RESUMO
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.
Assuntos
Gammaherpesvirinae/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Western Blotting , Bovinos , Linhagem Celular , Clonagem Molecular , DNA/química , Expressão Gênica/efeitos dos fármacos , Glutationa Transferase , Glicosídeo Hidrolases , Glicosilação , Rim , Dados de Sequência Molecular , Peso Molecular , Monensin/farmacologia , Oligossacarídeos/química , Coelhos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Tunicamicina/farmacologia , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/química , Vírion/metabolismoRESUMO
This study reports that in bovine herpesvirus 4, glycoprotein B (gB) is a heterodimer and a major component of the virion, unlike gBs of Epstein-Barr virus (gp110) and murine gammaherpesvirus 68, two other gammaherpesviruses. These are new characteristics with regard to the general features of gB in the Gammaherpesvirinae subfamily.