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 capsid protein VP5, was tagged with glutathione S-transferase and expressed by a recombinant baculovirus. The recombinant protein was subsequently purified to homogeneity, and its possible biological role in virus infection was investigated. Purified VP5 was able to bind mammalian cells but was not internalized, which indicates it is not involved in receptor-mediated endocytosis. The purified VP5 protein was shown to be able to permeabilize mammalian and Culicoides insect cells, inducing cytotoxicity. Sequence analysis revealed that VP5 possesses characteristic structural features (including two amino-terminal amphipathic helices) compatible with virus penetration activity. To assess the role of each feature in the observed cytotoxicity, a series of deleted VP5 molecules were generated, and their expression and biological activity was compared with the parental molecule. VP5 derivatives that included the two amphipathic helices exhibited cytotoxicity, while those that omitted these sequences did not. To confirm their role in membrane destabilization two synthetic peptides (amino acids [aa] 1 to 20 and aa 22 to 41) encompassing the two helices and an additional peptide representing the 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 higher activity than the adjacent peptide (aa 22 to 41). Purified VP5 was shown 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 vesicle subsequent to cell binding and endocytosis.
Hassan
S H
SH
Department of Infectious and Tropical Diseases, School of Hygiene and Tropical Medicine, London WC1E 7HT, England.
Wirblich
C
C
Forzan
M
M
Roy
P
P
D000818:Animals; D001665:Binding Sites; D001820:Bluetongue virus; D002213:Capsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D036022:Capsid Proteins; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D002463:Cell Membrane Permeability / Q000502:physiology*; D002522:Chlorocebus aethiops; D006224:Cricetinae; D015870:Gene Expression; D051379:Mice; D009842:Oligopeptides; D010455:Peptides; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D012996:Solutions; D013997:Time Factors; D014709:Vero Cells
D036022:Capsid Proteins; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012996:Solutions