key: cord-1021961-h6hx6mco
authors: Li, Yize; Renner, David M; Comar, Courtney E; Whelan, Jillian N; Reyes, Hanako M; Cardenas-Diaz, Fabian Leonardo; Truitt, Rachel; Tan, Li Hui; Dong, Beihua; Alysandratos, Konstantinos Dionysios; Huang, Jessie; Palmer, James N.; Adappa, Nithin D.; Kohanski, Michael A.; Kotton, Darrell N.; Silverman, Robert H; Yang, Wenli; Morrisey, Edward; Cohen, Noam A.; Weiss, Susan R
title: SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial derived cells and cardiomyocytes
date: 2020-09-25
journal: bioRxiv
DOI: 10.1101/2020.09.24.312553
sha: a916c04bccb53eaeb31912e852b346297db278bf
doc_id: 1021961
cord_uid: h6hx6mco

Coronaviruses are adept at evading and/or antagonizing double-stranded RNA-induced host antiviral pathways, including interferon signaling, OAS-RNase L and PKR while robust cytokine responses characterize severe coronavirus disease. Knowledge of how newly emerged SARS-CoV-2 interacts with these pathways is minimal. SARS-CoV-2 readily infects patient-derived nasal epithelial cells and induced pluripotent stem cell-derived alveolar type 2 cells(iAT2) and cardiomyocytes(iCM). Robust activation of interferons or RNase L is not observed, while PKR activation is evident in iAT2 and iCM. In SARS-CoV-2 infected Calu-3 and A549ACE2 lung derived cell lines, activation of all pathways is observed, similar to a mutant MERS-CoV lacking innate immune antagonists. Moreover, increased replication in RNASEL knockout A549ACE2 cells, implicates RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 is less adept at antagonizing these host defense pathways compared to other coronaviruses, the innate immune response is still generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2.

. To further investigate this very weak ISG induction, using cells from the same donors 146 as the IFN/ISG mRNA quantification, we assessed the phosphorylation of STAT1, a transcription 147 factor that is itself encoded by an ISG, which is primarily a key mediator of type I and type III IFN 148 signaling. Upon infection, STAT1 is phosphorylated before complexing with STAT2 and IRF9 to 149 form ISGF3, which translocates to the nucleus where it mediates the activation of ISG 150 transcription (Stark and Darnell, 2012) . Consistent with the weak activation of ISGs, there was no 151 evidence of phosphorylation of STAT1, likely due to low levels or transient phosphorylation (Fig   152   2C ). In addition, we did not detect PKR activation, as indicated by the absence of phosphorylated 153 PKR and eIF2a in SARS-CoV-2 infected cells, while PKR and eIF2a were clearly expressed (Fig   154   2C ). We also assessed activation of the OAS-RNase L pathway during SARS-CoV-2 infection of 155 cells from two of the same four donors. Since 28S and 18S ribosomal RNAs (rRNAs) are targeted 156 for degradation by activated RNase L, we can evaluate 28S and 18S rRNA integrity using a 157 Bioanalyzer as a readout for RNase L activation. The absence of any rRNA degradation in SARS-

CoV-2 infected cells (Fig 2E) indicated that RNase L was not activated despite abundant RNase 159 L protein expression (Fig 2C) . 

