FIG 7.
Nt-activated infections and uptake of HIV-1JRCSF(Ad) by HeLa-CD4/CCR5(Δ18) cells are blocked by inhibitors of clathrin-mediated endocytosis. (A to C) Inhibition of virus uptake by dynasore. Virions were adsorbed at 4°C to cells preincubated with serum-free medium containing 80 μM dynasore (bottom) or 0.4% DMSO alone (without dynasore [−dynasore]; top). After these treatments, cultures were either treated with pronase or not treated on ice and fixed immediately (A) or incubated at 37°C with or without 100 μM Nt and with or without dynasore for 1 (B) or 4 (C) h and then digested with pronase and fixed. Cells in fixed cultures were permeabilized and stained for p24 Gag using a p24 monoclonal antibody and goat antimouse Alexa 594, and images were obtained using deconvolution microscopy. Results of a representative experiment of six total experiments performed are shown. (D) Dynasore inhibition of wild-type HIV-1JRCSF uptake by cells expressing wild-type CCR5 (JC.53). The same experiment described for panels A to C was performed without Nt. Cultures that were fixed after 1 h at 37°C are shown. Conditions in which fixation of cultures occurred immediately after virus binding at 4°C or after 4 h of incubation at 37°C yielded results identical to those shown in panels A and C. (E) Dynasore and chlorpromazine inhibition of Nt-activated infections. Cells were pretreated with the inhibitors dynasore (dyn) or chlorpromazine (CPZ) or with vehicle (0.4% DMSO or 0.1% ethanol [EtOH]) and then adsorbed with HIV-1JRCSF(Ad) with or without inhibitors at 4°C. Cultures were then placed at 37°C for 1 h, at which time the inhibitors were washed away and inhibiting TAK779 was added. Controls revealed that the inhibitors were not cytotoxic under the conditions of our assays. Results of a single representative experiment (executed in duplicate) of two independent experiments performed are shown. (F) The same experiment described for panel E performed using wild-type HIV-1JRCSF and JC.53 cells. Relative infectivity values were generated by normalizing the titers obtained under each condition to the titers obtained in the presence of vehicles. (G) Dynasore inhibition is reversible. Transferrin-Alexa 488 uptake was assessed in HeLa-CD4/CCR5(Δ18) cells treated with 80 μM dynasore (middle), in cells treated with 0.4% DMSO alone (top), or in cells treated with dynasore and then washed free of inhibitor (bottom).