Expression and functional characterization of bluetongue virus VP5 protein: Role in cellular permeabilization

Segment 5 of bluetongue virus (BTV) serotype 10, which encodes the outer ca psid protein NTS, was tagged with glutathione S-transferase and expressed b y a recombinant baculovirus. The recombinant protein was subsequently purif ied to homogeneity, and its possible biological role in virus infection was investigated. Purified VP5 was able to bind mammalian cells but was not in ternalized, which indicates it is not involved in receptor-mediated endocyt osis. The purified VP5 protein was shown to be able to permeabilize mammali an and Culicoides insect cells, inducing cytotoxicity. Sequence analysis re vealed that VP5 possesses characteristic structural features (including two amino-terminal amphipathic helices) compatible with virus penetration acti vity. To assess the role of each feature in the observed cytotoxicity, a se ries of deleted VP5 molecules were generated, and their expression and biol ogical activity was compared with the parental molecule. VP5 derivatives th at included the two amphipathic helices exhibited cytotoxicity, while those that omitted these sequences did not. To confirm their role in membrane de stabilization two synthetic peptides (amino acids [aa] 1 to 20 and aa 22 to 41) encompassing the two helices and an additional peptide representing th e adjacent downstream sequences were also assessed for their effect on the cell membrane. Both helices, but not the downstream VP5 sequence, exhibited cytotoxicity with the most-amino-terminal helix (aa 1 to 20) showing a hig her activity than the adjacent peptide (aa 22 to 41). Purified VP5 was show n to readily form trimers in solution, a feature of many proteins involved in membrane penetration. Taken together, these data support a role for VP5 in virus-cell penetration consistent with its revelation in the entry vesic le subsequent to cell binding and endocytosis.