key: cord-0924252-d6k7zrgo authors: Geerling, Elizabeth; Pinski, Amanda N.; Stone, Taylor E.; DiPaolo, Richard J.; Zulu, Michael Z.; Maroney, Kevin J.; Brien, James D.; Messaoudi, Ilhem; Pinto, Amelia K. title: Roles of antiviral sensing and type I interferon signaling in the restriction of SARS-CoV-2 replication date: 2021-12-03 journal: iScience DOI: 10.1016/j.isci.2021.103553 sha: 9b466c184c328b715521f4dbb83bc13dc27ec74f doc_id: 924252 cord_uid: d6k7zrgo Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019. Few studies have compared replication dynamics and host responses to SARS-CoV-2 in cell lines from different tissues and species. Therefore, we investigated the role of tissue type and antiviral genes during SARS-CoV-2 infection in nonhuman primate (kidney) and human (liver, respiratory epithelial, gastric) cell lines. We report different viral growth kinetics and release among the cell lines despite comparable ACE2 expression. Transcriptoimcs revealed that absence of STAT1 in nonhuman primate cells appeared to enhance inflammatory responses without effecting infectious viral titer. Deletion of RL-6 in respiratory epithelial cells increased viral replication. Imparied infectious virus release was detected in Huh7 but not Huh7.5 cells, suggesting a role for RIG1. Gastric cells MKN45 exhibited robust antiviral gene expression and supported viral replication. Data here provide insight into molecular pathogenesis of and alternative cell lines for study SARS-CoV-2 infection. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a positive-sense, single-39 stranded RNA virus belonging to Coronaviridae family and Betacoronavirus genus (Lu et The 370 DEGs unique to MKN45 enriched to GO terms associated with antiviral defense (e.g., 284 "response to virus", "type I interferon signaling pathway"), leukocyte immunity (e.g., "leukocyte 285 differentiation"), cytokine production (e.g., "regulation of cytokine production") and apoptosis (e.g., 286 "positive regulation of cell death") ( Figure 5C ). Expression analysis revealed that DEGs enriching to GO 287 terms related to antiviral processes, cell death and cytokine production were primarily upregulated ( Figure 288 5D ). This included genes encoding type I and III interferon (e.g., IFNB, IFNL1/2/3), key components of T 289 cell-mediated immunity (e.g., CX3CL1, JAK3, LAG3) and pro-apoptotic mediators (e.g., APBB1, KLF11) 290 ( Figure 5D ). Similar to MKN45, DEGs unique to AGS also enriched to GO terms with roles in antiviral 291 immunity (e.g., "response to virus"; APOBEC3D, IFITM2), cell stress and protein unfolding (e.g., 292 J o u r n a l P r e -p r o o f "response to unfolded protein"; ASNS, NOX1) and leukocyte-mediated immunity (e.g., "T cell 293 proliferation"; LGALS9) ( Fig.4C,E) . Most unique DEGs detected in AGS were upregulated ( Figure 5E) . Cell susceptibility and permissivity to SARS-CoV-2 infection is regulated by species, tissue type, 297 receptor expression and mobilization of host antiviral defense. In particular, the expression of ACE2 and 298 genes involved in antiviral type I IFN signaling dictate the ability and kinetics of SARS-CoV-2 replication 299 in vitro and in vivo. There is an urgent need for more data to inform the development of novel antiviral 300 therapies against SARS-CoV-2 and other oncornaviruses using in vitro models. In this study, we examined 301 the replication kinetics of SARS-CoV-2 in nonhuman and human cell lines lacking or possessing intact type 302 I IFN signaling machinery. We further characterized transcriptional responses to understand molecular basis 303 for differences in cell permissiveness and replicative capacity of SARS-CoV-2. 304 We noted peaks in intracellular viral replication at 24 HPI while peaks in cell-free virus occurred 305 later (48-96 HPI) for most cell lines, suggesting a delay in viral egress. Despite similar levels of ACE2 306 protein expression and peak levels of intracellular viral RNA, levels of infectious virus particles were lower 307 in the supernatant of Huh7 cells compared to Vero WHO and Huh7.5 cells. Transcriptional analysis 308 revealed that these two cell lines are divergent in several pathways that could explain the differences in 309 infectious virus production. Chief amongst these differences are genes that play a critical role in innate 310 immune responses to microbes (e.g decreased expression of ISG-15, IL-32 and STAT-6) as well as lipid between uninfected Huh7.5 and Huh7 that could contribute to increased virus release in Huh7.5 cells. For 315 instance, expression of CAV1 was increased in Huh7.5. Caveolin 1 plays a critical role for virus trafficking, 316 assembly, and egress (Xing et al., 2020) . Similarly, levels of CXCL5 were increased in Huh 7.5 and levels 317 of this chemokine correlated with increased SARS-CoV-2 replication in a hACE-2 transgenic mouse 318 model(Liang et al., 2020). Increased expression of anti-apoptotic genes (e.g. BCL10) could also facilitate 319 viral production (Kvansakul, 2017 , Liang et al., 2015 . The role of these various changes in modulating virus 320 production still remains to be determined empirically using knock in and knock down experiment. Finally, 321 as described in these previous studies, we did not detect significant induction of ISGs or antiviral defense 322 genes, as expected given the absence of functional IFNA10, TRIM56 (both Huh7 and Huh7.5) and RIG-I 323 (Huh7.5 only). 324 Additionally, several genes encoding translation initiation/elongation factors and ribosomal subunits 325 (e.g., EIF4EBP, RPL10/11/12) were uniquely upregulated in Huh7 cells, suggesting that, despite adequate 326 J o u r n a l P r e -p r o o f protein synthesis machinery, Huh7 cells are incapable of supporting infectious virion production. This was 327 accompanied by the upregulation of inflammatory and chemotactic genes, suggesting a more a robust 328 antiviral response to SARS-CoV-2 in Huh7 cells than in Huh7.5 cells. Interestingly, genes related to 329 membrane morphology/dynamics and cell adhesion (e.g., NPC2, RAB11FIP1) were unique to Huh7.5 and 330 downregulated, suggesting viral egress potential may be altered in Huh 7.5. For instance, NPC2 has been 331 shown to interact with ORF8 and its suppression maybe required for virion morphogenesis, in line with 332 greater infectious virus titers in Huh7 associated with innate defense such as those involved inflammation (e.g., ANXA1), oxidative stress (e.g., 343 SOD1) and apoptosis (e.g., BAK1) was altered in both cell lines. The upregulation of pro-inflammatory and 344 pro-apoptotic genes in Vero WHO, but not Vero E6, suggests greater sensitivity of these cells to SARS-345 CoV-2 infection and NFκB-mediated, inflammatory responses. Additionally, both Vero E6 and Vero WHO 346 also downregulated genes related to protein-folding; a process commonly seen in viral infection (Paladino 347 et al., 2020) . These results collectively support the greater viral replication in Vero WHO, while the higher 348 levels of infectious SARS-CoV-2 in Vero E6 may be associated with the greater number of translation-and 349 cell cycle-associated genes. There has been concern that in vitro passaging can induce mutations in SARS-CoV-2 (Davidson et 393 294 upregulated in Huh7.5 308 downregulated in Huh7.5 J o u r n a l P r e -p r o o f SARS-CoV-2 cell tropism and 574 multiorgan infection Identification of Common Deletions in the Spike 578 Protein of Severe Acute Respiratory Syndrome Coronavirus 2 More Than 50 Long-Term Effects of COVID-19: A 581 Systematic Review and Meta-Analysis Genomic 586 characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and 587 receptor binding COVID-19) vs Patients With Influenza Orf6 hijacks Nup98 to block STAT nuclear import and antagonize interferon signaling Exogenous ACE2 expression allows refractory cell lines to support severe acute respiratory 602 syndrome coronavirus replication SARS-CoV-2 tropism, entry, replication, and propagation: Considerations for drug 606 discovery and development SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid 610 adaptation and cytopathology The genome landscape of the african green monkey kidney-derived vero 613 cell line The Role of Molecular 616 Chaperones in Virus Infection and Implications for Understanding and Treating COVID-19 SARS-CoV-2 variants 620 reveal features critical for replication in primary human cells Overcoming Culture 624 Restriction for SARS-CoV-2 in Human Cells Facilitates the Screening of Compounds Inhibiting 625 Viral Replication Analysis of ACE2 in 628 polarized epithelial cells: surface expression and function as receptor for severe acute respiratory 629 syndrome-associated coronavirus Suramin Inhibits