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Functional interactions of structural and NS proteins of hepatitis c virus

thesis
posted on 22.03.2017, 01:41 by Gouklani, Hamed
Hepatitis C virus (HCV) is a small enveloped virus with a positive-sense single stranded RNA genome. Based on its molecular genetic characteristics, the virus has been classified into the Hepacivirus genus of the family Flaviviridae. HCV is one of the major causes of chronic hepatitis which can lead to liver cirrhosis and hepatocellular carcinoma. According to the recent WHO published data, 123 million individuals are infected with HCV (approximately 3% of the world’s population) throughout the world. Due to its highly variable nature, HCV is classified into six major genotypes. The HCV genome encodes a single polyprotein that is cleaved to yield at least 10 mature proteins (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). The recently developed HCV cell culture system, based on the JFH1 strain of HCV, has provided an opportunity to study the role of the viral proteins in the complete HCV replication cycle in human hepatoma cells. How the viral proteins functionally interact during replication of HCV in cell culture is not completely understood. Passage of cell cultures transfected with HCV genomic RNA containing attenuating mutations allows for the selection of genomes with second site compensatory mutations that restore replication to wild type levels. Using this approach, the functional interactions of p7 and E2 with other viral proteins during HCV replication was investigated. A small protein of 63 amino acids, p7 is encoded at the junction of the structural and non-strucutural region. p7 is a highly hydrophobic, integral membrane protein and is classified in the viroporin family. In this thesis, it is shown that p7 is critical for production of viral particles and is implicated in a late step of particle assembly. Since the protein plays a critical role in the virus life cycle, chemical compounds that block p7 function are potential candidates for anti-viral therapy. In this thesis, a chimeric JFH1 genome that encodes the p7 protein of genotype (GT) 1b strain J4 was generated. The intergenotypic chimeric genome was nonviable in human hepatoma cells and infectious chimeric virions were only produced after cells harboring the chimeric genomes were passaged several times. To investigate the emergence of compensatory mutations in the viral proteins during cell passaging, the consensus sequences of the entire polyprotein coding regions of the wild type JFH1 and three chimeric viruses were determined. Sequence analysis revealed mutations in core, NS2, NS5A and NS5B. Reverse genetic analysis demonstrated that any one of the single mutations restored the infectivity of the defective chimeric genomes. These data suggest that there are critical genetic interactions between p7 with core, NS2, NS5A and NS5B. In addition, a stable physical interaction between p7 and NS2 is shown in a transient expression system. The HCV glycoproteins E1 and E2 are present on the surface of virions as a heterodimer that attach virions to host cell receptors and facilitate virus fusion and entry. HCV entry proceeds via attachment to glycosaminoglycans followed by binding to scavenger receptor type B class I, and the tetraspanin CD81. Recently, claudin-1 and occludin have emerged as additional receptors required for entry. E2 has a receptor binding domain (E2661RBD) that conatins three variable regions, hypervariable regions 1 (HVR1), HVR2 and the intergenotypic variable region (igVR). In this thesis, HVR1 of E2 was deleted in the context of full-length replication comptetent HCV. Deletion of HVR1 increases CD81-binding ability of the mutant and also increases its susceptibility to neutralizing antibody MAb 24. The infectivity of the HVR1 deleted virions was attenuated approximately 10-fold prior to accumulation of compensatory mutations. Sequencing of cDNA obtained from reverted virions revealed mutations in E1 (I262L) and E2 (N415D). Reverse genetic studies revealed that I262L improved the infectivity of HVR1 deleted virions 2.5 fold while N415D restored infectivity to wild type levels. These data suggest that mutations within E1 or E2 can compensate for the reduction in infectivity observed for HVR1 deleted viruses. In summary, this thesis demonstrates the importance of functional interactions between HCV proteins during virus morphogenesis and infectivity.

History

Principal supervisor

Heidi Drummer

Year of Award

2011

Department, School or Centre

Microbiology

Campus location

Australia

Course

Doctor of Philosophy

Degree Type

DOCTORATE

Faculty

Faculty of Medicine Nursing and Health Sciences