FIG 9.

ISG12a relies on the E3 ligase domain of SKP2 to restrict viral infection. (A) Ubiquitination assay and Western blotting of lysates from HEK293T cells expressing exogenous SKP2, HA-ubiquitin (HA-ub), and ISG12a domains (full-length, DII, DII-N, or DII-C). (B) Ubiquitination assay and Western blotting of lysates from HEK293T cells expressing exogenous ISG12a, HA-ub, and SKP2 domains [full-length, SKP2-N, SKP2-(N+L), or SKP2-DL]. (C) HLCZ01 cells were infected by HCV (MOI, 0.1) for 24 h followed by the transfection of the indicated plasmids for 48 h. NS5A, ISG12a, and the domains of SKP2 were analyzed by Western blotting. (D and E) Huh7.5 cells were infected by HCV (MOI, 0.1) for 24 h followed by the transfection of the indicated plasmids for 48 h. Intracellular HCV RNA (D) and extracellular infectious virus particles (E) were detected by real-time PCR and FFU assay, respectively. Error bars represented SD from triplicate experiments. *, P < 0.05; **, P < 0.01. V5, Flag, and HA were the tags used.