Mutations of the SL2 dimerization sequence of the hepatitis C genome abrogate viral replication (original) (raw)

Abstract

Stem-loop SL2 is a self-interacting palindromic sequence that has been identified within the hepatitis C virus genome (HCV). While, RNA dimerization of the HCV genome has been observed in vitro with short RNA sequences, the role of a putative RNA dimerization during viral replication has not been elucidated. To determine the effect of genomic dimerization on viral replication, we introduced mutations into SL2 predicted to disrupt genomic dimerization. Using surface plasmon resonance, we show that mutations within the SL2 bulge impact dimerization in vitro. Transfection of Huh7 cells with luciferase-encoding full-length genomes containing SL2 mutations abolishes viral replication. Luciferase expression indicates that viral translation is not or slightly affected and that the viral RNA is properly encapsidated. However, RT-qPCR analysis demonstrates that viral RNA synthesis is drastically decreased. In vitro synthesis experiments using the viral recombinant polymerase show that modifications of intra-molecular interactions have no effect on RNA synthesis, while impairing inter-molecular interactions decreases polymerase activity. This confirms that dimeric templates are preferentially replicated by the viral polymerase. Altogether, these results indicate that the dimerization of the HCV genomic RNA is a crucial step for the viral life cycle especially for RNA replication. RNA dimerization could explain the existence of HCV recombinants in cell culture and patients reported recently in other studies.

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Acknowledgments

We thank the structural biophysico-chemistry facility (UMS 3033/US001) of the Institut Européen de Chimie et Biologie (Pessac, France) for access to the Biacore T200 instrument that was acquired with the support of the Conseil Régional d’Aquitaine, the GIS-IBiSA, and the Cellule Hôtels à Projets of the CNRS. This work was supported by the Agence Nationale de Recherche contre le SIDA (ANRS), the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM) and the Université de Bordeaux. The authors would like to thank Dr. Clint Smith for critical review of the manuscript.

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Authors and Affiliations

  1. Univ. Bordeaux, Laboratoire MFP, 33076, Bordeaux, France
    Cyril Masante, Chloé Jaubert, Jacqueline Plissonneau, Lucie Besnard & Michel Ventura
  2. CNRS UMR 5234, Laboratoire MFP, 33076, Bordeaux, France
    Cyril Masante, Chloé Jaubert, Jacqueline Plissonneau, Lucie Besnard & Michel Ventura
  3. Univ. Bordeaux, Laboratoire ARNA, 33076, Bordeaux, France
    William Palau & Carmelo Di Primo
  4. INSERM, U869, Laboratoire ARNA, 33607, Pessac, France
    William Palau & Carmelo Di Primo

Authors

  1. Cyril Masante
  2. Chloé Jaubert
  3. William Palau
  4. Jacqueline Plissonneau
  5. Lucie Besnard
  6. Michel Ventura
  7. Carmelo Di Primo

Corresponding authors

Correspondence toMichel Ventura or Carmelo Di Primo.

Additional information

C. Masante and C. Jaubert are the joint first authors.

M. Ventura and C. Di Primo contributed equally to this work.

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Masante, C., Jaubert, C., Palau, W. et al. Mutations of the SL2 dimerization sequence of the hepatitis C genome abrogate viral replication.Cell. Mol. Life Sci. 72, 3375–3385 (2015). https://doi.org/10.1007/s00018-015-1893-3

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