Wang Jian Ben, Nixon Daniel E, McVoy Michael A
Department of Pediatrics, Medical College of Virginia Campus of Virginia Commonwealth University, P.O. Box 980163, Richmond, VA 23298-0163, USA.
J Virol. 2008 Mar;82(5):2394-404. doi: 10.1128/JVI.00063-07. Epub 2007 Dec 19.
Herpesvirus DNA replication proceeds via concatemeric replicative intermediates that are comprised of head-to-tail-linked genomes. Genome maturation is carried out by the terminase, a protein complex that mediates both insertion of concatemer DNA into capsids and its subsequent cleavage to release genomes within these capsids. This cleavage is sequence specific, but the governing cis-acting DNA sequences are only partially characterized. Two highly conserved motifs called pac1 and pac2 lie near the ends of herpesvirus genomes and are known to be critical for genome maturation. However, the potential importance of other sequences has not been fully investigated. We have undertaken to define all of the sequences necessary for efficient genome maturation for a herpesvirus by inserting ectopic cleavage sites into the murine cytomegalovirus genome and assessing their ability to mediate genome maturation. A combination of deletion and substitution mutations revealed that the minimal cleavage site is large ( approximately 180 bp) and complex. Sequences distal of pac1 (relative to the point of cleavage) were dispensable, suggesting that pac1 may be the sole cis-acting element on this side of the cleavage site. In contrast, a region distal to pac2 up to 150 bp from the point of cleavage was essential. Scanning substitutions revealed that the pac2 side of the cleavage site is complex and may contain multiple cis-acting sequence elements in addition to pac2. These results should facilitate the identification of trans-acting factors that bind to these elements and the elucidation of their functions. Such information will be critical for understanding the molecular basis of this complex process.
疱疹病毒DNA复制通过由头对头连接的基因组组成的串联复制中间体进行。基因组成熟由末端酶完成，末端酶是一种蛋白质复合物，介导串联体DNA插入衣壳并随后进行切割以释放这些衣壳内的基因组。这种切割是序列特异性的，但起控制作用的顺式作用DNA序列仅得到部分表征。两个高度保守的基序，称为pac1和pac2，位于疱疹病毒基因组末端附近，已知对基因组成熟至关重要。然而，其他序列的潜在重要性尚未得到充分研究。我们通过将异位切割位点插入鼠巨细胞病毒基因组并评估它们介导基因组成熟的能力，来确定疱疹病毒高效基因组成熟所需的所有序列。缺失和替代突变的组合表明，最小切割位点很大（约180 bp）且复杂。pac1远端的序列（相对于切割点）是可有可无的，这表明pac1可能是切割位点这一侧唯一的顺式作用元件。相比之下，pac2远端距切割点达150 bp的区域是必不可少的。扫描替代显示，切割位点的pac2一侧很复杂，除pac2外可能还包含多个顺式作用序列元件。这些结果应有助于鉴定与这些元件结合的反式作用因子并阐明其功能。此类信息对于理解这一复杂过程的分子基础至关重要。
