Bennett E R, Naujokas M, Hassell J A
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
J Virol. 1989 Dec;63(12):5371-85. doi: 10.1128/JVI.63.12.5371-5385.1989.
Replication of papovavirus DNA requires a functional replication origin, a virus-encoded protein, large T antigen, and species-specific permissive factors. How these components interact to initiate and sustain viral DNA replication is not known. Toward that end, we have attempted to identify the viral target(s) of permissive factors. The functionally defined replication origins of polyomavirus and simian virus 40, two papovaviruses that replicate in different species (mice and monkeys, respectively), are composed of two functionally distinct domains: a core domain and an auxiliary domain. The origin cores of the two viruses are remarkably similar in primary structure and have common binding sites for large T antigen. By contrast, their auxiliary domains share few sequences and serve as binding sites for cellular proteins. It seemed plausible, therefore, that if cellular permissive factors interacted with the replication origin, their targets were likely to be in the auxiliary domain. To test this hypothesis we constructed hybrid origins for DNA replication that were composed of the auxiliary domain of one virus and the origin core of the other and assessed their capacity to replicate in a number of mouse and monkey cell lines, which express the large T antigen of one or the other virus. The results of this analysis showed that the auxiliary domains of the viral replication origins could substitute for one another in DNA replication, provided that the viral origin core and its cognate large T antigen were present in a permissive cellular milieu. Surprisingly, the large T antigens of the viruses could not substitute for one another, regardless of the species of origin of the host cell, even though the two large T antigens bind to the same sequence motif in vitro. These results suggest that species-specific permissive factors do not interact with the origin-auxiliary domains but, rather, with either the origin core or the large T antigen or with both components to effect DNA replication.
乳头多瘤空泡病毒DNA的复制需要一个功能性复制起点、一种病毒编码蛋白——大T抗原以及物种特异性许可因子。这些组分如何相互作用以启动和维持病毒DNA复制尚不清楚。为此，我们试图确定许可因子的病毒靶点。多瘤病毒和猴病毒40是两种分别在不同物种（小鼠和猴子）中复制的乳头多瘤空泡病毒，其功能定义的复制起点由两个功能不同的结构域组成：一个核心结构域和一个辅助结构域。这两种病毒的起点核心在一级结构上非常相似，并且具有大T抗原的共同结合位点。相比之下，它们的辅助结构域几乎没有共享序列，并且作为细胞蛋白的结合位点。因此，似乎合理的是，如果细胞许可因子与复制起点相互作用，那么它们的靶点很可能在辅助结构域中。为了验证这一假设，我们构建了用于DNA复制的杂交起点，其由一种病毒的辅助结构域和另一种病毒的起点核心组成，并评估它们在一些表达一种或另一种病毒大T抗原的小鼠和猴细胞系中的复制能力。这一分析结果表明，病毒复制起点的辅助结构域在DNA复制中可以相互替代，前提是病毒起点核心及其同源大T抗原存在于允许的细胞环境中。令人惊讶的是，病毒的大T抗原不能相互替代，无论宿主细胞的来源物种如何，尽管这两种大T抗原在体外结合相同的序列基序。这些结果表明，物种特异性许可因子不与起点辅助结构域相互作用，而是与起点核心或大T抗原或与这两种组分相互作用以实现DNA复制。
