Novel viral immune interference mechanisms: HCV as a model system

丙型肝炎病毒(HCV)感染会导致肝纤维化、肝硬变和肝细胞癌 癌症，每年导致全球350,000人死亡，是最常见的病因 导致美国的肝移植。估计有17.4万名退伍军人感染了丙型肝炎病毒 退伍军人事务部是丙型肝炎病毒治疗的最大提供者。虽然有新的和 令人兴奋的丙型肝炎治疗，由于耐药性和获得问题，治疗不太可能 彻底根除丙型肝炎病毒感染。因此，需要一种疫苗。此外，患有疾病的人 丙型肝炎病毒感染可能会继续发展为肝细胞癌。在持续的丙型肝炎中 病毒特异性T细胞的感染、增殖和激活延迟且效率低下， 对乙肝疫苗接种以及细菌和血吸虫感染的免疫反应是 被压抑。这表明，丙型肝炎病毒感染抑制了T细胞功能，尽管 这一现象的机制尚不清楚。最近的数据显示，丙型肝炎病毒的RNA是从 感染外体中的肝细胞，可将丙型肝炎病毒RNA转移到T细胞。自从丙型肝炎病毒 不会在T细胞中复制，目前尚不清楚为什么病毒RNA会转移到这些细胞中。我们 发现丙型肝炎病毒基因组RNA被加工成病毒短RNA序列(VsRNA)， 减少蛋白酪氨酸磷酸酶E(PTPRE)的表达。PTPRE击倒 抑制淋巴细胞特异性Src激酶(LCK)的磷酸化和激活。 突变丙型肝炎病毒vsRNA序列恢复了LCK磷酸化和随后的TCR 刺激。靶向不同T细胞受体(CXCR4)恢复的TCR的丙型肝炎病毒RNA 信号转导和PTPRE蛋白表达，而CXCR4表达降低。因此，丙型肝炎病毒 似乎利用了细胞的microRNA机制将其基因组处理成vsRNA 减少PTPRE的表达并抑制TCR介导的信号转导。根据我们最近的调查 研究中，我们假设，在淋巴细胞和肝细胞中，丙型肝炎病毒与sRNAs靶标 影响T细胞功能和病毒复制的多种基因的表达，从而 提供关于免疫逃避的新见解，抗病毒治疗的潜在目标，以及(自 它们阻断T细胞的反应性)更好的疫苗策略。由于主要的知识差距 关于丙型肝炎病毒vsRNA，我们在三个具体目标上探索关键问题。首先，我们 将表征以PTPRE为靶点的丙型肝炎病毒RNA结构，并检查 介导vsRNA的生物发生。我们假设丙型肝炎病毒vsRNA使用非规范的miRNA。 产生vsRNA的机制。其次，我们将通过以下几个方面来刻画这个机制(S) 哪些丙型肝炎病毒vsRNA被传递到T细胞，潜在地识别新的治疗方法 目标。我们推测，转移可能涉及通过以下途径释放到血浆中的外体 感染的肝细胞。最后，我们将演示丙型肝炎病毒与srna的相关性。 此前在人类丙型肝炎病毒感染中发现。我们的初步数据显示PTPRE和 TCR信号在丙型肝炎病毒感染期间减少，并在治疗后恢复。 PTPRE降低的程度与丙型肝炎病毒vsRNA序列直接相关。越高的 与患者分离株中存在的PTPRE 3‘UTR的互补性百分比越大， PTPRE表达降低。在提案中，我们应该证明丙型肝炎病毒vS-RNA调节T细胞 很可能是体内肝细胞的功能。这些信息对于理解 丙型肝炎病毒的发病机制，并强调一个新的机制，可以作为治疗和治疗的靶点 预防这种全球病毒病原体，以及潜在的许多其他病原体。

Hepatitis C virus (HCV) infection causes liver fibrosis, cirrhosis, and hepatocellular carcinoma, leading to >350,000 deaths annually globally, and is the most common etiology leading to liver transplantation in the U.S. An estimated 174,000 veterans have HCV infection, and the VA is the largest provider of HCV care. Although there are new and exciting HCV treatments, due to resistance and access issues, therapy is not likely to eradicate HCV infection completely. Thus, a vaccine is needed. In addition, people with HCV infection may continue to develop hepatocellular carcinoma. In persistent HCV infection, proliferation and activation of virus-specific T cells is delayed and inefficient, and immune responses to HBV vaccination and bacterial and schistosomal infections are repressed. This suggests that HCV infection suppresses T cell function, although the mechanism for this is unknown. Recent data showed that HCV RNA is released from hepatocytes in infectious exosomes that can transfer HCV RNA to T cells. Since HCV does not replicate in T cells, it is unclear why viral RNA is transferred to these cells. We found that HCV genomic RNA is processed into a viral short RNA sequence (vsRNA) that reduces expression of protein tyrosine phosphatase type E (PTPRE). PTPRE knockdown inhibits phosphorylation and activation of the lymphocyte-specific Src kinase (LCK). Mutating the HCV vsRNA sequence restored LCK phosphorylation and subsequent TCR stimulation. Targeting HCV RNA to a different T cell receptor (CXCR4) restored TCR signaling and PTPRE protein expression, but reduced expression of CXCR4. Thus, HCV appears to exploit the cellular microRNA machinery to process its genome into vsRNAs that reduce PTPRE expression and inhibit TCR-mediated signaling. Based on our recent studies, we hypothesize that, in lymphocytes and hepatocytes, HCV vsRNAs target expression of multiple genes that influence T cell function and viral replication, and thus offer new insights into immune evasion, potential targets for antiviral therapies, and (since they block T-cell responsiveness) better vaccine strategies. Since major knowledge gaps remain concerning HCV vsRNAs we explore key questions in three specific aims. First, we will characterize HCV RNA structures that target PTPRE and examine cellular factors that mediate vsRNA biogenesis. We hypothesize that HCV vsRNA uses non-canonical miRNA mechanisms to generate the vsRNA. Secondly, we will characterize the mechanism(s) by which HCV vsRNAs are delivered to T cells, potentially identifying novel therapeutic targets. We hypothesize that transfer may involve exosomes released into plasma by infected hepatocytes. Finally, we will demonstrate the relevance of the HCV vsRNA we previously identified in human HCV infection. Our preliminary data show that PTPRE and TCR signaling are reduced during HCV infection, and restored following curative therapy. The level of PTPRE reduction correlated directly with the HCV vsRNA sequence. The higher the percent complementarity with the PTPRE 3'UTR present in the patient's isolate, the greater the reduction in PTPRE expression. In the proposal we should show that HCV vs-RNA regulates T cell and likely hepatocyte function in vivo. This information will be critical for understanding the pathogenesis of HCV and highlight a novel mechanism that could be targeted for the treatment and prevention of this global viral pathogen, and potentially numerous other pathogens.