FIG. 4.

Effects of NSs on minireplicon activity in mosquito and mammalian cells. Cells were transfected with pTM1-based or pT7Ac-based L expression vectors as appropriate and the minigenome plasmid pT7riboBUNMREN(−), along with plasmids expressing either N and NSs (pTM1-BUNS or pT7AcS) or N alone (pTM1-BUNN or pT7AcN), as indicated. pT7AcLuc was cotransfected as an internal control to standardize luciferase activities. (−) control, negative control without functional L protein. The amounts of DNA transfected were kept constant by adding the corresponding empty plasmid if necessary. Activities are expressed in light units (for BSR-T7/5) or as fold induction of Renilla luciferase (Ren luc) activity (for C6-IBT7/3). (A) Control experiment in BSR-T7/5 cells transfected with N- or N-plus-NSs-expressing pTM1- or pT7Ac-based expression plasmids and minireplicon components as described above. (B) C6-IBT7/3 cells transfected with N- or N-plus-NSs-expressing pT7Ac-based expression plasmids and minireplicon components as described above. (C) Control experiment in BSR-T7/5 cells transfected with NSs-expressing pTM1- or pT7Ac-based plasmids and minireplicon components as described above. (D) C6-IBT7/3 cells transfected or not with the pT7AcNSs plasmid and minireplicon components as described above. (E) Expression of NSs protein in transfected mosquito cells. C6-IBT7/3 cells were either transfected with 1 (lane 1) or 2 (lane 2) μg of pT7AcNSs or left untransfected (lane 3). Cells infected with wtBUN (lane 4) were used as a positive control. Following SDS-PAGE and blotting to a membrane, NSs protein was detected by using an NSs-specific antibody; different exposures were needed to visualize NSs in transfected and infected cells. The positions of NSs and molecular size standards (in kilodaltons) are indicated.