Parvovirus-Cell Interactions

描述(申请人提供)：腺相关病毒(AAV)需要较大DNA病毒的辅助病毒功能才能有效复制。虽然AAV复制所必需的五个腺病毒(Ad)辅助功能早在几年前就已确定，但它们在AAV感染中的作用细节仍不完整。我们最近的分析显示，在AAV复制过程中，Ad Helper功能所扮演的角色具有惊人的复杂性。已知AdE4Orf6在AAV第二链合成的早期限速步骤起作用。我们的进一步研究发现，AAV劫持了Ad E4Orf6/E1B 55 kDa E3泛素连接酶复合体供其使用；它有效地靶向AAV复制抑制因子的使能器以及令人惊讶的AAV蛋白本身。我们的发现是，不再能够灭活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PKR激活元件。此外，我们的研究表明，AAV参与了宿主的抗病毒反应，这是以前没有对任何细小病毒显示的。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AdE3 Ub连接酶(特异性目标I)和Ad VA RNA(特异性目标II)在AAV/Ad联合感染过程中的作用。然而，除了帮助解决助手功能如何促进AAV复制这一长期存在的问题外，我们的研究还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同感染的DNA病毒的共生，以及DNA病毒mRNA中的新的PKR激活元件如何发挥作用，参与宿主的抗病毒反应，并可能控制从没有助手的病毒潜伏期到存在时的表达和裂解复制的转换。对这最后一个领域的研究也应该阐明自治的细小病毒组和需要帮助的细小病毒组之间的基本差异，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.