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Mutations of the SL2 dimerization sequence of the hepatitis C genome abrogate viral replication.

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.

SUBMITTER: Masante C

PROVIDER: S-EPMC7079775 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Mutations of the SL2 dimerization sequence of the hepatitis C genome abrogate viral replication.

Masante Cyril C Jaubert Chloé C Palau William W Plissonneau Jacqueline J Besnard Lucie L Ventura Michel M Di Primo Carmelo C

Cellular and molecular life sciences : CMLS 20150328 17

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 tha ...[more]

PMID: 25822205

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Mutations of the SL2 dimerization sequence of the hepatitis C genome abrogate viral replication.

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Mutations of the SL2 dimerization sequence of the hepatitis C genome abrogate viral replication.

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