virus replicated efficiently, reaching a titer of 10 6 PFU/ml by 48hpi (Fig 3A) . Staining of cultures 169 with an anti-N antibody showed that most of the iAT2 cells were infected, without obvious 170 cytopathic effect (CPE) during infection (Fig 3B) . Notably, SARS-CoV-2 infection of iAT2 cells 171 was robust despite ACE2 expression being below the level of detection by immunoblotting (Fig   172   3C ). We observed activation of the PKR pathway as indicated by both PKR and eIF2a 173 phosphorylation by immunoblotting (Fig 3C) . We extracted RNA from infected iAT2 cells for RT-174 qPCR analysis, verified these cells were replicating virus by quantifying genome RNA copies (Fig   175   S1B ), and assessed IFN/ISG induction. As with the nasal cells, we observed weak induction of 176 IFN-b and IFN-l mRNA from mock infected and infected cells (Fig 3D) , while MDA5 (Roth-Cross 177 et al., 2008) a dsRNA sensor in the pathway leading to IFN production during coronavirus 178 expression, is not detected (Fig 3C) . We used the alphavirus Sindbis virus (SINV) as a positive 179 control, which we have previously shown induces robust activation of all dsRNA-induced 180 pathways (Comar et al., 2019) . Surprisingly, we observed greater increases in OAS2 and IFIT 181 mRNA expression by SARS-CoV-2 compared with SINV (Fig 3D) , but with minimal induction of 182 IFIH1 mRNA or protein (MDA5) (Fig 3C&D) . However, we did not observe phosphorylation of 183 STAT1 (Fig 3C) , as in the nasal cells above. Additionally, we did not observe any degradation of 184 rRNA in SARS-CoV-2 infected cells, and only slight degradation by SINV despite ample 185 expression of RNase L (Fig 3E) , suggesting minimal activation of RNase L in iAT2 cells in general. derived-cardiomyocytes (iCM). SARS-CoV-2 replicated robustly in these cells reaching titers of 191 approximately 10 6 PFU/ml by 48hpi (Fig 4A) , similar to replication in nasal and iAT2 cells. Cells 192 were stained with an antibody against cardiac troponin-T (cTnT) as a marker for cardiomyocytes,

and an antibody against the viral N protein to identify infected cells (Fig 4B) . In addition, we 194 detected clear cytopathic effect (CPE), in iCM, which differed from infected nasal and iAT2 cells.

This CPE included syncytia resulting from cell-to-cell fusion, which is typical of coronaviruses and 196 a result of intracellular cleavage of the viral spike protein by the host enzyme furin (Belouzard et 

Interestingly, while we observed detectable ACE2 protein expression in mock infected cells in two 199 independent experiments, we observed loss of ACE2 expression upon SARS-CoV-2 infection, 200 consistent with a recent study (Sharma et al., 2020) (Fig 4C) . We extracted RNA from mock 201 infected cells and cells infected with SARS-CoV-2 or SINV, verified that virus was replicating by 202 quantifying viral genome in intracellular RNA (Fig S1C) , and quantified expression of mRNAs for 203 IFNs and select ISGs. We found low levels of IFN/ISGs transcript in iCM similar to the nasal and 204 iAT2 cells (Fig D) , perhaps due to the undetectable levels of MDA5 and MAVS protein expression 205 in these cells (Fig 4C) . SINV also induced host mRNAs weakly, with the exception of IFN-l, in 206 these cells (Fig 4D) . We observed no degradation of rRNA, suggesting an absence of RNase L 207 activation in iCM with SARS-CoV-2 or SINV (Fig 4E) , despite clear infection with either virus (Fig   208   S1C ). This was not surprising as there was no RNase L detectable by immunoblot in these cells 209 (Fig 4C) . Finally, as in iAT2 cells, we observed phosphorylation of PKR and eIF2a, indicating that 210 the PKR antiviral pathway is activated (Fig 4C) . 

We aimed to further characterize the relationship between SARS-CoV-2 and dsRNA-induced host 217 response pathways, which were activated to variable levels in the different primary and iPSC 218 derived cells evaluated. We chose two respiratory epithelium-derived human cell lines, A549 and CoV-2 receptor ACE2 (Fig S3) . Therefore, we generated A549 cells expressing the ACE2 223 receptor (A549 ACE2 ) by lentiviral transduction, and used two single cell clones, C44 and C34, for 224 all experiments (Fig S3) . Both A549 ACE2 clones express high levels of ACE2 greater than the 225 endogenously expressed ACE2 in Calu-3 cells (Fig S3) and in the primary cells discussed above 226 (Fig 2-4) .

