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1 SARS Coronavirus ORF7a inhibits BST-2 virion tethering through a novel 1 mechanism of glycosylation interference 2 3 Justin K. Taylor a , Christopher M. Coleman a , Sandra Postel b , Jeanne M. Sisk a , John G. 4 Bernbaum c , Thiagarajan Venkatarajan a , Eric J. Sundberg a,b,d , Matthew B. Frieman a # 5 6 Department of Microbiology and Immunology, University of Maryland at Baltimore, 7 Baltimore, Maryland, USA a ; Institute of Human Virology, University of Maryland 8 School of Medicine, Baltimore, Maryland, USA b ; and Integrated Research Facility, 9 National Institutes of Health, Frederick, Maryland, USA c ; Department of Medicine, 10 University of Maryland School of Medicine, Baltimore, Maryland, USA d 11 12 Running Head: BST-2 restricts SARS-coronavirus 13 14 #Address correspondence to Matthew B. Frieman, [email protected]. 15 16 17 18 19 20 21 22 23 JVI Accepted Manuscript Posted Online 16 September 2015 J. Virol. doi:10.1128/JVI.02274-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

2015 SARS Coronavirus ORF7a inhibits BST-2 virion tethering through a novel mechanism of glycosylation interference

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Page 1: 2015 SARS Coronavirus ORF7a inhibits BST-2 virion tethering through a novel mechanism of glycosylation interference

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SARS Coronavirus ORF7a inhibits BST-2 virion tethering through a novel 1 mechanism of glycosylation interference 2 3 Justin K. Taylora, Christopher M. Colemana, Sandra Postelb, Jeanne M. Siska, John G. 4 Bernbaumc, Thiagarajan Venkatarajana, Eric J. Sundberga,b,d, Matthew B. Friemana# 5 6 Department of Microbiology and Immunology, University of Maryland at Baltimore, 7 Baltimore, Maryland, USAa; Institute of Human Virology, University of Maryland 8 School of Medicine, Baltimore, Maryland, USAb; and Integrated Research Facility, 9 National Institutes of Health, Frederick, Maryland, USAc; Department of Medicine, 10 University of Maryland School of Medicine, Baltimore, Maryland, USAd 11 12 Running Head: BST-2 restricts SARS-coronavirus 13 14 #Address correspondence to Matthew B. Frieman, [email protected]. 15 16 17 18 19 20 21 22 23

JVI Accepted Manuscript Posted Online 16 September 2015J. Virol. doi:10.1128/JVI.02274-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Abstract 24 Severe Acute Respiratory Syndrome (SARS) emerged in November 2002 as a case of 25 atypical pneumonia in China and the causative agent of SARS was identified as a 26 novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV). 27 Bone marrow stromal antigen 2 (BST-2; also known as CD317 or tetherin) was 28 initially identified as a pre-B-cell growth promoter but also inhibits the release of 29 the retrovirus human immunodeficiency virus type 1 (HIV-1) virions by tethering 30 budding virions to the host cell membrane. Further work has shown that BST-2 31 restricts the release of many other viruses, including the human coronavirus hCoV-32 229E, and many of these viruses encode BST-2 antagonists to overcome BST-2 33 restriction. Given the previous studies on BST-2, we aimed to determine if BST-2 has 34 the ability to restrict SARS-CoV and if SARS-CoV encodes any proteins that modulate 35 BST-2’s anti-viral function. Through an in vitro screen we identified four potential 36 BST-2 modulators encoded by SARS-CoV: PLPro, nsp1, ORF6, and ORF7a. As the 37 function of ORF7a in SARS-CoV replication was previously unknown, we focused our 38 study on ORF7a. We found that BST-2 does restrict SARS-CoV, but the loss of ORF7a 39 leads to a much greater restriction, confirming the role of ORF7a as an inhibitor of 40 BST-2. We further characterized the mechanism of BST-2 inhibition by ORF7a and 41 found that ORF7a localization changes when BST-2 is overexpressed and ORF7a 42 binds directly to BST-2. Finally, we also show that SARS-CoV ORF7a blocks the 43 restriction activity of BST-2 by blocking with the glycosylation of BST-2. 44 45 46

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Importance 47 The severe acute respiratory syndrome coronavirus (SARS-CoV) emerged from 48 zoonotic sources in 2002 and caused over 8000 infections and 800 deaths in 32 49 countries around the world. Identifying host factors that regulate SARS-CoV 50 pathogenesis is critical to understanding how this lethal virus causes disease. We 51 have found that BST-2 is capable of restricting SARS-CoV release from cells, 52 however we also identified a SARS-CoV protein that inhibits BST-2 function. We 53 show that the SARS-CoV protein, ORF7a, inhibits BST-2 glycosylation leading to loss 54 of BST-2’s anti-viral function. 55 56 Introduction 57 Severe acute respiratory syndrome coronavirus (SARS-CoV) was identified as the 58 causative agent of a 2003 outbreak of severe respiratory disease in the Guangdong 59 province of China resulting in 8096 cases, with 774 deaths, across 29 countries(1, 2). 60 SARS-CoV is an enveloped virus with a positive-sense, single stranded RNA genome 61 of roughly 30,000 nucleotides, encoding four structural proteins: spike (S), envelope 62 (E), membrane (M), and nucleocapsid (N)(3). N protein forms the nucleocapsid, 63 while E and M are minor virion membrane proteins. SARS-CoV entry into the cell is 64 mediated by S protein binding to angiotensin-converting enzyme 2 (ACE2) on the 65 cell surface(4). In addition to the structural proteins, SARS-CoV encodes several 66 non-structural and accessory proteins that promote SARS-CoV replication and 67 virulence(5). Some of the non-structural and accessory proteins function as outside 68 of replication as type-I interferon antagonists(6-8). 69

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ORF7a is a SARS-CoV encoded accessory protein that is composed of a type I 70 transmembrane protein that localizes primarily to the Golgi but can be found on the 71 cell surface (9, 10). SARS-CoV ORF7a overlaps ORF7b in the viral genome where 72 they share a transcriptional regulatory sequence (TRS). ORF7a has a 15 amino acid 73 (aa) N-terminal signal peptide, an 81 aa luminal domain, a 21 aa transmembrane 74 domain and a 5 aa cytoplasmic tail (9, 10). To investigate the role of ORF7a in SARS-75 CoV replication, an ORF7ab deletion virus was produced that replicated to similar 76 titer as wildtype SARS-CoV in vitro and in vivo(10-12). Characterization of ORF7a in 77 vitro demonstrated ORF7a-dependent induction of apoptosis in a caspase-78 dependent pathway(13-15). Analysis of ORF7a evolution during the SARS-CoV 79 outbreak identified several residues in ORF7a that were under positive selection as 80 SARS-CoV evolved during transmission from bat to palm civet to humans(16). These 81 data suggest that ORF7a is vital for SARS-CoV biology and has a yet unidentified role 82 in pathogenesis and disease. 83 Bone marrow stromal antigen 2 (BST-2; also known as CD317 or tetherin) 84 was initially identified as a pre-B-cell growth promoter(17, 18). However, BST-2 is 85 also a marker of type-I interferon producing cells (IPC) and is broadly expressed in 86 many cell types when treated with type-I interferon(19). BST-2 has an unusual 87 structure, with an N-terminal transmembrane domain and a C-terminal 88 glycosylphosphatidylinositol (GPI) anchor and two N-linked glycosylation sites in its 89 extracellular domain and exists as a disulfide-linked homodimer(20, 21). BST-2 90 traffics through the endoplasmic reticulum (ER) and Golgi, eventually localizing to 91 the surface and trans-Golgi network(20). Studies have shown evolutionary 92

