FIG. 4.

Nef enhances the infectivity of HIV-1 particles pseudotyped with the receptor for an avian retrovirus. (A) Nef does not affect the incorporation of Tva into Env-deficient HIV-1 particles. 293T cells were transfected with HXB/Env⁻/Nef⁺, HXB/Env⁻/Nef⁻, or HXB/Gag⁻, along with a vector expressing HA-tagged Tva. Progeny virions were pelleted and analyzed by Western blotting with anti-HA antibody to detect Tva and with anti-HIV-1 CA antibody to monitor particle production. The expression levels of Tva in the virus producer cells were also examined by Western blotting. (B) All forms of Tva-HA can be detected at the cell surface. Control and Tva-HA-expressing 293T cells were surface biotinylated with the membrane-impermeative reagent sulfo-NHS-LC-biotin, and proteins recovered on streptavidin beads from cell lysates and the unfractionated cell lysates were analyzed by Western blotting with anti-HA. (C) Effect of Nef on the infectivities of HIV-1(Tva) and HIV-1(EnvA) pseudotypes. To produce pseudotyped viral particles, 293T cells were transfected with HXB/Env⁻/Nef⁺ or HXB/Env⁻/Nef⁻, along with vectors expressing HA-tagged Tva or EnvA as indicated. Supernatants were normalized for RT activity and used to infect TZM-bl indicator cells expressing either EnvA or Tva, as appropriate. Infectious events were quantified by counting blue foci after staining with X-Gal. Error bars indicate standard deviations.