Human cells infected with SARS-CoV-2 exhibit a reduction in TRMT1 levels and perturbations in tRNA modification patterns. (A) Immunoblot analysis of lysates prepared from MRC-5-ACE2 human cells that were mock-infected or infected with SARS-CoV-2 at MOI of 5 for 24 or 48 hours. The immunoblot was probed with anti-TRMT1, actin, or SARS-CoV-2 nucleocapsid (N) antibodies. Circle represents endogenous full-length TRMT1. Asterisk (*) denotes a non-specific band. Size markers are noted in kiloDalton. (B) Quantification of TRMT1 signal intensity normalized to actin in the mock or SARS-CoV-2-infected cell lines. TRMT1 protein levels are expressed relative to mock-infected samples for each time point. (C) m2,2G levels in small RNAs isolated from MRC5 cells that were either mock-infected or infected with SARS-CoV-2 at MOI of 5 for 24 or 48 hours. m2,2G levels were normalized to A, C, G, and U. Samples were measured in biological replicates. Statistical significance for (B) and (C) was determined by two-way ANOVA with multiple comparisons test. ***p<0.001; ****p<0.0001; ns, non-significant. (D) Levels of the indicated RNA modifications in small RNAs isolated from MRC5 cells that were either mock-infected or infected with SARS-CoV-2 for 24 or 48 hours. RNA modification levels were normalized to A, C, G, and U. Y-axis represents the log2 fold change in the levels of the indicated tRNA modification between SARS-CoV-2 infected versus mock-infected MRC5 cells.

SARS-CoV-2 Nsp5 binds TRMT1 in human cells. (A) Schematic of human TRMT1 primary structure with predicted Nsp5 cleavage site. Mitochondrial targeting signal (MTS), methyltransferase (MT) domain and zinc finger motif are denoted. (B) Consensus sequence logo of cleavage sites in SARS-CoV-2 polyproteins. (C) Alpha-fold predicted structure of human TRMT1 with putative Nsp5 cleavage site denoted in magenta and arrowhead. (D) Immunoblot of input and streptactin purifications from human cells expressing empty vector, wildtype (WT) Nsp5, or Nsp5-C145A fused to the Strep tag without or with co-expression with TRMT1-FLAG. The immunoblot was probed with anti-Strep, FLAG and actin antibodies. Square represents TRMT1-FLAG, circle represents endogenous TRMT1. Size markers are noted to the left in kiloDalton. The experiment in (D) was repeated in Supplemental Figure 2.

Nsp5 expression induces cleavage of TRMT1 in human cells. (A) Immunoblot of lysates prepared from human 293T cells expressing GFP, Nsp5 or Nsp5-C145A. The immunoblot was probed with anti-TRMT1, Strep, or actin antibodies. Hours post represents the time post-transfection. Circle represents endogenous TRMT1. Arrow represents the N-terminal (N)-TRMT1 cleavage fragment. * denotes a non-specific band. Size markers to the left in kiloDalton. (B, C) Quantification of endogenous TRMT1 or N-terminal (N)-TRMT1 cleavage product in transfected cells. TRMT1 and N-TRMT1 signal was normalized to actin. (D) Immunoblot of lysates prepared from wildtype or TRMT1-knockout (KO) human cell lines expressing Nsp5. Experiments in (A) and (D) were repeated three times (see Source data).

Sequence-dependent cleavage of TRMT1 by SARS-CoV-2 Nsp5. (A) Immunoblot of lysates from human cells expressing empty vector, wildtype (WT) Nsp5-Strep, or Nsp5-C145A-Strep without or with co-expression with TRMT1-FLAG. The immunoblot was probed with anti-Strep, FLAG and actin antibodies. Square represents TRMT1-FLAG, * denotes a non-specific band, arrow represents N-terminal TRMT1 cleavage product and arrowhead indicates the C-terminal TRMT1 cleavage product. (B) Schematic of human TRMT1 with predicted Nsp5 cleavage site and Q530N mutation. (C) Immunoblot of lysates from human cells expressing empty vector, wildtype (WT) Nsp5-Strep, or Nsp5-C145A-Strep without or with co-expression with TRMT1-FLAG or TRMT1-FLAG Q530N. Experiments in (A) and (C) were repeated three times with comparable results (see Source data).

