Recognition and Cleavage of Human tRNA Methyltransferase TRMT1 by the SARS-CoV-2 Main Protease

The SARS-CoV-2 main protease (Mpro, or Nsp5) is critical for the production of functional viral proteins during infection and, like many viral proteases, can also target host proteins to subvert their cellular functions. Here, we show that the human tRNA methyltransferase TRMT1 can be recognized and cleaved by SARS-CoV-2 Mpro. TRMT1 installs the N2,N2-dimethylguanosine (m2,2G) modification on mammalian tRNAs, which promotes global protein synthesis and cellular redox homeostasis. We find that Mpro can cleave endogenous TRMT1 in human cell lysate, resulting in removal of the TRMT1 zinc finger domain. TRMT1 proteolysis results in elimination of TRMT1 tRNA methyltransferase activity and reduced tRNA binding affinity. Evolutionary analysis shows that the TRMT1 cleavage site is highly conserved in mammals, except in Muroidea, where TRMT1 is likely resistant to cleavage. In primates, regions outside the cleavage site with rapid evolution could indicate adaptation to ancient viral pathogens. Furthermore, we determined the structure of a TRMT1 peptide in complex with Mpro, revealing a substrate binding conformation distinct from the majority of available Mpro-peptide complexes. Kinetic parameters for peptide cleavage show that the TRMT1(526-536) sequence is cleaved with comparable efficiency to the Mpro-targeted nsp8/9 viral cleavage site. Mutagenesis studies and molecular dynamics simulations together indicate that kinetic discrimination occurs during a later step of Mpro-mediated proteolysis that follows substrate binding. Our results provide new information about the structural basis for Mpro substrate recognition and cleavage, the functional roles of the TRMT1 zinc finger domain in tRNA binding and modification, and the regulation of TRMT1 activity by SARS-CoV-2 Mpro. These studies could inform future therapeutic design targeting Mpro and raise the possibility that proteolysis of human TRMT1 during SARS-CoV-2 infection suppresses protein translation and oxidative stress response to impact viral pathogenesis.