FIG 3.

Vpr enhances Env processing/infectivity in MDMs via TET2- and IFITM3-dependent mechanisms. (A) MDMs transduced with lentiviral vectors with shRNA-mediated knockdown of TET2 and IFITM3 were infected with HIV-1 or HIV-1 ΔVpr viruses as described above. Western blot analysis of cell lysates was performed as in Fig. 1B. The gp120/gp160 ratio of HIV-1 from control MDMs is defined as 100%. (B) MDMs were transduced with lentivirus vectors with control shRNA, shTET2, and shIFITM3. Viral particles were purified by ultracentrifugation (over 20% sucrose) from the aforementioned MDMs infected with HIV-1 or HIV-1 ΔVpr viruses for 4 days, with NVP added at 2 dpi. Five nanograms of p24 per virion for each sample was added per lane for analysis of gp120, p24, and gp41 in virions. Western blotting was performed by using anti-gp120, p24, and gp41 antibodies. (C) The ratios of gp120/p24 and of gp41/p24 are summarized from three different donors. (D) Supernatants from MDMs infected with HIV-1 or HIV-1 ΔVpr viruses were used to infect TZM-bl indicator cells. The relative infectivity is defined as HIV-induced luciferase/ng (n = 3 donors).