A hepatitis C virus cis-acting replication element forms a long-range RNA-RNA interaction with upstream RNA sequences in NS5B.

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.
Journal of Virology (Impact Factor: 5.08). 08/2008; 82(18):9008-22. DOI: 10.1128/JVI.02326-07
Source: PubMed

ABSTRACT The genome of hepatitis C virus (HCV) contains cis-acting replication elements (CREs) comprised of RNA stem-loop structures located in both the 5' and 3' noncoding regions (5' and 3' NCRs) and in the NS5B coding sequence. Through the application of several algorithmically independent bioinformatic methods to detect phylogenetically conserved, thermodynamically favored RNA secondary structures, we demonstrate a long-range interaction between sequences in the previously described CRE (5BSL3.2, now SL9266) with a previously predicted unpaired sequence located 3' to SL9033, approximately 200 nucleotides upstream. Extensive reverse genetic analysis both supports this prediction and demonstrates a functional requirement in genome replication. By mutagenesis of the Con-1 replicon, we show that disruption of this alternative pairing inhibited replication, a phenotype that could be restored to wild-type levels through the introduction of compensating mutations in the upstream region. Substitution of the CRE with the analogous region of different genotypes of HCV produced replicons with phenotypes consistent with the hypothesis that both local and long-range interactions are critical for a fundamental aspect of genome replication. This report further extends the known interactions of the SL9266 CRE, which has also been shown to form a "kissing loop" interaction with the 3' NCR (P. Friebe, J. Boudet, J. P. Simorre, and R. Bartenschlager, J. Virol. 79:380-392, 2005), and suggests that cooperative long-range binding with both 5' and 3' sequences stabilizes the CRE at the core of a complex pseudoknot. Alternatively, if the long-range interactions were mutually exclusive, the SL9266 CRE may function as a molecular switch controlling a critical aspect of HCV genome replication.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Hepatitis C virus (HCV) is a small (approximately 55 to 65 nm), spherical, enveloped, hepatotropic RNA virus that causes acute and chronic hepatitis in humans. Persistent virus infection with HCV often leads to cirrhosis and hepatocellular carcinoma (HCC). At present there is neither a selective antiviral therapy nor a preventive vaccine. The only available treatment option is a long-acting pegylated-interferon-alpha, given in combination with nucleoside analog ribavirin, which is not very effective. Molecular studies of HCV began with the successful cloning of its genome in 1989. For many years, research to develop therapeutics was stalled by the inability to grow virus in tissue culture. A major milestone was achieved with the recent development of a robust cell culture system for HCV propagation. HCV proteins assemble and form replication complexes on modified host membranes, called as membranous webs. Even though HCV is detected and targeted by host immune mechanisms, it establishes and maintains a life-long persistent infection. HCV has evolved multiple strategies to survive and persist in hostile cellular environments; and the viral population is known to rapidly change during the course of a natural infection thereby escaping immune surveillance. Rapid mutations also help virus to survive by selecting for the variants which are resistant to antiviral drugs. Although precise mechanisms regulating HCV entry into hepatic cells via receptors remain unknown, HCV also has the capability of direct cell-to-cell transmission. The extremely complex and incompletely understood nature of the HCV lifecycle has complicated the discovery of new therapies. A complete understanding of the functional roles played by the HCV proteins during HCV lifecycle is vital for developing a successful cure. This review deals with current status of efforts in addressing these daunting tasks and challenges in developing therapeutics against chronic and rapidly changing hepatitis C virus.
    The Indian Journal of Medical Research 01/2010; 131:17-34. · 2.06 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: BACKGROUND: Hepatitis C virus (HCV) infection is widespread, abhorrently under-diagnosed, and radically under-treated. Globally, infection with HCV is a major cause of acute hepatitis and chronic liver disease. Therefore, novel HCV inhibitors are required for the treatment of the HCV infected patients. OBJECTIVE AND PERSPECTIVES: This review gives the detailed knowledge of upcoming therapy such as NS5B polymerase inhibitors that are urgently needed. CONCLUSION: In the past decade, intensive hard work has focused on the discovery of both structural and nonstructural inhibitors of the HCV NS5B polymerase. These demanding efforts have resulted in various promising agents advancing in clinical development with emphasis on clinical efficacy and impact for future combination studies.
    European review for medical and pharmacological sciences 05/2012; 16(5):667-71. · 1.09 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The complex structures that RNA molecules fold into play important roles in their ability to perform various functions in the cell. The structure and composition of viral RNA influences the ability of the virus to implement the various stages of the viral lifecycle and can influence the severity of the virus effects on the host. Although many individual secondary structures and some tertiary interactions of the Hepatitis C virus genome have previously been identified, the global 3D architecture of the full 9678 nucleotide genome still remains uncertain. One promising technique for the determination of the overall 3D structure of large RNA molecules is nanoimaging with Atomic Force Microscopy. In order to get an idea of the structure of the HCV genome, we imaged the RNA prepared in the presence of Mg2+, which allowed us to observe the compact folded tertiary structure of the viral genome. In addition, to identify individual structural elements of the genome, we imaged the RNA prepared in the absence of Mg2+, which allowed us to visualize the unfolded secondary structure of the genome. We were able to identify a recurring single stranded region of the genome in many of the RNA molecules which was about 58 nm long. This method opens up a whole new avenue for the study of the secondary and tertiary structure of long RNA molecules. This ability to ascertain RNA structure can aid in drawing associations between the structure and the function of the RNA in cells which is vital to the development of potential antiviral therapies.
    Journal of Nanomedicine & Nanotechnology 02/2014; S5(010):1-7. · 5.72 Impact Factor

Full-text (2 Sources)

Available from
May 19, 2014