title: Biogenesis of the vaccinia virus membrane: genetic and ultrastructural analysis of the contributions of the A14 and A17 proteins.
abstract: Vaccinia virus membrane biogenesis requires the A14 and A17 proteins. We show here that both proteins can associate with membranes co- but not posttranslationally, and we perform a structure function analysis of A14 and A17 using inducible recombinants. In the absence of A14, electron-dense virosomes and distinct clusters of small vesicles accumulate; in the absence of A17, small vesicles form a corona around the virosomes. When the proteins are induced at 12 h postinfection (hpi), crescents appear at the periphery of the electron-dense virosomes, with the accumulated vesicles likely contributing to their formation. A variety of mutant alleles of A14 and A17 were tested for their ability to support virion assembly. For A14, biologically important motifs within the N-terminal or central loop region affected crescent maturation and the immature virion (IV)→mature virion (MV) transition. For A17, truncation or mutation of the N terminus of A17 engendered a phenotype consistent with the N terminus of A17 recruiting the D13 scaffold protein to nascent membranes. When N-terminal processing was abrogated, virions attempted to undergo the IV-to-MV transition without removing the D13 scaffold and were therefore noninfectious and structurally aberrant. Finally, we show that A17 is phosphorylated exclusively within the C-terminal tail and that this region is a direct substrate of the viral F10 kinase. In vivo, the biological competency of A17 was reduced by mutations that prevented its serine-threonine phosphorylation and restored by phosphomimetic substitutions. Precleavage of the C terminus or abrogation of its phosphorylation diminished the IV→MV maturation; a block to cleavage spared virion maturation but compromised the yield of infectious virus.
authors.affiliation: Department of Microbiology & Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI, USA.
authors.forename: Bethany
authors.initials: B
authors.lastname: Unger
authors.forename: Jason
authors.initials: J
authors.lastname: Mercer
authors.forename: Kathleen A
authors.initials: KA
authors.lastname: Boyle
authors.forename: Paula
authors.initials: P
authors.lastname: Traktman
mesh_terms: D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly
chemical_list: C110264:A14L protein, Vaccinia virus; C081257:A17L protein, Vaccinia virus; D008565:Membrane Proteins; D050505:Mutant Proteins; D014759:Viral Envelope Proteins