FIG 5 .

NS2 K63-ubiquitinated lysine residues mediate HRS UIM binding and HCV assembly. (A) Analysis of interactions of WT and mutant HRS with NS2, NS5A, and TSG101 by PCAs. Data are presented as NLRs relative to the interaction with WT HRS. (B) Levels of HRS in lysates of cells transfected with the indicated plasmids. (C) Purified recombinant truncated NS2 (rNS2) (92 to 216 aa) was incubated with ubiquitin-hemagglutinin (UB-HA) for 20 or 60 min in the presence or absence of E1 and E2 enzymes and Huh-7.5 cell extract. Cell extracts incubated in the absence of NS2 served as controls. A representative membrane blotted with anti-NS2 antibodies is shown. Samples were run on the same gel, from which two lanes were cut out. (D) Lysates of HCV-transfected or naive Huh-7.5 cells were incubated with FLAG anti-K63 TUBE reagent followed by IP with anti-NS2 antibodies or IgG. Representative membranes blotted with antibodies against NS2, ubiquitin, FLAG, and actin are shown. (E and F) Huh-7.5 cells were cotransfected with WT or mutant GLuc-NS2 and ubiquitin and subjected to IP with anti-GLuc (E) or anti-NS2 (F) antibodies or IgG under denaturing conditions. Membranes were blotted with antibodies against ubiquitin, polyubiquitin-K63, GLuc (E), NS2 (F), and actin. Nonconsecutive lanes that were run on the same gel are shown in panel F. (G) Levels of NS2 in lysates of cells transfected with the indicated plasmids. (H) Analysis of interactions of WT or mutant NS2 with HRS by PCAs. Plotted data represent NLRs relative to WT NS2-HRS binding. (I) Cells were electroporated with WT or mutated NS2 bicistronic J6/H77NS2/JFH HCV RNA. HCV RNA replication was measured by luciferase assays at 6 and 72 h postelectroporation, and results are expressed as RLU. (J) Intra- and extracellular infectivity analyzed by luciferase assays in naive cells inoculated with clarified cell lysates and supernatants derived from the HCV-electroporated cells, respectively. Plotted data represent infectivity normalized to WT controls. Mean values and standard deviations of results of 3 independent experiments are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t test).