FIG 1.

Characterization of fluorescent protein-expressing proviral molecular clone of HIV-1 with T/F Env. (A) Cloning scheme of introducing T/F Env into fluorescent protein-expressing proviral molecular clone NLCI. LTR, long terminal repeat. (B) Viral production in transfected 293T cells was measured by p24 ELISA. Mean values from three independent experiments are shown. Error bars represent standard errors of the means. (C) Env expression in transfected 293T cells and Env incorporation into viral particles were examined by Western blotting. The amount of sample loaded was normalized to the sample p24 content. (D) Representative flow cytometry plots of transwell assay and cell-to-cell coculture system for comparison side by side. For the transwell assay, nucleofected Jurkat donor cells and MT4R5 target cells were separated into two compartments by a membrane with a pore size of 0.4 μm, which allows passage of cell-free virus particles. A cell-to-cell coculture infection was performed at the same time under similar conditions. To inhibit secondary rounds of infection, AZT (10 μM) was added 18 h postinfection. (E) Single-round cell-free infection in CD4⁺ CCR5-expressing MT4R5 cells. Cell-free infection was normalized to the p24 input. Del, deleted. (F) Single-round cell-to-cell infection levels of MT4R5 target cells from coculture of Jurkat donor cells transfected with NLCI constructs with T/F Env. Cell-to-cell infection was normalized to same percentage of donor transfection and the donor target ratio. Error bars represent results of three independent experiments.