The African swine fever virus prenyltransferase is an integral membrane trans-geranylgeranyl-diphosphate synthase.
In a previous study, it was shown that the protein encoded by the gene B318L of African swine fever virus (ASFV) is a trans-prenyltransferase that catalyzes in vitro the condensation of farnesyl diphosphate and isopentenyl diphosphate to synthesize geranylgeranyl diphosphate and longer chain prenyl diphosphates (Alejo, A., Yáñez, R. J., Rodríguez, J. M., Viñuela, E., and Salas, M. L. (1997) J. Biol. Chem. 272, 9417-9423). To investigate the in vivo function of the viral enzyme, we have determined, in this work, its subcellular localization and activity in cell extracts. Two systems were used in these studies: cells infected with ASFV and cells infected with a recombinant pseudo-Sindbis virus carrying the complete B318L gene. In this latter system, the trans-prenyltransferase was found to colocalize with the endoplasmic reticulum marker protein-disulfide isomerase, whereas in cells infected with ASFV, the viral enzyme was present in cytoplasmic viral assembly sites, associated with precursor viral membranes derived from the endoplasmic reticulum. In addition, after subcellular fractionation, the viral enzyme partitioned into the membrane fraction. Extraction of membrane proteins with alkaline carbonate and Triton X-114 indicated that the ASFV enzyme behaved as an integral membrane protein. The membrane enzyme synthesized predominantly all-trans-geranylgeranyl diphosphate from farnesyl diphosphate and isopentenyl diphosphate. These results indicate that the viral B318L protein is a trans-geranylgeranyl-diphosphate synthase, being the only enzyme of this type that is known to have a membrane localization.
Alejo
A
A
Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Andrés
G
G
Viñuela
E
E
Salas
M L
ML
D000358:African Swine Fever Virus / Q000201:enzymology* / Q000235:genetics; D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D000818:Animals; D002522:Chlorocebus aethiops; D004122:Dimethylallyltranstransferase / Q000235:genetics / Q000378:metabolism*; D004224:Diterpenes / Q000032:analysis; D051231:Farnesyltranstransferase; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D012845:Sindbis Virus / Q000201:enzymology / Q000235:genetics; D014709:Vero Cells; D014764:Viral Proteins / Q000378:metabolism
D004224:Diterpenes; D014764:Viral Proteins; D019883:Alkyl and Aryl Transferases; D004122:Dimethylallyltranstransferase; D051231:Farnesyltranstransferase