key: cord-0725635-kao36eo7
authors: Silvas, Jesus; Morales-Vasquez, Desarey; Park, Jun-Gyu; Chiem, Kevin; Torrelles, Jordi B.; Platt, Roy Neal; Anderson, Tim; Ye, Chengjin; Martinez-Sobrido, Luis
title: Contribution of SARS-CoV-2 accessory proteins to viral pathogenicity in K18 hACE2 transgenic mice
date: 2021-03-10
journal: bioRxiv
DOI: 10.1101/2021.03.09.434696
sha: 0737e64f4015a90add02e267a2900f84039681ec
doc_id: 725635
cord_uid: kao36eo7

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. To date, it is estimated that over 113 million individuals have been infected with SARS-CoV-2 and over 2.5 million human deaths have been recorded worldwide. Currently, three vaccines have been approved by the Food and Drug Administration for emergency use only. However much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse genetics system approach to successfully engineer recombinant (r)SARS-CoV-2, where we individually removed viral 3a, 6, 7a, 7b, and 8 ORF proteins, and characterized these recombinant viruses in vitro and in vivo. Our results indicate differences in plaque morphology, with ORF deficient (ΔORF) viruses producing smaller plaques than those of the wild-type (rSARS-CoV-2/WT). However, growth kinetics of ΔORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice with the ΔORF rSARS-CoV-2 identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while ΔORF7a, ΔORF7b and ΔORF8 rSARS-CoV-2 induced comparable pathology to rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse genetics system to generate rSARS-CoV-2 and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live-attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19. IMPORTANCE Despite great efforts put forward worldwide to combat the current coronavirus disease 2019 (COVID-19) pandemic, Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) continues to be a human health and socioeconomic threat. Insights into the pathogenesis of SARS-CoV-2 and contribution of viral proteins to disease outcome remains elusive. Our study aims to determine the contribution of SARS-CoV-2 accessory open reading frame (ORF) proteins in viral pathogenesis and disease outcome, and develop a synergistic platform combining our robust reverse genetics system to generate recombinant (r)SARS-CoV-2 with a validated rodent model of infection and disease. We demonstrated that SARS-CoV-2 ORF3a and ORF6 contribute to lung pathology and ultimately disease outcome in K18 hACE2 transgenic mice, while ORF7a, ORF7b, and ORF8 have little impact on disease outcome. Moreover, our combinatory platform serves as the foundation to generate attenuated forms of the virus to develop live-attenuated vaccines for the treatment of SARS-CoV-2.

Coronaviruses are enveloped, positive sense, single-stranded RNA 78 and had a 75% and 50% survival rate, respectively. Furthermore, both 125

ORF3a and ORF6 rSARS-CoV-2 had lower viral titers (10 2 PFU/ml) at 2 126 days post-infection (d p.i.) , and by 4 d p.i. were no longer detected in nasal 127 turbinates. In contrast, ORF6 viral replication in the lungs reached 10 5 128 PFU/ml at 2 d p.i. and only decreased by ~2-log 10 The SARS-CoV-2 genome, which was divided into 5 fragments and 156 chemically synthesized, was assembled into a single BAC that led to efficient 157 virus rescue after transfection into Vero E6 cells using LPF2000 (21). 158

Fragment 1 included the SARS-CoV-2 ORF accessory proteins. Using 159 standard gene engineering approaches, we systematically deleted, 160

individually, ORF3a, ORF6, ORF7a, ORF7b, or ORF8 from fragment 1 using 161 PCR and primer pairs containing BsaI type IIS restriction endonuclease sites. 162

After being confirmed by Sanger sequencing (data not shown), fragment 1 163 containing the individual deletions in the ORF3a, ORF6, ORF7a, ORF7b, or 164 ORF8 accessory proteins were reassembled into the BAC (Figure 1) . 165

Each BAC with individual deletions in the accessory ORFs were 167 transfected into Vero E6 cells for the recovery of ORF rSARS-CoV-2, 168 according to our previously described protocol (21). At 72 h post-transfection, 169 tissue culture supernatants (P0) were collected to inoculate fresh Vero E6 170 cells (P1). Supernatants were then collected from P1 at 72 hours post-171 infection (h p.i.) and viral titers, defined as plaque forming units/milliliter 172 (PFU/ml), were determined as previously described (21). To verify rescue of 173 each ORF rSARS-CoV-2, indirect immunofluorescence was performed using antibodies directed at the nucleocapsid (N) and spike (S) proteins ( Figure  175 2A). We next verified the individual deletion of each ORF in the rSARS-CoV-2 176 using RT-PCR procedures to amplify the viral N gene (control), and the 177 regions which cover the corresponding individual ORF deletions ( Figure 2B) . 178

