KIF3A is required for viral production by macrophages. (A) Schematic representation of the experimental design. (B) Immunoblot analysis of KIF3A and Tsg101 expression in macrophages transfected with the indicated siRNA. After 4 d, both siRNA specific for KIF3A significantly reduced its level of expression in primary macrophages. (C) Macrophages were infected by HIV-1 and transfected with siRNA as described in A. Cell viability was measured at d 4 p.i. using the CellTiter Glo kit and normalized to the control (siLuc). (D) Primary macrophages were infected with HIV Gag-iGFP ΔEnv pseudotyped with VSV-G and transfected with siRNA as described in A. This virus has a single cycle in macrophages and does not induce syncitia formation. Percentages of GFP+ macrophages were estimated by flow cytometry at d 4 p.i. (E) Infectivity of the virions produced by the macrophages subjected to the indicated siRNA was evaluated using the same amount of p24 (2 ng; see Materials and methods). (F) KIF3A depletion does not affect the early steps of infection. The reporter cell line TZM-bl was transfected with the indicated siRNA. 2 d later, cells were infected with HIV-1. Cells were washed 8 h later, reincubated for an additional 16 h, and assayed for β-gal activity, whose expression is driven by a Tat-sensitive promoter. (G) Dosage of p24 Gag in the 24-h culture supernatants harvested as indicated in A. *** indicates that the difference between the two histogram bars is statistically significant (P < 0.001). Experiments from D–F have been repeated at least two times and from B, C, and G at least three times in quadruplicate.