FIG 5.
Activation of HIV-1JRCSF(Ad) infection by Nt is reversible and can occur many hours after viruses have been adsorbed onto HeLa-CD4/CCR5(Δ18) cells. (A) Nt reversibility. HIV-1JRCSF(Ad) was adsorbed onto cells at 4°C, and 100 μM Nt was added upon warming to 37°C (squares) or after 2 h at 37°C (circles). In some cultures, Nt was washed out after 2 h of incubation at 37°C (asterisks). Kinetics of infection were also measured in HeLa-CD4 cells with wild-type CCR5 (triangles) or in CCR5(Δ18) cells in the absence of Nt (diamonds). This experiment was performed with HeLa-CD4 cells expressing moderate amounts of CCR5(Δ18) and gave similar results to those obtained in experiments performed with cells expressing large or small amounts of CCR5(Δ18). Results of a representative experiment of eight performed in duplicate are shown. Error bars are the ranges. (B and C) Nt activates HIV-1JRCSF(Ad) infections many hours after virus adsorption onto target cells. Viruses were bound to HeLa-CD4/CCR5(Δ18) cells at 4°C, and 100 μM Nt was added after 0, 2, or 5 h (B) or 0, 12, 24, 36, or 48 h (C) of incubation at 37°C. Error bars are the ranges. Titer values (104 focus-forming units [FFU] per ml) were plotted versus time using Microsoft Excel software. (D) Quantitative analysis of infectivity data from panels A, B, and C. The graph was obtained by plotting titer differences between cultures that had Nt added initially and after delays versus time after warming.