FIG. 1.

Basic vector for the analysis of Nef enhancement of infectivity. (A) The 5′ region of nef of the HIV-1 NL4-3 strain was replaced with oligonucleotides containing XhoI and NotI restriction sites to generate NL-Not. Then nef allele of NL4-3 amplified by PCR was inserted into the XhoI/NotI site to generate NL-Not-Nef. (B) Wild-type NL4-3, nef-defective NL4-3 (NL-Xh), NL-Not, and NL-Not inserted with NL4-3 nef (NL-Not-Nef) were transfected into 293T cells for propagation of virus. 293T cells were lysed and analyzed by immunoblotting with a mixture of monoclonal antibodies against p24^gag, gp41^env, and Nef. Molecular standards are shown on the left. (C) Serially diluted virus stocks were inoculated into MAGNEF cells plated on 96-well dishes. Forty-eight hours later, infected cells were identified by being stained with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. Infectivity was normalized by the quantity of p24^gag in the virus stocks. Experiments were done in triplicate and repeated three times. A representative result is shown with standard deviations.