Kirchmaier A L, Sugden B
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
J Virol. 1998 Jun;72(6):4657-66. doi: 10.1128/JVI.72.6.4657-4666.1998.
Replication of the Epstein-Barr viral (EBV) genome occurs once per cell cycle during latent infection. Similarly, plasmids containing EBV's plasmid origin of replication, oriP, are replicated once per cell cycle. Replication from oriP requires EBV nuclear antigen 1 (EBNA-1) in trans; however, its contributions to this replication are unknown. oriP contains 24 EBNA-1 binding sites; 20 are located within the family of repeats, and 4 are found within the dyad symmetry element. The site of initiation of DNA replication within oriP is at or near the dyad symmetry element. We have identified a plasmid that contains the family of repeats but lacks the dyad symmetry element whose replication can be detected for a limited number of cell cycles. The detection of short-term replication of this plasmid requires EBNA-1 and can be inhibited by a dominant-negative inhibitor of EBNA-1. We have identified two regions within this plasmid which can independently contribute to this replication in the absence of the dyad symmetry element of oriP. One region contains native EBV sequences within the BamHI C fragment of the B95-8 genome of EBV; the other contains sequences within the simian virus 40 genome. We have mapped the region contributing to replication within the EBV sequences to a 298-bp fragment, Rep*. Plasmids which contain three copies of Rep* plus the family of repeats support replication more efficiently than those with one copy, consistent with a stochastic model for the initiation of DNA synthesis. Plasmids with three copies of Rep* also support long-term replication in the presence of EBNA-1. These observations together indicate that the latent origin of replication of EBV is more complex than formerly appreciated; it is a multicomponent origin of which the dyad symmetry element is one efficient component. The experimental approach described here could be used to identify eukaryotic sequences which mediate DNA synthesis, albeit inefficiently.
在潜伏感染期间，爱泼斯坦-巴尔病毒（EBV）基因组每个细胞周期复制一次。同样，含有EBV质粒复制起点oriP的质粒每个细胞周期也复制一次。从oriP进行复制在反式作用中需要EBV核抗原1（EBNA-1）；然而，其对这种复制的作用尚不清楚。oriP包含24个EBNA-1结合位点；20个位于重复序列家族内，4个位于二联体对称元件内。oriP内DNA复制的起始位点在二联体对称元件处或其附近。我们鉴定出一种质粒，它含有重复序列家族但缺乏二联体对称元件，其复制在有限数量的细胞周期内可被检测到。这种质粒短期复制的检测需要EBNA-1，并且可被EBNA-1的显性负性抑制剂抑制。我们在这种质粒内鉴定出两个区域，在缺乏oriP的二联体对称元件时，它们可独立地对这种复制起作用。一个区域包含EBV B95-8基因组的BamHI C片段内的天然EBV序列；另一个区域包含猿猴病毒40基因组内的序列。我们已将EBV序列中对复制起作用的区域定位到一个298 bp的片段Rep*。含有三个Rep拷贝加上重复序列家族的质粒比含有一个拷贝的质粒更有效地支持复制，这与DNA合成起始的随机模型一致。含有三个Rep拷贝的质粒在有EBNA-1存在时也支持长期复制。这些观察结果共同表明，EBV的潜伏复制起点比以前认识到的更复杂；它是一个多组分起点，其中二联体对称元件是一个有效的组分。这里描述的实验方法可用于鉴定介导DNA合成的真核序列，尽管效率不高。