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conservation in three major surface patches of BST-2, near each of the two N-linked 93 glycosylation sites and in the C-terminal region(22). 94 The anti-viral effect of BST-2 was first identified when it was shown that 95 BST-2 inhibits the release of the retrovirus human immunodeficiency virus type 1 96 (HIV-1) virions by directly tethering budding virions to the host cell. BST-2 also 97 restricts the release of many other viruses, including alphaviruses, arenaviruses, 98 herpesviruses, paramyxoviruses and other retroviruses(23-26). BST-2 is thought to 99 restrict virus release by physically tethering the budding enveloped virion to the 100 plasma membrane(27) and a number of mechanism models have been proposed (28, 101 29). All of the BST-2 restriction models predict that BST-2 functions as a dimer, 102 interfacing through ectodomains that incorporate into both the viral envelope and 103 plasma membrane, however models vary in regards to the orientation of the GPI 104 anchor and transmembrane domain. BST-2 has not been shown to interact with any 105 specific viral surface protein, but rather functions as an embedded inter-membrane 106 physical tether. Therefore, BST-2 is thought to be able to restrict any membrane-107 budding enveloped virus(28, 29). Previous studies have shown that the ability to 108 form cysteine-linked dimers is necessary for BST-2 function, while conflicting 109 results concerning the importance of the N-linked glycosylation have been 110 reported(29, 30). More recently, it has been suggested that BST-2 is a virus sensor 111 during HIV-1 infection and induces a proinflammatory response through NFκB(31). 112 Given the lack of virus specificity in BST-2 restriction, numerous viruses 113 encode BST-2 antagonists to allow release of virions. The first such antagonist was 114 identified as HIV-1 accessory protein Vpu (27). HIV-1 Vpu binds BST-2 and causes β-115

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TrCP2-dependent degradation of BST-2 and efficient release of HIV-1 virions, 116 although it is not clear whether degradation occurs in the lysosome or 117 proteasome(32-34). Other viral antagonists of BST-2 include Chikungunya virus 118 nsp1, ebolavirus GP1,2, herpes simplex virus GP M, HIV-2 envelope glycoprotein, 119 Sendai virus glycoproteins, and simian immunodeficiency virus (SIV) nef and 120 envelope glycoproteins(23-26, 35-38). HIV-2 and SIV are closely related to HIV-1, 121 however, the envelope glycoproteins from HIV-2 and SIV antagonize BST-2 by 122 sequestration within the trans-Golgi network rather than degradation, suggesting 123 that different mechanisms of BST-2 antagonism exist for different viruses, even 124 within the same virus genus(35, 36). Another example is Ebolavirus GP1,2 which 125 antagonizes BST-2 through an unknown mechanism that does not involve surface 126 removal but still leads to BST-2 functional inhibition(39). 127 Unlike many enveloped viruses, which bud from the cell plasma membrane, 128 coronaviruses bud in the ER-Golgi intermediate compartment (ERGIC) and are 129 transported to the plasma membrane inside vesicles(40). However, it has recently 130 been shown that BST-2 restricts release to human coronavirus (hCoV)-229E, 131 suggesting that BST-2 can also restrict viruses that bud in the ERGIC and then are 132 released from the cell via vesicle fusion(37). 133 134 In this study we found that BST-2 restricts SARS-CoV virion egress by 135 tethering virions to the plasma membrane. We also identified several SARS-CoV 136 proteins that are putative modulators of BST-2 function. Focusing on ORF7a, we 137 found that ORF7a directly binds BST-2 and when co-expressed with BST-2, ORF7a 138

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localizes to the plasma membrane, rather than the ER and Golgi. Additionally, we 139 demonstrate that the interaction of ORF7a and BST-2 results in inhibition of BST-2 140 glycosylation leading to a reduced tethering function in cells and subsequent loss of 141 BST-2 anti-viral function. Together, these data indicate a novel role for SARS-CoV 142 ORF7a as an inhibitor of BST-2, as well as reveal a novel mechanism for altering the 143 function of BST-2. 144 145 Materials and Methods 146 147 Viruses and cells. icSARS-CoV and icSARS-ORF7abΔ-CoV were constructed as 148 previously described(41, 42). All virus stocks were stored at -80°C until ready to use. 149 Vero E6 cells were purchased from ATCC (catalog number CRL-1586; Manassas, VA) 150 and were grown in minimal essential medium (MEM) (Invitrogen, Carlsbad, CA) 151 with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA), 2mM L-152 Glutamine (Life Technologies, Grand Island, NY), and 1% penicillin/streptomycin 153 (Gemini Bioproducts, West Sacramento, CA). HEK293T cells were grown in 154 Dulbecco’s minimal essential medium (DMEM) (Invitrogen, Carlsbad, CA) with 10% 155 fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA), 2mM L-Glutamine 156 (Life Technologies, Grand Island, NY), and 1% penicillin/streptomycin (Gemini 157 Bioproducts, West Sacramento, CA). HEK293T ACE2 cells were a gift from David 158 Wentworth (J Craig Venter Institute) and were grown in HEK293T media 159 supplemented with 1mg/mL G418 (Corning, Manassas, VA). 160 161

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Plasmids. We received BST-2/Flag in pCAGGS as a gift from Sina Bavari(24). We 162 received ORF7a-Fc as a gift from Andrew Pekosz. The ORF3a, ORF3b, ORF6, ORF7a, 163 ORF8a, S, E, Membrane, N, and PLPro SARS-CoV plasmids were produced from 164 previous work(6, 7). The nonstructural proteins were cloned into the CAGGS/GFP 165 (green fluorescent protein) or CAGGS/HA (Hemagglutinin) vector for expression in 166 HEK293T cells as previously described(7). Amplicons were produced using the 167 primers shown in (Table 1). For each construct, an ATG start codon was added as 168 the first codon but no stop codon was included at the 3′ terminus of each ORF. 169 Rather, an HA or GFP tag was fused in frame to each ORF. The amplicons and vector 170 were digested with EcoRI/XmaI fragments for cloning, and all constructs were 171 verified by sequence analysis. 172 173 SARS-CoV growth curve. HEK293T/hACE2 cells were plated in a 24 well plate and 174 grown overnight at 37°C. Cells were transfected with 2 ul of Lipofectamine LTX 175 (Invitrogen, Carlsbad, CA) and 700 ng of BST-2 Flag in pCAGGS, ORF7a-HA or 176 MISSION pLKO.1-puro non-mammalian shRNA control plasmid (Sigma-Aldrich, St. 177 Louis, MO) according to the manufacturers’ instructions. For the glycosylation 178 mutant experiments, pCR3.1-EXN-tetherin-HA(463) and pCR3.1-EXN-179 tetherin(N65A/N92A)-HA(463) were kindly provided by Dr Paul Bieniasz(29). 24 180 hours post-transfection, HEK293T ACE2 cells were infected with icSARS-CoV or 181 icSARS-GFP-CoV at a multiplicity of infection (MOI) of 0.1. Supernatant was taken at 182 12, 24, and 36 hours post-infection to measure SARS-CoV titer by plaque assay on 183

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Vero E6 cells. Supernatant and cell lysate was also analyzed by Western blot. The 184 growth curve experiments were repeated twice with an n of 6 for each sample. 185 SARS-CoV RNA products of replication were assessed by RT-PCR. RNA from 186 cells infected with SARS-CoV for 24 hours was isolated using Trizol® reagent 187 (Ambion) according to the manufacturers’ instructions. RNA was converted to cDNA 188 using RevertAid RT-PCR (Thermo Scientific) according to the manufacturers’ 189 instructions and treated with RNase H (New England Biolabs) according to the 190 manufacturers’ instructions. Levels of SARS-CoV pp1a (forward primer: 191 GCCGTAGTGTCAGTATCATCACC; reverse primer: 192 AATAGGACCAATCTCTGTAAGAGCC) and N protein mRNA (forward primer: 193 CTCTTGTAGATCTGTTCTCTAAACGAAC; reverse primer: 194 TTACTGTACTAGCAAAGCAATATTGTCG) were quantified using Sybr® green PCR 195 master mix (Applied Biosystems) according to the manufacturers’ instructions and a 196 7500 fast Dx real-time PCR instrument (Applied Biosystems). Levels of SARS-CoV 197 RNA were quantified using the ΔΔCt method. Means and standard deviations were 198 calculated from 3 independent infections. 199 200 Electron Microscopy. Vero E6 cells were plated in a 24 well plate and grown 201 overnight at 37°C. Cells were transfected using Lipofectamine LTX (Invitrogen, 202 Carlsbad, CA) with BST-2 Flag in pCAGGS or MISSION pLKO.1-puro non-mammalian 203 shRNA control plasmid (Sigma-Aldrich, St. Louis, MO) according to the 204 manufacturers’ instructions. 24 hours post-transfection, Vero cells were infected 205 with icSARS-CoV or icSARS-GFP-CoV at an MOI of 10. At 24 hours post-infection cells 206

