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. 2024 May 30;12:RP90316. doi: 10.7554/eLife.90316

Figure 7. tRNA methyltransferase 1 (TRMT1) is required for efficient severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication in human cells.

(A) Immunoblot of lysates prepared from the indicated 293T TRMT1-knockout (KO) cell lines that were mock-infected (multiplicity of infection, MOI of 0) or infected with SARS-CoV-2 for 24 hr. 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). Statistical significance was determined in (B) and (C) by one-way ANOVA with Dunnett’s multiple comparisons test. (D) SARS-CoV-2 RNA copy number in control-wild-type (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. Statistical significance was determined by two-way ANOVA with Dunnett’s multiple comparisons test. *p<0.05; **p<0.01; ****p<0.0001; ns, non-significant.

Figure 7—source data 1. Raw uncropped immunoblots for Figure 7A.
Figure 7—source data 2. QRT-PCR measurements of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA for Figure 7D.

Figure 7.

Figure 7—figure supplement 1. Expression of nonstructural protein 5 (Nsp5) leads to cleavage of tRNA methyltransferase 1 (TRMT1)-wild-type (WT) re-expressed in TRMT1-knockout (KO) cells, but not TRMT1-Q530N.

Figure 7—figure supplement 1.

Immunoblot of lysates from human cells integrated with empty lentiviral vector or lentiviral expression vectors for wild-type (WT) TRMT1 or TRMT1-Q530N. The cell lines were transfected with either a 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, Asterisk (*) denotes a non-specific band, and arrow represents N-terminal TRMT1 cleavage product.
Figure 7—figure supplement 1—source data 1. Raw uncropped immunoblots for Figure 7—figure supplement 1.
Figure 7—figure supplement 2. Immunoblot analysis of lysates prepared from the indicated 293T cell lines expressing empty vector or ACE2.

Figure 7—figure supplement 2.

The immunoblot was probed with anti-ACE2 and actin.
Figure 7—figure supplement 2—source data 1. Raw uncropped immunoblots for Figure 7—figure supplement 2.