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

Figure 4. Sequence-dependent cleavage of tRNA methyltransferase 1 (TRMT1) by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural protein 5 (Nsp5).

(A) Immunoblot of lysates from human cells expressing empty vector, wild-type (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, Asterisk (*) 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, wild-type (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 as biological replicates with comparable results (see source data).

Figure 4—source data 1. Raw uncropped immunoblots for Figure 4.

Figure 4.

Figure 4—figure supplement 1. Detection of the C-terminal tRNA methyltransferase 1 (TRMT1) fragment produced by nonstructural protein 5 (Nsp5)-dependent cleavage in human cells.

Figure 4—figure supplement 1.

(A) Immunoblot of lysates from the indicated wild-type (WT) or TRMT1-knockout (K)O human 293T cells that were untransfected (-) (lanes 1 and 2) or transfected with empty vector, wild-type (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, wild-type (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 (B) represents non-specific detection of the Nsp5-C145A variant that exhibits extremely high levels of expression since it cannot self-cleave.
Figure 4—figure supplement 1—source data 1. Raw uncropped immunoblots for Figure 4—figure supplement 1.