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were fixed and analyzed by electron microscopy. For conventional ultrastructural 207 investigations, infected VERO E6 cells were fixed with 2.5% Glutaraldehyde (E.M. 208 Sciences, Warrington, PA) at 24 hours post-infection. After fixation for 72 hours, the 209 preserved cells were post-fixed in 1.0% Osmium Tetroxide (E.M. Sciences), en bloc 210 stained with 2.0% Uranyl Acetate, dehydrated in a series of graded ethanol, and 211 infiltrated and embedded in Spurr plastic resin (Tousimis Research, Rockville, MD). 212 Embedded blocks were sectioned using a Leica UC7 Ultramicrotome, collected thin-213 sections were mounted on 200 mesh copper grids, contrasted with Lead Citrate, and 214 subsequently viewed at 80 kV with a FEI Tecnai Twin Transmission Electron 215 Microscope. The scale bar shown on Figure 2 is 500nm. 216 217 BST-2:SARS-CoV accessory protein co-transfections. HEK293T cells were 218 transfected with 500 ng total DNA using Lipofectamine LTX (Invitrogen, Carlsbad, 219 CA) according to the manufacturers’ instructions. 100 ng of BST-2 Flag in pCAGGS, 220 200 ng or 400 ng of GFP- or HA-tagged SARS-CoV proteins, and MISSION pLKO.1-221 puro non-mammalian shRNA control plasmid (Sigma-Aldrich, St. Louis, MO) were 222 co-transfected into HEK293T cells. After 18 hours of expression, cells were lysed in 223 lysis buffer (20 mM Tris-HCL [pH 7.6], 150 mM NaCl, 1% NP-40, 0.5% SDS, 5 mM 224 EDTA, 1 protease inhibitor tablet). Lysate was combined with 2X Laemmli Sample 225 Buffer (Bio-Rad, Hercules, CA) before boiling and electrophoresis using Mini-226 PROTEAN TGX Gels (Bio-Rad, Hercules, CA). Protein expression was assessed using 227 rabbit anti-HA antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-GFP antibody 228 (Sigma-Aldrich, St. Louis, MO), mouse anti-Flag M2 antibody (Sigma-Aldrich, St. 229

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Louis, MO), and mouse anti-β-tubulin antibody (Sigma-Aldrich, St. Louis, MO). For 230 inhibition experiments cells were transfected as above and four hours post-231 transfection, media was removed and replaced with 20 nM Concanamycin A (Sigma-232 Aldrich, St. Louis, MO) or 500 nM MG-132 (Sigma-Aldrich, St. Louis, MO). Cell lysate 233 was collected after 18 hours of drug treatment. For time course experiments HEK-234 293T were transfected with 500 ng of ORF7a or control plasmid using 235 Lipofectamine LTX (Invitrogen, Carlsbad, CA) according to the manufacturers’ 236 instruction. After 6 hours of expression, media was replaced with fresh HEK293T 237 media. 22 hours post-transfection, cells were transfected with 500 ng of DNA, 100 238 ng of BST-2 plasmid and 400 ng control plasmid, using Lipofectamine LTX 239 (Invitrogen, Carlsbad, CA) according to the manufacturers’ instruction. Cell lysate 240 was collected as described above at 4, 8, 12, and 16 hours after the second 241 transfection. Expression was analyzed as described above. Deglyosylation was 242 performed using Glycopeptidase F (Takara, Mountain View, CA) according to the 243 manufacturer’s instructions for deglycosylating denatured proteins. The ratio of 244 glycosylated to unglycosylated was calculated by measuring density of the bands 245 with ImageJ (National Institute of Mental Health, Bethesda, MD). All of the 246 transfection experiments were repeated at least two times. 247 248 Anti-Flag immunoprecipitations. HEK293T cells were transfected with 1000 ng 249 total DNA using Lipofectamine LTX (Invitrogen, Carlsbad, CA). 500 ng of Flag-tagged 250 BST-2 and 500 ng of SARS PLPro-GFP, nsp1-GFP, ORF6-GFP, or ORF7a-HA or 251 MISSION pLKO.1-puro non-mammalian shRNA control plasmid (Sigma-Aldrich, St. 252

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Louis, MO) was co-transfected into HEK293T cells. After 18 h of expression, cells 253 were lysed in lysis buffer (20 mM Tris-HCL [pH 7.6], 150 mM NaCl, 1% NP-40, 0.5% 254 SDS, 5 mM EDTA, 1 protease inhibitor tablet), the extract was centrifuged for 10 min 255 at 4°C, and the supernatant was removed. EZ View Red Anti-Flag M2 Affinity Gel 256 beads (catalog number F2426; Sigma, St. Louis, MO) was added to each extract and 257 rotated overnight at 4°C. The extract was then washed twice with lysis buffer and 258 eluted using 0.1 M Glycine (pH 3.5). The elution was combined with 2X Laemmli 259 Sample Buffer (Bio-Rad, Hercules, CA) before boiling and electrophoresis using 260 Mini-PROTEAN TGX Gels (Bio-Rad, Hercules, CA). Protein levels were assessed using 261 rabbit anti-HA antibody (Sigma-Aldrich, St. Louis, MO) and mouse anti-Flag M2 262 antibody (Sigma-Aldrich, St. Louis, MO). Co-immunoprecipitation experiments were 263 performed twice. 264 265 Confocal microscopy. HEK293T cells were seeded onto microscope cover glass 266 (Fisher Scientific, Pittsburg, PA) pre-treated with fibronectin (Sigma-Aldrich, St. 267 Louis, MO) for 30 minutes. HEK293T cells were transfected with 500 ng total DNA 268 using Lipofectamine LTX (Invitrogen, Carlsbad, CA) according to the manufacturers’ 269 instructions. 250 ng of BST-2 Flag in pCAGGS, ORF7a-HA, and/or MISSION pLKO.1-270 puro Non-Mammalian shRNA Control Plasmid (Sigma-Aldrich, St. Louis, MO) were 271 transfected. At 24 hours posttransfection, cells were fixed with 4% formaldehyde 272 overnight at 4°C, then incubated in cold PBS for 10 minutes at room temperature 273 (RT). Each sample was permeabilized with permeabilization buffer (phosphate-274 buffered saline [PBS], 0.1% Triton X-100, 0.5% bovine serum albumin (BSA)) for 15 275

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minutes at RT and then blocked for 5 minutes using blocking buffer (PBS, 5% BSA). 276 The cells were washed using wash buffer (PBS, 1% BSA, 0.05% NP40) and then 277 stained for protein expression. Primary antibodies used were rabbit anti-HA 278 antibody (Sigma-Aldrich, St. Louis, MO) and mouse anti-Flag M2 antibody (Sigma-279 Aldrich, St. Louis, MO). Cells were incubated with primary antibodies diluted in 280 antibody dilution buffer (PBS, 1% BSA, 0.05% NP40, 2% normal goat serum) for 45 281 minutes at RT. Cells were washed three times with wash buffer and then incubated 282 while rocking for 30 minutes at room temperature with goat anti-rabbit IgG 283 conjugated with AMCA (Vector Laboratories, Burlingame, CA) and/or horse anti-284 mouse conjugated with Texas Red (Vector Laboratories, Burlingame, CA). Cells were 285 then washed with wash buffer 3 times and a final time with PBS for 30 minutes at 286 RT. For the ORF7a localization experiments the endoplasmic reticulum (ER) was 287 stained with Concanavalin A, Alexa Fluor® 594 Conjugate (Invitrogen, Carlsbad, CA) 288 and the Golgi was stained with BODIPY TR Ceramide complexed to BSA (Invitrogen, 289 Carlsbad, CA) according to the manufacturer’s instructions. The coverslips were 290 then mounted on slides using VECTASHIELD Hard Set Mounting Medium with DAPI 291 (Vector Laboratories, Burlingame, CA). Slides were analyzed by confocal microscopy 292 using a Zeiss LSM 510 microscope. Images were collated and adjusted using ImageJ 293 (National Institute of Mental Health, Bethesda, MD). 294 295 Flow Cytometry. 296 297

