Functional Profiles of Hepatitis C Virus Genome at Single Nucleotide Resolution

HCV已成为一个主要的人类健康问题，估计全世界有1.7亿人持续感染。HCV感染引起肝脏疾病，包括慢性肝炎、肝硬化和肝细胞癌，特别是在HIV感染者中。病毒性肝炎在艾滋病患者中进展更快，更具破坏性，已成为非艾滋病相关死亡的主要原因。旨在评估HCV根除对HCC发病率影响的研究表明，成功根除HCV可降低肝功能衰竭和HCC发展的风险。然而，无论是预防性还是治疗性疫苗都不能用于HCV感染。在最近批准这两种蛋白酶抑制剂之前，标准治疗是与干扰素（IFN-？）和利巴韦林，其仅提供有限的响应率。因此，有必要了解HCV对IFN的抗病毒功能产生耐药性的机制。已知I型干扰素（IFN）应答可以保护哺乳动物宿主免受病毒感染。然而，病毒已经进化到利用不同的策略来逃避多层免疫应答。因此，尽管诱导IFN应答，HCV仍在宿主中持续存在，从而约50%的HCV患者对IFN-？治疗为了研究HCV拮抗IFN应答的机制，拟议的研究将系统地绘制HCV序列，这对于使用高通量，定量，基因组规模的分析平台以单个氨基酸分辨率进行抗IFN功能至关重要。该方法背后的概念是随机诱变病毒基因组上的每个碱基对，在存在或不存在IFN-？的情况下选择突变体文库，并进行大规模平行测序以确定哪些突变在选择中减少，这可能是由于它们丧失了抗IFN功能。本研究的具体目的如下：（1）建立 （2）用IFN-？鉴定对抗IFN应答的关键病毒序列;（3） 表征单个突变体并验证HCV与IFN应答之间的相互作用。该研究有望以系统和无偏倚的方式绘制整个HCV基因组中负责其对IFN治疗耐药的病毒序列。这些序列的鉴定将提供知识，以了解为什么HCV可以逃避IFN应答并建立对治疗的抗性的原因。此外，这项研究将为疫苗开发提供深入的知识。在不存在IFN处理的情况下选择突变体文库还将揭示具有高遗传突变屏障的序列段，这将作为疫苗开发的新靶标。因此，它将提供新的抗病毒治疗策略，以克服病毒耐药性。

DESCRIPTION: HCV has emerged as a major human health concern with an estimate of 170 million people who are persistently infected worldwide. HCV infections cause liver diseases including chronic hepatitis, cirrhosis, and hepatocellular carcinoma, especially in HIV- infected persons. Viral hepatitis progresses faster and more devastating among AIDS patients, which has become the leading cause of non AIDS-related deaths. Studies aiming to evaluate the effect of HCV elimination on the incidence of HCC indicate that successful eradication of HCV can reduce the risk for failure of liver function and HCC development. However, neither prophylactic nor therapeutic vaccine is available for HCV infection. Before the recent approval of the two protease inhibitors, the standard treatment has been combination therapy with interferon (IFN-?) and ribavirin which only offers limited response rate. Therefore, there is a need for understanding the mechanisms by which HCV develops resistance to the antiviral function of IFN. It's known that type I interferon (IFN) response can defend mammalian host from virus infection. However, viruses have evolved to utilize different strategies to evade from multiple layers of immune response. As a result, HCV persist in the host despite induction of IFN response and thereby approximately 50% of HCV patients are resistant to IFN-? treatment. To study the mechanisms by which HCV antagonizes the IFN response, the proposed study will systematically map HCV sequences, which is critical for anti-IFN function at single amino acid resolution with a high-throughput, quantitative, genome-scale profiling platform. The concept behind this approach is to randomly mutagenize every base pair on the virus genome, select for the mutant library in the presence or absence of IFN-?, and perform massively parallel sequencing to determine which mutations are diminished in the selection, which is presumably due to their loss of anti-IFN function. The research specific aims are the following: (1) Establish a single nucleotide mutant HCV library with high complexity; (2) Screen the library with treatment of IFN-? to identify the viral sequences critical for counteracting the IFN response; (3) Characterize individual mutants and verify the interaction between HCV with IFN response. The study is expected to map the viral sequences in the entire HCV genome responsible for its resistance to IFN therapy in a systemic and unbiased manner. Identification of these sequences will provide knowledge in understanding the reasons why HCV can evade the IFN response and establish resistance to the treatment. Additionally, this study will provide a depth of knowledge for vaccine development. Selection of the mutant library in the absence of IFN treatment will also reveal the sequence stretches with high genetic barrier to mutate, which will serve as novel targets for vaccine development. Therefore, it will offer novel antiviral treatment strategies to overcome viral resistance.