A role for amino acids 212KLR214 of Ebola virus VP40 in VLP assembly and budding

Ebola virus VP40 is able to produce virus-like particles (VLPs) in the absence of other viral proteins. At least three domains within VP40 are thought to be required for efficient VLP release: the late (L) domain, membrane (M) association domain, and self-interacting (I) domain. While the L-domain of Ebola VP40 has been well characterized, the exact mechanism by which VP40 mediates budding through the M and I domains remains unclear. Based on the crystal structure of VP40, we targeted amino acids 212KLR 214 for mutagenesis since these residues are part of a loop connecting two beta sheets in the C-terminal region and thus are potentially important for overall structure and/or oligomerization of VP40. A series of alanine substitutions were generated in the 212KLR214 region of VP40, and these mutants were examined for VLP budding, intracellular localization, and oligomerization. Our results indicated that: (i) 212KLR 214 residues of V P40 were important for efficient release of VP40 VLPs, with Leu213 being most critical, (ii) VP40 212KLR214 mutants displayed altered patterns of cellular localization compared to that of VP40-WT, and (iii) self assembly of VP40 212KLR 214 mutants into oligomers was altered compared to that of VP40-WT. These results suggest that 212KLR214 residues of VP40 are important for proper assembly/oligomerization of VP40 which subsequently leads to efficient budding of VLPs.