Mechanism of the Antiviral Activity of BAF Against Poxvirus and HSV-1 Infection

 描述（由申请人提供）：关于细胞利用细胞质和细胞核中的外来DNA进行防御的机制，以及这些防御在感染期间如何被病毒对抗措施破坏，还有很多东西有待了解。为了解决这一知识缺口，我们的研究宿主防御外来DNA的重点痘病毒牛痘和HSV 1作为模式病原体。了解这些病毒如何完成DNA复制和转录将产生新的见解细胞用来应对所有外来DNA的防御机制。为了实现这一目标，我们对牛痘的研究发现，痘病毒B1激酶对病毒DNA复制是必不可少的，因为需要它来抑制细胞DNA结合蛋白BAF（自整合屏障因子）。如果未失活，BAF与 病毒DNA，并作为对牛痘DNA复制的防御。我们实验室最近的数据表明，BAF也可以作为HSV 1抑制剂，表明BAF可能会损害多种DNA病毒的生长。BAF的抗病毒活性可能是通过其压缩和聚集DNA的能力发生的，我们假设这种压缩可能有助于宿主防御的多种方式。例如，压实可能通过空间位阻限制DNA对病毒复制蛋白的可接近性，并促进其他抗病毒蛋白与外源基因组的结合。对BAF抗病毒活性的进一步研究将对其作用机制产生独特的见解。我们的中心假设是，BAF启动组装的DNA：蛋白质复合物在磷酸化调节的方式，提供了一个支架上的内在防御效应可以收敛。为了验证我们的假设，我们提出了三个目标。目的1）研究磷酸化和定位对BAF抑制痘病毒DNA复制的调控作用。这些研究将深入了解翻译后调节和定位如何提供调节BAF宿主防御活性的相互关联的机制。目的2）确定BAF-DNA复合物如何调节转录和DDR信号以保护基因组的完整性.了解BAF和DNA修复机制如何协调外源DNA的沉默，但被痘病毒逃避，将是这一目标的中心焦点。目的3）探讨BAF抗HSV-1感染的作用机制。我们的数据表明，BAF可以削弱HSV-1感染的方式调节的本地化和/或磷酸化，因此平行BAF的抗痘病毒活性的调制。这些研究将有助于深入了解BAF抗HSV-1活性的分子机制。在整个研究过程中，我们将比较和对比BAF如何对抗细胞质和核DNA病毒，这将是这些病原体的创新应用。作为这项工作的结果，我们将更好地了解BAF的贡献，这些核蛋白复合物和BAF本身是如何在病毒感染的调节。因此，我们将深入了解细胞机制，这些机制在以下能力方面重叠：1）通过压实使外源DNA沉默，2）即使在没有感染的情况下也保护基因组完整性。

 DESCRIPTION (provided by applicant): Much remains to be learned about mechanisms cells utilize to defend against foreign DNA, both in the cytoplasm and nucleus, as well as how these defenses are subverted by viral countermeasures during infection. To address this knowledge gap, our studies of host defenses against foreign DNA focus on the poxvirus vaccinia and HSV1 as model pathogens. Understanding how these viruses accomplish DNA replication and transcription will yield new insights into the defense mechanisms cells use to respond to all foreign DNA. Toward this goal, our studies of vaccinia have led to the discovery that the poxviral B1 kinase is essential for viral DNA replication because it is needed to inactivate the cellular DNA binding protein BAF (barrier to autointegration factor). If it is not inactivated, BAF binds to viral DNA and acts as a defense against vaccinia DNA replication. Recent data from our lab suggests that BAF can act as an HSV1 inhibitor as well, indicating BAF may impair growth of multiple DNA viruses. BAF's antiviral activity likely occurs via its ability to compact and aggregate DNA to which it binds; we postulate that this compaction may contribute to host defense in multiple ways. For example, compaction likely limits the accessibility of the DNA to viral replication proteins via steric hindrance, and facilitates binding of other antiviral protein to the foreign genomes. Further examination of BAF's antiviral activity will yield unique insights int its mechanism of action. Our central hypothesis is that BAF initiates the assembly of DNA:protein complexes in a phosphorylation-regulated manner, providing a scaffold on which intrinsic defense effectors can converge. To test our hypothesis we propose three aims. AIM 1) Determine how phosphorylation and localization both regulate BAF's repression of poxviral DNA replication. These studies will yield insights into how post-translational regulation and localization provide interconnected mechanisms of regulating BAF's host defense activity. AIM 2) Determine how BAF- DNA complexes modulate transcription and DDR signaling to protect genomic integrity. Understanding how BAF and DNA repair machinery coordinate the silencing of foreign DNA, but are eluded by poxviruses, will be a central focus of this aim. AIM 3) Determine the mechanism whereby BAF acts as an antiviral against HSV-1 infection. Our data demonstrate that BAF can impair HSV-1 infection in a manner regulated by localization and/or phosphorylation, thus paralleling how BAF's anti-poxviral activity is modulated. These studies will yield insights into the molecular mechanism of BAF's activity against HSV-1. Throughout the course of these studies, we will compare and contrast how BAF works against both a cytoplasmic and a nuclear DNA virus, which will be an innovative application of these pathogens. As an outcome of this work we will better understand BAF's contribution to these nucleoprotein complexes and how BAF itself is regulated during viral infection. Thus, we will gain insight into cellular mechanisms which overlap in their ability to 1) silence foreign DNA through compaction and 2) protect genome integrity even in the absence of infection.