Characterization of the vaccinia virus H3L envelope protein: Topology and posttranslational membrane insertion via the C-terminal hydrophobic tail

The vaccinia virus H3L open reading frame encodes a 324-amino-acid immunodo minant membrane component of virus particles. Biochemical and microscopic s tudies demonstrated that the H3L protein was expressed late in infection, a ccumulated in the cytoplasmic viral Factory regions, and associated primari ly with amorphous material near immature virions and with intracellular vir ion membranes. Localization of the H3L protein on the surfaces of viral par ticles and anchorage via the hydrophobic tail were consistent with its extr action by NP-40 in the absence of reducing agents, its trypsin sensitivity, its reactivity with a membrane-impermeable biotinylation reagent, and its immunogold labeling with an antibody to a peptide comprising amino acids 24 7 to 259, The H3L protein, synthesized in a coupled in vitro transcription/ translation system, was tightly anchored to membranes as determined by resi stance to Na2CO3 (pH 11) extraction and cytoplasmically oriented as shown b y sensitivity to proteinase K digestion. Further studies demonstrated that membrane insertion of the H3L protein occurred posttranslationally and that the C-terminal hydrophobic domain was necessary and sufficient for this to occur. These data indicated that the H3L protein is a member of the C-term inal anchor family and supported a model in which it is synthesized on free ribosomes and inserts into the membranes of viral particles during their m aturation.