Summary auto-generated
This study analyzed glycoproteins H and L of bovine herpesvirus-4 (BHV-4), a gammaherpesvirus. The researchers identified and sequenced the gH and gL genes, produced antibodies using glutathione S-transferase fusion proteins, and characterized the viral glycoproteins in infected cells and virions. BHV-4 gH was detected as a 110 kDa glycoprotein (gp110) containing complex N-linked oligosaccharides. BHV-4 gL was identified in multiple forms: a 14 kDa unglycosylated precursor, a 31-35 kDa intermediate form (gp31-35) with high mannose sugars, and mature 45-65 kDa forms (gp45-65) with complex oligosaccharides. Using immunoprecipitation and Western blotting, the researchers demonstrated that gH and gL form a stable complex both in infected cells and virus particles. The gp31-35 form represents early glycosylation in the rough endoplasmic reticulum, while gp45-65 results from further processing in the Golgi apparatus. This is the first detailed biochemical characterization of gH and gL homologues in a gammaherpesvirus other than Epstein-Barr virus.
Key findings
- BHV-4 gH is a 110 kDa glycoprotein with complex N-linked oligosaccharides modified in the Golgi apparatus
- BHV-4 gL exists in multiple processing forms: 14 kDa unglycosylated precursor, 31-35 kDa intermediate form with high mannose structures, and mature 45-65 kDa forms with complex oligosaccharides
- gH and gL form a stable complex in both infected cells and virions, demonstrated through co-immunoprecipitation
- gL processing involves initial glycosylation in the rough endoplasmic reticulum followed by Golgi-mediated maturation
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Abstract
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.