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

牛疱疹病毒4型（BHV-4）根据其基因组结构被归类为伽玛疱疹病毒，包括卡波西肉瘤相关疱疹病毒（KSHV）。从结膜炎、肺炎、子宫炎、皮肤病变、溃疡性乳头炎、肠炎、膀胱和瘤胃肿瘤等多种临床症状的动物中分离出BHV-4菌株。BHV-4有两种蛋白（Bo4和Bo5）参与植物同源结构域（Plant homeodomain， PHD）， PHD是一个由7个半胱氨酸和一个组氨酸组成的基序，以C4HC3共识排列。近年来，PHD结构域通过增加内吞速率和泛素化系统逃避宿主免疫系统，从而下调MHC I类表面表达。从BHV-4 cDNA文库中克隆BHV-4 Bo4和Bo5基因，并在293T细胞中表达。经Western blotting分析，这些蛋白是不稳定的。因此，我们将这些基因亚克隆到ha标记的egfp融合载体中。共形显微镜观察发现，两种n端融合蛋白均定位于细胞核内，c端融合蛋白均定位于细胞质内。此外，Bo4和Bo5的n端蛋白具有E3泛素连接酶活性来表达自泛素化，而c端蛋白则没有。免疫共沉淀实验表明，免疫沉淀- western blotting分析未检测到E2和E3蛋白复合物。这意味着具有共同抗ha标签抗体的Bo4和Bo5蛋白难以沉淀。有必要利用液相色谱法或超离心法分离络合物。

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.