Thursday, May 24, 2012

Analysis of Functional Differences between Hepatitis C Virus NS5A of Genotypes 1–7 in Infectious Cell Culture Systems

Analysis of Functional Differences between Hepatitis C Virus NS5A of Genotypes 1–7 in Infectious Cell Culture Systems

Troels K. H. Scheel, Jannick Prentoe, Thomas H. R. Carlsen, Lotte S. Mikkelsen, Judith M. Gottwein, Jens Bukh*

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre and Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark

Hepatitis C virus (HCV) is an important cause of chronic liver disease. Several highly diverse HCV genotypes exist with potential key functional differences. The HCV NS5A protein was associated with response to interferon (IFN)-α based therapy, and is a primary target of currently developed directly-acting antiviral compounds. NS5A is important for replication and virus production, but has not been studied for most HCV genotypes. We studied the function of NS5A using infectious NS5A genotype 1–7 cell culture systems, and through reverse genetics demonstrated a universal importance of the amphipathic alpha-helix, domain I and II and the low-complexity sequence (LCS) I for HCV replication; the replicon-enhancing LCSI mutation S225P attenuated all genotypes. Mutation of conserved prolines in LCSII led to minor reductions in virus production for the JFH1(genotype 2a) NS5A recombinant, but had greater effects on other isolates; replication was highly attenuated for ED43(4a) and QC69(7a) recombinants. Deletion of the conserved residues 414-428 in domain III reduced virus production for most recombinants but not JFH1(2a). Reduced virus production was linked to attenuated replication in all cases, but ED43(4a) and SA13(5a) also displayed impaired particle assembly. Compared to the original H77C(1a) NS5A recombinant, the changes in LCSII and domain III reduced the amounts of NS5A present. For H77C(1a) and TN(1a) NS5A recombinants, we observed a genetic linkage between NS5A and p7, since introduced changes in NS5A led to changes in p7 and vice versa. Finally, NS5A function depended on genotype-specific residues in domain I, as changing genotype 2a-specific residues to genotype 1a sequence and vice versa led to highly attenuated mutants. In conclusion, this study identified NS5A genetic elements essential for all major HCV genotypes in infectious cell culture systems. Genotype- or isolate- specific NS5A functional differences were identified, which will be important for understanding of HCV NS5A function and therapeutic targeting.

Author Summary
Hepatitis C virus (HCV) is a major public health burden and leads to chronic liver disease, including liver cirrhosis and liver cancer. Understanding the biological functions of the virus is crucial to the development of a vaccine and to improve current therapy through development of directly-acting antiviral compounds. The NS5A protein is a promising antiviral target, but much remains to be understood about its role in the viral life cycle. Great diversity among the seven major HCV genotypes poses challenges for broadly active inhibitors. Here we used infectious cell culture systems for NS5A of the seven major HCV genotypes, and demonstrated that all genotypes depended on the NS5A amphipathic alpha-helix, domain I, low-complexity sequence (LCS) I and domain II for viral replication. Interestingly, effects on replication and virus production by changes in LCSII and domain III varied greatly among NS5A isolates. Furthermore, we found that genotype 2 had evolved genotype-specific residues in domain I of importance for viral function. Thus, the highly diverse sequence of the NS5A protein reflected functional differences between HCV genotypes and isolates. Such differences will be important to consider in understanding HCV biology and for future development of antiviral compounds.

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