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For experiments to determine BST-2 surface expression, HEK293T cells were 298 transfected as above with BST-2 and ORF7a. After 18 hours of expression cells were 299 washed with PBS and dissociated with 0.05% Trypsin-EDTA (1X), phenol red 300 (Invitrogen, Carlsbad, CA). Cells were washed in HEK293T media to inactivate the 301 Trypsin and were resuspended in FACS buffer (PBS with 1% fetal bovine serum) 302 and stained for 20 minutes with APC anti-human CD317 Clone RS38E (Biolegend, 303 San Diego, CA) or control Mouse IgG1 APC (Becton Dickinson, Franklin Lakes, NJ). 304 Cells were then washed, resuspended in FACS buffer, and analyzed on a LSRII flow 305 cytometer (Becton Dickinson, Franklin Lakes, NJ). Data was analyzed using Flowjo 306 (Tree Star, Ashland, OR). 307 For experiments to determine mutant BST-2 surface expression, 293T cells 308 were plated 250,000 per well in 6-well plates, grown overnight at 370C with 5% CO2 309 and then transfected with 1 ug of each DNA (empty vector, empty vector + WT BST2, 310 empty vector + mt BST2, empty vector + ORF 7a, WT BST2 + ORF 7a, mt BST2 + ORF 311 7a). All following steps were performed at room temperature. At twenty-four hours 312 after transfection, cells were harvested using Cell Dissociation Buffer (Invitrogen). 313 Duplicate transfected wells were pooled and samples transferred to a 96-well plate. 314 Cells were pelleted at 2000 rpm for 2 minutes, fixed in 4% paraformaldehyde for 5 315 minutes, then washed with 10%FBS/PBS and pelleted as above. Samples were 316 divided into 2 aliquots then blocked with 10%FBS/PBS, or blocked/permeabilized 317 with 10% saponin in 10%FBS/PBS for 30 minutes. Cells were pelleted as above and 318 incubated in primary antibodies for 1 hour (HA antibody, Sigma H6908 and FLAG 319 antibody, Sigma F3165, 1:1000). Cells were washed 2 times with 10%FBS/PBS and 320

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pelleted as above. Secondary antibodies (FITC anti-rabbit, Vector Labs and Alexa 321 405 anti-mouse, ThermoFisher, 1:1000) were added to cells and incubation was 1 322 hour, followed by washing and pelleting as above. Cells were resuspended in PBS 323 and cell surface localization as well as total cell expression of BST2 and ORF 7a were 324 determined using an LSRII flow cytometer. Control (no DNA), BST2 or ORF 7a 325 transfected alone were used as compensation controls. Data was analyzed using 326 FlowJo and statistical analysis was generated from a t test generated using standard 327 errors based on results from three individual experiments. 328 329 Circular Dichroism (CD) of BST2 and ORF7a-Fc. 330 CD spectra ranging from 200-260nm were recorded in 10mM Sodium 331 phosphate buffer pH7.5 of 10μM BST2 expressed in HEK293T cells, 12μM BST2 332 expressed in E.coli cells and 8μM ORF7a-Fc expressed in HEK239T cells using a 333 JASCO J-810 instrument. CD melting curves were analyzed at 222nm by increasing 334 the temperature 1°C/min starting at 20°C. 335 336 Surface Plasmon Resonance. 21 μg of pCAGGS-T7/ORF7a-Fc was transfected into 337 HEK293T cells seeded in 100 mm dishes using Lipofectamine LTX (Invitrogen, 338 Carlsbad, CA) according to the manufacturers’ instructions. After 48 hours of 339 expression, supernatant was collected and purified using a HiTrap Protein A column 340 (GE Healthcare, Little Chalfont, Buckinghamshire, UK) according to the 341 manufacturer’s instructions. Purified ORF7a-Fc was subsequently dialyzed into PBS. 342 The codon-optimized sequence of the extracellular domain (residues 47-161) of 343

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BST-2 was cloned with a N-terminal His6- and a C-terminal Flag-tag into pET28b. 344 The protein was expressed at 19°C overnight in BL21(DE3)pLysS cells induced at an 345 OD600 of 0.6 with 0.4mM IPTG. The fusion protein was purified by nickel affinity 346 (Thermo Scientific, Pittsburg, PA), using a Mono Q™ 5/50GL anion exchange column 347 (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and finally separated on a 348 Superdex™ 200 10/300 GL gel filtration column (GE Healthcare, Little Chalfont, 349 Buckinghamshire, UK). The extracellular domain (residues 47-161) of BST-2 was 350 also cloned into the plGplus Vector (R&D Systems, Minneapolis, MN). The resulting 351 BST-2 protein includes a C-terminal His6-Flag-tag and due to the encoded stop 352 codon did not express as an Fc fusion protein. HEK293T cells in suspension culture 353 were transfected with this construct using polyethylenimine (Polysciences, Inc., 354 Warrington, PA). The cell culture supernatant was harvested 96 hours post-355 transfection and purified using nickel affinity (Thermo Scientific, Pittsburg, PA) 356 followed by gel filtration on a Superdex™ 200 10/300 GL column (GE Healthcare, 357 Little Chalfont, Buckinghamshire, UK). Direct binding of the extracellular domain of 358 ORF7a-Fc expressed in HEK293T cells to the extracellular domain of BST-2 359 expressed in E. coli or HEK293T cells was measured by surface plasmon resonance 360 (SPR) analysis using a Biacore T100 instrument (GE Healthcare, Little Chalfont, 361 Buckinghamshire, UK). 1000 RU of protein A was immobilized by amine coupling on 362 the surface of a CM5 sensor chip. Approximately 170 RU of human IgG (Sigma-363 Aldrich, St. Louis, MO) as negative control and also of ORF7a-Fc was captured on 364 flow cells 1 and 2, respectively. In single-cycle kinetics experiments, two-fold 365 dilutions from 80 to 5 μM of BST-2 were injected over the surfaces and the control-366

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subtracted response was recorded. HBS-EP was used as a running buffer and the 367 surfaces were regenerated with 20 mM HCl after each cycle. Steady-state analysis of 368 the data was performed using the Biacore T100 Evaluation Software 2.0.3. All of the 369 SPR experiments were repeated at least three times. 370 371 Statistical analysis. Growth curve titers were analyzed by t-test using the 372 Holm_Sidak method with alpha=5.0%. Prism (GraphPad Software Inc., La Jolla, CA) 373 was used to perform the analysis. 374 375 Results 376 377 SARS-CoV proteins antagonize BST-2 expression in vitro. Many enveloped 378 viruses, including the coronavirus hCoV-229E, encode proteins that counteract BST-379 2 (24, 27, 37). We hypothesized that the highly pathogenic SARS-CoV may also 380 inhibit BST-2 function. To investigate this hypothesis, HA- and GFP-tagged SARS-381 CoV proteins and BST-2 were co-transfected into HEK293T cells and BST-2 382 expression levels were assessed by Western blot. Four SARS-CoV proteins, non-383 structural protein 1 (nsp1), the papain-like protease domain of nsp3 (PLPro), ORF6, 384 and ORF7a altered BST-2 protein expression or molecular weight. Several proteins 385 encoded in the SARS-CoV genome have been shown to alter other anti-viral 386 response pathways during infection(43-53). Three of the proteins, papain-like 387 protease (PLPro), ORF6, and nsp1, have been previously shown to be interferon 388 antagonists(7, 8, 54). PLPro inhibits IRF3 and NFkB activation(54), ORF6 blocks 389

