Post-Transcriptional Regulation of Parvovirus Capsid Gene Expression

描述(申请人提供)：细小病毒AMDV和B19在动物DNA病毒中是独一无二的，因为它们只有一个启动子，因此它们的遗传多样性完全由转录后机制控制。由这些病毒产生的单一类别的前信使核糖核酸分子经历广泛的选择性剪接和多聚腺苷酸化，从而产生编程这种多样性的亚基因组信使核糖核酸分子。 控制衣壳蛋白的合成是B19组织趋向性的关键调节因素之一，并控制AMDV在感染宿主动物体内持续存在的能力。B19允许性感染的特征是转向增加衣壳产生，而AMDV在动物系统中持续感染的特点是衣壳蛋白合成水平受到控制，而且可能需要控制低水平的衣壳蛋白合成。所有其他细小病毒都有一个内部启动子，在病毒复制的合成阶段调节衣壳编码mRNA的产生，然而，这种机制对B19和AMDV是不可用的。通过转录后机制控制AMDV和B19衣壳编码基因的表达是这一应用的重点。 这两种病毒在基因组的中心都有一个有效的Polya位点。使用这个位点排除了将衣壳编码的ORFs包含到信使核糖核酸中，理解如何选择多腺苷或通读这个位点，是具体目标2和3的主题。我们建议识别控制这些事件的顺式序列和反式因子，控制它们选择性使用的机制，以及对于B19，检查这种选择在允许和不允许复制的细胞中是如何不同的。我们最近发现，单个剪接的AMDV mRNA既编码衣壳蛋白VP1和VP1，也编码其5‘端必需的小非结构蛋白NS2。因此，除了选择性剪接和多聚腺苷化，选择性翻译起始的特征对于调控AMDV衣壳蛋白的正确表达至关重要。在特定的目标1中，我们将从遗传和生化两个方面表征NS2基因中的顺式元件如何影响衣壳蛋白的翻译。 这些转录后过程如何从AMDV和B19编码的单个前mRNA分子中产生适当水平的衣壳蛋白，对于我们理解细小病毒的基因表达和细小病毒感染的生物学至关重要。然而，此外，这些病毒系统提供了非常容易处理的模型，用来从总体上了解这些基本的细胞机制。

DESCRIPTION (provided by applicant): The parvoviruses AMDV and B19 are unique among animal DNA viruses in that they have a single promoter, and so their genetic diversity is controlled exclusively by post-transcriptional mechanisms. A single class of pre-mRNA molecules generated by these viruses undergoes extensive alternative splicing and polyadenylation that generates sub-genomic mRNA molecules which program this diversity. Control of the synthesis of the capsid proteins is one of the key regulators of B19 tissue tropism, and governs the ability of AMDV to remain persistent in infected host animals. B19 permissive infection is characterized by a switch to increased capsid production, while persistence of AMDV infection in animal systems is characterized by, and likely requires, controlled low levels of capsid protein synthesis. All other parvoviruses have an internal promoter that regulates production of capsid-encoding mRNA during the synthesis phase of viral replication, however, this mechanism is not available to B19 and AMDV. Control of expression of the AMDV and B19 capsid-coding genes by post-transcriptional mechanisms is the focus of this application. Both viruses have an efficient polyA site in the center of the genome. Use of this site precludes inclusion of the capsid coding ORFs into mRNA, and an understanding of how the choice is made to either polyadenylate, or read-through this site, is the topic of Specific Aim 2 and 3. We propose to identify both the cis sequence and trans-factors that control these events, the mechanisms that govern their selective use, and for B19, exam how this choice is differently made in cells permissive or nonpermissive for replication. We have recently discovered that a single spliced AMDV mRNA encodes both capsid proteins VP1 and VP1, as well as an essential small non-structural protein NS2 at its 5'end. Thus, in addition to alternative splicing and polyadenylation, features of alternative translation initiation are critical for governing the proper expression of the AMDV capsid proteins. In Specific Aim 1, we will characterize how the cis-element in the NS2 gene affects translation of the capsid proteins, both genetically and biochemically. How these post-transcriptional processes generate appropriate levels of the capsid proteins from the single pre-mRNA molecules encoded by AMDV and B19, is critical to our understanding of parvovirus gene expression, and the biology of parvovirus infection. However, in addition, these viral systems provide very tractable models with which to learn much about these basic cellular mechanisms in general.