All the ORF rSARS-CoV-2, and rSARS-CoV-2/WT, produced a RT-PCR 179 product of approximately 1.2 kb corresponding to the N gene, whereas 180 amplified regions that cover the corresponding ORF deletions were smaller in 181 the ORF rSARS-CoV-2 as compared to rSARS-CoV-2/WT ( Figure 2B) , 182 demonstrating the deletion of the individual ORFs in the viral genomes. 183

Individual deletions of the viral proteins in the ORF rSARS-CoV-2 were 184 further confirmed by Sanger sequencing of PCR products ( Figure 2C ) and by 185 full sequencing of the viral genome (accession number PRJNA707072). 186

These data demonstrate that each ORF rSARS-CoV-2 contained the 187 individual deletion of their respective ORF accessory proteins. 188

We next proceeded to characterize each ORF rSARS-CoV-2 in vitro 190 Interestingly, we noticed an effect in the plaque phenotype of all ORF 196 rSARS-CoV-2 as compared to rSARS-CoV-2/WT in all times p.i. studied (24, 197 48, 72, and 96 h) ( Figure 3A ). Next, we compared the growth kinetics of the 198 ORF rSARS-CoV-2 to those of the rSARS-CoV-2/WT in Vero E6 cells. To this end, viral titers in the tissue culture supernatants from Vero E6 cells 200 infected (MOI, 0.01) with WT or ORF rSARS-CoV-2 collected at 12, 24, 48, 201 72, and 96 h p.i. were determined by plaque assay ( Figure 3B ). No 202 statistically significant differences between WT and ORF3a, ORF6, and 203

ORF8 rSARS-CoV-2 were observed at any times p.i., except for the 204 replication of ORF7a and ORF7b rSARS-CoV-2 that was significantly 205 different (~ 1 log 10 ) than those of rSARS-CoV-2/WT at 12 and 24 h p.i. 206

( Figure 3B) . Peak viral titers for all ORF rSARS-CoV-2 and rSARS-CoV-207 2/WT were observed between 24 and 48 h p.i., with viral titers decreasing (~ 1 208 log 10 ) at later times p.i., consistent with previous studies with SARS-CoV-2 209 natural isolates (26, 27) . Altogether, these results suggest that despite slight 210 differences in plaque phenotype, ORF rSARS-CoV-2 have similar replication 211 kinetics than rSARS-CoV-2/WT in Vero E6 cells. 212

Coronavirus ORF accessory proteins have been implicated as virulence 214 factors and contribute to both pathogenicity and disease outcome (12, 13, 20, 215 28-30) . Therefore, we proceeded to further investigate the contribution of 216 SARS-CoV-2 ORF3a, ORF6, ORF7a, ORF7b, and ORF8 to viral 217 pathogenicity and disease outcome in our previously established K18 hACE2 218 transgenic mouse model of SARS-CoV-2 infection and COVID-19 disease 219 (22). Four-to-six-week-old female mice (n=4) were mock (PBS)-infected, 220 infected (10 5 PFU) with rSARS-CoV-2/WT, or with each of the ORF rSARS-221

CoV-2 , and observed for 14 days for morbidity (body weight loss) and 222 mortality (survival) (Figure 4) . Our results indicate a similar decrease in body 223 weight percentage up to 5 d p.i., from which ORF3a and ORF7b rSARS-224

CoV-2 infected mice began to recover ( Figure 4A ). Interestingly, mice 225 infected with ORF6 and ORF7a rSARS-CoV-2 continued to loss body 226 weight until 7 and 8 d p.i., respectively, and start to recover ( Figure 4A ). All 227 mice infected with WT or ORF8 rSARS-CoV-2 succumbed to viral infection 228 by 6 and 7 d p.i., respectively ( Figure 4B ). Our continued observations of 229 mice infected with ORF3a, ORF6a, ORF7a, and ORF7b rSARS-CoV-2 230 identified survival rates of 75%, 50%, 25%, and 25%, respectively (Figure 231 nasal turbinates in all mice, with rSARS-CoV-2/WT reaching titers of up to 239 5X10 3 PFU/ml, while ORF6 rSARS-CoV-2 peaked at 5X10 2 PFU/ml. We only 240 detected ORF3a rSARS-CoV-2 (10 2 PFU/ml) in 50% of infected mice while 241

