Figure 3
From: BI-2 destabilizes HIV-1 cores during infection and Prevents Binding of CPSF6 to the HIV-1 Capsid
BI-2 prevents the binding of the cellular factor CPSF6 to HIV-1 CA-NC complexes. (A) The ability of CPSF6 to bind in vitro assembled HIV-1 CA-NC complexes in the presence of BI-2 was analyzed as previously described [22]. Briefly, extracts of 293 T cells transiently transfected with a CPSF6-FLAG construct (Input) were incubated with in vitro assembled HIV-1 or SIVmac CA-NC complexes in the presence BI-2 for 1 h. Subsequently, extracts were applied onto 70% sucrose cushion and centrifuged, and the pelleted fraction was collected (Pellet). Input and Pellet fractions were analyzed using anti-FLAG and anti-p24 antibodies. As control, the binding of CPSF6 to in vitro assembled HIV-1 CA-NC complexes was studied in the presence of PF74. Similar results were obtained in three independent experiments and a representative experiments is shown. To control for the bona fide origin of in vitro assembled SIVmac CA-NC complexes, we tested the ability of TRIM5α protein from Tamarin monkeys tagged with an HA epitope (TRIM5αTamarin-HA) to bind SIVmac capsid. (B) The ability of CPSF6 to bind in vitro assembled HIV-1 CA-NC complexes in the presence of increasing concentrations of BI-2 was analyzed (left panel). As a control, similar experiments were performed using increasing concentrations of PF74 (right panel). Similar results were obtained in three independent experiments and a representative experiments is shown. (C) To show that the interaction of CPSF6 with in vitro assembled HIV-1 CA-NC complexes is only dependent upon the capsid protein, we tested the ability of CPSF6 to bind in vitro assembled HIV-1 CA-NC complexes bearing the change N74D. Similar results were obtained in three independent experiments and a representative experiments is shown.