Bulk and single-cell gene expression profiling of SARS-CoV-2 infected human cell lines identifies molecular targets for therapeutic intervention

Abstract

The coronavirus disease 2019 pandemic, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an ongoing global health threat with more than two million infected people since its emergence in late 2019. Detailed knowledge of the molecular biology of the infection is indispensable for understanding of the viral replication, host responses, and disease progression. We provide gene expression profiles of SARS-CoV and SARS-CoV-2 infections in three human cell lines (H1299, Caco-2 and Calu-3 cells), using bulk and single-cell transcriptomics. Small RNA profiling showed strong expression of the immunity and inflammation-associated microRNA miRNA-155 upon infection with both viruses. SARS-CoV-2 elicited approximately two-fold higher stimulation of the interferon response compared to SARS-CoV in the permissive human epithelial cell line Calu-3, and induction of cytokines such as CXCL10 or IL6. Single cell RNA sequencing data showed that canonical interferon stimulated genes such as IFIT2 or OAS2 were broadly induced, whereas interferon beta (IFNB1) and lambda (IFNL1-4) were expressed only in a subset of infected cells. In addition, temporal resolution of transcriptional responses suggested interferon regulatory factors (IRFs) activities precede that of nuclear factor-κB (NF-κB). Lastly, we identified heat shock protein 90 (HSP90) as a protein relevant for the infection. Inhibition of the HSP90 charperone activity by Tanespimycin/17-N-allylamino-17-demethoxygeldanamycin (17-AAG) resulted in a reduction of viral replication, and of TNF and IL1B mRNA levels. In summary, our study established in vitro cell culture models to study SARS-CoV-2 infection and identified HSP90 protein as potential drug target for therapeutic intervention of SARS-CoV-2 infection.

A direct RNA-protein interaction atlas of the SARS-CoV-2 RNA in infected human cells

“…In agreement with recent transcriptome studies [5][6][7] , our proteome data suggest activation of interferon signaling upon SARS-CoV-2 infection. Among interferon-related genes, we observed significant upregulation of several major components of IFN signaling pathways, including STAT1 and IRF9, which together with STAT2 make up the ISGF3 complex, their upstream components TYK2 and JAK1, as well as their downstream targets IFIT1, IFIT3, IFITM3, OAS2, and ISG15 ( Figure 3a).…”

“…Only 10 of the 334 human proteins that were found to interact with viral proteins in uninfected cells, also bound directly to viral RNA in infected cells (Supplementary Table 2). Beyond the well-documented impact of viral infection on host cell gene expression [5][6][7] , which may lead to remodeling of observed interactions, these results further highlight the importance of discriminating between proteinprotein and RNA-protein interactions when dissecting the biology of SARS-CoV-2.…”

“…Two recent studies showed that CNBP activates the innate immune response and induces the expression of several pro-inflammatory cytokines, including Il-6 and Cxcl10 in vivo 78,79 . While our proteomics experiments did not yield peptides for these two CNBP-regulated cytokines, recent transcriptome studies demonstrated that IL-6 and CXCL10 were indeed induced in SARS-CoV-2 infected human cells [5][6][7] . In light of these results, we speculated that in addition to its previously described role in foreign DNA sensing, CNBP might recognize the SARS-CoV-2 RNA through direct binding, which may be linked to its role in transcriptionally activating specific innate immune genes.…”

“…p = 0.045; Supplementary Table 6). Newly emerging evidence indicates that these pathways are indeed highly relevant in the context of SARS-CoV-2 infections 5,[7][8][9][10]42 . Notably, inhibition of growth factor signaling through the MAPK pathway, which responds to and controls the production of pro-inflammatory cytokines, including TNFα and IL-6, was recently shown to modulate SARS-CoV-2 replication in infected cells [8][9][10] .…”

“…To understand this interplay between virus and host, it is essential to characterize with molecular detail which host proteins make direct contact with viral RNA and may function as host dependency factors or antiviral regulators. To date, studies on SARS-CoV-2 infected human cells have primarily focused on characterizing expression or modification changes in the host cell transcriptome [5][6][7] or proteome [8][9][10] . While several studies described protein-protein interactions of recombinantly expressed viral proteins in uninfected cells 10,11 , no study has comprehensively identified direct interactions between viral RNA and the host cell proteome in infected human cells.…”