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. 2022 Jan 21;25(1):103553.
doi: 10.1016/j.isci.2021.103553. Epub 2021 Dec 3.

Roles of antiviral sensing and type I interferon signaling in the restriction of SARS-CoV-2 replication

Elizabeth Geerling  1 , Amanda N Pinski  2 , Taylor E Stone  1 , Richard J DiPaolo  1 , Michael Z Zulu  2 , Kevin J Maroney  2 , James D Brien  1 , Ilhem Messaoudi  2 , Amelia K Pinto  1
Affiliations

Roles of antiviral sensing and type I interferon signaling in the restriction of SARS-CoV-2 replication

Elizabeth Geerling et al. iScience. .

Abstract

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. Transcriptomics 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. Impaired 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 studying SARS-CoV-2 infection.

Keywords: Immunology; Microbiology; Virology.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

None
Graphical abstract
Figure 1
Figure 1
Viral replication and growth kinetics, and ACE2 expression in immortalized nonhuman primate and human cell lines SARS-CoV-2 genome copy number per microliter in cells (A) or supernatants (B) as determined by qRT-PCR for human (Huh7/7.5, A549, AGS, MKN45) and nonhuman primate cell lines (Vero E6/WHO) infected at an MOI of 0.5 with SARS-CoV-2 and sampled at 24-h intervals. Total cellular RNA was extracted and normalized to an RNaseP and GADPH control as well as an in-house virus copy control. (C) Infectious virus in supernatant was quantified at 48 h by focus-forming assay (LOD = 10 FFU/ml). Quantification of ACE2 (D) mRNA (p = 0.048 AS versus Vero E6; p = 0.048 AGS versus Vero WHO) and (E) protein expression (p = 0.048 AGS versus Huh7.5; p = 0.004 AGS versus MKN45) by qRT-PCR and western blot, respectively, in infected cell lines.
Figure 2
Figure 2
Transcriptional analysis of SARS-CoV-2-infected VeroE6 and Vero WHO cell lines (A) Venn diagram of differentially expressed genes (DEGs) for SARS-CoV-2-infected Vero E6 and Vero WHO cell lines relative to uninfected samples. (B) Bubbleplot representing functional enrichment of DEGs unique to Vero E6 and Vero WHO. Color intensity of each bubble represents the –log(q-value), and the relative size of each bubble represents the number of DEGs belonging to the specified Gene Ontology (GO) term. (C and D) Heatmaps representing DEGs enriching to GO terms in (B) “positive regulation of cell death” and “response to oxidative stress” for (C) Vero E6 and (D) Vero WHO. Columns of all heatmaps represent the reads per kilobase per million mapped reads (rpkm) of an individual uninfected or infected sample. Range of colors for each heatmap is based on scaled and centered rpkm values of the represented DEGs. (E) Volcano plot of global gene expression changes shared between Vero E6 and Vero WHO. DEGs (average rpkm ≥5) are denoted in red. Exemplar DEGs are labeled. (F) Functional enrichment of DEGs shared by both Vero E6 and Vero WHO. Horizontal bars represent the number of genes enriching to each GO term with color intensity representing the negative log of the FDR-adjusted p value (-log(q-value)). Range of colors based on the GO terms with the lowest and highest –log(q-value) values. (G) Beanplots representing the expression (rpkm) of the indicated gene in uninfected and infected Vero E6 or Vero WHO cells. Median and standard deviation plotted.
Figure 3
Figure 3
Transcriptional analysis of SARS-CoV-2-infected Huh7 and Huh7.5 cell lines (A) Volcano plot of global gene expression changes in uninfected Huh7.5 relative to uninfected Huh7. DEGs (average rpkm ≥5) are denoted in red. Exemplar DEGs are labeled. (B) Venn diagram of differentially expressed genes (DEGs) for SARS-CoV-2-infected Huh7 and Huh7.5 cell lines relative to uninfected samples. (C) Bubbleplot representing functional enrichment of DEGs unique to Huh7 and Huh7.5. Color intensity of each bubble represents the –log(q-value) and the relative size of each bubble represents the number of DEGs belonging to the specified Gene Ontology (GO) term. (D–F) Heatmap in (D) represents GO term “myeloid leukocyte mediated immunity” unique to Huh7.5. Heatmaps representing DEGs from GO terms in (C) blood vessel development,” “response to wounding,” and “response to extracellular stimulus” for (E) Huh7 and (F) Huh7.5. Columns of all heatmaps represent the reads per kilobase per million mapped reads (rpkm) of an individual uninfected or infected sample. Range of colors per heatmap is based on scaled and centered rpkm values of the represented DEGs. (G) Volcano plot of global gene expression changes shared between infected Huh7 and Huh7.5. DEGs (average rpkm ≥5) are denoted in red. Exemplar DEGs are labeled. (H) Functional enrichment of DEGs shared by both Huh7 and Huh7.5. Horizontal bars represent the number of genes enriching to each GO term with color intensity representing the negative log of the FDR-adjusted p value (-log(q-value)). Range of colors based on the GO terms with the lowest and highest –log(q-value) values. (I) Beanplots representing the rpkm of the indicated gene in uninfected and infected Huh7 or Huh7.5 cells. Median values for each condition are indicated.
Figure 4
Figure 4
Transcriptional analysis of SARS-CoV-2-infected A549 wild-type and knockout cell lines (A) Bar graph of DEGs detected in SARS-CoV-2-infected wild-type (WT) and knockout (KO) A549 cell lines relative to uninfected counterparts. (B) Bubbleplot representing functional enrichment of WT infection DEGs. Color intensity of each bubble represents the –log(q-value), and the relative size of each bubble represents the number of DEGs belonging to the specified Gene Ontology (GO) term. (C) Heatmap representing all WT DEGs. Columns of heatmap represent the reads per kilobase per million mapped reads (rpkm) of an individual uninfected or infected sample. Range of colors per heatmap is based on scaled and centered rpkm values of the represented DEGs. (D) Venn diagram of DEGs in (A) for KO only. (E) GO network depicting functional enrichment of the 40 DEGs expressed by all KO using Metascape. Clustered nodes of identical color correspond to one GO term. Node size represents the number of DEGs associated with the GO term. Gray lines represent shared interactions between GO terms, with density and number indicating the strengths of connections between closely related GO terms.
Figure 5
Figure 5
Transcriptional analysis of SARS-CoV-2-infected AGS and MKN45 cell lines (A) Venn diagram of differentially expressed genes (DEGs) for SARS-CoV-2-infected AGS and MKN45 cell lines relative to uninfected samples. (B) Violin plot representing the log2(fold-change) of the eight DEGs shared by both cell lines. (C) Bubbleplot representing functional enrichment of DEGs unique to AGS and MKN45. Color intensity of each bubble represents the –log(q-value), and the relative size of each bubble represents the number of DEGs belonging to the specified Gene Ontology (GO) term. (D and E) Heatmaps of (D) DEGs unique to MKN45 and enriching to GO terms “response to virus” (orange), “positive regulation of cell death” (green), and “regulation of cytokine production” (blue), and (E) all DEGs unique to AGS. Columns of heatmaps represent the reads per kilobase per million mapped reads (rpkm) of an individual uninfected or infected sample. Range of colors per heatmap is based on scaled and centered rpkm values of the represented DEGs.
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References

