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

## TL;DR

Removing SAMHD1 limits SARS-CoV-2 infection in lung cells by reducing ACE2 levels, a key receptor for the virus.

## Contribution

SAMHD1 depletion restricts SARS-CoV-2 replication in lung epithelial cells via HNF1-dependent ACE2 suppression, independent of IFN signaling.

## Key findings

- SAMHD1 knockout reduces ACE2 expression in Calu-3 cells at both mRNA and protein levels.
- SAMHD1 KO impairs SARS-CoV-2 spike protein-mediated viral entry in lung epithelial cells.
- SAMHD1 regulates HNF1α and HNF1β, which are essential for ACE2 expression in Calu-3 cells.

## Abstract

Sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) restricts a broad spectrum of viruses through multifaceted mechanisms. It also limits spontaneous- and virus-induced innate immune responses by suppressing proinflammatory cytokine and type-I interferon (IFN-I) production. Some viruses escape SAMHD1 restriction and utilize SAMHD1-mediated innate immune suppression to establish effective infection through IFN antagonism. Our previous studies showed that SAMHD1 is a proviral factor facilitating replication of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in human macrophages, monocytic THP-1 and epithelial-like HEK293T cell lines by suppressing IFN responses. However, it is unclear about the function of SAMHD1 in lung epithelial cells during SARS-CoV-2 infection. Here, we report that SAMHD1 knockout (KO) restricts SARS-CoV-2 replication in lung epithelial Calu-3 cells by suppressing endogenous expression of the viral receptor angiotensin-converting enzyme 2 (ACE2) via hepatocyte nuclear factor 1-alpha (HNF1α) and HNF1β. Using pseudotyped SARS-CoV-2 and lentiviral vectors, we found that SARS-CoV-2 spike protein-mediated viral entry was suppressed in SAMHD1 KO Calu-3 cells. SAMHD1 KO repressed ACE2 expression in Calu-3 cells at mRNA and protein levels. Functional analyses revealed that HNF1α and HNF1β were crucial for the endogenous ACE2 expression in Calu-3 cells. Additionally, SAMHD1 KO led to a reduction in the expression levels and ACE2-promoting function of HNF1α and HNF1β. Inhibition of IFN antiviral response by baricitinib, a Janus kinase 1 and 2 (JAK 1/2) inhibitor, did not revert the suppression of SARS-CoV-2 in SAMHD1 KO Calu-3 cells. SAMHD1 knock-in and deoxynucleoside supplementation experiments indicated that SAMHD1 expression and dNTP pool balance collectively regulated HNF1-mediated ACE2 expression in Calu-3 cells. Our findings demonstrate that SAMHD1 depletion hinders HNF1-mediated ACE2 expression and SARS-CoV-2 replication in Calu-3 cells via a novel mechanism beyond its IFN-suppressive function.

During viral infection, SAMHD1 acts as a viral restriction factor and a suppressor of the innate immune system, controlling viral replication while also ensuring immune homeostasis. The innate immune suppressive function of SAMHD1 can be proviral for some viruses. SAMHD1 KO has been shown to hinder SARS-CoV-2 infection in human macrophages, THP-1 and HEK293T cell lines by promoting IFN response, but its role in lung epithelial cells is unclear. Here, we demonstrated that SAMHD1 KO impeded SARS-CoV-2 replication in human lung epithelial Calu-3 cells by downregulating the expression of the major viral receptor ACE2. We found that SAMHD1 KO suppressed HNF1-medaited ACE2 expression that was required for spike protein-mediated SARS-CoV-2 entry. However, inhibiting IFN signaling in SAMHD1 KO Calu-3 cells was not sufficient to revert SARS-CoV-2 replication. Restoring SAMHD1 expression and increasing dNTP intracellular pool in Calu-3 cells suggested multifaceted mechanisms contributing to the regulation of HNF1-mediated ACE2 expression. Our findings shed light on the differential proviral function of SAMHD1 in ACE2 expressing cells and suggest that SAMHD1 expression can facilitate SARS-CoV-2 infection beyond enhancing IFN antagonism.