Novel viral immune interference mechanisms: HCV as a model system

新型病毒免疫干扰机制：HCV 作为模型系统

• 批准号: 9898211
• 负责人: Jack T. Stapleton
• 金额: --
• 依托单位: IOWA CITY VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2021-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9898211
• 关键词: 3' Untranslated Regions; Antiviral Therapy; Biogenesis; Biological Models; CXCR4 gene; Caring; Cell physiology; Cells; Cessation of life; Cirrhosis; DNA Viruses; Data; E protein; Epidemic; Etiology; Event; Gene Expression; Gene Expression Regulation; Genes; Genome; Healthcare Systems; Hepatitis B Vaccination; Hepatitis C; Hepatitis C Therapy; Hepatitis C virus; Hepatocyte; Human; Immune; Immune Evasion; Immune response; In Vitro; Infection; Interleukin-2; Knowledge; Learning; Life Cycle Stages; Liver Fibrosis; Liver diseases; Lymphocyte; Lymphocyte Activation; Lymphocyte Function; Mediating; MicroRNAs; Mutate; Pathogenesis; Patient Isolators; Phosphorylation; Plasma; Prevention; Primary carcinoma of the liver cells; Process; Protein Tyrosine Phosphatase; Provider; RNA; RNA Sequences; RNA Viruses; Regulation; Resistance; Retroviridae; Signal Transduction; Signaling Protein; Structure; Surface; Syphilis; T-Cell Receptor; T-Lymphocyte; Untranslated RNA; Vaccines; Veterans; Viral; Viral Physiology; Virion; Virus Activation; Virus Replication; base; chronic infection; curative treatments; exosome; in vivo; insight; knock-down; liver transplantation; new therapeutic target; novel; novel strategies; novel therapeutics; particle; pathogen; pathogenic virus; protein expression; src-Family Kinases; targeted treatment; vaccine development; viral detection; viral genomics

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.

丙型肝炎病毒（HCV）感染可导致肝纤维化、肝硬化和肝细胞癌。 癌症，全球每年导致> 350，000例死亡，并且是最常见的病因 据估计，174，000名退伍军人患有HCV 感染，VA是HCV护理的最大提供者。虽然有新的和 令人兴奋的HCV治疗，由于耐药性和获取问题，治疗不太可能 彻底根除HCV感染。因此，需要疫苗。此外， HCV感染可继续发展为肝细胞癌。在持续性HCV 病毒特异性T细胞的感染、增殖和活化延迟且效率低下， 对HBV疫苗接种以及细菌和染色体感染的免疫应答是 压抑这表明HCV感染抑制了T细胞功能，尽管 其机制尚不清楚。最近的数据表明，HCV RNA是从 感染性外泌体中的肝细胞可以将HCV RNA转移到T细胞。由于HCV 虽然病毒RNA不能在T细胞中复制，但目前还不清楚为什么病毒RNA会转移到这些细胞中。我们 发现HCV基因组RNA被加工成病毒短RNA序列（vsRNA）， 降低蛋白酪氨酸磷酸酶E型（PTPRE）的表达。PTPRE敲低 抑制淋巴细胞特异性Src激酶（LCK）的磷酸化和活化。 突变HCV vsRNA序列恢复了LCK磷酸化和随后的TCR 刺激.将HCV RNA靶向不同的T细胞受体（CXCR 4）恢复TCR 信号传导和PTPRE蛋白表达，但减少CXCR 4的表达。因此，HCV 似乎利用细胞microRNA机制将其基因组加工成vsRNA 其降低PTPRE表达并抑制TCR介导的信号传导。根据我们最近的 研究中，我们假设，在淋巴细胞和肝细胞中，HCV vsRNA靶向 影响T细胞功能和病毒复制的多个基因的表达， 提供了新的见解免疫逃避，抗病毒治疗的潜在目标，并（因为 它们阻断了T细胞的反应性）更好的疫苗策略。由于重大的知识差距 关于HCV vsRNA，我们在三个具体目标中探索关键问题。一是 将表征靶向PTPRE的HCV RNA结构，并检查细胞因子， 介导vsRNA生物合成。我们假设HCV vsRNA使用非典型的miRNA 产生vsRNA的机制。其次，我们将通过以下方式描述该机制的特征： 其中HCV vsRNA被递送到T细胞，潜在地鉴定新的治疗方法， 目标的我们假设转移可能涉及外泌体释放到血浆中， 感染的肝细胞最后，我们将证明HCV vsRNA的相关性， 以前在人类HCV感染中发现。我们的初步数据显示，PTPRE和 TCR信号在HCV感染期间减少，并在治愈性治疗后恢复。 PTPRE降低水平与HCV vsRNA序列直接相关。越高 与患者分离株中存在的PTPRE 3 'UTR的互补性百分比越高， PTPRE表达减少。在这项提案中，我们应该证明HCV vs-RNA调节T细胞， 和体内肝细胞功能。这些信息对于理解 HCV的发病机制，并强调了一种新的机制，可以有针对性的治疗， 预防这种全球性的病毒病原体，以及潜在的许多其他病原体。