Transcriptomic analysis of sorted lung cells revealed a proviral activity of the NF-κB pathway towards SARS-CoV-2

Abstract

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-κB 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-κB 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-κB 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.

SARS-CoV-2 Nsp14 protein associates with IMPDH2 and activates NF-κB signaling

“…SARS-CoV-2 Nsp14 induces the NF-κB signaling and downstream cytokines, which may support the host cell proliferation and survival, or prevent cell apoptosis, thus benefiting viral replication ( 42 , 76 ). As the supportive evidence, a recent study showed that knockdown of NF-κB p50 or IL-8 indeed impairs SARS-CoV-2 viral RNA expression and its replication in A549-ACE2 cells ( 64 ). RIB and MPA are both FDA-approved drugs for treating HCV infection and transplant organ rejection, respectively.…”

“…IL-8 may attract neutrophils, stimulate granulocytes’ response to tissue damage, and generate ARDS-related micro thrombosis ( 62 , 63 ). IL-8 induction could initiate a positive feedback via autocrine of attracted neutrophils ( 63 ), which may also support viral replication of SARS-CoV-2 ( 64 ) and inhibit SARS-CoV-2 specific T-cell responses ( 65 ). In terms of possible molecular mechanisms, since IMPDH2 participates in regulating the host nucleotide metabolism ( 66 , 67 ), it may further modulate cellular stress response and downstream NF-κB activation ( 67 – 69 ).…”

Non-canonical contribution of human Dicer helicase domain in antiviral innate immune response

“…Indeed, SARS-CoV-2 is known to benefit from the upregulation of the inflammatory response, mainly through the NF-kB pathway, which is essential for the replication and propagation of the virus (Nilsson-Payant et al, 2021). A very recent study confirmed the proviral role of the NF-kB pathway in SARS-CoV-2 infected lung cells (Bhargava et al, 2023).…”