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）发展所需的致癌病毒，也与两种淋巴组织增生性疾病有关;原发性渗出性淋巴瘤和多中心Castleman病。目前，没有特异性的KSHV抗病毒药物或疫苗，并且目前对KSHV相关疾病的治疗不是依赖于重建免疫系统和使用细胞毒性剂。由于KS是一种艾滋病定义疾病，使用调节性免疫重建控制HIV/AIDS已被研究作为KS的可能治疗方法。虽然，即使有有效的抗逆转录病毒治疗（ART）和良好的控制艾滋病毒感染，许多患者仍然发展为进行性KS。因此，仍然迫切需要特异性、有效的抗KS疗法。这些抗病毒药物通过（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断裂解性感染来清除潜伏感染的储库，将显著增强当前的治疗。KSHV裂解性复制在KSHV感染的发病机制中起重要作用。因此，研究KSHV裂解性复制的分子调控机制对全面了解KSHV的发病机制至关重要。此外，抑制KSHV裂解性复制可能为开发新的抗病毒策略以抑制KS形成提供机会。我们有令人兴奋的数据证明了一种抑制KSHV裂解性复制的新方法。我们已经鉴定了一种小分子，其破坏病毒mRNA正确加工所需的基本病毒-宿主细胞相互作用，即KSHV ORF 57-细胞hTREX相互作用。使用这种小分子特异性破坏ORF 57-hTREX相互作用的能力现在提供了一种新的机会来研究ORF 57-hTREX相互作用在KSHV裂解复制中的其他作用，特别是确定ORF 57-hTREX相互作用在增强细胞RNA亚组的正确加工中的作用，所述细胞RNA亚组也被病毒ORF 57蛋白结合，并且被认为增强病毒复制。因此，我们将使用转录组学和蛋白质组学方法，在KSHV潜伏性与裂解性复制过程中，在存在或不存在小分子的情况下，评估细胞mRNA丰度和蛋白质产生的总体变化。这将鉴定通过ORF 57-hTREX相互作用稳定和加工的细胞转录物。进一步的分析将被执行，以检查ORF 57如何识别和结合细胞转录本的这一子集，并确定这些细胞转录本是否是病毒复制所必需的。此外，我们希望进一步研究破坏ORF 57-hTREX相互作用的小分子的抗病毒潜力。我们已经证明，称为CCT 018159的小分子可以抑制细胞培养系统中KSHV裂解性复制和感染性病毒体产生。下一阶段将是探索CCT 018159在人γ-疱疹病毒感染的适当小动物模型中的抑制活性。然而，尽管CCT 018159可用于这种情况，但小分子具有相对较高的代谢转换，这可能降低其疗效。因此，我们将使用药物化学方法修饰小分子以增强其抗病毒活性和药代动力学特性。然后将测试小分子和衍生物在体内环境中抑制鼠γ-2疱疹病毒、鼠γ疱疹病毒68复制的能力。

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.