Parvovirus-Cell Interactions

描述（由申请方提供）：腺相关病毒（AAV）需要来自较大DNA病毒的辅助病毒功能以进行有效复制。虽然五腺病毒（Ad）辅助功能所需的AAV复制几年前就确定了，其在AAV感染中的作用的细节是不完整的。我们最近的分析揭示了一个令人惊讶的复杂性的作用，广告辅助功能在AAV复制过程中发挥作用。 已知Ad E4 Orf 6在AAV第二链合成的早期限速步骤中起作用。我们更仔细的检查显示，AAV劫持了Ad E4 Orf 6/E1 B 55 kDa E3泛素连接酶复合物用于其使用;它有效地靶向AAV复制抑制剂的使能因子以及令人惊讶的AAV蛋白本身。我们的发现是，不再能抑制dsRNA活化的蛋白激酶R（PKR）的Ad VA RNA突变体不能支持AAV复制，这导致我们发现了AAV衣壳编码mRNA内结合并活化PKR的元件。这是第一个在DNA病毒产生的mRNA中描述的PKR激活元件。此外，我们的研究表明，AAV参与宿主的抗病毒反应，这在以前对于任何细小病毒都没有显示。本申请建立在这些观察的基础上。 我们的具体目标是了解在AAV/Ad共感染过程中Ad E3 Ub连接酶（特异性Aim I）和Ad VA RNA（特异性Aim II）的功能。然而，除了帮助解决辅助功能如何促进AAV复制的长期存在的问题之外，我们的研究将有助于解释共享病毒E3泛素连接酶如何有助于优化两种共感染DNA病毒的共生，以及DNA病毒mRNA内的新型PKR激活元件如何发挥作用，参与宿主抗病毒反应，并且可能控制从没有辅助病毒时的病毒潜伏期到存在辅助病毒时的表达和裂解性复制的转变。在这最后一个领域的研究也应该阐明自主和辅助需要组的细小病毒之间的基本差异，也有助于提高重组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.