N- and C-terminal TRMT1 cleavage fragments exhibit alterations in RNA binding and tRNA modification activity. (A) Schematic of wildtype TRMT1 and predicted TRMT1 fragments resulting from Nsp5 cleavage at Q530N. (B) Immunoblot analysis of anti-FLAG purifications from human cells expressing vector control, full-length TRMT1, or TRMT1 cleavage fragments fused to the FLAG tag. The immunoblot was probed with anti-FLAG and anti-actin antibodies. (C) Nucleic acid stain of RNAs extracted from the indicated input or purified samples after denaturing PAGE. The migration pattern of 5.8S rRNA (∼150 nt), 5S rRNA (∼120 nt) and tRNAs (∼70–80 nt) are denoted. (D) Immunoblot of TRMT1 expression in either control-WT or TRMT1-KO human 293T cell lines. (E, F) Representative gel of primer extension assays to monitor the presence of m2,2G in tRNA-Met-CAU or mt-tRNA-Ile-UAU from the cell lines transfected with the indicated TRMT1 constructs. D, dihydrouridine; m1G, 1-methylguanosine; >, labeled oligonucleotide used for primer extension. Protein-RNA purification was repeated with comparable results (see Source data for repeat).

The expression of TRMT1 affects the levels of SARS-CoV-2 RNA replication in human cells. (A) Immunoblot of lysates prepared from 293T control-wildtype (WT) or TRMT1-KO cell lines that were mock-infected (MOI of 0) or infected with SARS-CoV-2 at MOI of 0.2 or 0.4 for 24 hours. The immunoblot was probed with antibodies against TRMT1 or actin. Circle represents endogenous full-length TRMT1. Asterisk denotes a non-specific band. Size markers to the right in kiloDalton. (B) Normalized TRMT1 signal intensity relative to mock-infected cells (MOI of 0). (C) Immunoblot of lysates prepared from 293T control-wildtype (WT) or TRMT1-KO cell lines that were mock-infected (MOI of 0) or infected with SARS-CoV-2 at MOI of 0.1 or 5.0 for 24 hours. The immunoblot was probed with antibodies as in (A). (D) Normalized TRMT1 signal intensity relative to mock-infected cells (MOI of 0). (E, F) SARS-CoV-2 RNA copy number in control-WT or TRMT1-KO human 293T cell lines after infection at the indicated MOI for 24 hours. Viral copy number was measured by QRT-PCR and normalized to GAPDH. Samples were measured in triplicate. *p < 0.05; **p<0.01; ***p<0.001; ****p < 0.0001; ns, non-significant.

TRMT1 is required for efficient SARS-CoV-2 replication in human cells. (A) Immunoblot of lysates prepared from the indicated 293T TRMT1-KO cell lines that were mock-infected (MOI of 0) or infected with SARS-CoV-2 for 24 hours. The immunoblot was probed with antibodies against TRMT1 or actin. Square represents full-length TRMT1-FLAG. Asterisk denotes a non-specific band. Size markers to the left in kiloDalton. (B) Normalized TRMT1-WT signal intensity relative to mock-infected cells (MOI of 0). (C) Normalized TRMT1-Q530N signal intensity relative to mock-infected cells (MOI of 0). (D) SARS-CoV-2 RNA copy number in control-WT or TRMT1-KO human 293T cell lines after infection at the indicated MOI. Viral copy number was measured by QRT-PCR and normalized to GAPDH. *p < 0.05; **p<0.01; ****p < 0.0001; ns, non-significant.

