FIG 7.

PCV2 Cap lysine residues 164, 179, and 191 are the sites of ubiquitination by pMKRN1. (A) Schematic diagram of N- and C-terminal truncation mutants of PCV2 Cap. (B) Mapping the regions of PCV2 Cap that interact with pMKRN1. HEK293T cells were transfected with plasmids to express Flag-pMKRN1 and Cap truncation mutants fused with the GFP tag, and lysates were immunoprecipitated with an anti-GFP antibody, followed by immunoblotting using an anti-MKRN1 antibody, anti-GFP antibody, or anti-β-actin antibody. The middle white space divides the samples that were resolved by two SDS-PAGEs due to the limit of the gel lanes. (C) PCV2 Cap containing amino acid residues 162 to 198 is degraded by pMKRN1. HEK293T cells were transfected with plasmids to express Flag-pMKRN1 and the indicated Cap mutants, and Western blotting was used to detect the levels of Cap mutant expression with anti-HA antibodies. (D to F) K164, K179, and K191 were the ubiquitination sites of the PCV2 Cap protein. The Cap lysine residues at residues 164, 179, and 191 were replaced with alanine (D). HEK293T cells were transfected to express Flag-pMKRN1 and the indicated Cap lysine mutants, Western blotting was used to detect the expression levels of Cap or mutants using anti-Cap antibodies (E), and the ubiquitination of Cap mutants was detected (F). These results were confirmed in three independent experiments.