Vaccinia virus F13L protein with a conserved phospholipase catalytic motif induces colocalization of the B5R envelope glycoprotein in post-Golgi vesicles.
The wrapping of intracellular mature vaccinia virions by modified trans-Golgi or endosomal cisternae to form intracellular enveloped virions is dependent on at least two viral proteins encoded by the B5R and F13L open reading frames. B5R is a type I integral membrane glycoprotein, whereas F13L is an unglycosylated, palmitylated protein with a motif that is conserved in a superfamily of phospholipid-metabolizing enzymes. Microscopic visualization of the F13L protein was achieved by fusing it to the enhanced green fluorescent protein (GFP). F13L-GFP was functional when expressed by a recombinant vaccinia virus in which it replaced the wild-type F13L gene or by transfection of uninfected cells with a plasmid vector followed by infection with an F13L deletion mutant. In uninfected or infected cells, F13L-GFP was associated with Golgi cisternae and post-Golgi vesicles containing the LAMP 2 late endosomal-lysosomal marker. Association of F13L-GFP with vesicles was dependent on an intact phospholipase catalytic motif and sites of palmitylation. The B5R protein was also associated with LAMP2-containing vesicles when F13L-GFP was coexpressed, but was largely restricted to Golgi cisternae in the absence of F13L-GFP or when the F13L moiety was mutated. We suggest that the F13L protein, like its human phospholipase D homolog, regulates vesicle formation and that this process is involved in intracellular enveloped virion membrane formation.
Husain
M
M
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0445, USA.
Moss
B
B
D001692:Biological Transport; D006056:Golgi Apparatus / Q000821:virology*; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000502:physiology*; D018613:Microscopy, Confocal; D009154:Mutation; D010740:Phospholipases / Q000235:genetics; D011994:Recombinant Proteins; D014616:Vaccinia virus / Q000502:physiology*; D014759:Viral Envelope Proteins / Q000502:physiology*; D014764:Viral Proteins / Q000502:physiology*; D014779:Virus Replication
D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C074512:42kDa protein, Vaccinia virus; D010740:Phospholipases