Proteolytic processing of human cytomegalovirus glycoprotein B is dispensable for viral growth in culture.
Glycoprotein B (gB) of human cytomegalovirus (HCMV), which is considered essential for the viral life cycle, is proteolytically processed during maturation. Since gB homologues of several other herpesviruses remain uncleaved, the relevance of this property of HCMV gB for viral infectivity is unclear. Here we report on the construction of a viral mutant in which the recognition site of gB for the cellular endoprotease furin was destroyed. Because mutagenesis of essential proteins may result in a lethal phenotype, a replication-deficient HCMV gB-null genome encoding enhanced green fluorescent protein was constructed, and complementation by mutant gBs was initially evaluated in transient-cotransfection assays. Cotransfection of plasmids expressing authentic gB or gB with a mutated cleavage site (gB-DeltaFur) led to the formation of green fluorescent miniplaques which were considered to result from one cycle of phenotypic complementation of the gB-null genome. To verify these results, two recombinant HCMV genomes were constructed: HCMV-BAC-DeltaMhdI, with a deletion of hydrophobic domain 1 of gB that appeared to be essential for viral growth in the cotransfection experiments, and HCMV-BACDeltaFur, in which the gB cleavage site was mutated by amino acid substitution. Consistent with the results of the cotransfection assays, only the DeltaFur mutant replicated in human fibroblasts, showing growth kinetics comparable to that of wild-type virus. gB in mutant-infected cells was uncleaved, whereas glycosylation and transport to the cell surface were not impaired. Extracellular mutant virus contained exclusively uncleaved gB, indicating that proteolytic processing of gB is dispensable for viral replication in cell culture.
Strive
Tanja
T
Institut für Virologie der Philipps-Universität, 35037 Marburg, Germany.
Borst
Eva
E
Messerle
Martin
M
Radsak
Klaus
K
D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial; D003587:Cytomegalovirus / Q000254:growth & development* / Q000378:metabolism; D004269:DNA, Bacterial; D003673:Defective Viruses / Q000235:genetics / Q000254:growth & development; D045683:Furin; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D016296:Mutagenesis; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D013381:Subtilisins / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D010948:Viral Plaque Assay; D014779:Virus Replication
D004269:DNA, Bacterial; D008164:Luminescent Proteins; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus; D049452:Green Fluorescent Proteins; D013381:Subtilisins; D045683:Furin