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STAT1 nuclear import, and nsp1 blocks interferon beta induction by degrading host 390 mRNAs(6, 7, 54, 55). Because the function of ORF7a is unclear, we decided to further 391 study interactions between BST-2 and ORF7a. 392 393 icSARS-ORF7abΔ-CoV shows defects in replication compared to icSARS-CoV 394 when BST-2 is overexpressed. Since ORF7a affects BST-2 protein, we 395 hypothesized that an ORF7ab deletion SARS-CoV (icSARS-ORF7abΔ-CoV) would 396 show a greater defect in replication compared to WT SARS-CoV when BST-2 is 397 overexpressed. Since ORF7a and ORF7b have overlapping open reading frames, an 398 ORF7ab double deletion virus will be used for the infection experiments. No effect 399 on BST-2 was found when ORF7b was expressed alone in the assays in transfection 400 screens. We transfected HEK293T/hACE2 cells with BST-2 or a control plasmid 401 and 24 hours post-transfection we infected the cells with either icSARS-CoV or 402 icSARS-ORF7abΔ-CoV at an MOI of 0.1. HEK293T cells do not express endogenous 403 BST-2(27), so we were able to ensure that any effect was from the transfected BST-404 2 and not endogenous BST-2 expression. icSARS-CoV replicated to 1.10 x 105 405 PFU/mL, while in BST-2 expressing cells icSARS-CoV replicated to significantly 406 lower titers at 3.43 x 104 PFU/mL (Figure 1a, p<0.01). icSARS-ORF7abΔ-CoV was 407 also significantly restricted by BST-2 expression at 24 and 36 hours. In control 408 transfected cells icSARS-ORF7abΔ-CoV replicated to 7.37 x 104 PFU/mL and 4.80 x 409 104 PFU/mL at 24 and 36 hours respectively, while in BST-2 transfected cells 410 icSARS-ORF7abΔ-CoV replicated to significantly lower titers of 4.00 x 103 PFU/mL 411 (p<0.05) and 1.10 x 104 PFU/mL (p<0.001) at 24 and 36 hours, respectively (Figure 412

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1b). While BST-2 restricts SARS-CoV by a small, but significant amount, BST-2 413 restricts icSARS-ORF7abΔ-CoV by a much greater amount, suggesting that ORF7a 414 antagonizes BST-2. 415 We confirmed that BST-2 is not affecting another step in the SARS-CoV 416 replication cycle by assessing the accumulation of SARS-CoV RNA products of 417 replication in the presence of BST-2 at 24 hours post infection. There is no 418 significant effect of BST-2 expression on SARS-CoV pp1a (Figure 1c) or SARS-CoV N 419 mRNA (Figure 1d), regardless of ORF7a expression. These data suggest that BST-2 420 does not effect SARS-CoV RNA accumulation, even in the absence of ORF7a 421 expression. 422 423 icSARS-ORF7abΔ-CoV defect in replication is due to direct tethering of SARS-424 CoV virions to the plasma membrane. Since BST-2 has been shown to restrict 425 virus replication by directly tethering HIV-1 virions to the plasma membrane(27, 426 56), we sought to determine if overexpression of BST-2 was preventing release of 427 SARS-CoV and if icSARS-ORF7abΔ-CoV was more susceptible to BST-2 restriction. 428 To determine if BST-2 was affecting release, we transfected either BST-2 or a 429 control plasmid into VeroE6 cells and subsequently infected with either icSARS-CoV 430 or icSARS-ORF7abΔ-CoV at a MOI of 10. At 24 hours post-infection cells were fixed 431 and imaged using electron microscopy. When VeroE6 cells were transfected with 432 the control plasmid, both icSARS-CoV and icSARS-ORF7abΔ-CoV showed minimal 433 accumulation of virions at the plasma membrane (Figure 2). Transfection of BST-2 434 leads to a small accumulation of icSARS-CoV on plasma membrane (Figure 2, top 435

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left). BST-2 transfection shows a much greater effect on icSARS-ORF7abΔ-CoV, 436 which shows a large accumulation of virions at the plasma membrane (Figure 2, top 437 right). These results confirm that similar to many other viruses, BST-2 is restricting 438 SARS-CoV by preventing virus release. The increased effect of BST-2 on icSARS-439 ORF7abΔ-CoV further suggests that ORF7a acts as an inhibitor BST-2-mediated 440 restriction of SARS-CoV. 441 442 ORF7a expression leads to lower molecular weight BST-2 within the cells, but 443 not reduced BST-2 surface expression. Since ORF7a appears to be a BST-2 444 antagonist, we aimed to determine if SARS-CoV ORF7a causes BST-2 surface 445 removal and subsequent degradation, as seen in HIV-1 Vpu protein antagonism(33, 446 56). SARS-CoV ORF7a was co-transfected with increasing amounts of BST-2 to assay 447 the effect of ORF7a on BST-2 expression. Increasing the amount of ORF7a co-448 transfected with BST-2 leads to decreased levels of BST-2 expression and lower 449 molecular weight products, suggesting that BST-2 protein is affected by ORF7a 450 expression (Figure 3a). Next, we sought to determine if, similarly to HIV-1 Vpu, 451 expression of ORF7a leads to a reduction in BST-2 surface expression(56). To assay 452 the effect of ORF7a on BST-2 surface expression, we transfected BST-2 either alone 453 or in combination with an ORF7a expression plasmid to compare BST-2 surface 454 expression by flow cytometry. Untransfected cells exhibited little to no expression of 455 surface BST-2. Cells transfected with BST-2 alone were 88.2% positive with the 456 majority of cells in a highly expressing population and a smaller percentage in a 457 lower expressing population (Figure 3b). Interestingly, increasing amounts of 458

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ORF7a had no effect on surface expression of BST-2 (Figure 3b). These data 459 demonstrate that ORF7a co-expression leads to lower molecular weight BST-2 460 within cells, but does not lead to surface removal of BST-2, suggesting that ORF7a 461 may antagonize BST-2 through a mechanism other than surface removal. 462 463 Lysosomal and proteasomal inhibitors do not affect BST-2 antagonism by 464 ORF7a. While we did not observe ORF7a dependent removal of surface BST-2, we 465 did observe the appearance of lower molecular weight bands of BST-2, suggesting 466 degradation of intracellular BST-2. Many other viruses, such as HIV-1, antagonize 467 BST-2 by degradation through either the lysosome or proteasome and, thus, we 468 assessed whether lysosomal or proteasomal inhibitors could block BST-2 469 antagonism by ORF7a(32-34). First, to demonstrate that the concentration of 470 Concanamycin A (Con A) and MG-132 inhibit proteasome and lysosomal 471 degradation, respectively, in HEK293T cells, we treated cells and assayed for 472 Ubiquitin and LC3B levels by western blot (Figure 4A and B). As expected, MG-132 473 treatment increases total ubiquitin levels in the cell (Figure 4A) and Con-A 474 treatment blocks lysosomal degradation, as shown with an increase in the lower 475 weight LC3B product. To test for the effect of proteosomal or lysosomal effects on 476 BST-2 antagonism, we transfected HEK293T cells with BST-2 and ORF7a or a 477 control plasmid and at four hours post-transfection, replaced the media with media 478 containing either 20 nM Concanamycin A (to inhibit lysosomal degradation) or 500 479 nM MG-132 (to inhibit proteasome function). At 18 hours post-transfection, cells 480 were lysed and analyzed by Western blot to determine if BST-2 was degraded. After 481

