Targeting the RNA helicase, UAP56: understanding KSHV RNA processing mechanisms to novel antiviral approaches

卡波西肉瘤相关疱疹病毒(KSHV)是卡波西肉瘤(KS)发生发展所必需的致癌病毒，也与两种淋巴增生性疾病有关：原发性渗出性淋巴瘤和多中心Castleman病。目前，还没有特效的KSHV抗病毒药物或疫苗，目前对KSHV相关疾病的治疗不是依靠重建免疫系统和使用细胞毒药物来靶向治疗。由于KS是一种定义为艾滋病的疾病，通过调节免疫重建来控制艾滋病毒/艾滋病已被研究为KS的一种可能治疗方法。尽管如此，即使采用了有效的抗逆转录病毒疗法(ART)和良好控制的艾滋病毒感染，许多患者仍然发展为进展性KS。因此，目前仍迫切需要特效的抗KS疗法。这些抗病毒药物将通过(A)与ART联合作用，(B)抑制与移植免疫抑制相关的KSHV复制，或(C)通过重新激活病毒并同时阻止裂解性感染来清除潜伏感染的蓄水池，从而显著加强目前的治疗。KSHV裂解复制在KSHV感染的发病机制中起重要作用。因此，研究KSHV裂解复制调控的分子机制对于全面了解KSHV的致病机理是十分必要的。此外，抑制KSHV裂解复制可能为开发新的抗病毒策略来抑制KS的形成提供了机会。我们有令人兴奋的数据展示了一种抑制KSHV裂解复制的新方法。我们已经确定了一种小分子，它可以破坏病毒-宿主细胞之间的基本相互作用，即KSHV ORF57-细胞hTREX相互作用，这是正确处理病毒mRNAs所必需的。利用这种小分子特异性地破坏ORF57-hTREX相互作用的能力现在为研究ORF57-hTREX相互作用在KSHV裂解复制中的其他作用提供了一个新的机会，特别是确定ORF57-hTREX相互作用在促进细胞RNA子集的正确处理方面的作用，这些RNA也被病毒ORF57蛋白结合，并被认为促进病毒复制。因此，我们将使用转录和蛋白质组学方法，在KSHV潜伏复制和裂解复制过程中，在没有或存在小分子的情况下，评估细胞信使核糖核酸丰度和蛋白质产量的全球变化。这将识别通过ORF57-hTREX相互作用稳定和处理的细胞转录本。然后，将进行进一步的分析，以检查ORF57如何识别和结合这一细胞转录子集，并确定这些细胞转录本是否对病毒复制是必要的。此外，我们希望进一步研究破坏ORF57-hTREX相互作用的小分子的抗病毒潜力。我们已经证明，被命名为CCT018159的小分子可以在细胞培养系统中抑制KSHV的裂解复制和感染性病毒粒子的产生。下一阶段将探索CCT018159在适当的人类伽马疱疹病毒感染小动物模型中的抑制活性。然而，虽然CCT018159可以在这种情况下使用，但小分子的代谢周转率相对较高，这可能会降低其疗效。因此，我们将利用药物化学方法对该小分子进行修饰，以增强其抗病毒活性和药代动力学特性。然后，将在活体环境中测试小分子及其衍生物是否有能力抑制小鼠伽马-2疱疹病毒--小鼠伽马疱疹病毒68的复制。

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus required for the development of Kaposi's sarcoma (KS) and is also associated with two lymphoproliferative disorders; primary effusion lymphoma and multicentric Castleman's disease. At present, there are no specific KSHV antivirals or vaccines and current treatments for KSHV-associated diseases are not targeted relying on rebuilding the immune system and using cytotoxic agents. As KS is an AIDS-defining disease, controlling HIV/AIDS using regulated immune reconstitution has been investigated as a possible treatment for KS. Although, even with effective Antiretroviral Therapy (ART) and well-controlled HIV infection, many patients still develop progressive KS. Consequently, specific, efficacious anti-KS therapies are still urgently needed. These antivirals would significantly enhance current treatments by (a) working in combination with ART, (b) inhibiting KSHV replication associated with transplant immunosuppression or (c) clearing the latently-infected reservoir by reactivating the virus and simultaneously blocking lytic infection.Like all herpesviruses KSHV has two distinct life cycles, a persistence life-long infection (latency) and infectious productive cycle (lytic replication). KSHV lytic replication plays an important part in the pathogenesis of KSHV infection. Therefore, it is essential to study the molecular mechanisms which regulate lytic replication to fully understand KSHV pathogenesis. Moreover, inhibiting KSHV lytic replication may provide an opportunity to develop novel antiviral strategies to inhibit KS formation. We have exciting data demonstrating a novel approach to inhibit KSHV lytic replication. We have identified a small molecule which disrupts an essential virus-host cell interaction, the KSHV ORF57-cellular hTREX interaction, required for the correct processing of viral mRNAs. The ability to specifically disrupt the ORF57-hTREX interaction using this small molecule now presents a novel opportunity to investigate other roles of the ORF57-hTREX interaction in KSHV lytic replication, in particular determining the role of the ORF57-hTREX interaction in enhancing the correct processing of a subset of cellular RNAs, which are also bound by the viral ORF57 protein, and are thought to enhance virus replication. Therefore, we will assess the global changes in cellular mRNA abundance and protein production, using transcriptomic and proteomic approaches, during KSHV latent versus lytic replication in the absence or presence of the small molecule. This will identify cellular transcripts which are stabilised and processed by the ORF57-hTREX interaction. Further analysis will then be performed to examine how ORF57 recognises and binds this subset of cellular transcripts and determine if these cellular transcripts are essential for virus replication.In addition, we wish to further investigate the antiviral potential of the small molecule which disrupts the ORF57-hTREX interaction. We have shown that the small molecule termed, CCT018159, can inhibit KSHV lytic replication and infectious virion production in a cell culture system. The next stage will to be to explore the inhibitory activity of CCT018159 in an appropriate small animal model for human gamma-herpesvirus infection. However, although CCT018159 can be used in this setting, the small molecule has a relatively high metabolic turnover which may reduce its efficacy. Therefore, we will modify the small molecule to enhance its antiviral activity and pharmacokinetic properties using medicinal chemistry approaches. The small molecule and derivatives will then be tested for their ability to inhibit the replication of the murine gamma-2 herpesvirus, murine Gammaherpesvirus 68, in an in vivo setting.