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. Author manuscript; available in PMC: 2014 Aug 20.
Published in final edited form as: Cell. 2013 Aug 15;154(4):763–774. doi: 10.1016/j.cell.2013.07.015

Figure 3. Analysis of the VP40 CTD-to-CTD interface.

Figure 3

(A) The CTD-to-CTD interface between VP40 dimers is shown with the NTDs from each protomer colored light or dark blue and CTDs colored light or dark orange. Below, a view of the CTD-to-CTD interface around residues M241 and I307 (yellow) with the disruptive mutations M241R and I307R (black) modeled onto the orange CTD. (B) Western blot of cell lysates and purified VLPs from transfected cells. Disruptive CTD-to-CTD interface point mutants, VP40E-M241R, VP40E-I307R and VP40E-R134A/I307R, fail to bud VLPs (red arrows). The CTD control mutants, VP40E-T242R and VP40E-Q309R, successfully assemble and bud VLPs. (C) IFA of transfected cells show dimeric WT-VP40E and VP40E-R134A migrate to the cell membrane and bud VLPs (yellow arrows). In contrast, VP40E-M241R and VP40E-R134A/I307R, bearing point mutations to the CTD-to-CTD interface, similarly migrate to the cell membrane but do not bud VLPs. Patches of VP40E-R134A/I307R accumulated at the membrane are indicated by blue arrows. VP40E-I307R, which exclusively forms rings, does not migrate to the membrane, but instead forms perinuclear structures. (D) EM micrographs show WT-VP40E and VP40E-R134A cause typical membrane ruffling, assembly and budding of filamentous VLPs (black arrows). VP40E-M241R displays an exaggerated wild-type VP40 membrane ruffling morphology, yet the cell surface remains devoid of budding filamentous VLPs. VP40E-I307R and VP40E-R134A/I307R display no membrane ruffling, matrix assembly or budding activity. See also Figure S3 and S4. *Solid scale bars = 1 μm and the dashed scale bars = 500 nm.