We performed single step growth curves to measure replication of SARS-CoV-2 over the course 229 of one infectious cycle in A549 ACE2 cells, simian Vero-E6 cells, which are commonly used to 230 prepare SARS-CoV-2 stocks, and Calu-3 cells (clone HTB-55). SARS-CoV-2 replicated robustly 231 in A549 ACE2 and Vero-E6 cells (Fig 5A) but in comparison viral yields were lower in Calu-3 cells 232 (Fig 5B) . Since Calu-3 cells also support MERS-CoV infection, we compared SARS-CoV-2 233 replication to that of MERS-CoV (WT) and MERS-CoV-DNS4ab, a mutant deleted in host cell 234 antagonists NS4a, a dsRNA-binding protein, and NS4b, a 2'5'-phosphodiesterase that prevents 235 RNase L activation and nuclear translocation of NF-kB (Canton et al., 2018; Comar et al., 2019) .

Consistent with our previous work (Comar et al., 2019), MERS-CoV-DNS4ab reduced viral titers 237 from WT MERS-CoV levels, although they remained higher than SARS-CoV-2 titers (Fig 5B) . To 238 further understand the replication of SARS-CoV-2, we stained A549, Calu-3 and Vero-E6 cells at 239 24 hpi with antibodies against viral N protein and viral dsRNA, including additional Calu-3 staining 240 at 48 hpi since replication kinetics are slower (Fig 5C) . We observed cytopathic effect in all three 241 cell types, with N staining in the cytoplasm. Syncytia were observed in A549 ACE2 and Calu-3 cells,

but not in Vero cells (Fig 5C) . We also observed viral dsRNA localized to perinuclear foci as we 

We used RT-qPCR to quantify the induction of type I and type III IFNs and select ISGs at 24 and 247 48 hpi (Fig 6A) , as well as the intracellular viral genome copies to verify replication (Fig 6B) in 248 A549 ACE2 cells. Using SINV as a positive control, we found relatively low levels of both IFNb and 249 IFNl mRNA at 24 and 48 hpi by SARS-CoV-2, compared to SINV (Fig 6A) . Notably, IFN induction 250 was greater than observed in the nasal, iAT2, or iCM cells, possibly due to lower basal levels of

IFNb, but not IFNl, mRNA in the A549 ACE2 cells, which allow for greater fold changes over mock 252 infected cells (Fig S2) . As observed previously with SINV and MERS-CoV, SARS-CoV-2 induced 253 more IFNl than IFNb, typical of A549 cells (Comar et al., 2019). Levels of ISG mRNAs were 254 variable, with SARS-CoV-2 inducing moderate levels of OAS2 and IFIT1 mRNAs, but only late in 255 infection (48 hpi), similar to those induced by SINV at 24 hpi (Fig 6A) . We observed minimal 256 effects on mRNA levels of IFIH1 and the cytokine CXCL8 at both timepoints (Fig 6A) .

Furthermore, we did not detect any STAT1 phosphorylation at 24 hpi (Fig 6C) , which correlates 258 with weak ISG expression, suggesting defective IFN signaling downstream of IFN production. The 259 data shown in Fig 6 were derived from A549 ACE2 clone C44; similar data were obtained for a 260 second clone, C33 (Fig S4) .

We evaluated IFN/ISG responses in Calu-3 cells, which provided a second lung-derived cell line 263 that additionally supports both SARS-CoV-2 and MERS-CoV infection, allowing us to compare 264 host responses between the two lethal CoVs. We compared SARS-CoV-2 responses to both WT 265 MERS-CoV and mutant MERS-CoV-DNS4ab (Fig 7A) . Although we observed reduced MERS-

CoV-DNS4ab infectious virus production compared with WT MERS-CoV (Fig 5B) , we detected 267 similar intracellular viral genome levels of all three viruses (Fig 7B) . We found previously that CoV-DNS4ab by 48hpi (Fig 7A) . Similarly, SARS-CoV-2 generally induced more ISG mRNA than 274 WT MERS-CoV, and even more OAS2 mRNA than MERS-DNS4ab (Fig 7A) . Induction of CXCL8 275 was weak for all viruses (Fig 7A) . Notably, SARS-CoV-2 induced ISG mRNAs in Calu-3 (24hpi) 

