Analysis of the biochemical properties of, and complex formation between, glycoproteins H and L of the gamma2 herpesvirus bovine herpesvirus-4.
Genes encoding glycoprotein gH and gL homologues were localized in the genome of the gamma-herpesvirus bovine herpesvirus-4 (BHV-4). Both genes were sequenced and glutathione S-transferase fusion proteins were produced and used to immunize rabbits against the translation products of the two genes. The anti-gH serum recognized a protein with an apparent molecular mass (MM) of 110 kDa both in infected cells and in virions. This protein was sensitive to endo-beta-N-acetylglucosaminase-H (endoH) and endoglycosidase F-N-glycosidase F (endoF-PNGaseF) digestion. A protein with the same relative mobility was immunoprecipitated from infected cells radiolabelled with [3H]glucosamine which confirmed that this product (gp110), now designated BHV-4 gH, was glycosylated. Western blotting with the anti-gL serum detected in infected cells a product with an apparent MM ranging from 31-35 kDa and diffusely migrating protein species ranging from 45-65 kDa. Tunicamycin, monensin, endoH or endoF-PNGaseF treatments showed that both the 31-35 kDa and the 45-65 kDa proteins were glycosylated, gp31-35 being a precursor of the 45-65 kDa glycoprotein species. In radioimmunoprecipitation assays, the anti-gL serum immunoprecipitated from infected cells two glycosylated proteins with apparent MMs of 31-35 kDa (gp31-35) and 45-55 kDa (gp45-55). However a third glycoprotein, gp110, was also immunoprecipitated together with gp31-35 and gp45-55. gp110 and gp45-55 were subsequently confirmed to be virion glycoproteins corresponding to mature forms of BHV-4 gH and gL respectively. In addition, the present study clearly demonstrated complex formation between BHV-4 gH and gL both in virions and in infected cells.
Lomonte
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Laboratory of Virology-Immunology, Faculty of Veterinary Medicine, University of Liège, Belgium. p.lomonte@vir.gla.ac.uk
Filée
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Lyaku
J R
JR
Bublot
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Pastoret
P P
PP
Thiry
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D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D018142:Gammaherpesvirinae / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D005982:Glutathione Transferase; D006026:Glycoside Hydrolases; D006031:Glycosylation; D007668:Kidney; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008985:Monensin / Q000494:pharmacology; D009844:Oligosaccharides / Q000737:chemistry; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D014771:Virion / Q000378:metabolism
D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C107621:glycoprotein H, bovine herpesvirus 4; C107620:glycoprotein L, bovine herpesvirus 4; D014415:Tunicamycin; D004247:DNA; D008985:Monensin; D005982:Glutathione Transferase; D006026:Glycoside Hydrolases