High-resolution functional profiling of hepatitis C virus genome.
High-resolution functional profiling of hepatitis C virus genome.
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DOI：
10.1371/journal.ppat.1000182
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发表时间：
2008-10
期刊：
影响因子：
6.7
通讯作者：
Sun R
中科院分区：
文献类型：
--
作者：
Arumugaswami V;Remenyi R;Kanagavel V;Sue EY;Ngoc Ho T;Liu C;Fontanes V;Dasgupta A;Sun R
丙型肝炎病毒是世界范围内人类肝脏疾病的主要原因。最近发现的JFH-1分离物能够感染细胞培养物，为研究HCV复制开辟了新的途径。我们描述了一个高通量，定量，基因组规模，突变分析系统的发展，以研究HCV顺式元件和蛋白质结构域是病毒复制所必需的。在细胞培养中传代具有15个核苷酸随机插入的HCV文库，通过插入特异性荧光pcr分析检查每个基因组位置插入的影响。在基因组的9517个核苷酸中鉴定的2399个插入中，分别有374,111和1914个对病毒复制具有耐受性、减毒性和致死性。除了发现新的功能域外，该方法还确认了与以往研究一致的其他功能域。通过测试几个单独的突变病毒，结果得到了验证。此外，对3 '非翻译可变区域的分析揭示了病毒复制中的间隔器作用，证明了这种方法在功能发现中的实用性。HCV结构域的高分辨率功能分析为进一步的机制研究奠定了基础，并为设计候选疫苗提供了新的治疗靶点和拓扑信息。丙型肝炎病毒（HCV）是引起致命肝脏疾病的主要人类健康问题。目前还没有预防丙型肝炎病毒感染的疫苗。虽然丙型肝炎病毒早在20年前就被发现，但直到最近才在细胞培养条件下成功培养。HCV蛋白和调控元件子结构域在病毒生长过程中的作用尚不清楚。我们开发了一种突变分析方法，以高分辨率识别HCV子结构域的功能。一组含有15个核苷酸随机插入的HCV突变体在细胞培养中进行生长测试。利用毛细管基因分型技术和生物信息学技术分析了插入物的精确位置及其对病毒生长的影响。在总共鉴定的2399个HCV突变体中，374个突变体生长正常，111个突变体表现出生长减少，1914个突变体在细胞培养中不能生长。这种突变分析方法通过测试许多单个突变病毒得到了验证。本研究确定了病毒生长所需的几个HCV功能亚域，提出了新的治疗靶点。经鉴定具有生长降低特性的HCV突变病毒可用于设计候选疫苗。
Hepatitis C virus is a leading cause of human liver disease worldwide. Recent discovery of the JFH-1 isolate, capable of infecting cell culture, opens new avenues for studying HCV replication. We describe the development of a high-throughput, quantitative, genome-scale, mutational analysis system to study the HCV cis-elements and protein domains that are essential for virus replication. An HCV library with 15-nucleotide random insertions was passaged in cell culture to examine the effect of insertions at each genome location by insertion-specific fluorescent-PCR profiling. Of 2399 insertions identified in 9517 nucleotides of the genome, 374, 111, and 1914 were tolerated, attenuating, and lethal, respectively, for virus replication. Besides identifying novel functional domains, this approach confirmed other functional domains consistent with previous studies. The results were validated by testing several individual mutant viruses. Furthermore, analysis of the 3′ non-translated variable region revealed a spacer role in virus replication, demonstrating the utility of this approach for functional discovery. The high-resolution functional profiling of HCV domains lays the foundation for further mechanistic studies and presents new therapeutic targets as well as topological information for designing vaccine candidates. Hepatitis C virus (HCV) is a major human health concern that causes fatal liver diseases. Currently no vaccine is available to prevent HCV infection. Though the HCV was identified two decades ago, the virus has only recently been successfully grown in cell culture conditions. The role of HCV protein and regulatory element sub-domains during virus growth is poorly understood. We have developed a mutational analysis method to identify the function of HCV sub-domains at a high resolution. A collection of HCV mutants containing 15-nucleotide random insertions was tested for growth in cell culture. The precise location of the insertions and their effects on virus growth were analyzed by capillary genotyping technology and bioinformatics. Out of the total 2399 HCV mutants identified, 374 mutants grew normally, 111 mutants demonstrated reduced growth, and 1914 mutants failed to grow in cell culture. This mutational analysis method was validated by testing many individual mutant viruses. The present study identified several HCV functional sub-domains required for virus growth, presenting novel therapeutic targets. The HCV mutant viruses identified with the property of reduced growth can be used for designing vaccine candidates.
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