Hijacking hepatitis C viral replication with a non-coding replicative RNA

Bitard, Juliette; Chognard, Gaëlle; Dumas, Estelle; Rumi, Julie; Masante, Cyril; Mahias, K.; Astier-Gin, T.; Ventura, Michel

doi:10.1016/j.antiviral.2010.04.003

Cited by 2 publications

(4 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The templates used for in vitro transcription to produce the 5UTR-H2AE-5BSL-3UTR RNAs and the WT555, TD555 and Kistem555 RNAs were obtained using PCR with primers T7_5UTR_Start or T7_555_Start and 3UTR_Stop, which were designed to introduce a T7 RNA polymerase promoter ( Table 1). PCR and in vitro transcription have been described previously [25], and the purity and integrity of the RNAs were determined using capillary electrophoresis on a Bioanalyzer 2100 (Agilent).…”

Section: In Vitro Transcriptionmentioning

confidence: 99%

“…The Huh7-QR cells were obtained from cell line harboring the Rep5.1 RNA replicon (kindly provided by R. Bartenschlager). These cells were cured by a three-week treatment with 150 U/mL interferon-a until disappearance of the replicon RNA [25].…”

Section: Cell Culture and Transient Rna Transfectionmentioning

confidence: 99%

“…EGFP_S and EGFP_AS (Table 1) were designed to amplify a portion of the EGFP gene. The HCV RNA copy number was normalized to the SFRS4 cellular RNA, which is relatively stable in HCV-infected cells [25,27]. The SFRS4_S and SFRS4_AS primers ( Table 1) were used.…”

Section: Quantitative Rt-pcrmentioning

confidence: 99%

“…The SFRS4_S and SFRS4_AS primers ( Table 1) were used. Reactions were performed as described previously [25]. The copy numbers of NS3, EGFP and SFRS4 were determined by comparison with specific, serially diluted transcripts that were included in the RT-PCR analysis.…”

Section: Quantitative Rt-pcrmentioning

confidence: 99%

See 3 more Smart Citations

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

Masante

Jaubert

Palau

et al. 2015

Cell. Mol. Life Sci.

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: In Vitro Transcriptionmentioning

confidence: 99%

Section: Cell Culture and Transient Rna Transfectionmentioning

confidence: 99%

Section: Quantitative Rt-pcrmentioning

confidence: 99%

Section: Quantitative Rt-pcrmentioning

confidence: 99%

See 2 more Smart Citations

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

Masante

Jaubert

Palau

et al. 2015

Cell. Mol. Life Sci.

Self Cite

View full text Add to dashboard Cite

show abstract

Hepatitis C virus intragenomic interactions are modulated by the SLVI RNA structure of the core coding sequence

Ventura

Martin

Jaubert

et al. 2017

Journal of General Virology

View full text Add to dashboard Cite

Several RNA interactions are thought to play a role in the regulation of the hepatitis C virus (HCV) life cycle. Most of these interactions involve the 5BSL3.2 domain and therefore occur at the 3' end of the viral genomic RNA. A long-range interaction has also been described between 5BSL3.2 and the 5' untranslated region (UTR). Another interaction involves the SLVI stem loop of the core coding region and the 5'UTR. We aimed to analyse the role of this SLVI domain, which likely interferes with others interactions. By evaluating RNA stability, translation and RNA synthesis, we showed that the SLVI stem loop extensively modulates the effect of the interactions mediated by the 5BSL3.2 domain and strongly affects the IIId/5BSL3.2 interaction. Numerous interactions in HCV genomic RNA have been described in the UTRs and the coding sequence but their roles are poorly understood. We showed that the SLVI domain located in the core coding sequence plays an important role in the translation of the polyprotein, but also in the modulation of long-range RNA interactions centred on the 5BSL3.2 domain. The SLVI domain has been absent from most studies, especially from the extensively used subgenomic replicon; our data highlight the importance of this domain in the studies of these long-range interactions in the HCV life cycle.

show abstract

Hijacking hepatitis C viral replication with a non-coding replicative RNA

Cited by 2 publications

References 31 publications

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

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

Hepatitis C virus intragenomic interactions are modulated by the SLVI RNA structure of the core coding sequence

Contact Info

Product

Resources

About