High-Resolution Functional Profiling of Hepatitis C Virus Genome

被引:45
作者
Arumugaswami, Vaithilingaraja [1 ]
Remenyi, Roland [1 ]
Kanagavel, Vidhya [1 ]
Sue, Eric Yiang [1 ]
Ho, Tuyet Ngoc [1 ]
Liu, Chang [1 ]
Fontanes, Vanessa [2 ]
Dasgupta, Asim [2 ,3 ,4 ,5 ]
Sun, Ren [1 ,3 ,4 ]
机构
[1] Univ Calif Los Angeles, David Geffen Sch Med, Dept Mol & Med Pharmacol, Los Angeles, CA 90095 USA
[2] Univ Calif Los Angeles, David Geffen Sch Med, Dept Microbiol Immunol & Mol Genet, Los Angeles, CA 90095 USA
[3] Univ Calif Los Angeles, AIDS Inst, Los Angeles, CA 90024 USA
[4] Univ Calif Los Angeles, Jonsson Comprehens Canc Ctr, Los Angeles, CA 90024 USA
[5] Univ Calif Los Angeles, Inst Mol Biol, Los Angeles, CA 90024 USA
关键词
D O I
10.1371/journal.ppat.1000182
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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.
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页数:16
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