Fig. 3

CVB3 capsid protein VP1 undergoes ubiquitination. a HeLa cells were transiently transfected with HA-tagged ubiquitin (HA-Ub) or empty vector for 24 h, and then infected with CVB3 (MOI = 10) for 5 h. Co-IP was performed with an anti-HA antibody, followed by western blot analysis of HA-Ub using antibodies against HA-tag or VP1. b HeLa cells were infected with CVB3 (MOI = 10) for 5 h, followed by IP using anti-VP1 antibody. Western blotting was conducted using antibodies against Ub, VP1, or SQSTM1 as indicated. c Schematic illustration of the CVB3 polyprotein and the respective VP1-linked ubiquitinated fragments. Arrows depict sites of cleavage by virus-encoded proteinases. d HeLa cells were infected with CVB3 (MOI = 10) for 5 h in the presence or absence of a proteasome inhibitor MG132 (10 μM) or a lysosome inhibitor bafilomycin A1 (200 nM) as indicated. IP was conducted using an anti-VP1 antibody, followed by western blotting was performed using anti-K63-linked or anti-K48-linked Ub antibody and anti-VP1 antibody as indicated. Similar results were observed in three independent experiments