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

Abstract

The SARS-CoV-2 main protease (Mpro) 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 Mpro. 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 Mpro 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 Mpro recognizes the TRMT1 cleavage sequence, we determined the structure of a TRMT1 peptide in complex with Mpro, which reveals a substrate binding conformation distinct from the majority of available SARS-CoV-2 Mpro-peptide complexes. Kinetic parameters for peptide cleavage showed that while TRMT1(526-536) is cleaved much slower than the Mpro nsp4/5 autoprocessing sequence, it is proteolyzed 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 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.

Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease

“…Here, we demonstrate that TRMT1 tRNA modification enzyme is an endogenous cleavage target of the SARS-CoV-2 main protease. Our studies parallel the simultaneous work by D'Oliviera et al that has elucidated how the SARS-CoV-2 main protease recognizes the TRMT1 cleavage sequence (D'Oliviera et al, 2023;D′Oliviera et al, 2023). D'Oliviera et al have determined the structural basis for recognition of TRMT1 by the SARS-CoV-2 main protease, which has revealed a distinct binding mode for certain substrates of the main protease, including TRMT1.…”

Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease

“…Here, we demonstrate that TRMT1 tRNA modification enzyme is an endogenous cleavage target of the SARS-CoV-2 main protease. Our studies parallel the simultaneous work by D’Oliviera et al that has elucidated how the SARS-CoV-2 main protease recognizes the TRMT1 cleavage sequence ( D Oliviera et al, 2023 ). D’Oliviera et al have determined the structural basis for recognition of TRMT1 by the SARS-CoV-2 main protease, which has revealed a distinct binding mode for certain substrates of the main protease, including TRMT1.…”

SARS-CoV-2 main protease Nsp5 cleaves and inactivates human tRNA methyltransferase TRMT1

“…According to the primary sequence analysis and structure prediction by Alphafold, TRMT1 36–530 contains the S-adenosylmethionine (SAM) binding site, and the cleaved C-terminal portion is probably involved in tRNA binding. During the preparation and submission of this manuscript, two independent studies have been released on bioRxiv ( Oliviera et al., 2023 ; Zhang et al., 2023 ), both revealing the cleavage of TRMT1 by SARS-CoV-2 Nsp5 at Q530, which are in full agreement with our results. Very early after the virus enters human cells, the virus genome generates several non-structural proteins, among which Nsp1 has been widely reported to inhibit mRNA translation by blocking the mRNA entry channel ( Tidu et al., 2020 ).…”