FIG 2.
Saturation of HIV-1 Nef counteractive activity by Ser5 overexpression. (A) 293T cells were transfected with a fixed amount of HIV-1 WT or ΔNef proviral vector and the indicated amounts of 4 different Ser5 expression vectors, pBJ6-Ser5-HA, pBJ5-Ser5-HA, pBJ5-iHA-Ser5, and pMSCV-Ser5-FLAG. After 48 h, viruses were collected and normalized by p24Gag ELISA; viral infectivity was measured by infecting TZM-b1 cells. (B) The anti-HIV-1 activity of Ser5 expressed from the pBJ6 vector was determined in the presence or absence of VSV G pseudotyping. (C) J-TAg, J-TAg-KO, and J-TAg-KO-Ser5 cells were infected with WT or ΔNef HIV-1 by spinoculation. The viruses produced were normalized by p24Gag ELISA, and viral infectivity was determined after infection of TZM-b1 cells. Viruses were also purified by ultracentrifugation, and viral protein expression in the Jurkat cells and purified virions was analyzed by Western blotting. (D) The Ser5 expression levels in 293T cells from the different expression vectors were compared by Western blotting using an anti-Ser5 antibody. Numbers in the boxes indicate relative amounts of each vector used for transfection of 293T cells. The error bars represent SD from three experiments.