The role of HCV core protein in the regulation of HCV replication

Li, Dongsheng (2004). The role of HCV core protein in the regulation of HCV replication PhD Thesis, School of Molecular and Microbial Sciences, The University of Queensland.

       
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Author Li, Dongsheng
Thesis Title The role of HCV core protein in the regulation of HCV replication
School, Centre or Institute School of Molecular and Microbial Sciences
Institution The University of Queensland
Publication date 2004
Thesis type PhD Thesis
Supervisor Professor Eric Gowans
Professor John Mackenzie
Total pages 1v
Collection year 2004
Language eng
Subjects L
270303 Virology
780105 Biological sciences
Formatted abstract

The hepatitis C virus (HCV) is an hepatotropic RNA virus which is classified as the sole member of the Hepacivirus in the family Flaviviridae. HCV has a glycoprotein envelope and a capsid containing a plus-strand RNA. The genomic RNA contains a single open reading frame, encoding a polyprotein, flanked by untranslated regions (UTR) at the 5' and 3' ends. The 5' UTR functions as an intemal ribosome entry site (IRES) for the initiation of polyprotein translation that is processed into the individual structural and non-structural proteins.          

Infection with HCV leads to persistent infection in ~80% of infected individuals, with a subsequent risk of cirrhosis and hepatocellular carcinoma (HCC). The reason for the frequency of persistent infection is unclear. The development of antiviral compounds and preventative vaccines are hampered by the lack of an effective cell culture system and a convenient animal model.            

One of the significant features of HCV infection is that the replication of the virus is extremely poor. This may contribute to establish the persistent infection. It has been reported that the HCV IRES is efficient and that the low level replication might result from a self-modulation mechanism. However, current literature on this subject is highly contradictory and consequently, this study aimed to clarify the hypothesis that the HCV core protein regulates HCV IRES-directed translation.               

In vitro (cell free) and in vivo (in cells) translation regulation systems were developed to examine the effect of the HCV core protein on HCV IRES-dependent translation. Compared with the mutated core protein, the wild type-core protein was shown to inhibit both IRES- and cap-directed translation in a dose-dependent manner in a rabbit reticulocyte lysate (RRL) translation system. The HCV core protein expressed by a HCV core-recombinant vaccinia virus was shown to inhibit translation from capdependent and IRES elements derived from HCV, encephalomyocarditis virus (EMCV) and classical swine fever virus (CSFV) in CV-1 and HuH7 cells, but only inhibited HCV IRES function in HepG2 cells. The functional domain for translation inhibition was mapped to aa 1-20 of the HCV core protein and a synthetic peptide representing this sequence resulted in a dose-dependent translation inhibition in RRL. The peptide also caused an IRES RNA mobility shift in a peptide-RNA binding experiment. This peptide and another peptide identified from Ross River virus (RRV) core protein were also shown to inhibit replication of an HCV replicon.                

To investigate if the HCV IRES domain IV is responsible for translation regulation, two chimeric IRES elements were constructed to contain the reciprocal domain IV in the otherwise HCV and CFSV IRES elements. The HCV core protein specifically inhibited HCV IRES-directed translation in HepG2 cells, but had no effect on translation directed by the CSFV-, GBV-B- and the chimeric-IRES elements which contained the CSFV IRES domain I-III and the HCV IRES domain IV. In contrast, another specific inhibitor of the HCV IRES, vitamin B12, was shown to inhibit translation directed by all IRES elements which contained domain IV from the HCV IRES, and the GBV-B IRES. Thus the mechanism of translation inhibition by vitamin B12 and the core protein differ, and target different regions of the IRES.

This thesis demonstrated a mechanism to control the level of HCV replication by the core protein. The extremely low level of viral antigen may lead to immune tolerance and contribute to the establishment of persistent infection.  

Keyword Hepatitis C virus
RNA
Flaviviruses

 
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Created: Fri, 24 Aug 2007, 18:36:07 EST