Monahan S J, Grinstead L A, Olivieri W, Parris D S
Department of Medical Microbiology and Immunology, Ohio State University, 333 West Tenth Avenue, Columbus, Ohio, 43210, USA.
Virology. 1998 Feb 1;241(1):122-30. doi: 10.1006/viro.1997.8953.
Interactions between the herpes simplex virus type 1 (HSV-1) origin (ori)-binding protein (UL9) and two other components of the functional DNA replication complex have been observed. However, to date, no interaction between UL9 and a component of the DNA polymerase holoenzyme has been demonstrated. In this report, we demonstrate that UL9 and the DNA polymerase accessory protein (UL42) can form a stable complex in vitro as determined by coimmunoprecipitation with specific antibodies to each protein and by affinity chromatography using glutathione S-transferase (GST) fusion proteins. Complex formation does not require the presence of other viral proteins and occurs in the presence of ethidium bromide, indicating that UL9-UL42 interaction is DNA independent. Affinity beads charged with increasing concentrations of GST-42 fusion protein up to 5 microM bound increasing amounts of UL9 expressed by in vitro transcription/translation in rabbit reticulocyte lysates. Binding of N- and C-terminal portions of UL9 to GST affinity matrices revealed that the N-terminal 533 amino acids were sufficient for binding to GST-42, albeit at approximately a four- to six-fold reduced affinity compared to the full-length protein. No binding of a polypeptide containing the remainder of the UL9 C-terminal residues was observed. Thus the ori-binding protein, UL9, can physically associate with at least one member of each of the complexes (helicase/primase, DNA polymerase holoenzyme, single-stranded DNA-binding protein) required for origin-dependent DNA replication. These specific interactions provide a means by which the ordered assembly of HSV-1 DNA replication proteins at origins of replication can occur in the infected cell for initiation of viral DNA synthesis.
已观察到单纯疱疹病毒1型（HSV-1）的起始点（ori）结合蛋白（UL9）与功能性DNA复制复合体的其他两个组分之间存在相互作用。然而，迄今为止，尚未证实UL9与DNA聚合酶全酶的一个组分之间存在相互作用。在本报告中，我们证明，通过用针对每种蛋白质的特异性抗体进行共免疫沉淀以及使用谷胱甘肽S-转移酶（GST）融合蛋白进行亲和层析测定，UL9与DNA聚合酶辅助蛋白（UL42）在体外可形成稳定的复合体。复合体的形成不需要其他病毒蛋白的存在，并且在溴化乙锭存在的情况下也会发生，这表明UL9-UL42相互作用不依赖于DNA。用浓度不断增加的GST-42融合蛋白（最高达5 microM）包被的亲和珠结合了兔网织红细胞裂解物中通过体外转录/翻译表达的越来越多的UL9。UL9的N端和C端部分与GST亲和基质的结合表明，N端的533个氨基酸足以与GST-42结合，尽管与全长蛋白相比亲和力大约降低了4至6倍。未观察到含有UL9 C端其余残基的多肽有结合。因此，ori结合蛋白UL9可与起始点依赖性DNA复制所需的每种复合体（解旋酶/引物酶、DNA聚合酶全酶、单链DNA结合蛋白）的至少一个成员发生物理缔合。这些特异性相互作用提供了一种方式，通过这种方式，HSV-1 DNA复制蛋白在感染细胞的复制起始点处可进行有序组装，从而启动病毒DNA合成。