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treatment, lower molecular weight bands were still observed. Treatment with 482 neither Concanamycin A nor MG-132 blocked the ability of ORF7a to antagonize 483 BST-2 (Figure 4C). The far right 2 lanes contain a background band at a similar 484 molecular weight as HA-tagged ORF7a that does not affect the experiment. These 485 data demonstrate that the appearance of lower molecular weight bands of BST-2 is 486 not due to lysosomal or proteasomal degradation and suggests that ORF7a 487 antagonizes BST-2 through an alternative mechanism. 488 489 BST-2 colocalizes with and alters localization of SARS-CoV ORF7.. Since icSARS-490 ORF7abΔ-CoV is more susceptible to BST-2 restriction and ORF7a appears to cause 491 the appearance of a low molecular weight BST-2 band, we hypothesized that BST-2 492 may alter ORF7a localization within the cell. ORF7a was transfected into HEK293T 493 cells and the cells were stained for ORF7a, as well as ER and Golgi markers(9, 10). 494 ORF7a normally localizes to the Golgi and was also detectable in the ER, as would be 495 expected for a protein that passes through the ER to the Golgi (Figure 5a). To 496 determine if BST-2 and ORF7a co-localize, we performed confocal microscopy. 497 When transfected alone, ORF7a localizes primarily to the Golgi, whereas BST-2 498 localizes to the plasma membrane (Figure 5b). When BST-2 and ORF7a were co-499 transfected, ORF7a now appears to localize to the plasma membrane, coincident 500 with BST-2 (Figure 5b). These data suggest that BST-2 and ORF7a may be 501 interacting in cells. 502 503

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SARS-CoV ORF7a co-immunoprecipitates with BST-2. Having shown that ORF7a 504 both alters protein mobility and localizes to the plasma membrane when co-505 expressed with BST-2, we sought to determine if there is a molecular interaction 506 between the two proteins. We co-transfected BST-2 and ORF7a into HEK293T cells 507 and 18 hours post-transfection cells were lysed. We immunoprecipitated proteins 508 from transfected cells and immunoblotted for both BST-2 and ORF7a. We found 509 BST-2 and ORF7a present in both the input and the co-immunoprecipitation (Figure 510 6) suggesting an interaction between BST-2 and ORF7a, either directly or within a 511 larger multi-component complex. 512 513 Direct interaction between ORF7a and BST-2 is regulated by BST-2 514 glycosylation. To assess whether the extracellular domain of ORF7a interacts 515 directly with the extracellular domain of BST-2, we performed surface plasmon 516 resonance (SPR) analysis of ORF7a-BST-2 binding. SPR allows direct quantitation of 517 protein-protein interaction by measuring the affinity between two proteins. One 518 protein is immobilized on a sensor chip and the other is flowed over the sensor chip 519 in increasing concentrations. Binding of proteins causes changes in refraction, which 520 is detected and recorded as resonance units (RU). Affinity can then be calculated 521 from changes in RU(57). ORF7a with an Fc fusion tag (ORF7a-Fc) was expressed and 522 purified from HEK293T cells and BST-2 was expressed and purified from both E. coli 523 and HEK293T cells (Figure 7A). CD spectra of BST2 expressed in HEK239T and 524 E.coli cells both reveal the expected pattern for a protein containing primarily α–525 helical folds (Figure 7B). ORF7a-Fc, in contrast, shows the typical spectrum of 526

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proteins formed dominantly by β–sheets (Figure 7B). Melting temperatures were 527 deduced from the melting curves (Figure 7C) and tetrameric BST2 expressed in 528 E.coli cells has a slightly lower melting temperature of 61.95°C than dimeric BST2 529 expressed in HEK293T cells, 65.3°C (Figure 7C). These data suggests that both BST-530 2 and ORF-7a-Fc are folded correctly and were, therefore, used for the SPR analysis. 531 By SPR, we observed that unglycosylated BST-2 expressed in E. coli was able 532 to bind to ORF7a-Fc with an affinity (KD) of 10 μM (Figure 7D). Binding of 533 glycosylated BST-2 expressed in HEK293T cells, though, exhibited markedly weaker 534 responses in identical SPR experiments, which did not reach equilibrium and, 535 therefore, did not allow us to quantify an accurate KD for this interaction (Figure 7E). 536 We did attempt to fit the data to estimate the KD for this interaction and the binding 537 of ORF7a to glycosylated BST-2 is at least 4 times weaker than to unglycosylated 538 BST-2. These data indicate that ORF7a binds directly to unglycosylated BST-2 with 539 μM affinity and that the presence of N-linked glycosylation at positions 65 and 92 of 540 BST-2 significantly weakens this interaction. 541 542 ORF7a expression interferes with BST-2 glycosylation. Given that ORF7a-543 dependent BST-2 antagonism is unaffected by lysosomal or proteasomal inhibitors 544 and that ORF7a binds unglycosylated BST-2 with substantially higher affinity than 545 glycosylated BST-2, we hypothesized that ORF7a may bind to BST-2 before it is 546 glycosylated in the ER and interfere with glycosylation of BST-2. To determine if 547 ORF7a interferes with glycosylation, we transfected HEK293T cells with increasing 548 amounts of ORF7a. Co-transfection of increasing amounts of ORF7a leads to lower 549

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molecular weight bands of BST-2 in a dose-dependent manner (Figure 8). To 550 confirm that the lower molecular weight bands were unglycosylated, we treated 551 lysate from cells expressing BST-2 with glycopeptidase F. Previous studies have 552 shown that treatment with glycopeptidase F removes all the glycosylation from 553 BST-2(30). The BST-2 lysate treated with glycopeptidase F showed a shift to a lower 554 molecular weight with an identical size as the lower molecular weight band present 555 when BST-2 is co-transfected with ORF7a (Figure 8a). To further confirm that co-556 transfection of ORF7a leads to decreased levels of unglycosylated BST-2, we 557 measured the density of each band and calculated the ratio of glycosylated to 558 unglycosylated BST-2. As the levels of ORF7a increase, the levels of glycosylated 559 BST-2 decrease (Figure 8b). These data suggests that ORF7a interferes with 560 glycosylation of BST-2. 561 562 Unglycosylated BST-2 no longer restricts icSARS-ORF7abΔ-CoV release. 563 To confirm that glycosylation of BST-2 is necessary for restriction of icSARS-564 ORF7abΔ-CoV, we transfected 293T/ACE2 cells with a mutant of BST-2 that does 565 not undergo N-linked glycosylation (called N65A/N92A) (29). We confirmed 566 expression of the N65A/N92A mutant BST-2 by western blot, where we observed 567 only expression of the expected 19kDa unglycosylated form of BST-2 (Figure 9A). 568 We then confirmed that the N65A/N92A mutant was still able to localize to the cell 569 surface by quantifying the amount of WT and mutant BST-2 by flow cytometry on 570 non-permeabilized cells. Surface labeling shows that mutant BST-2 localization to 571 the plasma membrane is not significantly different from WT BST-2 surface 572

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expression (Figure 9B). We transfected cells with plasmids encoding WT and 573 N65A/N92A mutant BST-2 and then infected those cells with WT icSARS-Urbani or 574 icSARS-ORF7abΔ-CoV. The BST-2 plasmid used in these experiments has a HA tag 575 inserted at amino acid 463, so we first confirmed that HA/BST-2 is still able to 576 significantly (10-fold, p =.0046) restrict icSARS-ORF7abΔ-CoV compared to icSARS-577 Urbani (Figure 9C). However, there was no significant difference between 578 supernatant icSARS-Urbani and icSARS-ORF7abΔ-CoV in cells transfected with 579 N65A/N92A mutant BST-2 (Figure 9C, p =0.274), suggesting that N-linked 580 glycosylation is required for the BST-2 mediated restriction of icSARS-ORF7abΔ-CoV. 581 582 Discussion 583 Our studies further expand the role of BST-2 in restriction of enveloped viruses. We 584 screened selected genes from the SARS-CoV genome and identified four potential 585 BST-2 modulators, of which one was SARS-CoV ORF7a. While ORF7a has been 586 shown to induce apoptosis, a definitive role has not been established for ORF7a 587 during infection(13-15). Through overexpression, infection and transfection studies 588 we demonstrate that BST-2 blocks the release of SARS-CoV virions, that ORF7a 589 overcomes this inhibition and that ORF7ab deleted viruses display increased 590 sensitivity to BST-2. Importantly, the inhibition of BST-2 is not by protein 591 degradation, but by inhibiting its activity through inhibiting glycosylation at two key 592 sites on the protein that are required for its anti-viral function. We demonstrate that 593 a BST-2 mutant protein with the two glycosylation sites removed, still traffics to the 594 plasma membrane, but is unable to inhibit SARS-CoV release. Our data also 595