We found that SARS-CoV-2 promotes rRNA degradation minimally at 24 hpi and more clearly at 285 48 hpi in A549 ACE2 , using SINV as a positive control (Fig 8A) . Evaluation of RNase L activation in (Fig 8B) . We also observed activation of PKR as 291 indicated by phosphorylation of PKR and downstream eIF2a, in both A549 ACE2 cells (Fig 8C) and 

The A549 ACE2 cells were valuable in that they provided a system with intact innate immune 299 responses that was also amenable to CRISPR-Cas9 engineering. Thus, we used the A549 ACE2 

A549 ACE2 cell lines by western blot (Fig 9A) and compared replication of SARS-CoV-2 in MAVS 305 KO, RNASEL KO and PKR KO cells with levels in WT A549 ACE2 cells (Fig 9B) . Interestingly, there 306 was little effect on SARS-CoV-2 replication with MAVS or PKR expression absent. In RNASEL 307 KO cells at 48 hpi, virus replication was two-to four-fold higher compared to WT A549 ACE2 cells 308 (Fig 9B) . While the difference in replication between RNASEL KO and WT was not extensive, it 309 was statistically significant in three independent experiments. Additionally, infected RNASEL KO 310 cells exhibited strikingly more CPE as compared with WT, PKR KO, or MAVS KO cells, as 311 demonstrated by crystal violet-staining of infected cells (Fig 9C) . We also assessed rRNA 312 degradation and, as expected, found that rRNA remained intact in the RNASEL KO A549 ACE2 313 cells, which further validated these cells. However, rRNA was degraded in PKR or MAVS KO 314 cells, indicating RNase L activation in both of these cell types (Fig 9D) . These data are consistent replication in infected nasal cell culture, we found that SARS-CoV-2 replicates to higher titer than 351 MERS-CoV, and the time period for shedding of virus is much longer (Fig 2A) . We suggest that 398 Zhao et al., 2011) . We found that SARS-CoV-2, like other betacoronaviruses, induced limited 399 amounts of type I and type III IFN mRNAs, although this was somewhat variable among the cell 400 types examined. Using SINV as a control for robust activation of IFN, we detected low levels of 401 type I and type III IFN mRNA in nasal cell, iAT2 cells, and iCM (Fig 2-4) . However, we observed 402 higher levels of OAS2, an ISG, relative to SINV in iAT2 cells (Fig 3D) . As we have observed (Fig S2A) . As major barrier cells, we speculate that this may be important for 409 protection as these cells are more exposed to infectious agents in the environment. Indeed, it is 410 well documented that type III IFNs serve as an added defense for epithelial cells, which may 414 Zhou et al., 1997) , which is possibly to protect the heart from excessive inflammation.

In A549 ACE2 cells, SARS-CoV-2 induced low levels of IFNl and IFNb mRNAs and somewhat 417 higher ISG mRNA by 48 hpi, as compared with SINV (Fig 6A) . We observed greater increases 418 in IFN induction in Calu-3 compared to A549 ACE2 (Fig 7A) , which may be at least partially due to 419 higher basal levels of IFNs in the Calu-3 cells (Fig S2) . Calu-3 cells were employed to directly 

We found that SARS-CoV-2 was unable to prevent activation of RNase L and PKR, although to infected iAT2 (Fig 3C) and iCM (one/two experiments) (Fig 4C) , but not in nasal cells (Fig 2C) .

