Envelope formation is blocked by mutation of a sequence related to the HKD phospholipid metabolism motif in the vaccinia virus F13L protein.
The outer envelope of the extracellular form of vaccinia virus is derived from Golgi membranes that have been modified by the insertion of specific viral proteins, of which the major component is the 37-kDa, palmitylated, nonglycosylated product of the F13L gene. The F13L protein contains a variant of the HKD (His-Lys-Asp) motif, which is conserved in numerous enzymes of phospholipid metabolism. Vaccinia virus mutants with a conservative substitution of either the K (K314R) or the D (D319E) residue of the F13L protein formed only tiny plaques similar to those produced by an F13L deletion mutant, were unable to produce extracellular enveloped virions, and failed to mediate low-pH-induced fusion of infected cells. Membrane-wrapped forms of intracellular virus were rarely detected in electron microscopic images of cells infected with either of the mutants. Western blotting and pulse-chase experiments demonstrated that the D319E protein was less stable than either the K314R or wild-type F13L protein. Most striking, however, was the failure of either of the two mutated proteins to concentrate in the Golgi compartment. Palmitylation, oleation, and partitioning of the F13L protein in Triton X-114 detergent were unaffected by the K314R substitution. These results indicated that the F13L protein must retain the K314 and D319 for it to localize in the Golgi compartment and function in membrane envelopment of vaccinia virus.
Roper
R L
RL
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892-0445, USA.
Moss
B
B
D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002459:Cell Fusion; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002586:Cesium; D002712:Chlorides; D002522:Chlorocebus aethiops; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017830:Octoxynol; D010641:Phenotype; D010743:Phospholipids / Q000378:metabolism; D011092:Polyethylene Glycols; D011817:Rabbits; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism* / Q000502:physiology* / Q000648:ultrastructure; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D010948:Viral Plaque Assay; D014771:Virion; D019065:Virus Assembly
D002712:Chlorides; D008565:Membrane Proteins; D010743:Phospholipids; D014759:Viral Envelope Proteins; C084759:p37 protein, Vaccinia virus; D002586:Cesium; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; C028019:cesium chloride