Analysis for selective ubiquitination of host immune proteins by Bovine Herpes virus type4 proteins

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Bovine herpesvirus type 4 (BHV-4) is classified as a gammaherpesvirus including of Kaposi's sarcoma-associated herpesvirus (KSHV) on the basis of its genomic structure. BHV-4 strains have been isolated from animals with a variety of clinical signs, such as conjunctivitis, pneumonia, metritis, skin lesions, ulcerative mammillitis, enteritis, and tumors of the urinary bladder and rumen. BHV-4 has two kinds of proteins (Bo4 and Bo5) involved in Plant homeodomain (PHD) that is a motif characteristically defined by seven cysteins and a histidine arranged in a C4HC3 consensus. Recently, PHD domains are related with down-regulation of MHC class I surface expression by increasing the rate of endocytosis and by ubiquitination system to escape from host immune systems.We cloned BHV-4 Bo4 and Bo5 genes from BHV-4 cDNA library and expressed each protein in 293T cells. These proteins were unstable by Western blotting analysis. We, therefore, subcloned the genes into HA-tagged EGFP-fusion vectors. Both N-termini fusion proteins were localized in the nuclear and those of C-termini fusion proteins were localized into the cytoplasm by conforcal microscopy analysis. In addition, N-termini proteins of Bo4 and Bo5 have E3 ubiquitin ligase activities to express autoubiquitination but not C-termini proteins. The experiments of coimmunoprecipitation show that the complex of E2 and E3 proteins was not detected by immunoprecipitation-Western blotting analysis. This means that it is hard to precipitate Bo4 and Bo5 proteins with common anti-HA tag antibody. It is necessary that we may make use of liquid chromatography or ultracentrifugation methods to isolate complex.