The SARS-CoV-2 main protease (M^pro or Nsp5) is critical for production of viral proteins during infection and, like many viral proteases, also targets host proteins to subvert their cellular functions. Here, we show that the human tRNA methyltransferase TRMT1 is recognized and cleaved by SARS-CoV-2 M^pro. TRMT1 installs the N²,N²-dimethylguanosine (m2,2G) modification on mammalian tRNAs, which promotes cellular protein synthesis and redox homeostasis. We find that M^pro can cleave endogenous TRMT1 in human cell lysate, resulting in removal of the TRMT1 zinc finger domain. Evolutionary analysis shows the TRMT1 cleavage site is highly conserved in mammals, except in Muroidea, where TRMT1 is likely resistant to cleavage. TRMT1 proteolysis results in reduced tRNA binding and elimination of tRNA methyltransferase activity. We also determined the structure of an M^pro-TRMT1 peptide complex that shows how TRMT1 engages the M^pro active site in an uncommon substrate binding conformation. Finally, enzymology and molecular dynamics simulations indicate that kinetic discrimination occurs during a later step of M^pro-mediated proteolysis following substrate binding. Together, these data provide new insights into substrate recognition by SARS-CoV-2 M^pro that could help guide future antiviral therapeutic development and show how proteolysis of TRMT1 during SARS-CoV-2 infection impairs both TRMT1 tRNA binding and tRNA modification activity to disrupt host translation and potentially impact COVID-19 pathogenesis or phenotypes.

Wnt/β-catenin signaling directs animal development and tissue renewal in a tightly controlled, cell- and tissue-specific manner. In the mammalian central nervous system, the atypical ligand Norrin controls angiogenesis and maintenance of the blood-brain barrier and blood-retina barrier through the Wnt/β-catenin pathway. Like Wnt, Norrin activates signaling by binding and heterodimerizing the receptors Frizzled (Fzd) and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), leading to membrane recruitment of the intracellular transducer Dishevelled (Dvl) and ultimately stabilizing the transcriptional coactivator β-catenin. Unlike Wnt, the cystine knot ligand Norrin only signals through Fzd4 and additionally requires the co-receptor Tetraspanin12 (Tspan12); however, the mechanism underlying Tspan12-mediated signal enhancement is unclear. It has been proposed that Tspan12 integrates into the Norrin-Fzd4 complex to enhance Norrin-Fzd4 affinity or otherwise allosterically modulate Fzd4 signaling. Here, we measure direct, high-affinity binding between purified Norrin and Tspan12 in a lipid environment and use AlphaFold models to interrogate this interaction interface. We find that Tspan12 and Fzd4 can simultaneously bind Norrin and that a pre-formed Tspan12/Fzd4 heterodimer, as well as cells co-expressing Tspan12 and Fzd4, more efficiently capture low concentrations of Norrin than Fzd4 alone. We also show that Tspan12 competes with both heparan sulfate proteoglycans and LRP6 for Norrin binding and that Tspan12 does not impact Fzd4-Dvl affinity in the presence or absence of Norrin. Our findings suggest that Tspan12 does not allosterically enhance Fzd4 binding to Norrin or Dvl, but instead functions to directly capture Norrin upstream of signaling.