FIG 3.

NS3/4A/2K cleavage events correlate with physical changes to NS3 and NS3-4A. The proteinase K sensitivity of YFV polyprotein products was used to assess physical changes. (A) SW13 cells were infected with vTF7-3 and transfected to express the sig2A-5* or sig2A-5 polyprotein. After 4 h of metabolic labeling, cells were permeabilized by digitonin and then treated with or without proteinase K (+pk and –pk, respectively). NS3-containing products were analyzed as described in the legend to Fig. 1A. (B) SW13 cells transduced or not to express DNAJC14 were infected with YFV (MOI = 10) and 8 h later permeabilized with digitonin to separate the soluble (S) and insoluble (P) fractions. Proteins precipitated from the insoluble fraction were treated or not with proteinase K. Western blot analysis was performed using NS3- and calnexin-specific antibodies. Digestion of calnexin and protection of the N-terminal luminal fragment served as controls for protease activity and membrane integrity, respectively. (C) DNAJC14 interacts with NS4A. 293T cells were cotransfected with plasmids expressing myc-tagged DNAJC14 along with the YFV NS4A-GFP fusion protein. Expression of GFP served as a control. Total cell lysates and anti-myc-immunoprecipitated products were blotted with anti-GFP antibody. LC, antibody light chain. The results presented in each panel in panels B and C are representative of those from three independent experiments. The numbers to the left of the gels are molecular masses (in kilodaltons).