Exploring herpesvirus exonucleases as potential antiviral targets

世界上超过90%的人口对九种或更多人类疱疹病毒(HHV)中的三种或更多呈血清阳性， 它具有建立可重新激活的终身潜伏感染的能力。重新启动HHV 感染在免疫功能低下的患者中尤为严重，导致播散性生命威胁 感染。虽然到目前为止许多抗HHV药物的发现工作都集中在核苷/潮汐HHV 聚合酶抑制物，疱疹病毒编码的大量必需复制蛋白 为抗病毒治疗提供极好的新靶点。需要新的药物来更好地预防病理性 重新激活以及病毒脱落和传播给新宿主的后遗症。对新模式的需求 治疗HHV感染的疗法的出现强调了更彻底地了解HHV的重要性 DNA复制。除了七种基本的单纯疱疹病毒(HSV)复制蛋白外 我们已经证明了病毒碱性核酸酶UL12对病毒的产生也是必不可少的 可以被包装成传染性病毒的DNA。UL12与HSV单链DNA结合蛋白ICP8相互作用 形成能促进单链退火(SSA)的双组分重组酶(exo/SSAP)。我们有 提示单纯疱疹病毒使用一种不同寻常的DNA复制机制，与 比真核细胞或其他真核病毒更强的噬菌体。事实上，所有细菌、原生动物、 通过串联体形成进行复制的植物、昆虫和哺乳动物编码一个类似的exo/SSAP复合体。 我们假设ICP8和UL12促进了一系列反应，在这些反应中UL12切除了dsDNA，留下了3‘ ICP8识别的单链DNA悬垂。然后，ICP8促进单链DNA的退火热变成活跃的复制 通过病毒DNA聚合酶(UL30)促进DNA合成。UL30由两个功能组件组成 结构域：一种3‘到5’的核酸外切酶，PolExo，起到校对和催化聚合酶结构域的作用 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UL12和聚合酶的两种活性是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 以及它们来自其他HHV的同源化合物作为新的抗病毒疗法的靶点，并引起了人们的关注 文库设计的目的是为了能够结合在An和PolExo活性位点中发现的双金属结合基序。 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，可以抑制一种或多种核酸外切酶 活性以及在某些情况下针对聚合酶活性本身的额外活性。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复制的机制并继续 我们努力开发广谱抗病毒药物，以抑制AN、PolExo和/或POL。可以 抑制这些靶点中的一个以上有望增加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.