FIG. 1.

Nef enhances the infectivity but not the formation of CD4-chemokine receptor pseudotypes. (A) 293T cells were transfected with HXB/Env⁻/Nef⁺ or HXB/Env⁻/Nef⁻, along with a HIV-1 vector encoding GFP and either pCD4_ΔCT together with pcDNA6/CXCR4-FLAG (left) or pEnv_HXB (right). Progeny virions were normalized for RT activity and used to infect HeLa cells that had been transfected with vectors expressing either Env_HXBΔCT (left), or CD4 together with CXCR4-FLAG (right). Infectivities were evaluated by counting GFP-positive foci. Values represent the average from three parallel infections, and error bars correspond to one standard deviation. (B) HeLa cells expressing Env_HXB were infected with Nef⁺ and Nef⁻ CD4_ΔCT/CXCR4 receptor pseudotypes produced as described for panel A, and GFP-positive infected cells were quantified by flow cytometry. The percentage of infected cells in each panel is indicated. (C) Nef does not affect the incorporation of CD4_ΔCT or of CXCR4-FLAG into Env-deficient HIV-1 particles. Aliquots of the receptor-pseudotyped viruses used for the infections shown in panel A were pelleted through sucrose and analyzed by Western blotting with anti-FLAG, anti-CD4, anti-HIV-1 CA, and anti-Nef antibodies. A sample derived from cells cotransfected with pCD4_ΔCT, pcDNA6/CXCR4-FLAG, and a Gag-deficient HIV-1 provirus was included as a background control.