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Methylation of the mRNA 5′ cap by cellular methyltransferases enables efficient translation and avoids recognition by innate immune factors. Coronaviruses encode viral 2′‐O‐methyltransferases to shield their RNA from host factors. Here, we generate recombinant SARS–CoV‐2 harboring a catalytically inactive 2′‐O‐methyltransferase Nsp16, Nsp16mut, and analyze viral replication in human lung epithelial cells. Although replication is only slightly attenuated, we find SARS–CoV‐2 Nsp16mut to be highly immunogenic, resulting in a strongly enhanced release of type I interferon upon infection. The elevated immunogenicity of Nsp16mut is absent in cells lacking the RNA sensor MDA5. In addition, we report that Nsp16mut is highly sensitive to type I IFN treatment and demonstrate that this strong antiviral effect of type I IFN is mediated by the restriction factor IFIT1. Together, we describe a dual role for the 2′‐O‐methyltransferase Nsp16 during SARS–CoV‐2 replication in avoiding efficient recognition by MDA5 and in shielding its RNA from interferon‐induced antiviral responses, thereby identifying Nsp16 as a promising target for generating attenuated and highly immunogenic SARS–CoV‐2 strains and as a potential candidate for therapeutic intervention.

The deoxynucleotide triphosphate (dNTP) hydrolase SAMHD1 inhibits retroviruses in non-dividing myeloid cells. Although antiviral activity towards DNA viruses has also been demonstrated, the role of SAMHD1 during cytomegalovirus (CMV) infection remains unclear. To determine the impact of SAMHD1 on the replication of CMV, we used murine CMV (MCMV) to infect a previously established SAMHD1 knockout mouse model and found that SAMHD1 inhibits the replication of MCMV in vivo. By comparing the replication of MCMV in vitro in myeloid cells and fibroblasts from SAMHD1-knockout and control mice, we found that the viral kinase M97 counteracts SAMHD1 after infection by phosphorylating the regulatory residue threonine 603. The phosphorylation of SAMHD1 in infected cells correlated with a reduced level of dNTP hydrolase activity and the loss of viral restriction. Together, we demonstrate that SAMHD1 acts as a restriction factor in vivo and we identify the M97-mediated phosphorylation of SAMHD1 as a previously undescribed viral countermeasure. SAMHD1 inhibits murine cytomegalovirus replication in vivo and its activity is counteracted by the viral kinase M97. Phosphorylation of SAMHD1 by M97 correlates with reduced dNTP hydrolase activity and a loss of viral restriction in infected cells.

The host restriction factor sterile alpha motif and histidine–aspartate domain-containing protein 1 (SAMHD1) is an important component of the innate immune system. By regulating the intracellular nucleotide pool, SAMHD1 influences cell division and restricts the replication of viruses that depend on high nucleotide concentrations. Human cytomegalovirus (HCMV) is a pathogenic virus with a tropism for non-dividing myeloid cells, in which SAMHD1 is catalytically active. Here we investigate how HCMV achieves efficient propagation in these cells despite the SAMHD1-mediated dNTP depletion. Our analysis reveals that SAMHD1 has the capability to suppress HCMV replication. However, HCMV has evolved potent countermeasures to circumvent this block. HCMV interferes with SAMHD1 steady-state expression and actively induces SAMHD1 phosphorylation using the viral kinase pUL97 and by hijacking cellular kinases. These actions convert SAMHD1 to its inactive phosphorylated form. This mechanism of SAMHD1 inactivation by phosphorylation might also be used by other viruses to overcome intrinsic immunity. Human cytomegalovirus antagonizes the antiviral activity of sterile alpha motif and histidine–aspartate domain-containing protein 1 (SAMHD1) in macrophages by deploying the viral kinase pUL97 and hijacking cellular kinases.