Functional Analyses of Epstein-Barr virus DNA replication machinery consisting of viral elongation factors

EBV编码7个病毒基因，这些基因对orilyt依赖性DNA复制至关重要。BZLF1蛋白是一种orilyt结合蛋白，也是一种裂解反激活因子。BALF5基因编码DNA Pol催化亚基，BMRF1基因编码DNA Pol辅助亚基。BALF2基因编码单链dna结合蛋白。BBLF4、BSLFI和BBLF2/3基因编码的其余三种蛋白的功能尚未证实，但预测分别为解旋酶、引物酶和解旋酶-引物酶相关蛋白。我们在感染的昆虫细胞中构建了表达这三个基因产物的重组杆状病毒。用重组杆状病毒AcBBLF4、AcBSLF1或AcBBLF2/3感染的昆虫细胞分别产生90、89和76 k的蛋白。这些产物在Western blots中被鉴定为BBLF4， BSLF1和BBLF2/3基因产物，使用抗每种蛋白特异性抗体。抗BSLF1蛋白特异性抗体能免疫沉淀BSLF1蛋白，但不与BBLF4或BBLF2/3蛋白直接反应。该抗体从三次感染这些重组杆状病毒的昆虫细胞提取物中沉淀出BSLF1、BBLF4和BBLF2/3蛋白。此外，利用双感染细胞提取物进行的实验表明，BSLF1蛋白分别与BBLF4和BBLF2/3蛋白相互作用。此外，用组氨酸标记的BBLF2/3蛋白通过金属亲和树脂柱纯化BBLF4和BSLF1蛋白。这些结果表明EBV BBLF4、BSLF1和BBLF2/3蛋白至少通过BSLF1蛋白形成一个三方复合体。

EBV encodes seven viral genes that are essential for the oriLyt-dependent DNA replication. The BZLF1 protein is an oriLyt-binding protein and acts also the lytic transactivator. The BALF5 gene encodes the DNA Pol catalytic subunit and the BMRF1 gene encodes the DNA Pol accessory subunit. A single-stranded DNA-binding protein is encoded by the BALF2 gene . The functions of the remaining three proteins encoded by the genes BBLF4 , BSLFI, and BBLF2/3 are not demonstrated but predicted to be helicase, primase, and helicase-primase associated proteins, respectively. We have constructed recombinant baculoviruses which express the products of each of these three genes in infected insect cells. Insect cells infected with the recombinant baculovirus, AcBBLF4, AcBSLF1, or AcBBLF2/3, produced each protein of 90, 89, or 76 k, respectively. These products were identified as BBLF4, BSLF1, and BBLF2/3 gene products in Western blots using anti- each of the protein-specific antibodies. The anti-BSLF1 protein-specific antibody could immunoprecipitate the BSLF1 protein, but did not react directly with BBLF4 or BBLF2/3 protein. The antibody precipitated the BSLF1, BBLF4, and BBLF2/3 proteins from the extracts of the insect cells triply infected with these recombinant baculoviruses. In addition, experiments performed by using the extracts from doubly infected cells indicated that the BSLF1 protein interacts separately with each of the BBLF4 and BBLF2/3 proteins. Furthermore, both of the BBLF4 and BSLF1 proteins were purified via the histidine-tagged BBLF2/3 protein through the column of metal affinity resine. These results indicate that the EBV BBLF4, BSLF1, and BBLF2/3 proteins form a tripartite complex at least via BSLF1 protein.