APOBEC4 Enhances the Replication of HIV-1
Fig 5
Presence of A4 does not affect HIV-1 infectivity.
HIV-1 reporter virus NL-Luc R-E- (VSV-G) was produced in 293T cells in the presence of increasing amounts of A4 (no tag) and A4-HA (C-terminal HA-tag). A4 and A4-HA increase in a dose-dependent manner both (a) the virus-encoded luciferase activity and (b) the expression of intracellular viral capsid (p24) in the transfected virus producing cells as demonstrated by immunoblot analysis (same cell lysates used in (a) and (b)). Error bars indicate standard deviation. (c) Immunoblot analysis of intracellular viral p24 (capsid) expression. Similar as in (a) and (b), NL-Luc R-E-/VSV-G was co-transfected with increasing amounts of HA-A4 plasmid (N-terminal HA-tag), as indicated. Immunoblots of cells were probed with anti-p24 (capsid) antibody. A4-HA expression in transfected cells was detected by immunoblotting using anti-HA antibody. Anti-tubulin (tub) antibody served as loading control. α, anti. (d) Relative viral luciferase activity in cells co-transfected with A4-HA and HIV-1 plasmids, as in (a). Summary of 28 independent experiments, median indicated. A4-HA was transfected in increasing amounts. (e) Equal volumes of supernatants of cells co-transfected with NL-Luc R-E-/VSV-G and increasing amounts of A4-HA were used to infect HOS cells. Intracellular luciferase activities were determined in infected cells; summary of 16 experiments (a subset of the experiments shown in (d)), median is indicated. (f) A subset of samples (seven experiments) used in (e) was quantified for RT concentrations. RT normalized supernatants of cells co-transfected with NL-Luc R-E-/VSV-G and increasing amounts of A4-HA were used to infect HOS cells. Intracellular luciferase activities determined in infected cells, median is indicated. (d—f) Statistical evaluation of reporter luciferase activity data was performed by means of a multifactorial ANOVA.