Fg. Da Fonseca et al., Characterization of the vaccinia virus H3L envelope protein: Topology and posttranslational membrane insertion via the C-terminal hydrophobic tail, J VIROLOGY, 74(16), 2000, pp. 7508-7517
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.