ORF7b rSARS-CoV-2 replicated up to 3X10 3 PFU/ml in 75% of infected 242 mice. Only 50% of mice had detectable levels (range between 0.5-1X10 3 243 PFU/ml) of ORF8 rSARS-CoV-2 ( Figure 5A ). Interestingly, out of all the 244 ORF rSARS-CoV-2 tested, ORF7a replicated in the nasal turbinate to 245 levels (5X10 4 PFU/ml) higher than those observed with rSARS-CoV-2/WT. By 246 ranging between 5X10 1 to 5X10 2 PFU/ml. ORF7a, ORF7b and ORF8 249 rSARS-CoV-2 replicated to lower levels (~5X10 1 PFU/ml) than rSARS-CoV-250 2/WT ( Figure 5A ). In the lungs WT, ORF6 and ORF7a rSARS-CoV-2 were 251 detected at levels of ~10 5 PFU/ml at 2 d p.i. (Figure 5B ). Viral titers of 252

ORF7b and ORF8 (~10 4 PFU/ml) and ORF3a (~0.5X10 2 PFU/ml) rSARS-253

CoV-2 were lower than those of rSARS-CoV-2/WT ( Figure 5B) 2 ORFs 3a, 6, 7a, 7b, and 8 accessory proteins to viral pathogenesis. 297

In this study, we generated rSARS-CoV-2 deficient in ORFs 3a, 6, 7a, 7b, 298 or 8 accessory proteins (Figure 1 ) and characterized them both in vitro 299 (Figures 2 and 3) and in vivo (Figures 4-6) . Importantly, we have been able 300 to demonstrate the ORF deficient nature of these rSARS-CoV-2 (Figure 2) . 301

During our initial in vitro characterization, we first identified that ORF proteins 302 contributed to early dissemination and formation of detectable viral plaques in 303

Vero E6 cell monolayers: viruses lacking 3a, 7a, 7b, and 8 ORF proteins 304 developed smaller plaques than rSARS-CoV-2/WT (Figure 3) . This was a 305 surprising finding, as we only observed ~1 log 10 difference in growth kinetics 306 between the WT and any of the ORF rSARS-CoV-2 (Figure 3) . 307

To further determine the contribution of each ORF accessory protein in 308 SARS-CoV-2 pathogenesis, we used our recently established K18 hACE2 309 transgenic mouse model (Figure 4) (22) . This particular in vivo study is the 310 first, to our knowledge, that analyzes the contribution of SARS-CoV-2 ORF 311

We observed a broad range of morbidity and survival outcomes between each 313 of the ORF rSARS-CoV-2 as compared to rSARS-CoV-2/WT (Figure 4) . 314 SARS-CoV-2 ORF accessory proteins are encoded in the following order: 3a, 315 6, 7a, 7b, and 8. Our data collectively showed greatest survival in mice 316 infected with ORF3a (75%) and 100% mortality with the ORF8 rSARS-317

CoV-2. In this regard, a natural SARS-CoV-2 variant with a deletion in the 318 ORF8 has been recently isolated from patients presenting COVID-19 319 symptoms (20). Thus, our results with the ORF8 rSARS-CoV-2 in the K18 320 hACE2 transgenic mouse model correspond to those observed in people 321 infected with a natural ORF8 SARS-CoV-2 isolate (20). Our findings with ORF3a and ORF6 rSARS-CoV-2 warrant further characterization as there 323 is limited insight into mechanistic exploitation of host pathways by ORF3a (12, 324 13, 30, 35, 36) . Since it is well known that SARS-CoV-2 ORF6 is a potent 325 inhibitor of the host innate immune response (11, 29), we were expecting low 326 viral load and an increased immune response; however, since SARS-CoV N 327 protein also inhibits host immune responses (15, 38), it is plausible that 328 SARS-CoV-2 N protein may have a similar function and is responsible of 329 counteracting host innate immune and inflammatory responses. Even though 330 our studies did not analyze cytokine and chemokine production, the gradual 331 recovery of mice infected with ORF6 rSARS-CoV-2 are suggestive of viral 332 clearance over time. Analysis of innate and adaptive immune responses 333 would be important, as well as modulation of IFN secretion, ion signaling 334 channels, and cellular apoptotic and/or necrosis pathways, to determine the 335 contribution of these ORF accessory proteins in viral pathogenesis in vivo. 336 This is currently the focus of our ongoing in vivo studies with these ORF 337 rSARS-CoV-2. Overall, similar to what was described with rSARS-CoV, our 338 study highlights that SARS-CoV ORFs 3a, 3b, 6, 7a, and 7b had no significant 339 impact on viral replication in vivo (31), but ORF 3a seems to be involved in 340 virulence, as its absence decreases SARS-CoV-2 virulence. 341

