Characterizing the function and structural organization of the 5' tRNA-like motif within the hepatitis C virus quasispecies

Pirón, María; Beguiristain, Nerea; Nadal, Anna; Martínez‐Salas, Encarnación; Gómez, Jordi

doi:10.1093/nar/gki290

Cited by 31 publications

(39 citation statements)

References 44 publications

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“…The RNase P ribozyme from Synechocystis sp., which does not require the CCA sequence at the 39end of the tRNA precursor for its hydrolysis (Pascual and Vioque 1999), also recognizes the hepatitis C virus (HCV) IRES in vitro (Sabariegos et al 2004). However, no cleavage of the viral RNA in infected or transfected cells has been observed by the endogenous nuclear RNase P (Piron et al 2005), consistent with the fact that the viral cycle occurs in the cytoplasm of infected cells. The biological significance of the RNase P recognition motif within IRES elements is still unknown.…”

Section: Introductionsupporting

confidence: 75%

Tailoring the switch from IRES-dependent to 5′-end-dependent translation with the RNase P ribozyme

Fernández

Martínez‐Salas²

2010

RNA

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Translation initiation driven by internal ribosome entry site (IRES) elements is dependent on the structural organization of the IRES region. We have previously shown that a structural motif within the foot-and-mouth-disease virus IRES is recognized in vitro as substrate for the Synechocystis sp. RNase P ribozyme. Here we show that this structure-dependent endonuclease recognizes the IRES element in cultured cells, leading to inhibition of translation. Inhibition of IRES activity was dependent on the expression of the active ribozyme RNA subunit. Moreover, expression of the antisense sequence of the ribozyme did not inhibit IRES activity, demonstrating that stable RNA structures located upstream of the IRES element do not interfere with internal initiation. RNAs carrying defective IRES mutants that were substrates of the ribozyme in vivo revealed an increased translation of the reporter in response to the expression of the active ribozyme. In support of RNA cleavage, subsequent analysis of the translation initiation manner indicated a switch from IRES-dependent to 59-end-dependent translation of RNase P target RNAs. We conclude that the IRES element is inactivated by expression in cis of RNase P in the cytoplasm of cultured cells, providing a promising antiviral tool to combat picornavirus infections. Furthermore, our results reinforce the essential role of the structural motif that serves as RNase P recognition motif for IRES activity.

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Section: Introductionsupporting

confidence: 75%

Tailoring the switch from IRES-dependent to 5′-end-dependent translation with the RNase P ribozyme

Fernández

Martínez‐Salas²

2010

RNA

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“…In another FMDV IRES mutant bearing a CGCCC substitution at the most apical RAAA loop, a new product of the same length as in the GNRA substitution mutant was observed, indicating that this effect was not exclusively observed in the GUAG mutant. A related example was reported for HCV, in which a natural variant containing a single nucleotide substitution resulted in an enhanced second cut in a nearby residue by human RNase P (Piron et al 2005).…”

Section: Discussionmentioning

confidence: 86%

Characterization of a cyanobacterial RNase P ribozyme recognition motif in the IRES of foot-and-mouth disease virus reveals a unique structural element

Serrano

Gómez²,

Martínez‐Salas³

2007

RNA

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Translation initiation driven by internal ribosome entry site (IRES) elements is dependent on the structural organization of the IRES region. Picornavirus IRES are organized in structural domains, in which the terminal stem-loops participate in functional RNA-protein interactions. However, the mechanistic role performed by the central domain during internal initiation has not been elucidated yet. Here we show that the foot-and-mouth-disease virus IRES contains a structural motif that serves in vitro as substrate for the Synechocystis sp. RNase P ribozyme, a structure-dependent endonuclease that participates in tRNA precursor processing. Recognition of the IRES substrate was dose dependent, required high magnesium concentration, and resulted in the formation of cleavage products with 59 phosphate and 39 hydroxyl ends. Mapping of the core recognition motif indicated that it overlapped with the apical region of the central domain. Two IRES constructs containing nucleotide substitutions in the apical region of the central domain that reorganized RNA structure displayed an altered pattern of cleavage by the cyanobacterial ribozyme generating new cleavage events in nearby residues. From these data it is inferred that the central domain of the IRES region has evolved a tRNA structural mimicry that renders it a substrate for RNase P ribozyme reaction. Recognition of this motif was affected in defective IRES mutants with a local RNA structure reorganization, suggesting that its structural preservation is required for IRES activity.

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“…Further, the development of combination therapies using antiviral compounds with different specificities would additionally contribute to preventing the emergence of resistant viral pools [1,14,15]. The functional characterization of HH363 -10, which simultaneously targets two highly conserved regions that are required for the preservation of the IRES activity [18,36,37], clearly shows the advantage of combining two inhibitory activities in a single molecule to achieve a strong effect, making our strategy an excellent starting point for the development of new therapeutic tools. The chimeric RNA HH363 -10 strongly inhibited HCV IRES-dependent translation in vitro, with an IC 50 of 150 nM.…”

Section: Discussionmentioning

confidence: 99%

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

Romero-López

Díaz-González

Berzal‐Herranz

2007

Cell. Mol. Life Sci.

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Hepatitis C virus (HCV) translation initiation depends on an internal ribosome entry site (IRES). We previously identified an RNA molecule (HH363-10) able to bind and cleave the HCV IRES region. This paper characterizes its capacity to interfere with IRES function.Inhibition assays showed that it blocks IRES activity both in vitro and in a human hepatoma cell line. Although nucleotides involved in binding and cleavage reside in separate regions of the inhibitor HH363-10, further analysis demonstrated the strongest effect to be an intrinsic feature of the entire molecule; the abolishment of any of the two activities resulted in a reduction in its function. Probing assays demonstrate that HH363-10 specifically interacts with the conserved IIIf domain of the pseudoknot structure in the IRES, leading to the inhibition of the formation of translationally competent 80S particles.The combination of two inhibitory activities targeting different sequences in a chimeric molecule may be a good strategy to avoid the emergence of resistant viral variants.

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Characterizing the function and structural organization of the 5' tRNA-like motif within the hepatitis C virus quasispecies

Cited by 31 publications

References 44 publications

Tailoring the switch from IRES-dependent to 5′-end-dependent translation with the RNase P ribozyme

Tailoring the switch from IRES-dependent to 5′-end-dependent translation with the RNase P ribozyme

Characterization of a cyanobacterial RNase P ribozyme recognition motif in the IRES of foot-and-mouth disease virus reveals a unique structural element

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

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