However, we did not detect rRNA degradation indicative of RNase L activation in these cell types 443 (Fig 2E, 3E, 4E) . Activation of both RNase L and PKR were observed in A549 ACE2 and Calu-3 444 cells during infection with SARS-CoV-2 (Fig 8) . (Fig 9B&C) . In contrast, we found that PKR KO had no effect on viral titer. This is (Fig 1) . Interestingly, we observed possible RNase L-induced apoptosis in the SARS-

CoV-2 infected A549 ACE2 WT, MAVS KO, and PKR KO cells, when compared with mock infected 486 counterparts (Fig 9C) . However, RNASEL KO cells displayed the most cell death among the four 487 cell lines, suggesting that virus-induced cell lysis in the RNASEL KO cells where viral titers are 488 highest (Fig 9B) is more detrimental to cells than RNase L-induced programmed cell death. 

Our future studies will focus on identifying specific innate immunity antagonists among lineage b 512 betacoronavirus accessory proteins as well as conserved nsp proteins. The recent development Reverse Transcriptase Kit (Applied Biosystems). cDNA was amplified using specific RT-qPCR 692 primers (see Table below ), iQÔ SYBR Ò Green Supermix (Bio-Rad), and the QuantStudioÔ 3

PCR system (Thermo Fisher). Host gene expression displayed as fold change over mock-infected 

Emerging Pathogens (SRW, YL)

NIH grants U01HL148857, R01HL087825, U01HL134745 and

VA administration grant CX001617 (NAC); NIH grants U01TR001810

Perelman School of Medicine to the iPSC Core and by NIH grant U01TR001810. DMR was 743 supported in part by T32-AI055400 and CEC was supported in part by

BD 749 Writing -Original Draft: SRW, JNW 750 Writing -Review & Editing

An important role for type III interferon 761 (IFN-lambda/IL-28) in TLR-induced antiviral activity

Middle East Respiratory Syndrome

Cell-type-specific 768 effects of RNase L on viral induction of beta interferon

The Ebola virus VP35 protein inhibits activation of 772 interferon regulatory factor 3

Activation of the SARS coronavirus spike 775 protein via sequential proteolytic cleavage at two distinct sites

Murine coronavirus receptors are 779 differentially expressed in the central nervous system and play virus strain-dependent roles in 780 neuronal spread

Activation of RNase L by murine coronavirus in myeloid cells is dependent on 784 basal Oas gene expression and independent of virus-induced interferon

Imbalanced Host Response to SARS-CoV-2 Drives 788 Development of COVID-19

Evolutionary origins of the SARS-CoV-2 sarbecovirus lineage responsible for the 792 COVID-19 pandemic

MERS-CoV 4b protein interferes 796 with the NF-kappaB-dependent innate immune response during infection

A study of the interferon antiviral mechanism: apoptosis activation by the 2-801 5A system

RNase L activates the NLRP3 inflammasome during viral infections

Dysregulated Type I Interferon and Inflammatory Monocyte-Macrophage Responses 809 Cause Lethal Pneumonia in SARS-CoV-Infected Mice

IFN-I response timing 813 relative to virus replication determines MERS coronavirus infection outcomes

Association of Cardiac Infection With 948 SARS-CoV-2 in Confirmed COVID-19 Autopsy Cases

How Genomic Information has been Used to Deal with Past 952 Outbreaks and the COVID-19 Pandemic

Large, Diverse Population Cohorts of hiPSCs and 992 Derived Hepatocyte-like Cells Reveal Functional Genetic Variation at Blood Lipid

Coronaviruses post-SARS: update on replication and 996 pathogenesis

Mechanisms of type-I-and type-II-interferon-mediated signalling

Multiorgan and Renal Tropism of 1003 SARS-CoV-2

Innate immune response of human alveolar type II cells infected with severe 1007 acute respiratory syndrome-coronavirus

Endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus 1012 type 2 spike-mediated entry

Middle East Respiratory Coronavirus 1016 Accessory Protein 4a Inhibits PKR-Mediated Antiviral Stress Responses

Murine coronavirus mouse hepatitis virus 1020 is recognized by MDA5 and induces type I interferon in brain macrophages/microglia

Interferon-inducible antiviral effectors

High 1027 Contagiousness and Rapid Spread of Severe Acute Respiratory Syndrome Coronavirus 2. 1028 Emerging infectious diseases