SARS-CoV-2 Infection in Cell Culture by Interfering with Early Steps of the 633 Replication Cycle SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are 636 generated in vitro during propagation in TMPRSS2-deficient cells Cell entry 638 mechanisms of SARS-CoV-2 641 Localization of endogenous furin in cultured cell lines An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in 648 human airway epithelial cell cultures and multiple coronaviruses in mice SARS-CoV-2 suppresses IFNbeta production mediated by 652 NSP1, 5, 6, 15, ORF6 and ORF7b but does not suppress the effects of added interferon Viral encounters with 2',5'-oligoadenylate synthetase and RNase L during the 655 interferon antiviral response Regulating intracellular antiviral defense and permissiveness to hepatitis C 658 virus RNA replication through a cellular RNA helicase, RIG-I Type III Interferons Restrict SARS-CoV-2 Infection of Human Airway Epithelial Cultures Genome Sequence of the Parainfluenza Virus 5 Strain That Persistently Infects AGS Cells How RIG-I like receptors activate MAVS Evasion of Type I Interferon by SARS-CoV-2 Caveolae/Caveolin-1 in Virus Infections. Viruses RIG-I triggers a signaling-abortive anti-SARS-CoV-2 defense in human lung 675 cells Virus replication in engineered human cells that 678 do not respond to interferons AGS and other 680 tissue culture cells can unknowingly be persistently infected with PIV5; a virus that blocks 681 interferon signalling by degrading STAT1 SARS-CoV-2 nsp13, nsp14, nsp15 and orf6 684 function as potent interferon antagonists TMPRSS2 and TMPRSS4 promote SARS-CoV-2 688 infection of human small intestinal enterocytes Specific 690 ACE2 expression in small intestinal enterocytes may cause gastrointestinal symptoms and injury 691 after 2019-nCoV infection Clinical Characteristics of Children with Coronavirus 696 Disease A pneumonia outbreak 701 associated with a new coronavirus of probable bat origin CHINA 704 NOVEL CORONAVIRUS, I. & RESEARCH, T. 2020. A Novel Coronavirus from Patients with 705 Pneumonia in China SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway 719 Lead contact 801Further information and requests for resources and reagents should be dreicted to and will be fulfilled by 802 J o u r n a l P r e -p r o o f hACE2 expression was measured by qRT-PCR using Taqman primer and probe sets from IDT (assay ID 829 Hs.PT.58.27645939). SARS-CoV-2 viral burden was measured by qRT-PCR using Taqman primer and 830 probe sets from IDT with the following sequences: Forward 5' GAC CCC AAA ATC AGC GAA AT 3', 831Reverse 5' TCT GGT TAC TGC CAG TTG AAT CTG 3', Probe 5' ACC CCG CAT TAC GTT TGG TGG 832 ACC 3'. Synthesized hACE2 RNA was used as a copy control to quantify the number of hACE2 molecules 833 present in each sample. Similarly, a SARS-CoV-2 copy number control (available from BEI) was used to 834 quantify SARS-CoV-2 genomes. were identified using edgeR was used with the following criteria: genes with median rpkm of ≥5, a false 899 discovery rate (FDR) corrected p-value ≤ 0.05 and a log2fold change ≥ 1. To identify commonly regulated 900 genes with adjustments for cell-specific differences, edgeR was performed with GLM capabilities. Batch-901 effects were adjusted for using the Limma R package. 902Functional enrichment of DEGs was performed using Metascape to identify relevant Gene Ontology 903 (GO) biological process terms 78 . Heatmaps, bubbleplots, Venn diagrams and violin plots were generated 904 using R packages ggplot2 and VennDiagrams. GO network plots were generated in Cytoscape (Version 905 3.5.1). Graphs were generated using GraphPad Prism software (version 8). 906 Kruskal-Wallis test with multiple comparisons was used to compare ACE2 mRNA and protein expression 909 among cell lines. Standard deviation displayed on all bar graphs. p-value ≤ 0.05*, p-value ≤ 0.01**. 910 MAFK PLPP3 ID3 IL11 SLC39A10 CCN1 ID2 TNFSF15 ASNS C11orf86 CACNG6 ID1 PHGDH SFRP1 CD55 DYNLT3 TYMS ZWINT H3C13 H4C9 ADH1C H2AC12 H4C4 H4C1 NUSAP1 H3C1 TOP2A CCNB1 RACGAP1 H2BC10 H2AC4 KIF22 H4C15 H2AC18 NCAPD2 H2BC5 H2BC14 H2BC6 PRC1 H2BC18 HABP2 TIMELESS MYBL2 H2AC14 TK1 PBK H4C13 UBE2C MKI67 CENPF H2AC13 STMN1 H2BC3 H3C14 MELK H3C15 CCNB2 TPX2 PTTG1 CDC20 H2AC21 H4C14 MCM2 H2AC15 MUC5AC H3C12 E2F1 H4C12 H2BC21 H3C10 CCNA2 H2BC17 H2BC11 INCENP LCN2 H2AX H2AC19 H2BC4 H2AC11 H2BC13 uninfected infected