Parvovirus-Cell Interactions

描述（由申请人提供）：腺相关病毒（aav）需要来自较大DNA病毒的辅助病毒功能才能有效复制。虽然腺病毒（Ad）复制所需的五种辅助功能多年前就被确定，但它们在AAV感染中的作用细节尚不完整。我们最近的分析显示，在AAV复制过程中，Ad辅助功能的作用具有惊人的复杂性。已知Ad E4Orf6在AAV第二链合成的早期限速步骤中起作用。我们的进一步研究表明，AAV劫持了Ad E4Orf6/E1B 55kDa E3泛素连接酶复合物以供其使用；它既能有效地靶向AAV复制抑制剂的激活因子，也能靶向AAV蛋白本身。我们发现，不能再失活dsRNA激活的蛋白激酶R （PKR）的Ad VA RNA突变体不能支持AAV复制，这导致我们在AAV衣壳编码mRNA中发现了一个结合并激活PKR的元件。这是在DNA病毒产生的mRNA中描述的第一个pcr激活元件。此外，我们的研究表明，AAV参与宿主抗病毒反应，这在以前的任何细小病毒中都没有发现。这个应用程序建立在这些观察的基础上。我们的具体目标是了解在AAV/Ad合并感染过程中，Ad E3 ub连接酶（specific Aim I）和Ad VA RNA （specific Aim II）是如何起作用的。然而,除了帮助解决长期的问题,辅助函数如何促进AAV复制,我们的研究将有助于解释如何共享一个病毒E3 ubiquitin-ligase可以帮助优化两个co-infecting DNA病毒的共生关系,以及小说PKR——激活元素在一个信使rna的DNA病毒可能功能,参与宿主抗病毒反应,或许从病毒延时控制开关在没有辅助表达和裂解复制它的存在。最后一个领域的研究也应该阐明自主和需要帮助者的细小病毒群之间的基本差异，并有助于改进重组AAV载体的生产。

DESCRIPTION (provided by applicant): Adeno-associated viruses (AAVs) require helper-virus functions from larger DNA viruses for efficient replication. Although the five adenovirus (Ad) helper functions necessary for AAV replication were identified years ago, details of their role in AAV infection are incomplete. Our recent analysis revealed a surprising complexity in the roles that the Ad helper functions play during AAV replication. Ad E4Orf6 was known to function at an early rate-limiting step in AAV second-strand synthesis. Our closer examination revealed that AAV hijacks the Ad E4Orf6/E1B 55kDa E3 ubiquitin ligase complex for its use; it efficiently targets both an enabler of an inhibitor of AAV replication as well as, surprisingly, AAV proteins themselves. Our finding that Ad VA RNA mutants that could no longer inactivate the dsRNA- activated protein kinase R (PKR) could not support AAV replication led to our discovery of an element within the AAV capsid coding mRNA that binds and activates PKR. This was the first PKR-activating element described within an mRNA generated by a DNA virus. Further, our studies demonstrated that AAV engages the host antiviral response, which had not previously been shown for any parvovirus. This application builds on these observations. Our specific objectives are to understand how the Ad E3 Ub-ligase (Specific Aim I) and Ad VA RNA (Specific Aim II) function during AAV/Ad co-infection. However, in addition to helping solve the long standing issue of how helper-functions facilitate AAV replication, our studies will help explain how sharing a viral E3 ubiquitin-ligase can help optimize the symbiosis of two co-infecting DNA viruses, and how a novel PKR- activating element within an mRNA of a DNA virus might function, engage the host antiviral response, and perhaps govern a switch from viral latency in the absence of helper to expression and lytic replication in its presence. Studies in this last area should also shed light on basic differences between the autonomous and helper-requiring groups of parvoviruses, and also help improve recombinant AAV vector production.