The NF-κB Transcriptional Footprint Is Essential for SARS-CoV-2 Replication

Abstract

SARS-CoV-2, the etiological agent of COVID-19, is characterized by a delay in Type I interferon (IFN-I)-mediated antiviral defenses alongside robust cytokine production. Here we investigate the underlying molecular basis for this imbalance and implicate virus-mediated activation of NF-κB in the absence of other canonical IFN-I-related transcription factors. Epigenetic and single cell transcriptomic analyses show a selective NF-κB signature that was most prominent in infected cells. Disruption of NF-κB signaling through the silencing of the NF-κB transcription factors p65 or p50 resulted in loss of virus replication that was rescued upon reconstitution. These findings could be further corroborated with the use of NF-κB inhibitors, which reduced SARS-CoV-2 replication in vitro . These data suggest that the robust cytokine production in response to SARS-CoV-2, despite a diminished IFN-I response, is the product of a dependency on NF-κB for viral replication. IMPORTANCE The COVID-19 pandemic has caused significant mortality and morbidity around the world. Although effective vaccines have been developed, large parts of the world remain unvaccinated while new SARS-CoV-2 strains keep emerging. Furthermore, despite extensive efforts and large-scale drug screenings, no fully effective antiviral treatment options have been discovered yet. Therefore, it is of the utmost importance to gain a better understanding of essential factors driving SARS-CoV-2 replication in order to be able to develop novel approaches to target SARS-CoV-2 biology.

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

“…One consequence of this drastic shutoff is the suppression of expression of innate immune genes, such as IFN type I and type III. In agreement with previous RNA-seq studies performed in bulk population of infected A549-ACE2 cells [19,38] and kidney HEK293T-ACE2 cells [59], our transcriptomic profiling combined with analysis of mRNA levels and IFN secretion showed that infected cells failed to mount an antiviral response. Besides global gene expression reduction in host cells, SARS-CoV-2 has evolved numerous mechanisms to specifically counteract the IFN induction and signaling pathways [14].…”

“…2C). This difference explains why IL32 was not identified as an up-regulated gene in previous RNA-seq analysis performed in mixed population of infected A549-ACE2 cells [19,38]. Similarly, the expression of ITGAM, TRAF1, WAKMAR2, AL132990.1 and XLOC_007519 showed a modest increase of mRNA abundances in the bulk population and a significant increase in S + cells, as compared to mock-infected cells (Fig.…”

SARS-CoV-2 infection results in lasting and systemic perturbations post recovery

“…To corroborate the clinical validity of the SARS-CoV-2 acute hamster data, we compared our RNA-seq analyses to published results from lungs of COVID-19 deceased individuals that still had high viral loads at the time of death (Figure 2A-C) (Butler et al, 2021). In agreement with the published data, we find transcriptional signatures from both groups were dominated by a marked upregulation of the IFN-I response as well as TNFα signaling via NFκB (Blanco-Melo et al ., 2020; Hoagland et al ., 2021; Nilsson-Payant et al ., 2021).…”

“…These differences likely reflect the unique biologies of the viruses. SARS-CoV-2 generates significantly more double stranded RNA (dsRNA) during its life cycle as a result of sgRNA production (Nilsson-Payant et al ., 2021; Perlman et al, 1986). Given the immunogenicity and stability of dsRNA, it seems reasonable to postulate that comparable levels of replication would result in a more robust immune response to SARS-CoV-2 as compared to IAV.…”

“…interleukin 6, IL6) and chemokines (i.e. CXCL10) (Blanco-Melo et al ., 2020; Lucas et al, 2020; Nilsson-Payant et al, 2021). As a result of these dynamics, high levels of neutrophils and monocytes amass in the respiratory tract as the virus propagates in an environment with minimal antiviral defense engagement - further exacerbating the inflammatory environment.…”