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

Figure 6. The expression of tRNA methyltransferase 1 (TRMT1) affects the levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA replication in human cells.

(A) Immunoblot of lysates prepared from 293T control-wild-type (WT) or TRMT1-knockout (KO) cell lines that were mock-infected (multiplicity of infection, MOI of 0) or infected with SARS-CoV-2 at MOI of 0.2 or 0.4 for 24 hr. 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). Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test. (C) Immunoblot of lysates prepared from 293T control-wild-type (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 hr. 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 hr. Viral copy number was measured by QRT-PCR and normalized to GAPDH. Samples were measured in triplicate. Statistical significance was determined by two-way ANOVA with Šídák’s multiple comparisons test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, non-significant.

Figure 6—source data 1. Raw uncropped immunoblots for Figure 6.
Figure 6—source data 2. QRT-PCR measurements of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA.

Figure 6.

Figure 6—figure supplement 1. Human 293T cell lines expressing ACE2 can be infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Figure 6—figure supplement 1.

(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 (multiplicity of infection, 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.
Figure 6—figure supplement 1—source data 1. Raw uncropped immunoblots for Figure 6—figure supplement 1.