Royal Reader Proteins: role in KSHV RNA processing to novel antiviral approaches

Royal Reader Proteins：在 KSHV RNA 加工和新型抗病毒方法中的作用

• 批准号: MR/X000060/1
• 负责人: Adrian Whitehouse
• 金额: $ 103.18万
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FX000060%2F1
• 关键词: Royal; Reader; Proteins; role; KSHV

KSHV is an oncogenic virus required for the development of Kaposi's sarcoma (KS) and several lymphoproliferative diseases. There are no specific KSHV antivirals or vaccines and current treatments are not targeted, relying on rebuilding the immune system or using cytotoxic agents. As KS is an AIDS-defining disease, controlling HIV/AIDS using immune reconstitution has been investigated as a KS treatment. However, even with effective Antiretroviral Therapy (ART) and well-controlled HIV infection, many patients still develop progressive KS. HIV-KS patients can also exhibit a worsening of symptoms upon ART, developing KS-associated immune reconstitution inflammatory syndrome, now a major contributor to KSHV-associated deaths. Consequently, anti-KSHV therapies are urgently needed. KSHV has two distinct life cycles, a persistence life-long infection (latency) and an infectious productive cycle (lytic replication). Lytic replication plays an essential role in KSHV pathogenesis. Therefore, it is essential to study the molecular mechanisms which regulate lytic replication, leading to new opportunities for novel antiviral strategies.We have exciting data showing that KSHV manipulates the host cell N6-methyladenosine (m6A) pathway to promote lytic replication. m6A is the most abundant internal modification of mRNAs. Importantly, m6A exerts its influence over all aspects of RNA metabolism by recruiting effector proteins, m6A readers, which direct the m6A-modified RNA towards distinct biological fates. m6A is now thought to regulate a wide range of physiological process and aberrant m6A is implicated in multiple human diseases. Therefore, KSHV manipulation of m6A presents a novel opportunity to examine how m6A dysregulation impacts gene expression and how this leads to disease. Moreover, it provides an opportunity to develop novel therapeutic approaches. We highlight that KSHV manipulation of the host m6A pathway has a pro-viral effect. The KSHV transcriptome is heavily m6A-modified and this helps to stabilise specific viral mRNAs by recruiting a new group of m6A reader proteins, known as Royal proteins. These results are reinforced by experiments showing that depletion of the Royal protein, SND1, has a dramatic effect on KSHV ORF50 transcript stability and resulting virus replication. In addition, transcriptome-wide m6A-seq analysis highlights that cellular mRNAs are differentially m6A-modified which strongly correlates with an increase in their expression during infection.We will now address the following key questions: (i) We will examine what specific features within KSHV mRNAs, apart from m6A modification, enables different Royal proteins to bind preferentially to specific m6A-modified KSHV RNAs. We will use transcriptome-wide analysis to identify the groups of m6A-modified RNAs bound by each Royal protein and characterise any consensus sequences or motifs in these RNAs. Moreover, we will determine if RNA structure enhances Royal protein selectivity. (ii) We will determine why some cellular mRNAs are differentially m6A-modified during the course of infection. It may be the case that these RNAs are required by KSHV to enhance its replication or they are a response by the host cell upon infection. (iii) We have identified RNA-binding small molecules which can preferentially bind to the m6A-modified form of the KSHV ORF50 transcript and reduce KSHV lytic replication. We will fully characterise the activity of these inhibitory small molecules and optimise their function using pharmacophore-based design.In summary, we will identify novel ways KSHV manipulates the host to enhance its own replication and provide a better understanding of how the dynamic m6A RNA modification regulates gene expression. This will impact on our understanding of the emerging role of m6A in cell and developmental processes, the development of human disease and provide new strategies for therapeutic intervention of an important humuan pathogen.

KSHV 是一种致癌病毒，是卡波西肉瘤 (KS) 和多种淋巴组织增生性疾病发展所必需的。没有特定的 KSHV 抗病毒药物或疫苗，目前的治疗也没有针对性，依赖于重建免疫系统或使用细胞毒性药物。由于 KS 是一种 AIDS 定义的疾病，因此已将使用免疫重建来控制 HIV/AIDS 作为 KS 治疗方法进行了研究。然而，即使采用有效的抗逆转录病毒治疗 (ART) 和 HIV 感染得到良好控制，许多患者仍会出现进行性 KS。 HIV-KS 患者在 ART 后也可能表现出症状恶化，发展为 KS 相关的免疫重建炎症综合征，目前是 KSHV 相关死亡的主要原因。因此，迫切需要抗 KSHV 疗法。 KSHV 有两个不同的生命周期：持续性终生感染（潜伏期）和感染性生产周期（裂解复制）。裂解复制在 KSHV 发病机制中起着重要作用。因此，有必要研究调节裂解复制的分子机制，从而为新型抗病毒策略带来新的机会。我们有令人兴奋的数据表明，KSHV 操纵宿主细胞 N6-甲基腺苷 (m6A) 途径来促进裂解复制。 m6A 是最丰富的 mRNA 内部修饰。重要的是，m6A 通过招募效应蛋白（m6A 阅读器）对 RNA 代谢的各个方面发挥影响，这些蛋白将 m6A 修饰的 RNA 引导至不同的生物命运。 m6A 现在被认为可以调节广泛的生理过程，并且异常的 m6A 与多种人类疾病有关。因此，KSHV 对 m6A 的操纵提供了一个新的机会来研究 m6A 失调如何影响基因表达以及如何导致疾病。此外，它还提供了开发新治疗方法的机会。我们强调 KSHV 对宿主 m6A 途径的操纵具有促病毒作用。 KSHV 转录组经过大量 m6A 修饰，这有助于通过招募一组新的 m6A 读取蛋白（称为 Royal 蛋白）来稳定特定的病毒 mRNA。实验证实了这些结果，表明 Royal 蛋白 SND1 的缺失对 KSHV ORF50 转录稳定性和由此产生的病毒复制具有显着影响。此外，全转录组 m6A-seq 分析强调，细胞 mRNA 进行了差异性 m6A 修饰，这与其感染期间表达的增加密切相关。我们现在将解决以下关键问题：（i）我们将检查 KSHV mRNA 中除了 m6A 修饰之外的哪些特定特征，使不同的 Royal 蛋白能够优先与特定的 m6A 修饰的 KSHV RNA 结合。我们将使用全转录组分析来识别与每个 Royal 蛋白结合的 m6A 修饰的 RNA 组，并表征这些 RNA 中的任何共有序列或基序。此外，我们将确定 RNA 结构是否增强 Royal 蛋白的选择性。 (ii) 我们将确定为什么一些细胞 mRNA 在感染过程中发生不同的 m6A 修饰。情况可能是 KSHV 需要这些 RNA 来增强其复制，或者它们是宿主细胞感染后的反应。 (iii)我们已经鉴定出RNA结合小分子，它们可以优先结合m6A修饰形式的KSHV ORF50转录本并减少KSHV裂解性复制。我们将充分表征这些抑制性小分子的活性，并使用基于药效团的设计优化其功能。总之，我们将确定 KSHV 操纵宿主增强自身复制的新方法，并更好地理解动态 m6A RNA 修饰如何调节基因表达。这将影响我们对 m6A 在细胞和发育过程、人类疾病发展中的新作用的理解，并为重要的人类病原体的治疗干预提供新策略。