Figure 3
CHMP2-CHMP4 and CHMP2-VPS4 interactions contribute to EIAV release. Effects on EIAV release and infectivity of CHMP4 depletion/re-expression (A) and CHMP2 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), an siRNA that depleted ALIX (lane 2, positive control) or siRNAs that depleted CHMP4A and CHMP4B (lanes 3–6), together with an empty vector control (lanes 1–3), or with vectors expressing either siRNA-resistant wild type CHMP4B (lane 4) or CHMP4B proteins with mutations that impair binding to ALIX (CHMP4BL217A,W220A, denoted “ALIX-”, lane 5) or CHMP2A (CHMP4B104EVLK107/AAAA, denoted “CHMP2-”, lane 6). (B) EIAV producer cells were transfected with a control siRNA (lane 1), an siRNA that depleted ALIX (lane 2, positive control) or siRNAs that depleted CHMP2A and CHMP2B (lanes 3–6), together with an empty vector control (lanes 1–3), or with vectors expressing either siRNA-resistant wild type CHMP2A (lane 4) or CHMP2A proteins with mutations that impair binding to CHMP4B (CHMP2AR24A,R27A,R31A, denoted “CHMP4-”, lane 5) or VPS4 (CHMP2AL216D,L219D, denoted “VPS4-”, lane 6). Other panels are equivalent to the corresponding panels in Figure 1. Error bars in the top panel show the range from the mean of two independent repetitions of the experiment, performed in parallel.