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

Abstract

is crucial for tRNA structure and function. TRMT1 is localized in the nucleus, cytoplasm, and mitochondria. Its mitochondrial localization is mediated by an N-terminal mitochondrial targeting sequence ( MTS ) of ∼35 residues ( Dewe et al., 2017 ) . However, it is not known whether TRMT1 is a bone-fide substrate of Nsp5. Moreover, the consequence of the potential cleavage remains unclear. Here, we expressed and purified a recombinant SARS-CoV-2 Nsp5 with a C-terminal His 6 tag and an MTS-deleted TRMT1 with an N-terminal FLAG and a C-terminal His 6 tag ( TRMT1 35 ) using Escherichia coli ( Figure 1 A; Supplementary Figure S1 A and B ) . Western blot analysis using anti-FLAG antibodies showed that the amount of TRMT1 35 decreased after incubation with Nsp5 in a dose-dependent manner, accompanied by the appearance of a digested product of ∼58 kDa ( Figure 1 B ) , suggesting that the cleavage site is in the latter part of TRMT1. However, the C-terminal cleavage product with a lower molecular weight was not observed using anti-His 6 antibodies. Coomassie blue staining using the same protein samples and digestion conditions also showed a clear cleaved fragment after incubation with Nsp5 ( Supplementary Figure S1 C ) . To confirm the digestion of TRMT1 by SARS-CoV-2 Nsp5 in vivo , we co-expressed TRMT1 35 ( with N-terminal HA and C-terminal FLAG tags ) and 3 × Myc-tagged Nsp5 ( Figure 1 A ) in HEK293T cells. Indeed, western blot analysis using anti-HA antibodies showed the generation of a digested product of ∼58 kDa, while the cleaved fragment with the lower molecular mass was likewise invisible ( Figure 1 C ) . Considering the high sequence identity ( ∼96% ) between SARS-CoV-2 Nsp5 and SARS-CoV Nsp5, we further purified SARS-CoV Nsp5 ( Supplementary Figure S1 D ) . Consistently, SARS-CoV Nsp5 was also able to digest TRMT1 35 in vitro with a high efficiency ( Figure 1 D ) , indicating that TRMT1 cleavage is a conserved mechanism among different CoVs. To better understand whether TRMT1 digestion is mediated by the protease activity of Nsp5, the C145 residue of SARS-CoV-2 Nsp5 was replaced by alanine, resulting in a C145A mutant that was defective in cleavage ( Figure 1 E ) . The C145A mutant is a well-known variant that was constructed to show the contribution of the C145–H41 catalytic dyad to substrate cleavage ( Jin et al., 2020 ) . The C145A mutant exhibits the same three-dimensional structure as the wild-type enzyme ( Hsu et al., 2005 ) . These data clearly demonstrated that Nsp5 is able to cleave TRMT1 in vitro and in vivo via its protease activity. Subsequently, we intended to identify the cleavage site in TRMT1. Nsp5 digests all protein substrates at conserved glutamine residues. NetCorona 1.0, a website predicting Nsp5 cleavage sites ( Kiemer et al., 2004 ) , showed that Q530 was the only confident candidate with a high score of 0.942, while other glutamine residues ( such as Q584 ) were excluded ( Supplementary Figure S2 A ) . This prediction was roughly consistent with the molecular mass of the digested product in both in vitro ( Figure 1 B ) and in vivo ( Figure 1 C ) assays. To experimentally validate the predicted cleavage site, we mutated Q530 to alanine in the TRMT1 35 eukaryotic expression construct. In parallel, a Q584A mutant was also constructed for comparison. Immunoblotting with anti-HA antibodies showed that the cleavage of TRMT1 35 Q530A was abolished ( Figure 1 F ) . According Dear Editor, Coronavirus disease 2019 ( COVID-19 ) , caused by severe acute respiratory syndrome coronavirus 2 ( SARS-CoV-2 ) , has become a serious emergency to global public health since its outbreak in 2019. SARS-CoV-2 is an enveloped, positive-sense, single-strand RNA virus, belonging to the Betacoronavirus genus of the Coronaviridae family ( Wu et al., 2020 ) including SARS-CoV, SARS-CoV-2, MERS-CoV, etc. After viral entry, the RNA genome first encodes 16 nonstructural proteins, including papain-like protease and Nsp5 ( also called the main protease ) , which are essential for viral replication and transcription ( Bai et al., 2022 ) . Nsp5 is a 33-kDa cysteine protease whose most frequently found recognition sequence is LQˆ ( S/A/G ) . Many protein–protein interactions between the virus and the host have been identified ( Gordon et al., 2020a ) . Only a small number of interactions between host proteins and the viral protein Nsp5 have been experimentally validated ( Wenzel et al., 2021 ) . Few reports have linked the role of host protein cleavage by Nsp5 beyond immune evasion. An interaction between catalytically inactive Nsp5 mutant ( C145A ) and tRNA methyltransferase 1 ( TRMT1 ) has been described previously ( Gordon et al., 2020a , b ) . TRMT1 ( 659 amino acids ) catalyzes the formation of N 2 -methylguanosine ( m 2 G ) or N 2 , N 2 -dimethylguanosine ( m 2,2 G ) at position 26 of most human tRNAs, which