Figure 4

EIAV release requires VPS4 ATP, MIM1, and MIM2 binding activities. Effects on EIAV release and infectivity of VPS4 depletion/re-expression. Viral titers (top panel), and western blots showing levels of virion-associated Gag proteins (panel 2, “Virus”), and the designated intracellular viral and cellular proteins in 293T cells expressing EIAV (lower panels “Cell”). EIAV producer cells were transfected with a control siRNA (lane 1), an siRNA that depleted ALIX (lane 2, positive control) or siRNAs that depleted VPS4A and VPS4B (lanes 3–8), together with an empty vector control (lanes 1–3), or with vectors expressing either siRNA-resistant wild type VPS4B (lane 4) or VPS4B proteins with inactivating mutations within the ATPase binding site (VPS4B_K180Q, denoted “ATPase-”, lane 5), the MIM1 binding site (VPS4B_L66D, denoted “MIM1-”, lane 6), the MIM2 binding site (VPS4B_A15D, denoted “MIM2-”, lane 7), or both MIM1 and MIM2 binding sites (VPS4B_L66D,A15D, denoted “MIM1/2-”, lane 8). Other panels are equivalent to the corresponding panels in Figure 1. The arrow and asterisk in the anti-VPS4A panel designate bands that correspond to endogenous VPS4A and a band that arises owing to antibody cross-reactivity with overexpressed VPS4B, respectively. Error bars in the top panel show the range from the mean of two independent repetitions of the experiment, performed in parallel.