines were propagated at 37°C with 5% CO2 in Dulbecco’s Minimal Essential Medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS), sodium pyruvate (1 mM), 1% (v/v) non-essential amino acids, and 50 μg/mL gentamicin. Briefly, HEK293 monolayers were infected at equal

MOIs (approximately 10 PFU/cell) of SVHR, CHIKV or MAYV and allowed to incubate for 20 hours. Supernatants were titrated on BHK cells by the standard plaque assay to determine the virus titer. As a measure of the knockout cells’ ability to replicate an unrelated enveloped virus,

Influenza A/PRF/8/34 virus was used to infect the wild type and ∆SNX5 HEK293 cells.

Infectious virus titer was determined by plaque assay. Both ∆SNX5 HEK293 cells and wild-type cells produced an equivalent amount of infectious virus after 24 hrs.

3.7 Acknowledgments

The authors would like to thank Dr. Way Fountain (ECBC), Ms. Rebecca Braun (ECBC),

Elisabeth Vicente (Excet/ECBC), Dr. Edward Hofmann (Excet/ECBC), and Dr. Eric Calvo

(NIAID) for continued scientific and administrative support. Because JMCR is a government employee and this is a government work, the work is in the public domain in the United States.

Notwithstanding any other agreements, the NIH reserves the right to provide the work to

PubMedCentral for display and use by the public, and PubMedCentral may tag or modify the work consistent with its customary practices. You can establish rights outside of the U.S. subject to a government use license. We would also like to thank Dr. Richard Hardy (Indiana University) for the Sindbis virus anti-nsP2 Ab, Dr. Suchetana Mukhopadhyay (Indiana University) for the

Sindbis virus anti-C antibody and Dr. Scott Laster for the influenza virus and MDCK cells . RH is supported by the Clayton Foundation for Research, Carson City, NV. Conclusions and opinions presented here are those of the authors and are not the official policy of the US Army,

ECBC, or the US Government. Information in this report is cleared for public release and distribution is unlimited.

CHAPTER 4

Summary and Future Perspectives

Collectively, this research provides additional evidence to the idea that alphaviruses can enter the cell independently of pH and these studies identify new host cell proteins that have previously unidentified roles in the replication cycles of alphaviruses.

The first chapter of research sets the stage for the possibility that observations seen in the history of alphavirus research using strains of virus in a line of study that have undefined or low infectivities (high particle-to-PFU ratios) entry by receptor-mediated endocytosis may function as a means of increasing viral fitness. Entry of viruses with low infectivities by receptor- mediated endocytosis could ensure a greater opportunity of establishing a productive infection. It could be possible that entry pathways involving biologically relevant infectivities could vary between primary and secondary infection as well as primary and secondary infections in differing tissue types. Identifying a biologically relevant infectivity is paramount for extending observations seen in tissue culture to those that actually occur in vivo during infection.

Understanding any and all pathways used by viruses for entry is critically important for the development of antiviral strategies. Treatment using antiviral strategies targeting entry should be designed to inhibit all potential pathways used for entry as any un-drugged pathway could allow for a productive infection. These studies should be conducted for both mammalian and insect hosts as alphaviruses can infect both types of organisms. These studies would lead to a better understanding of how viral infectivity affects the pathways used for entry in respective host cell.

The second chapter of research redefines the role of the V-ATPase and shifts the paradigm once held that alphaviruses only enter a host cell by receptor-mediated endocytosis to a paradigm that an alphavirus with optimal infectivity can enter a host cell by a pathway that is independent of pH. For many years, it has been thought that the V-ATPase merely functioned as an enzyme that decreased the pH of endosomal compartments, serving to induce a structural

96 rearrangement of the surface glycoproteins into a fusion-competent state. However according to observations made during our studies, the V-ATPase has a much more diverse function during

Alphavirus infection and replication. It appears, according to our observations, that BRSV mutations in the genome allows for BAF-dependent blockages in viral genome replication and that after the genome is translated, these same mutations allow for bypass of BAF-dependent blockages in egress. The next logical steps in this line of study would be to investigate the biochemical mechanisms that allow for these bypasses. To accomplish this, differences in the structure of the viral genomes can be characterized using Selective 2’-Hyroxylation Analyzed by

Primer Extension coupled with Mutational Profiling (SHAPE-MaP) (204) as differences in the structure of the genome could recruit a different compliment of host cell proteins used in replication (205). Additionally, cross-linking of host cell proteins during infection to the viral

RNA or to the viral structural proteins followed by LC-MS/MS could begin to identify differences in the compliment of host cell proteins that are used during replication and egress.

Our method for studying entry could also be supplemented by using RNAScope, a relatively new in situ hybridization technology (206). For this experiment, cells would be infected with SVHR or BRSV in the presence or absence of BAF. Using a time-course scheme, cells would then be fixed and hybridized with the RNAScope probes along with anti-SVHR structural polyclonal antibodies. The secondary antibody would be selected to have a differing color than the

RNAScope probes so that discrimination between the two signals would be possible (207). This experiment would provide an opportunity to localize the virus particles infecting a cell and viral

RNA using confocal laser scanning microscopy.

The third chapter of research set out to identify novel host cell proteins that contribute to viral infection. For this study, we purified mature SVHR virus particles from infected

97 monolayers of mammalian and insect origin. The purified virus was was interrogated by LC-

MS/MS to identify proteins that remained associated with the virus after egress from the cell as any proteins that remained associated with the virus may have a function in the viral replication cycle. The strategy of identifying host cell proteins involved in viral replication is intended to identify new, potential targets for antiviral therapies. This strategy is intended to reduce the incidence of resistance to antivirals by reducing the selective pressure associated with targeting viral proteins (120, 121). To that end, associated with the purified virus we identified 187 mammalian proteins and 51 insect proteins. Additionally, we detected nsP2 associated with the mature virus. The roles of RBM3 and SNX5 were examined for their impact in alphavirus replication using HEK293 knockdown cell lines. SNX5, but not RBM3, was shown to have a critical function in RNA replication for all viruses tested. In this same study, nsP2 was detected in the mature Sindbis virus particle. The function of nsP2 in the virus particle remains elusive.

To further our efforts in identifying host cell proteins that have a critical impact on virus replication, the remaining proteins in this study should be systematically validated. The validated proteins can then undergo a screening against commercial small molecule libraries to identify candidate drug compounds for pharmaceutical development. Finally, the function of nsP2 should be elucidated. Creating a temperature-sensitive mutant and performing shifts to the non- permissive temperature at various stages of infection can accomplish this goal.

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