Reverse genetics with a full-1032 length infectious cDNA of the Middle East respiratory syndrome coronavirus

Human iPSC-Derived Cardiomyocytes Are Susceptible to SARS-CoV-2 1037 Infection

Association of Cardiac Injury With Mortality in Hospitalized Patients With COVID-19 in

Release of severe acute 1045 respiratory syndrome coronavirus nuclear import block enhances host transcription in human 1046 lung cells

Middle east respiratory syndrome coronavirus 4a protein is a double-1050 stranded RNA-binding protein that suppresses PACT-induced activation of RIG-I and MDA5 in 1051 the innate antiviral response

Continuous and Discontinuous RNA 1054 Synthesis in Coronaviruses

The JAK-STAT pathway at twenty

Identification of 1059 adult mouse neurovirulence determinants of the Sindbis virus strain AR86

Rapid reconstruction of SARS-CoV-2 using a 1064 synthetic genomics platform

Middle East Respiratory Syndrome Coronavirus NS4b 1068 Protein Inhibits Host RNase L Activation

Association between 1071 interleukin-8 concentration in nasal secretions and severity of symptoms of experimental 1072 rhinovirus colds

Proteomics. Tissue-based map of the 1077 human proteome. Science

Coronavirus 1080 Endoribonuclease and Deubiquitinating Interferon Antagonists Differentially Modulate the Host 1081 Response during Replication in Macrophages

Receptor recognition by novel 1084 coronavirus from Wuhan: An analysis based on decade-long structural studies of SARS

Coronavirus pathogenesis and the emerging pathogen 1088 severe acute respiratory syndrome coronavirus. Microbiology and molecular biology reviews : 1089 MMBR 69

Zika Virus Production Is Resistant to 1092

Evasion of type-I interferon by SARS-CoV-2

An Infectious cDNA Clone of SARS-CoV-2. Cell host & 1099 microbe

Proteolytic activation of the spike protein at a novel RRRR/S 1102 motif is implicated in furin-dependent entry, syncytium formation, and infectivity of 1103 coronavirus infectious bronchitis virus in cultured cells

Generation of iPSCs as a Pooled Culture Using Magnetic Activated Cell Sorting of Newly 1107 Reprogrammed Cells

Mouse hepatitis 1110 coronavirus A59 nucleocapsid protein is a type I interferon antagonist

Cell-type-specific activation of the oligoadenylate synthetase-1115

RNase L pathway by a murine coronavirus

Antagonism of the interferon-induced OAS-RNase L pathway by murine coronavirus 1119 ns2 protein is required for virus replication and liver pathology

Cell-type-specific type I 1123 interferon antagonism influences organ tropism of murine coronavirus

Interferon action and apoptosis are defective in mice devoid of 1128 2',5'-oligoadenylate-dependent RNase L

A pneumonia outbreak associated with a new coronavirus of probable bat origin. 1132 Nature. 1133 1134 three independent experiments. Scale bar = 100µm. (C) At 48 hours post infection, cells were 1179 lysed and proteins were analyzed by immunoblotting with antibodies as indicated. Data shown are 1180 from one representative experiment of two independent experiments

CoV-2) hpi, total RNA was harvested, and the mRNA expression level of IFNB

CT values were normalized to 18S rRNA to 1183 generate DCT values (DCT = CT gene of interest -CT 18S rRNA). Fold change over mock values 1184 were calculated by subtracting mock infected DCT values from virus infected DCT values, 1185 displayed as 2 -Δ(ΔCt)

Statistical significance was determined by Student t test (*, P < 0

Data shown are from one representative experiment of two independent 1188 experiments. (E) Total RNA was harvested at 16 (SINV) or 48 (SARS-CoV-2) hpi and rRNA integrity 1189 determined by Bioanalyzer. The position of 28S and 18S rRNA and indicated. Data shown are from 1190 one representative experiment of two independent experiments

