FIG. 2.

Expression of native NS3, NS3 HID, and NS3 HIID mutant proteins. HeLa T4⁺ cells were infected with VVTF-7 at a multiplicity of 10, transfected in the presence of Lipofectin with either a plasmid containing the wild-type NS3 gene, i.e., pGEM10.10, or with the indicated HID mutant NS3 plasmids (Table 2), and labeled with [³⁵S]methionine as described in Materials and Methods. Infected cells were lysed and immunoprecipitated with an NS3-monospecific polyclonal antiserum, and the proteins were analyzed by SDS-PAGE (15% polyacrylamide) and autoradiography. For the unmodified gene, the positions of the glycosylated GNS3 and nonglycosylated NS3 and NS3A bands are indicated. The mutant genes gave bands corresponding to the expected NS3 and NS3A deletion products.