Sequence-independent targeting of transmembrane proteins synthesized within vaccinia virus factories to nascent viral membranes.
The primary membrane of vaccinia virus, as well as those of other poxviruses, forms within a discrete cytoplasmic factory region. We recently determined the existence of an operative pathway from the endoplasmic reticulum within the virus factory to nascent viral membranes and demonstrated that a viral protein could be diverted from this pathway to Golgi membranes by the addition of COPII-binding sites (M. Husain, A. S. Weisberg, and B. Moss, Proc. Natl. Acad. Sci. USA, 103:19506-19511, 2006). Here we describe an investigation of the structural features that are required for transit of proteins to the viral membrane. Deletion of either the N-terminal domain or the C-terminal cytoplasmic tail from the conserved A9 protein did not prevent its incorporation into viral membranes, whereas deletion of the transmembrane domain resulted in its distribution throughout the cytoplasm. Nevertheless, replacement of the A9 transmembrane domain with the corresponding region of a nonpoxvirus transmembrane protein or of a vaccinia virus extracellular envelope protein allowed viral membrane targeting, indicating no requirement for a specific amino acid sequence. Remarkably, the epitope-tagged A9 transmembrane domain alone, as well as a heterologous transmembrane domain lacking a poxvirus sequence, was sufficient for viral membrane association. The data are consistent with a sequence-independent pathway in which transmembrane proteins that are synthesized within the virus factory and lack COPII or other binding sites that enable conventional endoplasmic reticulum exiting are incorporated into nascent viral membranes.
Husain
Matloob
M
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Weisberg
Andrea S
AS
Moss
Bernard
B
D000595:Amino Acid Sequence; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D007211:Indoles; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000648:ultrastructure; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D014616:Vaccinia virus / Q000378:metabolism*; D014763:Viral Matrix Proteins / Q000378:metabolism*; D014771:Virion / Q000378:metabolism*
D005456:Fluorescent Dyes; D007211:Indoles; D008565:Membrane Proteins; D014763:Viral Matrix Proteins; C007293:DAPI