Overall, this study demonstrates the robustness of our BAC-based 342 reverse genetics approach to generate rSARS-CoV-2, including those with 343 deletions of ORF accessory proteins, and provides information on the 344 contribution of ORFs 3a, 6, 7a, 7b, and 8 accessory proteins in viral fitness in 345 vitro (Vero E6 cells) and in vivo, in our recently established K18 hACE2 (22). Importantly, information from this study also provides novel insights for 348 the generation of attenuated forms of SARS-CoV-2 for the development of with 5% (v/v) fetal bovine serum, FBS (VWR) and 100 units/ml penicillin-366 streptomycin (Corning). 367

The BAC harboring the entire viral genome of SARS-CoV-2 USA-369 WA1/2020 (Accession No. MN985325) was described previously (21). 370

Deletion of individual accessory ORF proteins was achieved in viral fragment 371 1 by using inverse PCR and primer pairs containing a BsaI type IIS restriction 372 endonuclease site. All the primer sequences are available upon request. 373

Fragments including the individual deletion of accessory ORF proteins were 374 reassembled into the BAC using BamHI and RsrII restriction endonucleases. 375

Virus rescues were performed as previously described (21) using the SARS-CoV N protein cross-reactive monoclonal antibody 1C7C7, 417 as previously described (39) . 418

Total RNA from Vero E6 cells (10 6 

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SARS-CoV-2 nsp13, nsp14, 634 nsp15 and orf6 function as potent interferon antagonists

ORF3a mutation associated with higher 637 mortality rate in SARS-CoV-2 infection

Severe acute respiratory 640 syndrome coronavirus group-specific open reading frames encode 641 nonessential functions for replication in cell cultures and mice

Using reverse 644 genetics to manipulate the NSs gene of the Rift Valley fever virus MP-645 12 strain to improve vaccine safety and efficacy

Attenuation 649 of pathogenic Rift Valley fever virus strain through the chimeric S-650 segment encoding sandfly fever phlebovirus NSs or a dominant-651 negative PKR

The role of 653 reverse genetics systems in determining filovirus pathogenicity

ORF3a: Mutability and function

ORF3a of the COVID-19 virus SARS-CoV-2 blocks HOPS 659 complex-mediated assembly of the SNARE complex required for 660 autolysosome formation

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Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Inhibits 668

Type I Interferon Production by Interfering with TRIM25-Mediated RIG-I 669

Generation and 672 Characterization of recombinant SARS-CoV-2 expressing reporter 673 genes

Genome organization of WT and ORF rSARS-CoV-2: SARS

CoV-2 genome includes ~29.8 kb nucleotides among which ~21.5 kb encodes 691 the ORF1a and ORF1b replicase. The rest of the ~8.3 kb viral genome 692 encodes the structural spike (S), envelope (E), matrix (M), and nucleocapsid 693 (N) proteins

Individual deletions of the ORF accessory proteins were introduced into the 695 BAC for rescue of rSARS-CoV-2. Schematic representation not drawn to 696 scale

Rescue of ORF rSARS-CoV-2. A) IFA: Vero E6 cells (24-well 698 plate format, 10 5 cells/well, triplicates) were mock-infected or infected (MOI of 699 3) with WT

cells were fixed and immunostained with a cross-reactive 701 polyclonal antibody against SARS-CoV N protein and a cross-reactive 702 monoclonal antibody against SARS-CoV S protein (3B4)

-well plate format, 10 6 cells/well) were mock-infected or infected (MOI 706 of 0.1) with WT and ORF rSARS-CoV-2 and total RNA was

Regions in the viral genome corresponding with the deletion were 708 amplified and the N gene was amplified as an internal control. All the products 709 (PFU/ml) and immunostaining using the cross-reactive SARS-CoV 1C7C7 N 742 protein monoclonal antibody. Viral titers in the nasal turbinate (A) and lungs 743 (B) are shown. Symbols represent data from individual mouse, and bars the 744 geometric means of viral titers

PFU/ml). ND, not detected

@, not detected in 1 mouse

mice: &, not detected in 3 mice. Negative results of the PBS-infected mice 747 are not plotted

Gross pathology analysis of lungs from K18 hACE2 transgenic 749 mice infected with WT and ORF rSARS-CoV-2: Lungs from 6-to-8-week-750 old female K18 hACE2 transgenic mice (n=8/group) mock (PBS)-infected or 751 infected (i.n.) with 10 5 PFU of WT or ORF rSARS-CoV-2 were harvested at 2 752 (n=4/group) or 4 (n=4/group) d p.i. and imaged (A), and scored for viral 753 induced lesions (B). Calculation of total lung surface area affected by viral 754