Published on None in volume None.

PubMed ID: 36865253

DOI: https://doi.org/10.1101/2023.02.20.529306

Abstract:

The SARS-CoV-2 main protease (M pro ) plays a crucial role in the production of functional viral proteins during infection and, like many viral proteases, can also target and cleave 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 M pro . TRMT1 installs the N2,N2-dimethylguanosine (m2,2G) modification at the G26 position of mammalian tRNA, which promotes global protein synthesis, cellular redox homeostasis, and has links to neurological disability. We find that M pro can cleave endogenous TRMT1 in human cell lysate, resulting in removal of the TRMT1 zinc finger domain that is required for tRNA modification activity in cells. Evolutionary analysis shows that the TRMT1 cleavage site is highly conserved in mammals, except in Muroidea, where TRMT1 may be resistant to cleavage. In primates, regions outside of the cleavage site with rapid evolution could indicate possible adaptation to ancient viral pathogens. To visualize how M pro recognizes the TRMT1 cleavage sequence, we determined the structure of a TRMT1 peptide in complex with M pro , which reveals a substrate binding conformation distinct from the majority of available SARS-CoV-2 M pro -peptide complexes. Kinetic parameters for peptide cleavage showed that while TRMT1(526-536) is cleaved much slower than the M pro nsp4/5 autoprocessing sequence, it is proteolyzed with comparable efficiency to the M pro -targeted nsp8/9 viral cleavage site. Mutagenesis studies and molecular dynamics simulations together indicate that kinetic discrimination occurs during a later step of M pro -mediated proteolysis that follows substrate binding. Our results provide new information about the structural basis for M pro substrate recognition and cleavage that could help inform future therapeutic design and raise the possibility that proteolysis of human TRMT1 during SARS-CoV-2 infection may impact protein translation or oxidative stress response and contribute to viral pathogenesis.