SAMHD1 depletion restricts SARS-CoV-2 infection by suppressing HNF1-dependent ACE2 expression in lung epithelial cells

SAMHD1 protein is a restriction factor against broad spectrum of viruses through dNTPase- and dNTPase-independent mechanisms. Moreover, it limits spontaneous- and virus-induced innate immune responses by suppressing proinflammatory cytokine and IFN-I production. Some viruses that escape the restriction functions of SAMHD1 may utilize SAMHD1-mediated innate immune suppression to establish effective infection by better IFN antagonism. Previously, we found that, instead of being a restriction factor, SAMHD1 was a proviral factor facilitating SARS-CoV-2 replication in human macrophages and immortalized human kidney cells HEK293T by suppressing IFN response. However, it is unclear about the function of SAMHD1 to the primary target and producer cells of SARS-CoV-2. In the current study, we found that SAMHD1 was a proviral factor to facilitate SARS-CoV-2 replication in Calu-3 cells, which were lung epithelial cells endogenously expressing viral entry factors such as ACE2 and TMPRSS2. Unlike human macrophages and HEK293T cells, inhibition of IFN antiviral response by baricitinib, a JAK1/2 inhibitor, did not revert the suppression of SARS-CoV-2 in SAMHD1 knockout Calu-3 cells. Through the experiments with pseudotyped viruses, we found that spike-protein mediated viral entry was suppressed in SAMHD1 knockout Calu-3 cells. Through mRNA-seq, we found that SAMHD1 knockout repressed ACE2 expression of Calu-3 cells at mRNA and protein levels. Knockdown experiments revealed that HNF1a and HNF1ß were crucial for the endogenous expression of ACE2 in Calu-3 cells. Additionally, SAMHD1 knockout led to a reduction in the expression levels and ACE2-promoting function of HNF1a and HNF1ß. This suggested that SAMHD1 facilitates HNF1-mediated ACE2 expression and SARS-CoV-2 replication in ACE2-expressing cells via a mechanism independent of its IFN- suppressive function Overall design: To gain mechanistic insight, mRNA-seq was performed to profile the transcriptome of SAMHD1 knockout Calu3 cells (KO) and compared with that of the control Calu-3 cells (Ctrl). Two clones of KO cells were generated through CRISPR/Cas9 with one single guide RNA having different target sequences. Ctrl cells were mock control treated similarly as the KO cells but were transduced with empty vector. 8 x 105 Calu-3 Ctrl, KO1 and KO2 cells were seeded in 6 well plates in biological triplicate. Cells were cultured in 2mL DMEM/F12 with 20% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. At 20 hours after seeding, cells were washed once with DPBS and lysed with RLT plus lysis buffer. Cellular RNA was purified with RNeasy Plus kit following manufacturer's instructions. All RNA samples got RNA integrity Number (RIN) scores equal to or more than 9.9 as determined by Agilent BioAnalyzer by Genomics Division of Iowa Institute of Human Genetics (University of Iowa). RNA samples were processed through Illumina Stranded mRNA Prep (illumina) and sequenced by Element Aviti24 using Cloudbreak FS-High output flow cell at 150 cycle (2x75) by the Genomics Division.