EM analyses of EIAV budding defects in the absence of ALIX, CHMP2A/B and CHMP4A/B proteins. (A) EIAV infectivity and release in control cells (lane 1) or cells depleted of ALIX, CHMP2, or CHMP4 proteins (lanes 2–4, respectively). Panels show 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”). Virus-producing cells were treated with control siRNAs (lane 1) or siRNAs that depleted ALIX (lane 2), CHMP2A and CHMP2B (lane 3), or CHMP4A and CHMP4B (lane 4). Bottom panels show cellular levels of the designated ESCRT protein following treatment with the different siRNAs. Integrated band intensities are shown for virus release (panel 2, CA band intensities for the shown blot, relative to the control lane). (B) Thin section transmission EM images showing: cell-associated EIAV virions from cells depleted of ALIX, CHMP2 or CHMP4 proteins (panels 1–3, respectively), representative examples of the different virion phenotypes (panel 4), and an expanded view showing the phenotypes of virions associated with cells depleted of CHMP4 proteins (panel 5). Green and red arrows in panels 3 and 5 highlight virions with a multi-lobed or tubular phenotypes, respectively. Scale bars are 100 nm (white bars) or 20 nm (black bars). TEM images of EIAV virions released from control cells are shown in Additional file 4: Figure S3. (C) Relative frequencies (percentage) of the different phenotypes seen for cell-associated EIAV virions associated with control cells (blue) or cells depleted of ALIX, CHMP2A/B, or CHMP4A/B (red, green and purple, respectively).