Pepstatin a

Human vaginal epithelial cell line VK2/E6E7 cells (ATCC CRL-2616) were cultured in defined keratinocyte serum-free medium (SFM) (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C and 5% CO₂. HIV-1 IIIB-infected H9 cell line [61 (link),62 (link),63 (link)] was obtained from the NIH AIDS Research and Reference Reagent Program (NIH-ARRRP). The HIV-1 IIIB virus was harvested from these H9 cells. pCH040.c/2625, a full-length transmitted/founder (T/F) HIV-1 subtype B infectious molecular clone [64 (link)] and HEK 293T cells were obtained from NIH-ARRRP. The HIV-1 CH040 virus was produced by transfecting HEK 293T cells with the pCH040.c/2625 plasmid using polyethylenimine (PEI) as described previously [65 ]. Filipin, mouse monoclonal anti-actin antibody, DMSO, and MPA were purchased from Sigma (St. Louis, MO, USA). LysoSensor Green DND-189 was purchased from Thermo Fisher Scientific. LC3B antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). Pepstatin A, leupeptin and E-64 were purchased from AG Scientific (San Diego, CA, USA).

HIV-1 proviral constructs pNL4-3 [62] (link), and pYU2 [63] (link) along with zidovudine (AZT)were obtained from the NIH-ARRRP. Transfection of the dominant negative Rab11 mutant (DN Rab11, Addgene) was performed using Lipofectamine 2000 (Invitrogen) following the manufacturer's manual. Western blot analysis was carried out as previously described [64] (link). Colchicine, dynasore hydrate, DEAE-dextran hydrochloride and (E)-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL) were purchased from Sigma. Lysosomal inhibitors pepstatin A, leupeptin and E-64 were purchased from AG Scientific. anti-GFP antibody was purchased from Santa Cruz Biotechnology, Inc. The HIV-1 p24 monoclonal antibody [65] (link) was obtained from NIH-ARRRP. Transferrin was purchased from Invitrogen and staining was performed using the manufacturer's protocol.