Viral infectivity measurement of supernatants collected from the indicated cell lines infected with SARS-CoV-2 for 24 hours. (A) Viral titer of supernatants collected from the indicated cell lines infected with SARS-CoV-2. Infectious titer was determined by TCID50 endpoint dilution assay in VeroE6 cells and expressed in focus forming units per mL of supernatant (FFU/mL). (B) Infectivity of SARS-CoV-2 particles generated from cell lines in (A). Infectivity of viral particles was calculated with the formula [(FFU/mL)/(viral genomic RNA copies/mL)], and expressed in FFU per 100 genomic copies.

LC-MS analysis of m2,2G levels in small RNAs isolated from MRC5 cells that were either mock-infected or infected with SARS-CoV-2 at MOI of 5 for 24 or 48 hours. m2,2G levels were normalized to guanosine. Samples were measured in biological replicates. Statistical significance was determined by two-way ANOVA with multiple comparisons test. *p < 0.05; ns, non-significant.

Immunoblot of input and streptactin purifications from human cells expressing empty vector, wildtype (WT) Nsp5, or Nsp5-C145A fused to the Strep tag without or with co-expression with TRMT1-FLAG. The immunoblot was probed with anti-TRMT1, Strep, or actin antibodies. Circle represents endogenous TRMT1.

Detection of the C-terminal TRMT1 fragment produced by Nsp5-dependent cleavage in human cells. (A) Immunoblot of lysates from the indicated wildtype (WT) or TRMT1-KO human 293T cells that were untransfected (-) (lanes 1 and 2) or transfected with empty vector, wildtype (WT) Nsp5-Strep, or Nsp5-C145A-Strep expression plasmids (lanes 3 through 5). The blot was probed with an antibody detecting residues 609-659 of TRMT1. Circle represents endogenous TRMT1. (B) Immunoblot of lysates from human cells that were transfected with TRMT1-FLAG or TRMT1-FLAG Q530N expression plasmids along with empty vector, wildtype (WT) Nsp5-Strep, or Nsp5-C145A-Strep expression plasmids. The blot was probed with an antibody detecting residues 609-659 of TRMT1. Circle represents endogenous TRMT1, square represents TRMT1-FLAG, and arrowhead indicates the C-terminal TRMT1 cleavage product. Asterisks denote bands that are still detectable in the TRMT1-KO cell line. The band at ∼35 kDa in lanes 3 and 6 of Supplemental Figure 3B represents non-specific detection of the Nsp5-C145A variant that exhibits extremely high levels of expression since it cannot self-cleave.

Confocal microscopy images of 293T cells transiently transfected with constructs expressing TRMT1 or TRMT1 fragments fused with green fluorescent protein (GFP). Mitochondria were identified using mitochondrion-targeted red fluorescent protein (Mito-RFP) and nuclear DNA was stained with Hoechst. Overlap of red mitochondria and green GFP signal is displayed in the Merge panels.

Human 293T cell lines expressing ACE2 can be infected by SARS-CoV-2. (A) Immunoblot analysis of lysates prepared from the indicated 293T cell lines expressing empty vector or ACE2. The immunoblot was probed with anti-ACE2 and actin. (B) Immunoblot analysis of lysates prepared from 293T-ACE2 cell lines that were mock-infected (MOI 0) or infected with SARS-CoV-2 at the indicated MOI. The blot was probed against the SARS-CoV-2 nucleocapsid (N) and actin.

Expression of Nsp5 leads to cleavage of TRMT1-WT re-expressed in TRMT1-KO cells, but not TRMT1-Q530N. Immunoblot of lysates from human cells integrated with empty lentiviral vector or lentiviral expression vectors for wildtype (WT) TRMT1 or TRMT1-Q530N. The cell lines were transfected with either vector or a construct expressing Nsp5-Strep. The immunoblot was probed with anti-TRMT1, Strep, and actin antibodies. Square represents TRMT1-FLAG, circle represent endogenous TRMT1, * denotes a non-specific band, and arrow represents N-terminal TRMT1 cleavage product.

Immunoblot analysis of lysates prepared from the indicated 293T cell lines expressing empty vector or ACE2. The immunoblot was probed with anti-ACE2 and actin.