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. 2010 Jul 28;84(19):10241–10253. doi: 10.1128/JVI.00585-10

FIG. 2.

FIG. 2.

Investigation of mechanisms by which Mov10 inhibits HIV-1 replication. (A) Effect of Mov10 overexpression on infectivity of NL4-3 and HDV-EGFP. 293T cells were cotransfected with pNL4-3 (8 μg) or pHDV-EGFP (8 μg) and pHCMV-G (4 μg) and increasing amounts of F-Mov10 expression plasmid. Serial dilutions of culture supernatants were used to infect TZM-bl cells, and luciferase activity was determined. The labels indicate the ratio of F-Mov10 to HIV-1 DNA used for transfections. The data are plotted as relative infectivities, with the control virus (empty vector) set to 100%. Error bars represent standard errors from four independent experiments. (B) Interactions between Mov10 and A3G do not affect viral infectivity. 293T cells were cotransfected with pHDV-EGFP (3.33 μg) and pHCMV-G (0.67 μg), and increasing amounts (0 to 0.67 μg) of F-A3G expression plasmid were present during virus production in either the absence or presence of 0.67 μg of the F-Mov10 expression plasmid. Culture supernatants containing 5 ng of p24 CA were used to infect TZM-bl cells, and luciferase activity was determined. For viruses with different amounts of F-A3G but no F-Mov10, the data are plotted as relative infectivities, with the control virus (empty vector) set to 100%. For viruses with different amounts of F-A3G and a fixed amount of F-Mov10, the data are plotted as relative infectivities, with the virus having no F-A3G but with F-Mov10 set to 100%. Error bars represent standard errors from two independent experiments. (C) mRFP-Mov10 expression in target cells does not inhibit HIV-1 infection. Plasmids expressing mRFP-tagged P body-associated proteins were transfected into 293T cells; 48 h later, the transfected cells were infected with VSV-G pseudotyped pHDV-EGFP, and the proportions of mRFP− and mRFP+ cells that were EGFP+ were determined by FACS analysis. An mRFP expression vector was used as the control (set to 100%). Error bars represent standard errors from three independent experiments. (D) Effect of F-A3G and F-Mov10 on proteolytic processing of HIV-1 Gag. 293T cells were cotransfected with pHDV-EGFP (10 μg) and empty vector, F-A3G (2 μg), or F-Mov10 (4 μg) expression plasmids. Virions were lysed, and the virion proteins (35 or 25 ng of p24 CA) were analyzed by Western blotting using an anti-p24 CA antibody.