FIG. 3.

Vpx blocks HIV-1 Rev NLS- and SV40 NLS-mediated nuclear import. (A) Import of the SV40 NLS and HIV-1 Rev NLS were impaired in the presence of Vpx expression in HeLa cells. Similarly, Vpx translocation was inhibited by the expression of the SV40 NLS as well as the HIV-1 Rev NLS, which suggests that Vpx may utilize the same pathway that is utilized by the SV40 NLS and Rev NLS, as indicated by its competitive inhibition. Vpx expression plasmids were cotransfected with either the SV40 NLS or the HIV-1 Rev NLS in Cos-7 cells, and the cellular localization of GFP fusion proteins was visualized directly after fixing of the transfected cells with 3% paraformaldehyde; Vpx was visualized by indirect immunofluorescence using anti-Vpx monoclonal antibody followed by an Alexa fluor 488-conjugated secondary antibody and epifluorescence. (B) Overexpression of the C-terminal Vpx65-75 NLS interfered with the nuclear import of both the full-length and N-terminal NLS of Vpx.