FIG. 2.

W95 and E160 are essential for VP40 homooligomerization. (A) Interaction of VP40 in a mammalian two-hybrid assay. 293 cells were transfected with 500 ng each of plasmids encoding a GAL4 binding domain (pBind) and a VP16 activation domain (pAct) (negative control), pBind-Id and pAct-myo (positive control), or fusion proteins consisting of the GAL4 binding domain or the VP16 domain and VP40-WT or VP40-mutants, as indicated. Five hundred nanograms of a GAL4-driven luciferase reporter construct was also cotransfected. Cells were harvested 48 h after transfection, and luciferase reporter activity, reflecting interaction of VP40, was measured. (B) In vitro oligomerization of VP40. VP40-WT, VP40-WA, VP40-EA, and VP40-WEA were expressed as fusion proteins with glutathione S-transferase (GST) in bacteria and purified via the GST moiety. The GST was subsequently cleaved off using a specific protease, and the purified VP40 was cross-linked using glutaraldehyde. VP40 was detected after SDS-PAGE and Western blotting using VP40-specific antibodies. Monomeric and oligomeric forms of VP40 are labeled. The asterisk indicates uncleaved GST-VP40. (C) Quantitative analysis of cross-linked VP40. The average percentages of the different monomeric and oligomeric forms of VP40 after cross-linking from 3 independent experiments are shown.