FIG. 1.

Genetic structure and initial characterization of Wt and F⁻ viruses. (A) Location of the cPPT and central termination sequence (CTS) within HIV-1 NL4-3. The 10 (cPPT-D) nucleotide changes underscored in box F⁻ were introduced into Wt NL4-3 by site-directed mutagenesis. These changes have been previously described as sufficient to abrogate central flap formation in NL4-3 and several other isolates of HIV-1 (18, 29, 44). Mutant (F⁻) and Wt replication-competent viruses were generated by transient transfection of 293FT cells. (B) Schematic diagram of denv(Wt), a new NL4-3-based reporter virus that expresses the enhanced green fluorescent protein fused to firefly luciferase (EGFPLuc) in place of HIV Env. An endoplasmic reticulum retention sequence (KDEL) (33) is present at the 3′ end of the EGFPLuc coding sequence. The cPPT-D mutation was introduced into this vector to provide a corresponding denv(F⁻) reporter virus genome. (C) 293FT cells were infected with denv(Wt) or denv(F⁻) viruses and visualized for GFP expression by fluorescent microscopy (magnification, ×20) at 3 days postinfection. (D) 293FT cells were infected with denv(Wt) and denv(F⁻) viruses, and quadruplicate wells were harvested daily for a luciferase assay. Mean values ± standard deviations of quadruplicate infections are shown.