Mechanistic Studies of the Function of Desmosterol Homeostasis in HCV Replication

去莫甾醇稳态在 HCV 复制中的作用机制研究

• 批准号: 8652834
• 负责人: Valerie Anne Agustino Villareal
• 金额: $ 5.51万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-15 至 2016-03-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8652834
• 关键词: Adverse effects; Affect; Alternative Splicing; Anabolism; Antiviral Agents; Autoradiography; Biological Assay; Cells; Characteristics; Cholesterol; Chronic Hepatitis; Chronic Hepatitis C; Cirrhosis; Collaborations; Complex; Dactinomycin; Data; Desmosterol; Detergents; Endoplasmic Reticulum; Environment; Enzymes; Epidemic; Event; Fluorescence; Genes; Genome; Genotype; Goals; Hepatitis C virus; Homeostasis; Immunoblotting; Immunofluorescence Microscopy; Immunoprecipitation; In Vitro; Incubated; Integration Host Factors; Label; Lipids; Liver diseases; Mass Spectrum Analysis; Measurement; Measures; Messenger RNA; Methods; Microscopy; Molecular; Mutation; Nonstructural Protein; Northern Blotting; Organelles; Outcome; Pathway interactions; Peptide Hydrolases; Post-Translational Protein Processing; Primary carcinoma of the liver cells; Process; Proteins; RNA; RNA Viruses; RNA replication; Raman Spectrum Analysis; Reagent; Regulation; Replicon; Reporting; Research; Research Design; Resistance; Ribonucleases; Ribonucleotides; Site; Staging; Sterols; Therapeutic Agents; Transcript; Treatment Efficacy; Universities; Viral; Viral Proteins; Virus; Virus Inhibitors; Virus Replication; anti-hepatitis C; base; cholesterol biosynthesis; design; hepatoma cell; imaging modality; improved; in vitro Assay; inhibitor/antagonist; novel; public health relevance; replicase; research study; tool; traditional therapy; two-photon; vaccine development; viral RNA

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is an enveloped, plus-stranded RNA virus that chronically infects 180 million people, making them susceptible to cirrhosis, chronic hepatitis (liver disease), and hepatocellular carcinomas. Despite the HCV epidemic, challenges to the development of vaccines and broad-spectrum anti-HCV agents include the existence of multiple genotypes, low efficacy, and the characteristic high mutation rate of RNA viruses. While recently approved directly-acting viral inhibitors are expected to improve treatment efficacy, rapid resistance to these virally-targeted inhibitors has been reported, extending the need for novel antivirals for treatment of chronic HCV. Antivirals that target host factors required for HCV replication may provide an attractive alternative with several advantages over more traditional therapies that target viral factors, including higher barriers to resistance. Understanding the molecular mechanisms underlying productive HCV replication and how HCV manipulates cellular pathways is therefore critical to the design of host-targeted anti-HCV strategies. We recently discovered that HCV causes a specific, ten-fold increase in the abundance of intracellular desmosterol, the penultimate precursor of cholesterol in the Bloch branch of cholesterol biosynthesis, without perturbing the steady-state abundance of cholesterol or other sterols in the pathway. The HCV-induced perturbation of desmosterol homeostasis appears to be critical for HCV replication since inhibition of desmosterol synthesis severely inhibits HCV by decreasing steady-state HCV RNA replication. Here, we propose research designed to elucidate molecular mechanisms by which HCV causes the steady-state accumulation of desmosterol and to understand the functional significance of desmosterol in steady-state HCV genome replication. In Aim 1, we will use immunoprecipitation, immunofluorescence microscopy, and steady state mRNA and protein analyses to investigate whether HCV regulates cellular enzymes involved in late stage cholesterol biosynthesis. In Aim 2, we will use in vitro replication assays and steady state lipid and mRNA analyses to determine the functional significance of desmosterol in the formation, activity, or stability of HCV replicaton complexes (RCs). Last, since steady-state measurements of lipid abundance in cell lysates provide no information regarding the localization of desmosterol in the presence of HCV replication, in Aim 3, we will utilize stimulated Raman spectroscopy imaging methods to examine the subcellular localization of deuterated desmosterol in cells replicating HCV. In particular, we will investigate whether desmosterol accumulates in distinct puncta versus whether it is distributed throughout the cell or remains proximal to the ER. Collectively, these experiments provide unique opportunities to study and validate desmosterol biosynthesis as a potential anti-HCV target and to explore a heretofore uncharacterized mechanism of regulation in cholesterol biosynthesis.

描述（由申请人提供）：丙型肝炎病毒（HCV）是一种有包膜的正链RNA病毒，长期感染1.8亿人，使他们易患肝硬化、慢性肝炎（肝病）和肝细胞癌。尽管丙型肝炎病毒流行，但疫苗和广谱抗丙型肝炎病毒药物开发面临的挑战包括多种基因型的存在、低功效以及RNA病毒特有的高突变率。虽然最近批准的直接作用的病毒抑制剂有望提高治疗效果，但已报道对这些病毒靶向抑制剂的快速耐药性，从而扩大了对用于治疗慢性HCV的新型抗病毒药的需求。靶向HCV复制所需的宿主因子的抗病毒药物可能提供一种有吸引力的替代方案， 与靶向病毒因子的更传统疗法相比，它具有更大的优势，包括更高的耐药性屏障。因此，了解丙型肝炎病毒高效复制的分子机制以及丙型肝炎病毒如何操纵细胞途径对于设计靶向宿主的抗丙型肝炎病毒策略至关重要。我们最近发现，HCV导致细胞内桥甾醇丰度增加10倍，桥甾醇是胆固醇生物合成Bloch分支中胆固醇的倒数第二个前体，而不干扰途径中胆固醇或其他甾醇的稳态丰度。HCV诱导的桥甾醇稳态的扰动似乎对HCV复制至关重要，因为抑制桥甾醇合成通过降低稳态HCV RNA复制来严重抑制HCV。在这里，我们提出的研究旨在阐明HCV导致稳态积累的桥甾醇的分子机制，并了解稳态HCV基因组复制的桥甾醇的功能意义。在目标1中，我们将使用免疫沉淀，免疫荧光显微镜，稳态mRNA和蛋白质分析，以调查是否HCV调节细胞酶参与晚期胆固醇生物合成。在目标2中，我们将使用体外复制测定和稳态脂质和mRNA分析来确定桥甾醇在HCV复制复合物（RC）的形成、活性或稳定性中的功能意义。最后，由于细胞裂解物中脂质丰度的稳态测量没有提供关于在HCV复制存在下桥甾醇的定位的信息，在目的3中，我们将利用受激拉曼光谱成像方法来检查复制HCV的细胞中氘代桥甾醇的亚细胞定位。特别是，我们将调查是否桥甾醇积累在不同的斑点与它是否分布在整个细胞或仍然接近ER。总的来说，这些实验提供了独特的机会，研究和验证桥甾醇生物合成作为一个潜在的抗HCV的目标，并探索迄今为止未表征的调节胆固醇生物合成的机制。