Membrane topology of the vaccinia virus A17L envelope protein

The formation of a lipoprotein membrane within specialized areas of the cyt oplasm is the first visible step in poxvirus morphogenesis. The A17L viral protein, an essential nonglycosylated membrane component, was predicted to have four centrally located alpha-helical membrane-spanning domains. The ge ne was expressed as a 23-kDa protein in a cell-free transcriplion/translati on system containing canine pancreatic microsomes. The N- and C-terminal en ds of the membrane-associated protein were susceptible to proteinase digest ion, whereas the central region was resistant, consistent with a model in w hich the first and fourth hydrophobic domains are membrane spanning. This t opology was supported by the sizes of the major proteinase-resistant membra ne-associated products of genes containing one or more deleted hydrophobic domains and by evidence that the C-terminus was intraluminal and glycosylat ed on deletion of the second, third, and fourth domains, the third and four th domains, or just the fourth domain. Moreover, glycosylation also occurre d when an N-glycosylation site was introduced into the second hydrophobic d omain of the full-length A17L protein. The data indicated a predominant top ology in which the N- and C-termini are cytoplasmic, the first and fourth h ydrophobic domains span the microsomal membrane, and the second and third h ydrophobic domains are intraluminal. This arrangement has important implica tions for interactions of the A17L protein with other membrane components.