EIAV release requires an interaction between ALIX and CHMP4B. Effects on EIAV release and infectivity of ALIX depletion/re-expression (A) and CHMP4B depletion/re-expression (B). 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”). (A) EIAV producer cells were transfected with a control siRNA (lane 1) or an siRNA that depleted ALIX (lanes 2–4), together with an empty vector control (lanes 1 and 2), or with a vector expressing either siRNA-resistant wild type ALIX (lane 3) or an ALIX protein with a mutation that impairs CHMP4B binding (ALIXI212D, “CHMP4-”, lane 4). (B) EIAV producer cells were transfected with a control siRNA (lane 1) or siRNAs that depleted CHMP4A/B (lanes 2–4), together with an empty vector control (lanes 1 and 2), or with a vector expressing siRNA-resistant wild type CHMP4B (lane 3) or a CHMP4B protein with mutations that impair ALIX binding (CHMP4BL217A,W220A, denoted “ALIX-”, lane 4). Other panels are equivalent to the corresponding panels in Figure 1. Error bars in the top panels show the range from the mean of two independent repetitions of the experiments, performed in parallel.