title: Similarities in the induction of post-Golgi vesicles by the vaccinia virus F13L protein and phospholipase D.
abstract: Intracellular mature vaccinia virions are wrapped by cisternae, derived from virus-modified trans-Golgi or endosomal membranes, and then transported via microtubules to the cell periphery. Two viral proteins, encoded by the F13L and B5R open reading frames, are essential for the membrane-wrapping step. Previous transfection studies indicated that F13L induces the formation of post-Golgi vesicles that incorporate the B5R protein and that this activity depends on an intact F13L phospholipase motif. Here we show that the F13L protein has a general effect on the trafficking of integral membrane proteins from the Golgi apparatus, as both the vaccinia virus A36R protein and the vesicular stomatitis virus G protein also colocalized with the F13L protein in vesicles. In addition, increased expression of cellular phospholipase D, which has a similar phospholipase motif as, but little amino acid sequence identity with, F13L, induced post-Golgi vesicles that contained B5R and A36R proteins. Butanol-1, which prevents the formation of phosphatidic acid by phospholipase D and specifically inhibits phospholipase D-mediated vesicle formation, also inhibited F13L-induced vesicle formation, whereas secondary and tertiary alcohols had no effect. Moreover, inhibition of phospholipase activity by butanol-1 also reduced plaque size and decreased the formation of extracellular vaccinia virus without affecting the yield of intracellular mature virus. Phospholipase D, however, could not complement a vaccinia virus F13L deletion mutant, indicating that F13L has additional virus-specific properties. Taken together, these data support an important role for F13L in inducing the formation of vesicle precursors of the vaccinia virus membrane via phospholipase activity or activation.
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Matloob
authors.initials: M
authors.lastname: Husain
authors.forename: Bernard
authors.initials: B
authors.lastname: Moss
mesh_terms: D000438:Alcohols / Q000494:pharmacology; D002460:Cell Line; D022162:Cytoplasmic Vesicles / Q000821:virology*; D015967:Gene Expression Regulation, Viral; D006056:Golgi Apparatus / Q000502:physiology / Q000821:virology*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014616:Vaccinia virus / Q000187:drug effects / Q000472:pathogenicity / Q000502:physiology; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000438:Alcohols; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C084759:p37 protein, Vaccinia virus; D049452:Green Fluorescent Proteins; D010739:Phospholipase D