    1. Alexopoulou L., Holt A.C., Medzhitov R., Flavell R.A. Recognition of double-stranded RNA and activation of Nf-Kappab by toll-like receptor 3. Nature. 2001;413:732–738. - PubMed
    1. Barranco S.C., Townsend C.M., Jr., Casartelli C., Macik B.G., Burger N.L., Boerwinkle W.R., Gourley W.K. Establishment and characterization of an in vitro model system for human adenocarcinoma of the stomach. Cancer Res. 1983;43:1703–1709. - PubMed
    1. Beauclair G., Streicher F., Chazal M., Bruni D., Lesage S., Gracias S., Bourgeau S., Sinigaglia L., Fujita T., Meurs E.F., et al. Retinoic acid inducible gene I and protein kinase R, but not stress granules, mediate the proinflammatory response to yellow fever virus. J. Virol. 2020;94 - PMC - PubMed
    1. Bollavaram K., Leeman T.H., Lee M.W., Kulkarni A., Upshaw S.G., Yang J., Song H., Platt M.O. Multiple sites on Sars-Cov-2 spike protein are susceptible to proteolysis by cathepsins B, K, L, S, and V. Protein Sci. 2021;30:1131–1143. - PMC - PubMed
    1. Chatel-Chaix L., Cortese M., Romero-Brey I., Bender S., Neufeldt C.J., Fischl W., Scaturro P., Schieber N., Schwab Y., Fischer B., et al. Dengue virus perturbs mitochondrial morphodynamics to Dampen innate immune responses. Cell Host Microbe. 2016;20:342–356. - PMC - PubMed