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Is single-strand conformation polymorphism analysis of the full 5' untranslated region an adequate approach to study hepatitis C virus quasispecies distribution?

ABSTRACT: Single-strand conformation polymorphism (SSCP) analysis is used by many laboratories to study the quasispecies distribution of the hepatitis C virus (HCV). Here we question the validity of this experimental approach, as conclusions are drawn from the analysis of the migration patterns of two ssDNA molecules and not from RNA. Using previously characterized mutants of the HCV 5' untranslated regions, we show that contrary to what has been predicted, SSCP migration patterns of DNA amplicons with differences in their nucleotide sequences generated from the full 5' UTR of HCV are not necessarily unique.

SUBMITTER: Vera-Otarola J

PROVIDER: S-EPMC2738205 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Is single-strand conformation polymorphism analysis of the full 5' untranslated region an adequate approach to study hepatitis C virus quasispecies distribution?

Vera-Otarola Jorge J Barría María Inés MI León Ursula U Carvallo Pilar P Soza Alejandro A López-Lastra Marcelo M

Journal of virology 20090624 17

Single-strand conformation polymorphism (SSCP) analysis is used by many laboratories to study the quasispecies distribution of the hepatitis C virus (HCV). Here we question the validity of this experimental approach, as conclusions are drawn from the analysis of the migration patterns of two ssDNA molecules and not from RNA. Using previously characterized mutants of the HCV 5' untranslated regions, we show that contrary to what has been predicted, SSCP migration patterns of DNA amplicons with di ...[more]

PMID: 19553315

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Project description:The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. In this study, the relationship between NS5A mutations and selection pressures before and during antiviral therapy and virologic response to therapy were investigated. Full-length NS5A clones were sequenced from 20 HCV genotype 1-infected patients in a prospective, randomized clinical trial of IFN induction (daily) therapy and IFN plus ribavirin combination therapy. Pretreatment NS5A nucleotide and amino acid phylogenies did not correlate with clinical IFN responses and domains involved in NS5A functions in vitro were all well conserved before and during treatment. A consensus IFN sensitivity-determining region (ISDR(237-276)) sequence associated with IFN resistance was not found, although the presence of Ala(245) within the ISDR was associated with nonresponse to treatment in genotype 1a-infected patients (P<0.01). There were more mutations in the 26 amino acids downstream of the ISDR required for PKR binding in pretreatment isolates from responders versus nonresponders in both HCV-1a- and HCV-1b-infected patients (P<0.05). In HCV-1a patients, more amino acid changes were observed in isolates from IFN-sensitive patients (P<0.001), and the mutations appeared to be concentrated in two variable regions in the C terminus of NS5A, that corresponded to the previously described V3 region and a new variable region, 310 to 330. Selection of pretreatment minor V3 quasispecies was observed within the first 2 to 6 weeks of therapy in responders but not nonresponders, whereas the ISDR and PKR binding domains did not change in either patient response group. These data suggest that host-mediated selective pressures act primarily on the C terminus of NS5A and that NS5A can perturb or evade the IFN-induced antiviral response using sequences outside of the putative ISDR. Mechanistic studies are needed to address the role of the C terminus of NS5A in HCV replication and antiviral resistance.

Project description:Mouse hepatitis virus (MHV), a member of the Coronaviridae, contains a polyadenylated positive-sense single-stranded genomic RNA which is 31 kb long. MHV replication and transcription take place via the synthesis of negative-strand RNA intermediates from a positive-strand genomic template. A cis-acting element previously identified in the 3' untranslated region binds to trans-acting host factors from mouse fibroblasts and forms at least three RNA-protein complexes. The largest RNA-protein complex formed by the cis-acting element and the lysate from uninfected mouse fibroblasts has a molecular weight of about 200 kDa. The complex observed in gel shift assays has been resolved by second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis into four proteins of approximately 90, 70, 58, and 40 kDa after RNase treatment. Specific RNA affinity chromatography also has revealed the presence of a 90-kDa protein associated with RNA containing the cis-acting element bound to magnetic beads. The 90-kDa protein has been purified from uninfected mouse fibroblast crude lysates. Protein microsequencing identified the 90-kDa protein as mitochondrial aconitase. Antibody raised against purified mitochondrial aconitase recognizes the RNA-protein complex and the 90-kDa protein, which can be released from the complex by RNase digestion. Furthermore, UV cross-linking studies indicate that highly purified mitochondrial aconitase binds specifically to the MHV 3' protein-binding element. Increasing the intracellular level of mitochondrial aconitase by iron supplementation resulted in increased RNA-binding activity in cell extracts and increased virus production as well as viral protein synthesis at early hours of infection. These results are particularly interesting in terms of identification of an RNA target for mitochondrial aconitase, which has a cytoplasmic homolog, cytoplasmic aconitase, also known as iron regulatory protein 1, a well-recognized RNA-binding protein. The binding properties of mitochondrial aconitase and the functional relevance of RNA binding appear to parallel those of cytoplasmic aconitase.

Dataset Information

Is single-strand conformation polymorphism analysis of the full 5' untranslated region an adequate approach to study hepatitis C virus quasispecies distribution?

Publications

Is single-strand conformation polymorphism analysis of the full 5' untranslated region an adequate approach to study hepatitis C virus quasispecies distribution?

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