FIG 2.

DNAJC14 overexpression affects cleavage at the 4A/2K site but not the NS4B/5 site. (A) Schematic of YFV NS regions used in the vaccinia virus expressing bacteriophage T7-based trans-cleavage assays. (Top) The NS3-5*** (3-5***) construct encodes NS3-5₃₅₆ and also contains the protease active-site mutation along with the indicated mutations at the NS3/4A and NS4B/5 cleavage sites, which block cleavage. The wild-type version is not shown. (Bottom) The NS4B-5₃₅₆ substrate used in trans cleavage assays along with the active NS2B-3₁₈₁ (2B-3₁₈₁) protease. For additional details, see Fig. 1B. (B) trans-Cleavage analyses were conducted in vTF7-3-infected SW13 cells that had previously been transduced or not to express DNAJC14. YFV proteins were coexpressed by transfection of a combination of plasmids carrying the parental NS3-5 polyprotein, the triple mutant NS3-5***, the NS4B-5 (4B-5) region, and the cofactor protease NS2B-3, as indicated. Cells were labeled with [³⁵S]methionine for 4 h. Labeled NS3- or NS4B-containing proteins were analyzed as described in the legend to Fig. 1A. The data are representative of those from two independent experiments. The numbers to the left of the gel are molecular masses (in kilodaltons). Asterisks indicate bands of interest. (C) Schematic summary of the effect of DNAJC14 overexpression on cleavage events. Arrows, relative accumulation or reduction in protein levels.