Targeting transfer RNA-derived fragments during KSHV infection

卡波西氏肉瘤相关疱疹病毒（KSHV）是卡波西氏肉瘤（KS）发展所必需的一种致癌病毒。目前还没有特异性的KSHV抗病毒药物或疫苗，目前针对KSHV相关疾病的治疗也不是靶向性的，而是依赖于重建免疫系统和使用细胞毒性药物。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。像所有疱疹病毒一样，KSHV有两个不同的生命周期，一个是持续的终身感染（潜伏期），另一个是感染产生周期（裂解复制）。裂解复制在KSHV发病机制中起重要作用。因此，研究调控裂解复制的分子机制是十分必要的。此外，抑制KSHV裂解复制可能为开发抑制KS形成的新型抗病毒策略提供机会。我们有令人兴奋的数据表明，KSHV操纵一组最近分类的非编码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，参与多种生物学途径，作为基因表达的调节剂。毫不奇怪，在包括癌症、代谢和神经系统疾病和感染在内的许多人类疾病中都发现了异常的tRF水平。然而，如何调节tRF生物发生以及tRF失调如何导致疾病仍有待确定。因此，KSHV操纵tRF水平提供了一个新的机会来研究tRF失调如何影响基因表达以及这如何导致疾病。此外，逆转kshv介导的tRF失调的作用可以减少病毒复制，这表明它有可能成为一种新的抗病毒策略。已经报道了五种结构类型的tRF，我们的初步结果表明KSHV特异性地失调了两种不同的tRF亚型。首先，我们观察到3'- trf的特异性下调，由于它们的小尺寸和与Argonaute蛋白的关联，我们认为它们可能以类似mirna的方式在感染过程中调节基因表达。我们将确定这些失调的3'- trf的mRNA靶标，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i- trf的细胞RBP，并评估RBP隔离对病毒复制和转录组基因表达的影响。此外，我们将研究KSHV在感染期间如何操纵tRF水平的新机制。最后，我们表明逆转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感染细胞系中tRF失调的最佳方法和3D肿瘤模型，以验证该方法作为一种新的抗病毒策略。总之，该项目将确定KSHV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作用的理解，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。

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.