FIGURE 6.

Analysis of polyprotein processing in constructs containing NS5A second site compensatory mutations. A, Western blot analysis of lysates from HepG2 cells transduced with baculovirus vectors engineered to deliver replicon transcripts into cells using a tetracycline-responsive promoter. Transcripts introduced include [FA]neo and [P43VV]neo (lanes 1 and 2) as well as those that possess the P1′–P3′ QGG and VGG second site compensatory mutations, either alone ([P43ST/QGG]neo and [P43ST/VGG]neo) (lanes 3 and 6), in combination with the P43VV mutation ([P43VV/QGG]neo and [P43VV/VGG]neo) (lanes 5 and 8), or in combination with the P3V mutation ([P43SV/QGG]neo and [P43SV/VGG]neo) (lanes 4 and 7). A control lysate was derived from cells transduced with BACtTA only (lane M). Cell extracts were probed by Western blot for expression of HCV proteins (NS5B, NS5A, NS4B, and NS3) or neomycin phosphotransferase (NPT). Where present, arrows indicate the position of the full-length NS3 and NS5A, with other bands present representing products from internal cleavage or cross reactive cell antigens. B, HepG2 cells were transduced with the same baculovirus constructs as in A and metabolically labeled for 15 min with [³⁵S]Met/Cys prior to cell lysis. NS5A-FLAG- and NS5A-FLAG-containing precursors were immunoprecipitated by anti-FLAG mAb, separated on 10% SDS-PAGE, and visualized by a PhosphorImager. The positions of different HCV polyprotein products are indicated by arrows. Mock lysate comes from BACtTA-only-transduced cells. C, graphical representation of the data from B showing the proportion of uncleaved NS4B5A precursor products in cells from two separate experiments and expressed as a percentage of total NS5A-containing products.