Equine herpesvirus type 1 (EHV-1) glycoprotein K is required for efficient cell-to-cell spread and virus egress.
The function of the equine herpesvirus type 1 (EHV-1) glycoprotein K (gK) homologue was investigated. Deletion of 88% of the UL53-homologous open reading frame in EHV-1 strain RacH resulted in a severe growth defect of the gK-negative virus (HDeltagK) as reflected by a significant decrease in the production of infectious virus progeny on RK13 cells. The HDeltagK virus induced only minute plaques, was unable to form syncytia, and its penetration efficiency into RK13 cells was reduced by approximately 40%. To further analyze gK function and intracellular trafficking, gK of strain RacH was replaced by a C-terminally truncated gK-green fluorescent protein fusion protein (gK-GFP). The generated recombinant virus was shown to replicate well on non-complementing cells, and virus penetration and syncytium formation were comparable to parental RacH. A reduction in plaque size and slightly decreased intra- and extracellular virus titers, however, were observed. The gK-GFP fusion protein was expressed with early-late kinetics, and multiple forms of the protein exhibiting M(r)s between 50,000 and 85,000 were detected by Western blot analysis. The various gK-GFP forms were shown to be N-glycosylated, associated with membranes of the Golgi apparatus, and were incorporated into extracellular virions. Complete processing of gK-GFP was only observed within the context of viral infection. From the results, we concluded that EHV-1 gK is required for efficient virus growth in vitro and that the carboxy-terminal amino acids are not required for its function, because the gK-GFP fusion protein was able to complement for EHV-1 growth in the absence of authentic gK.
Institute for Medical Microbiology, Infectious and Epidemic Diseases, Ludwig-Maximilians-Universität München, D-80539 Munich, Germany. toni.neubauer@micro.vetmed.uni-muenchen.de
Antonie
A
Neubauer
Nikolaus
N
Osterrieder
D000818:Animals; D002460:Cell Line; D017353:Gene Deletion; D015726:Giant Cells / Q000502:physiology; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D004861:Herpesvirus 1, Equid / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C076117:UL4 protein, Equine herpesvirus 1; D049452:Green Fluorescent Proteins