Targeting transfer RNA-derived fragments during KSHV infection

KSHV 感染期间靶向转移 RNA 衍生片段

• 批准号: MR/V009478/1
• 负责人: Adrian Whitehouse
• 金额: $ 84.01万
• 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: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FV009478%2F1
• 关键词: Targeting; transfer; RNA; derived; fragments

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus required for the development of Kaposi's sarcoma (KS). 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 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. Like all herpesviruses KSHV has two distinct life cycles, a persistence life-long infection (latency) and infectious productive cycle (lytic replication). Lytic replication plays an important part in KSHV pathogenesis. Therefore, it is essential to study the molecular mechanisms which regulate lytic replication. Moreover, inhibiting KSHV lytic replication may provide an opportunity to develop novel antiviral strategies to inhibit KS formation. We have exciting data demonstrating that KSHV manipulates a recently classified group of non-coding RNAs, termed transfer RNA-derived small RNA fragments (tRFs), to enhance lytic replication. tRFs are derived from precursor or mature tRNAs and are implicated in multiple biological pathways, functioning as regulators of gene expression. Not surprisingly abnormal tRF levels are identified in a wide range of human diseases, including cancer, metabolic and neurological diseases and infections. However, it remains to be determined how tRF biogenesis is regulated and how tRF dysregulation contributes to disease. Therefore, KSHV manipulation of tRF levels presents an novel opportunity to examine how tRF dysregulation impacts gene expression and how this leads to disease. Moreover, reversing the effect of KSHV-mediated tRF dysregulation reduces virus replication, suggesting it has potential as a novel antiviral strategy.Five structural categories of tRFs have been reported and our preliminary results demonstrate that KSHV specifically dysregulates two distinct tRF subtypes. Firstly, we observe a specific downregulation of 3'-tRFs, and due to their small size and association with Argonaute proteins we believe they may function in a similar manner to miRNAs to regulate gene expression during infection. We will identify mRNA targets of these dysregulated 3'-tRFs and then assess their role in virus replication. In contrast, we observe an upregulation of a second tRF subset, i-tRFs. i-tRFs fail to associate with Argonaute proteins and instead contain conserved RNA binding protein (RBP) motifs, suggesting they function as RBP sponges. We will identify cellular RBPs which bind these upregulated i-tRFs and assess what effect RBP sequestration has on virus replication and transcriptome-wide gene expression. Moreover, we will investigate novel mechanisms of how KSHV manipulates tRF levels during infection. Finally, we show that reversing the effect of tRF dysregulation has a detrimental effect on virus replication. Therefore, we will optimise the best approach to reverse tRF dysregulation in KSHV-infected cell lines and a 3D tumour model to validate this approach as a novel antiviral strategy. In summary, this project will identify novel ways KSHV can manipulate the host cell to enhance its own replication and provide a better understanding of how tRFs regulate gene expression. This will impact on our understanding of the emerging role of tRFs in cell and developmental processes, the development of human disease and provide new strategies for therapeutic intervention of an important human pathogen.

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。没有特异性的KSHV抗病毒药物或疫苗，目前对KSHV相关疾病的治疗没有针对性，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。像所有疱疹病毒一样，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）。裂解复制在KSHV发病机制中发挥着重要作用。因此，有必要研究调控裂解性复制的分子机制。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表明，KSHV操纵最近分类的一组非编码RNA，称为转移RNA衍生的小RNA片段（tRFs），以增强裂解复制。tRF来源于前体或成熟的tRNA，并参与多种生物学途径，作为基因表达的调节剂发挥作用。毫不奇怪，在广泛的人类疾病中发现了异常的tRF水平，包括癌症、代谢和神经疾病以及感染。然而，它仍然有待确定如何调节tRF生物合成和如何tRF失调有助于疾病。因此，KSHV操纵tRF水平提供了一个新的机会，研究tRF失调如何影响基因表达，以及如何导致疾病。此外，逆转KSHV介导的tRF失调的影响，减少病毒复制，这表明它有潜力作为一种新的抗病毒strategy.Five结构类别的tRF已被报道，我们的初步结果表明，KSHV特异性失调两个不同的tRF亚型。首先，我们观察到3 '-tRFs的特异性下调，并且由于它们的小尺寸和与Argonaute蛋白的关联，我们相信它们可能以与miRNA类似的方式发挥作用，以在感染期间调节基因表达。我们将确定这些失调的3 '-tRFs的mRNA靶点，然后评估它们在病毒复制中的作用。相反，我们观察到第二个tRF子集i-tRF的上调。i-tRFs不能与Argonaute蛋白结合，而是含有保守的RNA结合蛋白（RBP）基序，表明它们具有RBP海绵的功能。我们将鉴定结合这些上调的i-tRF的细胞RBP，并评估RBP螯合对病毒复制和转录组范围基因表达的影响。此外，我们还将研究KSHV在感染过程中如何操纵tRF水平的新机制。最后，我们发现逆转tRF失调的影响对病毒复制有不利影响。因此，我们将优化逆转KSHV感染细胞系和3D肿瘤模型中tRF失调的最佳方法，以验证这种方法作为一种新的抗病毒策略。总之，该项目将确定KSHV可以操纵宿主细胞以增强其自身复制的新方法，并更好地了解tRFs如何调节基因表达。这将影响我们对tRFs在细胞和发育过程中的新兴作用的理解，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。

项目成果

Dysregulation of the miR-30c/DLL4 axis by circHIPK3 is essential for KSHV lytic replication.

• DOI: 10.15252/embr.202154117
• 发表时间: 2022-05-04
• 期刊: EMBO REPORTS
• 影响因子: 7.7
• 作者: Harper, Katherine L.;Mottram, Timothy J.;Anene, Chinedu A.;Foster, Becky;Patterson, Molly R.;McDonnell, Euan;Macdonald, Andrew;Westhead, David;Whitehouse, Adrian
• 通讯作者: Whitehouse, Adrian