Figure 6

PRMT6 automethylation is necessary for its HIV-1 restriction activity. The indicated forms of PRMT6 plasmid were co-transfected with HIV-1 pNL4.3 proviral DNA into 293T cells. At 48 hours after transfection, cell culture fluids containing virus were collected, quantified by QPCR (A) or by HIV RT activity assay (B), and titrated onto TZM-bl cells (C, D). The amount of transfected PRMT6 expression, i.e. not protein stability, was quantified by Western blots with anti-Myc and anti-actin antibodies followed by densitometric analysis normalized against actin (E). Endogenous levels of PRMT6 in HeLa cells (lane 2), TZM-bl cells (lane 3) and 293T cells (lane 4) were compared to transfected PRMT6 in HeLa cells (lane 1) (F). 15 μg of protein from whole cell extracts were loaded into each well used for analysis in the Western blots in (E) and (F). Actin was used as a loading control. Infectivity was measured by luciferase assay at 48 hours after infection. The control experiment was transfection of pNL4.3 with an empty plasmid (referred to on the figure as pNL4.3 only). Each experiment was performed in triplicate on three separate occasions, with similar results being obtained each time. T-tests were performed for both (A) and (B), showing that only PRMT6-WT was significantly different than the control *: p < 0.05. Two-way ANOVA was performed for (C) and (D), showing that only PRMT6-WT statistically inhibited HIV replication *: p < 0.001.