FIG. 2.

Analysis of culture-adapted virus. (A) Immunoblot analysis of SG3^IN2 and culture-adapted viruses. Supernatants from SupT1 cell cultures infected with the CF-65, CF-131, and SG3 viruses were collected for analysis. Culture supernatants were also collected for analysis from 293T cells transfected with pSG3^S-RT and pSG3^IN2. Virus was concentrated by ultracentrifugation (125,000 × g, 2 h) through cushions of 20% sucrose. Viral pellets were lysed, and immunoblots were prepared and probed with anti-CA monoclonal antibody or serum from an HIV-2-infected individual. (B) Analysis of viral cDNA synthesis in infected cells; 500 ng (p24 antigen equivalents) of CF-65, CF-131, SG3, SG3^S-RT, and SG3^IN2 was used to infect 5 × 10⁵ JC53 cells for 4 h at 37°C. The SG3^S-RT and SG3^IN2 viruses were obtained by transfection of 293T cells. Eighteen hours after infection, total DNA extracts were prepared, and 20 ng of each was analyzed by PCR for the R-U5 DNA product of reverse transcription. A standard curve was created with standards ranging from 5 to 5,000 DNA copies of pSG3. PCR-amplified DNA was resolved on a 1.5% agarose gel and stained with ethidium bromide, and the R-U5 product was quantified as described in Materials and Methods.