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Journal of Virology, August 2000, p. 7508-7517, Vol. 74, No. 16
0022-538X/00/$04.00+0

Characterization of the Vaccinia Virus H3L Envelope Protein: Topology and Posttranslational Membrane Insertion via the C-Terminal Hydrophobic Tail

Flávio G. da Fonseca,dagger Elizabeth J. Wolffe, Andrea Weisberg, and Bernard Moss*

Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0445

Received 22 March 2000/Accepted 18 May 2000

The vaccinia virus H3L open reading frame encodes a 324-amino-acid immunodominant membrane component of virus particles. Biochemical and microscopic studies demonstrated that the H3L protein was expressed late in infection, accumulated in the cytoplasmic viral factory regions, and associated primarily with amorphous material near immature virions and with intracellular virion membranes. Localization of the H3L protein on the surfaces of viral particles and anchorage via the hydrophobic tail were consistent with its extraction 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 247 to 259. The H3L protein, synthesized in a coupled in vitro transcription/translation system, was tightly anchored to membranes as determined by resistance to Na2CO3 (pH 11) extraction and cytoplasmically oriented as shown by 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-terminal anchor family and supported a model in which it is synthesized on free ribosomes and inserts into the membranes of viral particles during their maturation.

* Corresponding author. Mailing address: 4 Center Dr., MSC 0445, NIH, Bethesda, MD 20892-0455. Phone: (301) 496-9869. Fax: (301) 480-1147. E-mail: bmoss{at}nih.gov.

dagger Present address: Laboratório de Vírus, Departamento de Microbiologia, Instituto de Ciências Biológicas da Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.

Journal of Virology, August 2000, p. 7508-7517, Vol. 74, No. 16
0022-538X/00/$04.00+0


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