title: The gene 10 (UL49.5) product of equine herpesvirus 1 is necessary and sufficient for functional processing of glycoprotein M.
abstract: The functional cooperation of equine herpesvirus 1 (EHV-1) glycoprotein M (gM) and the gene 10 (UL49.5) product was analyzed. Transient-transfection experiments using gM and UL49.5 expression plasmids as well as RK13 cell lines constitutively expressing UL49.5 (RK49.5) or gM (RKgM) demonstrated that the endo-beta-N-acetylglucosaminidase H (endo H)-resistant mature form of gM was detectable only after coexpression of the two proteins. Deletion of the EHV-1 UL49.5-homologous gene 10 in strain KyA resulted in a small-plaque phenotype and up to 190-fold-reduced virus titers. The growth defects of the mutant KyA Delta 49.5 virus, which were very similar to those of a gM-negative KyA virus, could be completely compensated for by growth of the mutant virus on RK49.5 cells or by repairing the deletion of gene 10 in the revertant virus KyA Delta 49.5R. Analysis of cells infected with the UL49.5-negative EHV-1 demonstrated that gM was not transported to the trans-Golgi network in the absence of the UL49.5 product. In contrast, gM was efficiently transported and processed to the endo H-resistant mature form in KyA Delta 49.5-infected RK49.5 cells. Furthermore, radioimmunoprecipitation experiments demonstrated that gM maturation was observed only if a 10,000-M(r) protein was coprecipitated with gM in KyA- or KyA Delta 49.5R-infected cells or virions. This protein was absent in cells infected with Ky Delta 49.5 or KyA Delta gM, suggesting that it was the EHV-1 UL49.5 product. Taken together, our results demonstrate that the expression of the EHV-1 UL49.5 product is necessary and sufficient for gM processing and that it is required for efficient virus replication.
authors.affiliation: Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Rudolph
authors.forename: Christian
authors.initials: C
authors.lastname: Seyboldt
authors.forename: Harald
authors.initials: H
authors.lastname: Granzow
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Osterrieder
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D017353:Gene Deletion; D006031:Glycosylation; D004861:Herpesvirus 1, Equid / Q000235:genetics / Q000254:growth & development* / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D014162:Transfection; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D014759:Viral Envelope Proteins; D014764:Viral Proteins; C418522:glycoprotein M, Equid herpesvirus 1