McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, USA.
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, USA
J Virol. 2014 Aug;88(15):8490-503. doi: 10.1128/JVI.00742-14. Epub 2014 May 14.
The maintenance of latent Kaposi's sarcoma-associated herpesvirus (KSHV) genomes is mediated in cis by their terminal repeats (TR). A KSHV genome can have 16 to 50 copies of the 801-bp TR, each of which harbors a 71-bp-long minimal replicator element (MRE). A single MRE can support replication in transient assays, and the presence of as few as two TRs appears to support establishment of KSHV-derived plasmids. Why then does KSHV have such redundancy and heterogeneity in the number of TRs? By determining the abilities of KSHV-derived plasmids containing various numbers of the TRs and MREs to be established and maintained in the long term, we have found that plasmids with fewer than 16 TRs or those with tandem repeats of the MREs are maintained inefficiently, as shown by both their decreased abilities to support formation of colonies and their instability, resulting in frequent rearrangements yielding larger plasmids during and after establishment. These defects often can be overcome by adding the Epstein-Barr virus (EBV) partitioning element, FR (i.e., family of repeats), in cis to these plasmids. In addition we have found that the spacing between MREs is important for their functions, too. Thus, two properties of KSHV's origin of latent replication essential for the efficient establishment and maintenance of viral plasmids stably are (i) the presence of approximately 16 copies of the TR, which are needed for efficient partitioning, and (ii) the presence of at least 2 MRE units separated by 801 bp of center-to-center spacing, which are required for efficient synthesis.
KSHV is a human tumor virus that maintains its genome as a plasmid in lymphoid tumor cells. Each plasmid DNA molecule encodes many origins of synthesis. Here we show that these many origins provide an essential advantage to KSHV, allowing the DNAs to be maintained without rearrangement. We find also that the correct spacing between KSHV's origins of DNA synthesis is required for them to support synthesis efficiently. The identification of these properties illuminates plasmid replication in mammalian cells and should lead to the development of rational means to inhibit these tumorigenic replicons.
潜伏性卡波西肉瘤相关疱疹病毒 (KSHV) 基因组的维持是通过其末端重复序列 (TR) 顺式介导的。KSHV 基因组可以有 16 到 50 个 801bp 的 TR，每个 TR 都含有一个 71bp 长的最小复制元件 (MRE)。单个 MRE 可以支持瞬时测定中的复制，而仅存在两个 TR 似乎就足以支持源自 KSHV 的质粒的建立。那么，KSHV 的 TR 数量为何具有如此大的冗余性和异质性呢？通过确定含有不同数量的 TR 和 MRE 的源自 KSHV 的质粒在长期内建立和维持的能力，我们发现少于 16 个 TR 的质粒或 MRE 串联重复的质粒维持效率低下，这表现在它们支持形成菌落的能力降低以及不稳定性，导致在建立和建立后频繁发生重排，产生更大的质粒。这些缺陷通常可以通过在这些质粒中顺式添加 EBV 分区元件 FR（即重复家族）来克服。此外，我们还发现 MRE 之间的间隔对其功能也很重要。因此，对于有效建立和稳定维持病毒质粒至关重要的 KSHV 潜伏复制原点的两个特性是：(i) 存在大约 16 个 TR 拷贝，这对于有效分区是必需的，以及 (ii) 存在至少 2 个 MRE 单位，它们之间的中心到中心间隔为 801bp，这对于有效合成是必需的。
KSHV 是一种人类肿瘤病毒，它将其基因组作为质粒维持在淋巴肿瘤细胞中。每个质粒 DNA 分子编码许多合成原点。在这里，我们表明这些许多起源为 KSHV 提供了一个重要的优势，允许 DNA 无重排地维持。我们还发现，KSHV 的 DNA 合成原点之间的正确间隔对于它们有效地支持合成是必需的。这些特性的确定阐明了哺乳动物细胞中的质粒复制，应该会导致开发出合理的抑制这些致癌复制子的方法。
