Figure 2

HIV containing MLV capsid is largely TRN-SR2 independent, HIV with MLV integrase is not. (A) HeLaP4 cells depleted of TRN-SR2 (siTRN-SR_2) and control cells (mock and siTRN-SR_2MM) were infected with VSV-G pseudotyped HIV-1 single-round virus, MLV vector, or with the chimeric viruses MHIV-mMA12CA or MHIV-mIN. In MHIV-mMA12CA the HIV MA and CA proteins are replaced by the MLV MA, CA and p12 proteins. In MHIV-mIN the HIV IN protein is replaced by MLV IN. Three days post infection cells were lysed and Fluc activity was measured and normalized to the total amount of protein in the cell lysates. Results are shown as the mean values of relative light units per μg protein (Fluc RLU/μg protein) ± SD compared to mock transfected cells and represent 2 independent experiments each performed in triplicate. The arrows indicate the relative inhibition of infectivity in TRN-SR2 depleted cells compared to mismatch siRNA tranfected cells. (B) Direct interactions between recombinant GST-TRN-SR2 and His₆-tagged HIV-1 IN or MLV IN were measured by AlphaScreen. As negative controls for binding to GST-TRN-SR2 both His₆-Gα_oA and His₆-Roc-COR were used. 10 nM of GST-TRN-SR2 was incubated with different concentrations of His₆-tagged proteins and complexes were bound to glutathione donor beads and nickel-chelate acceptor beads. Light emission was measured using an EnVision Multilabel Reader. The apparent equilibrium dissociation constants (K_d) were calculated with GraphPad Prism 5 and are indicated on the graphs.