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

KSHV是卡波西肉瘤（KS）和几种淋巴增生性疾病发展所必需的致瘤病毒。目前还没有特异性的KSHV抗病毒药物或疫苗，目前的治疗也不是靶向性的，而是依赖于重建免疫系统或使用细胞毒性药物。由于KS是艾滋病的定义性疾病，利用免疫重建控制HIV/AIDS已被研究作为一种KS治疗方法。然而，即使有有效的抗逆转录病毒治疗（ART）和良好控制的艾滋病毒感染，许多患者仍然发展为进行性KS。HIV-KS患者在抗逆转录病毒治疗后也会出现症状恶化，出现与ks相关的免疫重建炎症综合征，这是目前导致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，一个持续的终身感染（潜伏期）和一个感染生产周期（裂解复制）。裂解复制在KSHV发病机制中起重要作用。因此，有必要研究调控裂解复制的分子机制，为开发新的抗病毒策略提供新的机会。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读取器）对RNA代谢的各个方面施加影响，这些效应蛋白引导m6A修饰的RNA走向不同的生物学命运。m6A现在被认为调节广泛的生理过程，异常的m6A与多种人类疾病有关。因此，KSHV操纵m6A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这如何导致疾病。此外，它还为开发新的治疗方法提供了机会。我们强调KSHV操纵宿主m6A通路具有前病毒作用。KSHV转录组被大量m6A修饰，这有助于通过招募一组新的m6A解读蛋白（称为Royal蛋白）来稳定特定的病毒mrna。这些结果被实验进一步证实，表明Royal蛋白SND1的缺失对KSHV ORF50转录物的稳定性和由此产生的病毒复制有显著影响。此外，转录组范围内的m6A-seq分析强调，细胞mrna是m6a修饰的差异，这与感染期间其表达的增加密切相关。我们现在将解决以下关键问题：(i)我们将研究除了m6A修饰之外，KSHV mrna中的哪些特定特征使不同的Royal蛋白能够优先结合特定的m6A修饰的KSHV rna。我们将使用转录组分析来鉴定与每种Royal蛋白结合的m6a修饰的rna组，并描述这些rna中任何一致的序列或基序。此外，我们将确定RNA结构是否增强了Royal蛋白的选择性。（ii）我们将确定为什么一些细胞mrna在感染过程中会被不同的m6a修饰。可能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一种反应。（iii）我们已经发现了rna结合小分子，这些小分子可以优先结合m6a修饰的KSHV ORF50转录物，并减少KSHV裂解复制。我们将充分表征这些抑制性小分子的活性，并利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在细胞和发育过程中的新兴作用的理解，以及人类疾病的发展，并为一种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.