Inhibition of hepatitis C virus internal ribosome entry site-mediated translation by an RNA targeting the conserved IIIf domain

Romero-López, Cristina; Díaz-González, R.; Berzal‐Herranz, Alfredo

doi:10.1007/s00018-007-7345-y

Cited by 30 publications

(60 citation statements)

References 32 publications

(58 reference statements)

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“…The resulting complexes were resolved in 5% native polyacrylamide gels in TB buffer (100 m m Tris–HCl pH 8.3, 86 m m boric acid) at room temperature. Gels were dried, scanned using a Storm 820 Phosphorimager (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and analysed using Image Quant v.5.2 software (Molecular Dynamics, Sunnyvale, CA, USA) [26]. Dissociation constant ( K d ) values were calculated using Sigma Plot v.8.02 software (Systat Software Inc., San José, CA, USA) and adjusted with the equation y = ( B max x )/( K d + x ) as previously described [33], where y is the percentage of complexed aptamer, B max is the amplitude of the reaction, and x is the concentration of the substrate HCV‐CRE 194 RNA.…”

Section: Methodsmentioning

confidence: 99%

RNA aptamer‐mediated interference of HCV replication by targeting the CRE‐5BSL3.2 domain

Marton¹,

Romero-López²,

Berzal‐Herranz³

2012

Journal of Viral Hepatitis

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Summary. The RNA genome of hepatitis C virus (HCV) contains multiple conserved structural RNA domains that play key roles in essential viral processes. A conserved structural component within the 3′ end of the region coding for viral RNA‐dependent RNA polymerase (NS5B) has been characterized as a functional cis‐acting replication element (CRE). This study reports the ability of two RNA aptamers, P‐58 and P‐78, to interfere with HCV replication by targeting the essential 5BSL3.2 domain within this CRE. Structure‐probing assays showed the binding of the aptamers to the CRE results in a structural reorganization of the apical portion of the 5BSL3.2 stem‐loop domain. This interfered with the binding of the NS5B protein to the CRE and induced a significant reduction in HCV replication (≈50%) in an autonomous subgenomic HCV replication system. These results highlight the potential of this CRE as a target for the development of anti‐HCV therapies and underscore the potential of antiviral agents based on RNA aptamer molecules.

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Section: Methodsmentioning

confidence: 99%

RNA aptamer‐mediated interference of HCV replication by targeting the CRE‐5BSL3.2 domain

Marton¹,

Romero-López²,

Berzal‐Herranz³

2012

Journal of Viral Hepatitis

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“…Among ribozymes tested in vitro were hammerhead constructs targeted at the apical loop of subdomain IIIb (HCV-195) 52, 53 or the subdomain IIIf (HH363). 56–58 Conjugated chemical nucleases consisting of RNA-hydrolyzing imidazole derivatives tethered to guide oligonucleotides were shown to inhibit in vitro IRES-driven translation at sub-μM IC 50 values. 61 However, testing of the conjugates in HCV-infected cells suggested that the inhibition was due to the antisense effect of the guide oligonucleotide and not through cleavage by the attached imidazole.…”

Section: Strategies For Inhibition Of the Hcv Iresmentioning

confidence: 99%

Hepatitis C Virus Translation Inhibitors Targeting the Internal Ribosomal Entry Site

et al. 2013

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The internal ribosome entry site (IRES) in the 5′ untranslated region (UTR) of the hepatitis C virus (HCV) genome initiates translation of the viral polyprotein precursor. The unique structure and high sequence conservation of the 5′ UTR render the IRES RNA a potential target for the development of selective viral translation inhibitors. Here, we provide an overview of approaches to block HCV IRES function by nucleic acid, peptide and small molecule ligands. Emphasis will be given to the IRES subdomain IIa which currently is the most advanced target for small molecule inhibitors of HCV translation. The subdomain IIa behaves as an RNA conformational switch. Selective ligands act as translation inhibitors by locking the conformation of the RNA switch. We review synthetic procedures for inhibitors as well as structural and functional studies of the subdomain IIa target and its ligand complexes.

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“…Electrophoresis was performed at 4°C over 5 h at 15 mA in TBM buffer (45 mM TrisHCl at pH 8.3, 43 mM boric acid, and 0.1 mM MgCl 2 ). Gels were dried, scanned, and analyzed as previously described (Romero-Ló pez et al 2007). K d values were calculated using Sigma Plot 8.02 software according to the equation y = (B max x)/(K d + x) (PuertaFernández et al 2005), where y is the percentage of complexed inhibitory RNA, B max is the amplitude of the reaction, x is the concentration of the target RNA, and K d the dissociation constant.…”

Section: Binding Assaysmentioning

confidence: 99%

A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

Romero-López¹,

Berzal‐Herranz²

2009

RNA

112

146

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The RNA genome of the hepatitis C virus (HCV) contains multiple conserved structural cis domains that direct protein synthesis, replication, and infectivity. The untranslatable regions (UTRs) play essential roles in the HCV cycle. Uncapped viral RNAs are translated via an internal ribosome entry site (IRES) located at the 59 UTR, which acts as a scaffold for recruiting multiple protein factors. Replication of the viral genome is initiated at the 39 UTR. Bioinformatics methods have identified other structural RNA elements thought to be involved in the HCV cycle. The 5BSL3.2 motif, which is embedded in a cruciform structure at the 39 end of the NS5B coding sequence, contributes to the three-dimensional folding of the entire 39 end of the genome. It is essential in the initiation of replication. This paper reports the identification of a novel, strand-specific, long-range RNA-RNA interaction between the 59 and 39 ends of the genome, which involves 5BSL3.2 and IRES motifs. Mutants harboring substitutions in the apical loop of domain IIId or in the internal loop of 5BSL3.2 disrupt the complex, indicating these regions are essential in initiating the kissing interaction. No complex was formed when the UTRs of the related foot and mouth disease virus were used in binding assays, suggesting this interaction is specific for HCV sequences. The present data firmly suggest the existence of a higher-order structure that may mediate a protein-independent circularization of the HCV genome. The 59-39 end bridge may have a role in viral translation modulation and in the switch from protein synthesis to RNA replication.

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Inhibition of hepatitis C virus internal ribosome entry site-mediated translation by an RNA targeting the conserved IIIf domain

Cited by 30 publications

References 32 publications

RNA aptamer‐mediated interference of HCV replication by targeting the CRE‐5BSL3.2 domain

RNA aptamer‐mediated interference of HCV replication by targeting the CRE‐5BSL3.2 domain

Hepatitis C Virus Translation Inhibitors Targeting the Internal Ribosomal Entry Site

A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

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