SARS-CoV-2. iCM were mock 1193 infected or infected at MOI=1 with SARS-CoV-2 or SINV. (A) At indicated times, supernatants 1194 were collected and virus quantified by plaque assay on Vero-E6 cells. Values are means ± SD 1195 (error bars). Data shown are one representative experiment from at least three independent 1196 experiments. (B) At 48 hpi, iCM were fixed with 4% PFA and permeabilized, the expression of 1197 SARS-CoV-2 N (green) of and

Channels are merged with DAPI nuclear staining. Images shown are representative from 1199 three independent experiments. Scale bar = 50µm. (C) At 16 (SINV) or 48 (SARS-CoV-2) hpi, 1200 cells were lysed and proteins were analyzed by immunoblotting with antibodies as indicated

Immunoblots were performed at least two times and one representative blot is shown

SARS-CoV-2) hpi, total RNA was harvested, the mRNA expression level of IFNB, total RNA were calculated by RT-qPCR standard curve generated using a digested plasmid 1254 encoding SARS-CoV-2 nsp12 or plasmid encoding a region of MERS-CoV orf1ab. Values are 1255 means ± SD (error bars)

0.01; ns = not significant). (C) At 24 hpi, Calu-3 cells were lysed and proteins harvested

All data are one 1258 representative experiment of three independent experiments

SARS-CoV-2 infection leads to activation of RNase L and PKR in A549 ACE2 and 1261

Calu-3 cells. A549 ACE2 and Calu-3 cells were mock infected or infected with SARS-CoV-2

ΔNS4ab at MOI=5. Total RNA was harvested from A549 ACE2 cells (A) or

At 24 hpi, A549 ACE2 cells (C) or Calu-3 cells (D) were lysed 1265 and proteins harvested for analysis by immunoblotting using the indicated antibodies. All data are 1266 one representative experiment of three independent experiments

Replication of SARS-CoV-2 is restricted by RNase L independent of PKR or

Indicated genes were knocked out (KO) from one clone of A549 ACE2 cells using CRISPR

Statistical significance was determined by 1275 two-way ANOVA (****, P < 0.0001; ns = not significant). Data are one representative experiment 1276 from at least three independent experiments. (C) Indicated cell lines were mock treated or infected 1277 with SARS-CoV-2 at MOI=1. At 48 hpi, cells were fixed with 4% PFA and stained with 1% crystal 1278 violet as a marker for live cells. The image is one representative experiment from two independent A549 ACE2 cell line clone (C34). (A) Vero-E6 or A549 ACE2 cells were infected with SARS-CoV-2 at 1310 MOI=1 and supernatant harvested at indicated times post infection. Infectious virus was quantified 1311 by plaque assay on Vero-E6 cells. Values are means ± SD (error bars). (B) A549 ACE2 cells (C34) 1312 were mock infected or infected with SARS-CoV-2 or SINV at MOI=5 and total RNA total RNA 1313

Fold change over mock values 1316 were calculated by subtracting mock infected DCT values from virus infected DCT values, 1317 displayed as 2 -Δ(ΔCt) . Statistical significance for each gene was determined by one-way ANOVA 1318

Technical replicates were averaged, the 1319 means for each replicate displayed, ± SD (error bars). (C&D) A549 ACE2 cells were infected at

MOI=5, lysed at 24 hpi, and proteins harvested for analysis by immunoblotting using the indicated 1321 antibodies. (E) A549 ACE2 cells were infected at MOI=1 (SINV) or MOI=5 (SARS-CoV-2) and total 1322 RNA harvested at 24 (SINV) or 24 and 48 (SARS-CoV-2) hpi

28S and 18s rRNA bands are indicated. All data are representative of two or three 1324 independent experiments

We thank Nicholas Parenti for technical help and Dr. Nikki Tanneti for reading the manuscript.

This work was supported by NIH grants AI140442 and supplement for SARS-CoV-2 (SRW),

Channels are merged with DAPI nuclear staining. Images shown are representative from at least