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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. Synopsis: Replication of SARS–CoV‐2 harboring inactive Nsp16 is attenuated and very sensitive to type I IFN treatment. Nsp16 counteracts intrinsic restriction and sensing mechanisms, rendering Nsp16‐deficient viruses good candidates for live‐attenuated vaccine development. SARS–CoV‐2 Nsp16mut shows attenuated replication.SARS–CoV‐2 Nsp16mut replication is highly IFN sensitive.Nsp16 protects SARS–CoV‐2 from IFIT1 restriction.Nsp16 shields SARS–CoV‐2 from MDA5 sensing. [ABSTRACT FROM AUTHOR]

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'‐O‐ribose cap needed for viral immune escape. We find that the host cap 2'‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19. Synopsis: A target‐based drug repurposing approach to inhibit SAM‐dependent methyltransferases (Mtases) shows that the host MTase MTr1 can compensate for loss of the viral encoded NSP16 MTase. Targeting both MTr1 and NSP16 is needed to suppress SARS‐CoV‐2 replication. Host 2'‐O‐ribose methyltransferase MTr1 is a novel host factor of SARS‐CoV‐2 and compensates for the loss of NSP16.Concomitant inhibition of MTr1 and NSP16 is required to efficiently suppress SARS‐CoV‐2 replication.Target‐based drug repurposing identified a bispecific MTr1/NSP16 inhibitor tubercidin, which is highly antiviral against SARS‐CoV‐2.Host‐directed SAM‐cycle inhibitors, exerting indirect broad spectrum methyltransferase inhibition, are potent antivirals against SARS‐CoV‐2 with additional immunomodulatory and fibrosis‐curbing functions. [ABSTRACT FROM AUTHOR]