Exploring herpesvirus exonucleases as potential antiviral targets

超过90%的世界人口对九种人类疱疹病毒（HHV）中的三种或更多种呈血清阳性， 其具有建立可被重新激活的终身潜伏感染的能力。HHV再激活 感染在免疫功能低下的患者中特别严重，导致危及生命的播散性 感染.尽管迄今为止许多抗HHV药物的发现工作都集中在核苷/核苷酸类HHV 聚合酶抑制剂，疱疹病毒编码的大量必需复制蛋白 为抗病毒治疗提供了极好的新靶点。需要新的药物来更好地预防病理性 再激活的后遗症以及病毒脱落和传播到新宿主。新模式的必要性 治疗HHV感染的治疗方法强调了更全面了解HHV的重要性 DNA复制。除了通过免疫组织化学鉴定的七种基本单纯疱疹病毒（HSV）复制蛋白外， 我们和其他人，我们已经表明，病毒碱性核酸酶UL 12也是生产病毒 可以包装成传染性病毒的DNA。UL 12与HSV ssDNA结合蛋白ICP 8相互作用， 形成可促进单链退火（SSA）的双组分重组酶（exo/SSAP）。我们有 这表明HSV使用了一种不寻常的DNA复制机制，这种机制与 噬菌体比真核细胞或其他真核病毒。事实上，所有细菌，原生动物， 通过多联体形成复制的植物、昆虫和哺乳动物编码类似的exo/SSAP复合物。 我们假设ICP 8和UL 12促进了一系列反应，其中UL 12切除dsDNA，留下3'端的DNA片段。 由ICP 8识别的ssDNA突出端。然后，ICP 8促进ssDNA退火成活性复制 叉以促进病毒DNA聚合酶（UL 30）的DNA合成。UL 30由两个功能模块组成 结构域：一种3'至5'核酸外切酶，PolExo，在校对和催化聚合酶结构域中起作用 在病毒DNA复制过程中延伸引物所需的。虽然DNA复制的SSA模型是一致的 根据现有的证据，关于UL 12和聚合酶的两种活性如何发挥作用的问题仍然存在 在DNA合成过程中。在平行研究中，Wright和Weller实验室对AN和PolExo都感兴趣 及其来自其他HHV的同源物作为新型抗病毒治疗的靶点， 文库设计用于其接合在AN和PolExo活性位点中发现的双金属结合基序的能力。 我们已经确定了几种先导化合物，它们是有效的抗病毒药物，并抑制一种或多种核酸外切酶。 活性和在某些情况下针对聚合酶活性本身的附加活性。在本提案中，我们将使用 我们的先导化合物作为分子探针来研究病毒DNA复制的机制， 我们致力于开发抑制AN、PolExo和/或Pol的广谱抗病毒剂。 预期抑制这些靶点中的一个以上将增加对抗病毒药物耐药性的屏障。

Over 90% of the world’s population is seropositive for three or more of the nine human herpesviruses (HHVs), which possess the ability to establish lifelong latent infections that can be reactivated. Reactivation of HHV infections is particularly serious in immunocompromised patients leading to disseminated life-threatening infections. Although many of the anti HHV drug discovery efforts to date have focused on nucleoside/tide HHV polymerase inhibitors, the large number of essential replication proteins encoded by the herpesviruses provide excellent novel targets for antiviral therapy. New agents are needed to better prevent pathological sequelae of reactivation as well as viral shedding and transmission to new hosts. The need for new modalities of therapeutics to treat HHV infections underscores the importance of a more thorough understanding of HHV DNA replication. In addition to the seven essential herpes simplex virus (HSV) replication proteins identified by us and others, we have shown that the viral alkaline nuclease UL12 is also essential for the production of viral DNA that can be packaged into infectious virus. UL12 interacts with the HSV ssDNA binding protein ICP8 to form a two-component recombinase (exo/SSAP) that can promote single strand annealing (SSA). We have suggested that HSV uses an unusual mechanism of DNA replication that has more in common with bacteriophage than eukaryotic cells or other eukaryotic viruses. In fact, all DNA viruses of bacteria, protozoa, plants, insects and mammals that replicate through concatemer formation encode a similar exo/SSAP complex. We hypothesize that ICP8 and UL12 promote a series of reactions in which UL12 resects dsDNA leaving a 3’ ssDNA overhang that is recognized by ICP8. ICP8 then promotes annealing of the ssDNA to an active replication fork to promote DNA synthesis by the viral DNA polymerase (UL30). UL30 is comprised of two functional domains: a 3’ to 5’ exonuclease, PolExo, that plays a role in proofreading and the catalytic polymerase domain required for extending primers during viral DNA replication. While the SSA model for DNA replication is consistent with available evidence, questions remain about how UL12 and the two activities of the polymerase function during DNA synthesis. In parallel studies, the Wright and Weller labs have been interested in both AN and PolExo and their othologs from other HHVs as targets for novel antiviral therapeutics and have generated focused libraries designed for their ability to engage a two-metal binding motif found in both AN and PolExo active sites. We have identified several lead compounds that are potent antivirals and inhibit one or more of the exonuclease activities and in some cases additional activity against polymerase activity itself. In this proposal we will use our lead compounds as molecular probes to study mechanisms of viral DNA replication and to continue our efforts to develop broad spectrum antiviral agents that inhibit AN, PolExo and/or Pol. Agents that can inhibit more than one of these targets would be expected to increase the barrier to antiviral drug resistances.