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

KSHV是一种致癌病毒，卡波西肉瘤（KS）和几种淋巴组织增生性疾病的发展所需的。没有特定的KSHV抗病毒药物或疫苗，目前的治疗方法没有针对性，依赖于重建免疫系统或使用细胞毒性剂。由于KS是一种艾滋病定义的疾病，使用免疫重建控制HIV/AIDS已被研究作为KS治疗。然而，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。HIV-KS患者在ART后也可表现出症状恶化，发展为KS相关的免疫重建炎症综合征，现在是KSHV相关死亡的主要原因。因此，迫切需要抗KSHV治疗。KSHV有两个不同的生命周期，持续性终身感染（潜伏期）和感染性生产周期（裂解复制）。裂解性复制在KSHV的发病机制中起重要作用。因此，有必要研究的分子机制，调节裂解复制，导致新的抗病毒策略的新机会。我们有令人兴奋的数据显示，KSHV操纵宿主细胞N6-甲基腺苷（m6 A）途径，以促进裂解复制。m6 A是mRNA中最丰富的内部修饰。重要的是，m6 A通过招募效应蛋白（m6 A阅读器）对RNA代谢的各个方面施加影响，这些效应蛋白将m6 A修饰的RNA导向不同的生物学命运。m6 A现在被认为调节广泛的生理过程，并且异常m6 A涉及多种人类疾病。因此，KSHV操纵m6 A提供了一个新的机会来研究m6 A失调如何影响基因表达以及如何导致疾病。此外，它还提供了开发新的治疗方法的机会。我们强调KSHV操纵宿主m6 A通路具有前病毒效应。KSHV转录组被m6 A严重修饰，这有助于通过招募一组新的m6 A阅读蛋白（称为皇家蛋白）来稳定特定的病毒mRNA。这些结果通过实验得到加强，该实验显示皇家蛋白SND 1的缺失对KSHV ORF 50转录物稳定性和所导致的病毒复制具有显著影响。此外，全转录组m6 A-seq分析强调，细胞mRNAs的差异m6 A修饰，这与它们在感染过程中的表达增加密切相关。我们现在将解决以下关键问题：（i）我们将研究KSHV mRNAs中除了m6 A修饰外，还有哪些特定特征使不同的皇家蛋白能够优先结合特定的m6 A修饰的KSHV RNA。我们将使用全转录组分析来鉴定由每个皇家蛋白结合的m6 A修饰的RNA组，并在这些RNA中识别任何共有序列或基序。此外，我们将确定RNA结构是否增强了皇家蛋白的选择性。(ii)我们将确定为什么一些细胞的mRNA是差异m6 A修饰在感染过程中。可能是KSHV需要这些RNA来增强其复制，或者它们是宿主细胞在感染时的反应。(iii)我们已经鉴定了RNA结合小分子，其可以优先结合KSHV ORF 50转录物的m6 A修饰形式并减少KSHV裂解复制。我们将充分验证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操纵宿主以增强其自身复制的新方法，并更好地了解动态m6 A RNA修饰如何调节基因表达。这将影响我们对m6 A在细胞和发育过程中的新作用的理解，人类疾病的发展，并为重要的人类病原体的治疗干预提供新的策略。

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.