KIF3A is dispensable for HIV-1 production by infected T cells. (A) Schematic representation of the experimental design. (B) Immunoblot analysis of KIF3A and α-tubulin expression in Jurkat T cells expressing the indicated shRNA. After 3 d, all shRNA specific for KIF3A reduced its level of expression down to less than 20% in Jurkat T cells. (C) Dosage of p24 Gag in 20-h culture supernatants of NL4-3 ΔEnv-infected Jurkat T cells harvested as indicated in A. Values presented have been corrected to the number of HIV-infected cells present in each sample, as calculated using CellTiter Glo (for cell viability) and anti-Gag staining analyzed by flow cytometry (for percentage of infection). (D) Confocal micrographs of primary T cells infected with VSV-G–pseudotyped HIV-1 NL4-3 for 24 h and stained for the indicated markers. Note that Gag distribution is highly polarized at the plasma membrane, whereas KIF3A distribution is totally different. Bars, 5 µm. (E) Ultrathin cryosection of Jurkat T cells infected with HIV-1 NL4-3 and immunogold labeled for Pr55Gag with PAG10. Bar, 200 nm.