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demonstrate that, unlike HIV-1 Vpu, which removes BST-2 protein from the surface 596 and induces degradation(32, 33, 56), SARS-CoV ORF7a does not remove BST-2 from 597 the plasma membrane 598 599 We have confirmed the interaction of ORF7a and BST-2 using multiple assays 600 including immunoprecipitation, co-localization and surface plasmon resonance, 601 which show that ORF7a directly binds to unglycosylated BST-2, but not glycosylated 602 BST-2. Previous studies have suggested that glycosylation of BST-2 is required for 603 BST-2 anti-viral activity(29) and the amino acid residues surrounding the N-linked 604 glycosylation sites are evolutionarily conserved in BST-2 suggesting that these 605 amino acids may be important for BST-2 function(22). We further demonstrated 606 that N-linked glycosylation is required for the restriction of SARS-CoV lacking 607 ORF7a, suggesting that the blocking of glycosylation by ORF7a is directly 608 responsible for the antagonism of BST-2. BST-2 N-linked glycosylation has been 609 proposed to effect the HIV-1 restriction activity of BST-2(27, 29, 33, 56, 58), 610 however, we have demonstrated for the first time that a virus encodes a BST-2 611 antagonist that inhibits BST-2 glycosylation, providing a potential mechanism for 612 other viral putative BST-2 antagonists. 613 614 Taken together, the data suggests that ORF7a may function by binding to and 615 preventing N-linked glycosylation of BST-2, preventing the tethering of SARS-CoV 616 virions to the cytoplasmic membrane after they are released from the cell. We 617 hypothesize that while BST-2 is trafficking through the ER and Golgi to the surface, 618

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ORF7a and BST-2 interact in the Golgi, where the extracellular domain of ORF7a 619 binds the unglycosylated extracellular domain of BST-2 and either directly prevents 620 glycosylation of BST-2 or binds to the evolutionarily conserved sites and as a side-621 effect blocks N-linked glycosylation. SARS-CoV virions form in the ERGIC during 622 virion maturation and it is yet to be determined if ORF7a or BST-2 are present in 623 those compartments. Potentially BST-2 is binding newly released SARS-CoV 624 virions at the plasma membrane, however, most models of BST-2 function predict 625 that BST-2 is inserted into the membrane the virion as it forms(20), so we would 626 predict that BST-2 will first interact with SARS-CoV virions in the ERGIC. 627 628 While a variety of enveloped viruses encode BST-2 antagonists, those antagonists 629 function by different mechanisms. HIV-1 Vpu and Kaposi-Sarcoma Herpesvirus K5 630 both ubiquitinate BST-2, leading to surface removal and subsequent lysosomal 631 degradation(27, 56, 59). HIV-2 Env also removes BST-2 from the surface, but rather 632 than being degraded, BST-2 is relocated to the trans-Golgi network and cannot 633 function as a cytoplasmic membrane tether(36). SIV Env removes BST-2 from the 634 surface through BST-2 internalization by endocytosis(38, 60). Ebolavirus GP1,2 635 does not remove BST-2 from the surface, but antagonizes BST-2 through an as yet 636 unknown mechanism(39). The diverse mechanisms of known BST-2 antagonists 637 demonstrate that viruses have independently evolved many different ways of 638 antagonizing BST-2, an important restriction factor for any enveloped virus. It is 639 possible that all enveloped viruses encode, in most cases undiscovered, BST-2 640 antagonists that act by a variety of mechanisms. 641

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642 In this study we have identified BST-2 as a potential inhibitor of SARS-CoV release. 643 Our studies suggest that SARS-CoV ORF7a antagonizes the function of BST-2 by 644 interfering with its N-linked glycosylation while binding it in the Golgi and then 645 trafficking with it from the Golgi to the plasma membrane. From this we predict that 646 therapeutics designed to inhibit the interaction between BST-2 and ORF7a may 647 inhibit virus growth in vitro and in vivo. 648 649 Acknowledgements 650 This work was supported by the Division of Intramural Research of the National 651 Institute of Allergy and Infectious Diseases (NIAID) with grant RO1AI1095569 652 (MBF) and by NIAID grant R01AI087452 (EJS). 653 654 655 656 657 658 659 660 661 662 663 664

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665 SARS-CoV Gene Forward Primer/EcoR1 Site

nsp1 5'-GATCGAATTCACCATGGAGAGCCTTGTTCTTGGTGTCA-3'

nsp4 5'-GATCGAATTCACCATGAAGATTGTTAGTACTTGTTTTA-3'

nsp5 5'-GATCGAATTCACCATGAGTGGTTTTAGGAAAATGGCAT-3'

nsp6 5'-GATCGAATTCACCATGGGTAAGTTCAAGAAAATTGTTA-3'

nsp7 5'-GATCGAATTCACCATGTCTAAAATGTCTGACGTAAAGT-3'

nsp8 5'-GATCGAATTCACCATGGCTATTGCTTCAGAATTTAGTT-3'

nsp9 5'-GATCGAATTCACCATGAATAATGAACTGAGTCCAGTAG-3'

nsp10 5'-GATCGAATTCACCATGGCTGGAAATGCTACAGAAGTAC-3'

nsp13 5'-GATCGAATTCACCATGAGGCTGTAGGTGCTTGTGTATTGT-3'

nsp14 5'-GATCGAATTCACCATGGCAGAAAATGTAACTGGACTTTTT-3'

nsp15 5'-GATCGAATTCACCATGAGTTTAGAAAATGTGGCTTATAAT-3'

nsp16 5'-GATCGAATTCACCATGGCAAGTCAAGCGTGGCAACCAG-3'

SARS-CoV Gene Reverse Primer/XmaI Site

nsp1 5'-GATCCCCGGGACCTCCATTGAGCTCACGAGTGAGT-3'

nsp4 5'-GATCCCCGGGCTGCAGAACAGCAGAAGTGATTGAT-3'

nsp5 5'-GATCCCCGGGTTGGAAGGTAACACCAGAGCATTGT-3'

nsp6 5'-GATCCCCGGGCTGTACAGTAGCAACCTTGATACAT-3'

nsp7 5'-GATCCCCGGGCTGAAGAGTAGCACGGTTATCGAGC-3'

nsp8 5'-GATCCCCGGGCTGTAGTTTAACAGCTGAGTTGGCT-3'

nsp9 5'-GATCCCCGGGCTGAAGACGTACTGTAGCAGCTAAA-3'

nsp10 5'-GATCCCCGGGCTGCATCAAGGGTTCGCGGAGTTGG-3'

nsp13 5'-GATCCCCGGGTTGTAATGTAGCCACATTGCGACGTGGTAT-3'

nsp14 5'-GATCCCCGGGCTGTAACCTGGTAAATGTATTCCACAGGTT-3'

nsp15 5'-GATCCCCGGGTTGTAGTTTTGGGTAGAAGGTTTCAACATG-3'

nsp16 5'-GATCCCCGGGGTTGTTAACAAGAATATCACTTGAA-3' 666 Table 1: SARS-CoV non-structural protein cloning primers. Cloning primers 667 used in this study 668 669 670 671 672 673

