Transcriptomic analysis of sorted lung cells revealed a proviral activity of the NF-?B pathway towards SARS-CoV-2 (RNA-Seq total)

Investigations of cellular responses to viral infection are commonly performed on mixed populations of infected and uninfected cells or using single-cell RNA sequencing, leading to inaccurate and low-resolution gene expression interpretations. Here, we performed deep polyA+ transcriptome analyses and novel RNA profiling of SARS-CoV-2 infected lung epithelial cells, sorted based on the expression of the viral spike (S) protein. Infection caused a massive reduction in mRNAs and lncRNAs, including transcripts coding for antiviral factors, such as interferons (IFN). This absence of IFN signaling probably explained the poor transcriptomic response of bystander cells co-cultured with S+ ones. NF-?B pathway and the inflammatory response escaped the global shutoff in S+ cells. Functional investigations revealed the proviral function of the NF-?B pathway and the antiviral activity of CYLD, a negative regulator of the pathway. Thus, our transcriptomic analysis on sorted cells revealed additional genes that modulate SARS-CoV-2 replication in lung cells. Overall design: Technical triplicates of 2 biological replicates of human alveolar basal epithelial cells transduced with a vector expressing human ACE2 (A549-ACE2) were infected with SARS-CoV-2 (BetaCoV/France/IDF0372/2020), at MOI of 1 during 24h.Cells were FACS-sorted based on the expression of the viral spike (S) protein to identify infected (S+) and bystander (S-). Total RNA sequencing was performed. Mock infected cells were used as control. Following transcript assembly, differential expression analysis was performed between mock, bystander and infected cells.