Transcriptomic landscapes of SARS-CoV-2-infected and bystander lung cells reveal a selective upregulation of NF-κB-dependent coding and non-coding proviral transcripts

Detailed knowledge of cellular networks that are modulated by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to understand viral replication and host response. So far, transcriptomic analyses of interactions between SARS-CoV-2 and cells were performed on mixed populations of infected and uninfected cells or using single-cell RNA sequencing, both leading to inaccurate or low-resolution gene expression interpretations. Moreover, they generally focused on annotated messenger RNAs (mRNAs), ignoring other transcripts, such as long non-coding RNAs (lncRNAs) and unannotated RNAs. Here, we performed deep polyA+ transcriptome analyses of lung epithelial A549 cells infected with SARS-CoV-2, which were sorted based on the expression of the viral protein spike (S). To increase the sequencing depth and improve the robustness of the analysis, the samples were depleted of viral transcripts. Infection caused a massive reduction in mRNAs and lncRNAs, including transcripts coding for antiviral innate immune proteins, such as interferons (IFNs). This absence of IFN response probably explains the poor transcriptomic response of bystander cells co-cultured with spike positive (S+) ones. NF-{kappa}B and inflammatory response were among the pathways that escaped the global shutoff in S+ cells. In agreement with the RNA-seq analysis, inflammatory cytokines, but not IFNs, were produced and secreted by infected cells. Functional investigations revealed the proviral function of the NF-kB subunit p105/p50 and some of its known target genes, including IL32 and IL8, as well as the lncRNA ADIRF-AS1, which we identified as a novel NF-kB target gene. Thus, analyzing the polyA+ transcriptome of sorted populations of infected lung cells allowed unprecedented identification of cellular functions that are directly affected by infection and the recovery of coding and non-coding genes that contribute to SARS-CoV-2 replication.

Identification of DAXX As A Restriction Factor Of SARS-CoV-2 Through A CRISPR/Cas9 Screen

Interferon restricts SARS-CoV-2 replication in cell culture, but only a handful of Interferon Stimulated Genes with antiviral activity against SARS-CoV-2 have been identified. Here, we describe a functional CRISPR/Cas9 screen aiming at identifying SARS-CoV-2 restriction factors. We identified DAXX, a scaffold protein residing in PML nuclear bodies known to limit the replication of DNA viruses and retroviruses, as a potent inhibitor of SARS-CoV-2 and SARS-CoV replication in human cells. Basal expression of DAXX was sufficient to limit the replication of SARS-CoV-2, and DAXX over-expression further restricted infection. In contrast with most of its previously described antiviral activities, DAXX-mediated restriction of SARS-CoV-2 was independent of the SUMOylation pathway. SARS-CoV-2 infection triggered the re-localization of DAXX to cytoplasmic sites and promoted its degradation. Mechanistically, this process was mediated by the viral papain-like protease (PLpro) and the proteasome. Together, these results demonstrate that DAXX restricts SARS-CoV-2, which in turn has evolved a mechanism to counteract its action.