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. Author manuscript; available in PMC: 2008 Nov 24.
Published in final edited form as: Dev Cell. 2008 Jul;15(1):62–73. doi: 10.1016/j.devcel.2008.05.014

Figure 6. The VPS4B MIT MIM1 and MIM2 Binding Surfaces Both Contribute to Efficient HIV-1 Budding and Replication.

Figure 6

Western blots 1 and 2 show simultaneous siRNA depletion of endogenous VPS4A and VPS4B (Compare lanes 1 and 2) and re-expression of exogenous siRNA-resistant wild type and mutant VPS4B proteins (blot 1, Lanes 3−7). Western blot 3 shows an α–Tubulin loading control. Western blots 4 and 5 show levels of the viral Gag protein and its processed MA and CA products in the cytoplasm (Cell, panel 4, note that all cells were transfected with a proviral HIV-1 expression construct), and released as viral particles (Virus). Panel 6 shows viral titers, as assayed in single cycle replication assays of the culture supernatants. The experiment shows that simultaneous depletion of both human VPS4 isoforms blocks HIV-1 release and infectivity (panels 5 and 6, compare lanes 1 and 2), that reintroduction of a wild type, siRNA-resistant VPS4B protein construct significantly rescues virus release and titer (compare lanes 2 and 3), and that virus release and titer are inhibited by the VPS4B K180Q (lane 4, positive control), L66D (lane 5, inhibited MIM1 binding), A15D (lane 6, inhibited MIM2 binding) and L66D,A15D (lane 7, inhibited MIM1 and MIM2 binding).