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Figure Legends 674 675 Figure 1: icSARS-CoV and icSARS-ORF7abΔ-CoV infection of cells with and 676 without BST-2 expression. (A and B) HEK293T ACE2 cells were transfected with 677 BST-2 Flag in pCAGGS or a control plasmid. 24 hours post-transfection, HEK293T 678 ACE2 cells were infected with icSARS-CoV (A) or icSARS-ORF7abΔ-CoV (B) at an 679 MOI of 0.1. Supernatant and cell lysate was taken at 12, 24, and 36 hours post-680 infection. Virus was titered from supernatant taken at 12, 24, and 36 hours. (B) RNA 681 extracted from icSARS and icSARS-ORF7abΔ-CoV infected HEK293T ACE2 cells was 682 analyzed by realtime PCR for genomic RNA levels (C) or leader containing N mRNA 683 (D) as a signature of replicating virus. * significant at p < 0.05, ** significant at P < 684 0.01, *** significant at P < 0.001. Data shown is representative of two independent 685 experiments. 686 687 Figure 2: BST-2 tethers SARS-CoV to plasma membrane 688 BST-2 or control plasmid was transfected into VeroE6 cells and infected with either 689 icSARS-CoV or icSARS-ORF7abΔ-CoV at an MOI of 10. At 24 hours post-infection 690 cells were fixed and imaged by electron microscopy. Transfection of BST-2 results in 691 a large increase in icSARS-ORF7abΔ-CoV virions retained at the surface compared to 692 control transfected cells. Scale bars equal 500nm. 693 694 Figure 3: ORF7a expression leads to lower molecular weight BST-2 within the 695 cells, but not reduced BST-2 surface expression. 696

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BST-2 was transfected into HEK293T cells with increasing amounts of ORF7a. 18 697 hours post-transfection cells were either lysed and analyzed by western blot or 698 stained with an anti-BST-2 antibody conjugated to APC and analyzed using flow 699 cytometry. Untransfected cells did not express BST-2. BST-2 transfected cells 700 showed high BST-2 expression and co-transfection of ORF7a and BST-2 lead to 701 decreased levels of the high molecular weight BST-2 band and increasing levels of a 702 lower molecular weight BST-2 band in a dose-dependent manner (A), but did not 703 lead to reduced surface expression of BST-2 (B). Data shown is representative of 704 two independent experiments. 705 706 Figure 4: Proteasomal and Lysosomal inhibitors effect on ORF7a antagonism 707 of BST-2. (A and B) HEK293T cells were treated with either Concanamycin A 708 (ConA) or MG-132 to demonstrate the concentrations used are inhibitory or 709 proteasome or lysosomal degradation, respectively. Lysate was analyzed by 710 western blot for with antibodies against Ubiquitin (A) and LC3B (B) to demonstrate 711 efficacy of compounds. (C) Increasing amounts of ORF7a was transfected into 712 HEK293T cells. Cells were subsequently treated with the lysosome inhibitor, 713 Concanamycin A, or proteasome inhibitor, MG132. Neither inhibitor prevented BST-714 2 antagonism by ORF7a. Data shown is representative of three independent 715 experiments. 716 717 718 Figure 5: ORF7a Co-localizes with BST-2. HEK293T cells were transfected with 719 BST-2, ORF7a, or both. (A) BST-2 was stained with mouse anti-Flag primary and 720

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goat anti-mouse Texas Red secondary. ORF7a was stained with rabbit anti-HA 721 primary and horse anti-rabbit AMCA secondary. Localization was analyzed with 722 confocal microscopy (merged images shown as yellow). (B) When BST-2 and ORF7a 723 are co-transfected, the two proteins display overlapping signal as seen in the 724 merged image (shown as yellow). 725 726 727 Figure 6: ORF7a Co-Immunoprecipiates with BST-2. HEK293T cells were 728 transfected with sham, ORF7a/HA and BST-2/Flag separately or together. After 729 expressing for 18 hours, cells were lysed and analyzed by western blot for 730 expression (A). BST-2 was immunoprecipitated with anti-Flag beads. Bound protein 731 was eluted and analyzed by Western Blot (B). BST-2 was detected with a mouse 732 anti-Flag M2 antibody. ORF7a was detected with rabbit anti-HA antibodies. ORF7a 733 was detected in the elution from the co-immunoprecipitation suggesting an 734 interaction between ORF7a and BST-2. Data shown is representative of two 735 independent experiments. Asterisk denotes a non-specific band. 736 737 Figure 7: Binding of ORF7a to BST-2. (A) Purified BST-2 expressed in E. coli and 738 HEK293T cells was stained with Coomassie. BST-2 expressed in E. coli has a lower 739 molecular weight than BST-2 expressed in HEK293T cells due to lack of 740 glycosylation. (B) CD spectra and melting curves (C) of BST2 and ORF7a-Fc. CD 741 spectra of 10μM BST2 expressed in HEK293T cells, 12μM BST2 expressed in E.coli 742 cells and 8μM ORF7a-Fc expressed in HEK239T cells. (D) Shown are representative 743 sensograms obtained in SPR experiments analyzing direct interaction of ORF7a-Fc 744

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with unglycosylated BST-2 expressed in E. coli (D) and glycosylated BST-2 745 expressed in HEK293T cells (E). For SPR experiments, ORF7a-Fc was captured via 746 immobilized protein A on a CM5 chip. Single-cycle kinetics were performed by 747 injection of 5μM, 10μM, 20μM, 40μM and 80μM of BST-2. Data shown is 748 representative of three independent experiments. 749 750 Figure 8: ORF7a interferes with glycosylation of BST-2 751 BST-2 was transfected into HEK293T cells with increasing amounts of ORF7a. 18 752 hours post-transfection cells were lysed and analyzed by western blot. (A) 753 Increasing levels ORF7a lead to increased levels of a lower molecular weight band of 754 BST-2, which we hypothesized to be unglycosylated BST-2. To confirm that the 755 lower molecular weight band was unglycosylated BST-2, we treated lysate from 756 BST-2 transfected cells with Glycopeptidase F, which deglycosylates proteins. When 757 treated with Glycopeptidase F, BST-2 shifts down to the same molecular weight as 758 the lower band of BST-2 co-transfected with ORF7 suggesting ORF7a leads to 759 increased levels of unglycosylated BST-2. (B) Density of each band was measured 760 and the ratio of glycosylated to unglycosylated BST-2 was calculated and graphed. 761 Data shown is representative of three independent experiments. 762 763 Figure 9: Unglycosylated BST-2 fails to inhibit SARS-CoV egress. 764 HEK293T/ACE2 cells were transfected with either control plasmid, wildtype HA-765 tagged BST-2 or a mutant HA-tagged BST-2 containing N65A and N92A mutations. 766 (A) Expression levels of wildtype BST-2 and N65A/N92A BST-2 were analyzed by 767

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western blot with anti-HA antibody and anti-Tubulin as a loading control. 768 N65A/N92A BST-2 runs noticeably slower due to its loss of glycosylation. (B) 769 HEK293T/ACE2 cells were transfected with each plasmid and levels of BST-2 770 protein on the surface of cells was analyzed by flow cytometry with an anti-HA 771 antibody. The percent of surface expression of BST-2(WT) transfected cells is 772 graphed displaying surface localization of the B65A/N92A mutant BST-2 (C) 773 HEK293T/ACE2 cells were transfected with each plasmid and infected with either 774 icSARS-CoV or icSARS-ORF7abΔ-CoV. Cell supernatants were analyzed by plaque 775 assay and graphed as the % of wildtype icSARS-CoV released. Notice the loss of 776 inhibition of icSARS-ORF7abΔ-CoV release in the mutant BST-2 transfected cells 777 compared to wildtype BST-2 transfected cells. *** equals a P value <0.005 778 779 780 781 782 783 784 785 786 787 788 789 790

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791 792 793 References 794 795 1. Organization WH. 2003. WHO | Summary of probable SARS cases with onset 796 of illness from 1 November 2002 to 31 July 2003 WHO. 797 2. Drosten C, Preiser W, Günther S, Schmitz H, Doerr HW. 2003. Severe 798 acute respiratory syndrome: identification of the etiological agent. Trends in 799 molecular medicine 9:325-327. 800 3. Marra MA, Jones SJM, Astell CR, Holt RA, Brooks-Wilson A, Butterfield 801

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1023

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