Zhang C, Trottier M, Chen C, Guo P
Department of Pathobiology and Purdue Cancer Center, Purdue University, West Lafayette, Indiana 47907, USA.
Virology. 2001 Mar 15;281(2):281-93. doi: 10.1006/viro.2000.0771.
During replication, the lengthy genome of dsDNA viruses is translocated with remarkable velocity into the limited space within the preformed procapsid. We previously found that a viral-encoded RNA (pRNA) played a key role in bacterial virus phi29 DNA translocation. Design of mutant pRNA sets containing two and three inactive mutant pRNAs, respectively, led to the conclusion that the stoichiometry of pRNA in DNA packaging is the common multiple of 2 and 3. Together with studies using binomial distribution of mutant and wild-type pRNA, it has been confirmed that six pRNAs of phi29 form a hexagonal complex to drive the DNA translocating machine. These findings have brought about commonality between viral DNA packaging and other universal DNA/RNA-riding processes including DNA replication and RNA transcription. Chemical modification was used to compare the structures of active and inactive as well as free and procapsid-bound pRNA. Our results explain why certain pRNA mutants are inactive in DNA packaging while remaining competent in procapsid binding, since the mutations were located in a domain involved in DNA translocation that is dispensable for procapsid binding. A mutant pRNA that had reduced procapsid binding was revealed to have a structural alteration within the procapsid-binding region that may account for the binding deficiency. Chemical probing of procapsid-bound pRNA revealed a large area of protection, while a 3-base bulge, C(18)C(19)A(20), was accessible to chemicals. A pRNA with a deletion of this 3-base bulge was fully competent to form dimers, bind procapsids, and inhibit phi29 virion assembly in vitro; however, its activity in DNA packaging and virion assembly was completely lost. The results suggest that this bulge is not involved in procapsid binding but may interact with other DNA-packaging components. A computer model showing the location of the CCA bulge was presented.
在复制过程中，双链DNA病毒的冗长基因组以惊人的速度被转运到预先形成的原衣壳内有限的空间中。我们之前发现一种病毒编码的RNA（pRNA）在噬菌体phi29 DNA转运中起关键作用。分别设计包含两个和三个无活性突变pRNA的突变pRNA组，得出DNA包装中pRNA的化学计量是2和3的公倍数的结论。结合使用突变型和野生型pRNA的二项分布的研究，已证实phi29的六个pRNA形成六边形复合物以驱动DNA转运机器。这些发现揭示了病毒DNA包装与其他通用的DNA/RNA搭载过程（包括DNA复制和RNA转录）之间的共性。使用化学修饰来比较活性和无活性以及游离和原衣壳结合的pRNA的结构。我们的结果解释了为什么某些pRNA突变体在DNA包装中无活性而在原衣壳结合方面仍具有能力，因为这些突变位于参与DNA转运的结构域中，而该结构域对于原衣壳结合是可有可无的。一个原衣壳结合减少的突变pRNA被发现其在原衣壳结合区域内有结构改变，这可能解释了结合缺陷。对原衣壳结合的pRNA进行化学探测发现有大面积的保护区域，而一个3碱基凸起C(18)C(19)A(20)可被化学物质接触到。一个缺失此3碱基凸起的pRNA完全能够形成二聚体、结合原衣壳并在体外抑制phi29病毒体组装；然而，其在DNA包装和病毒体组装中的活性完全丧失。结果表明这个凸起不参与原衣壳结合，但可能与其他DNA包装成分相互作用。展示了一个显示CCA凸起位置的计算机模型。
