Liquid-crystal NMR structure of HIV TAR RNA bound to its SELEX RNA aptamer reveals the origins of the high stability of the complex

Melckebeke, Hélène Van; Devany, Matthew; Primo, Carmelo Di; Beaurain, François; Toulmé, Jean‐Jacques; Bryce, David L.; Boisbouvier, Jérôme

doi:10.1073/pnas.0712121105

Cited by 43 publications

(58 citation statements)

References 58 publications

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“…However, most studies suggest that inhibition of virus infection by aptamers targeting different viral proteins can be attributed to suppression of viral protein by the aptamers themselves (30)(31)(32)(33). Consistent with these reports, our study showed that the aptamers for core protein do not induce IFN-␤ and ISGs, indicating that inhibition of infectious virus production by core aptamers is not due to the innate immune response.…”

Section: Figsupporting

confidence: 80%

Inhibition of Hepatitis C Virus Production by Aptamers against the Core Protein

Shi

Gao

et al. 2014

J Virol

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bHepatitis C virus (HCV) core protein is essential for virus assembly. HCV core protein was expressed and purified. Aptamers against core protein were raised through the selective evolution of ligands by the exponential enrichment approach. Detection of HCV infection by core aptamers and the antiviral activities of aptamers were characterized. The mechanism of their anti-HCV activity was determined. The data showed that selected aptamers against core specifically recognize the recombinant core protein but also can detect serum samples from hepatitis C patients. Aptamers have no effect on HCV RNA replication in the infectious cell culture system. However, the aptamers inhibit the production of infectious virus particles. Beta interferon (IFN-␤) and interferon-stimulated genes (ISGs) are not induced in virally infected hepatocytes by aptamers. Domains I and II of core protein are involved in the inhibition of infectious virus production by the aptamers. V31A within core is the major resistance mutation identified. Further study shows that the aptamers disrupt the localization of core with lipid droplets and NS5A and perturb the association of core protein with viral RNA. The data suggest that aptamers against HCV core protein inhibit infectious virus production by disrupting the localization of core with lipid droplets and NS5A and preventing the association of core protein with viral RNA. The aptamers for core protein may be used to understand the mechanisms of virus assembly. Core-specific aptamers may hold promise for development as early diagnostic reagents and potential therapeutic agents for chronic hepatitis C.

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Section: Figsupporting

confidence: 80%

Inhibition of Hepatitis C Virus Production by Aptamers against the Core Protein

Shi

Gao

et al. 2014

J Virol

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“…Van Melckebeke et al (28) Taken together, the results obtained by fluorescence, optical analysis, and native PAGE provide evidence that both loop-loop and stem-stem interactions can modulate the conversion of the (CAG) 10 and (CTG) 10 hairpins to the (CAG) 10 /(CTG) 10 duplex. Therefore, we considered that these two modes of interaction represent two independent mechanisms by which the complementary hairpins can convert to duplex.…”

Section: Discussionmentioning

confidence: 62%

Mechanistic Studies of Hairpin to Duplex Conversion for Trinucleotide Repeat Sequences

Figueroa

Delaney

2010

Journal of Biological Chemistry

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The expansion of a trinucleotide repeat sequence, such as CAG/CTG, has been pinpointed as the molecular basis for a number of neurodegenerative disorders. It has been proposed that as part of the expansion process, these repetitive sequences adopt non-B conformations such as hairpins. However, the prevalence of these hairpins and their contributions to the DNA expansion have not been well defined. In this work, we utilized a molecular beacon strategy to examine the stability of the (CAG) 10 hairpin and also its behavior in the presence of the complementary (CTG) 10 hairpin. We find that the two hairpins represent kinetically trapped species that can coexist but irreversibly convert to duplex upon thermal induction. Furthermore, as monitored by fluorescence and optical analysis, modifications to the base composition of either the loop or stem region have a profound effect on the ability of the trinucleotide repeat hairpins to convert to duplex. Additionally, the rate of duplex formation is also reduced with these loop and stem-modified hairpins. These results demonstrate that the trinucleotide repeat hairpins can convert to duplex via two independent mechanisms as follows: the loop-loop interactions found in kissing hairpins or the stem-stem interactions of a cruciform.

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“…This conclusion is somehow surprising. Previous works have shown that stable kissing complexes can only be formed through Watson-Crick base-pairing between the partially or totally complementary sequences of apical loops, i.e., in a stem-loop context (Westhof et al 1988;Lee and Crothers 1998;Mujeeb et al 1998;Kim and Tinoco 2000;Ennifar et al 2001;Lebars et al 2008;Van Melckebeke et al 2008). The intermolecular loop-loop helix is stacked between the two stem helices.…”

Section: Discussionmentioning

confidence: 99%

Direct evidence for RNA–RNA interactions at the 3′ end of the Hepatitis C virus genome using surface plasmon resonance

Palau

Masante

Ventura

et al. 2013

RNA

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Surface plasmon resonance was used to investigate two previously described interactions analyzed by reverse genetics and complementation mutation experiments, involving 5BSL3.2, a stem-loop located in the NS5B coding region of HCV. 5BSL3.2 was immobilized on a sensor chip by streptavidin-biotin coupling, and its interaction either with the SL2 stem-loop of the 3 ′ end or with an upstream sequence centered on nucleotide 9110 (referred to as Seq9110) was monitored in real-time. In contrast with previous results obtained by NMR assays with the same short RNA sequences that we used or SHAPE analysis with longer RNAs, we demonstrate that recognition between 5BSL3.2 and SL2 can occur in solution through a kissing-loop interaction. We show that recognition between Seq9110 and the internal loop of 5BSL3.2 does not prevent binding of SL2 on the apical loop of 5BSL3.2 and does not influence the rate constants of the SL2-5BSL3.2 complex. Therefore, the two binding sites of 5BSL3.2, the apical and internal loops, are structurally independent and both interactions can coexist. We finally show that the stem-loop SL2 is a highly dynamic RNA motif that fluctuates between at least two conformations: One is able to hybridize with 5BSL3.2 through loop-loop interaction, and the other one is capable of self-associating in the absence of protein, reinforcing the hypothesis of SL2 being a dimerization sequence. This result suggests also that the conformational dynamics of SL2 could play a crucial role for controlling the destiny of the genomic RNA.

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Liquid-crystal NMR structure of HIV TAR RNA bound to its SELEX RNA aptamer reveals the origins of the high stability of the complex

Cited by 43 publications

References 58 publications

Inhibition of Hepatitis C Virus Production by Aptamers against the Core Protein

Inhibition of Hepatitis C Virus Production by Aptamers against the Core Protein

Mechanistic Studies of Hairpin to Duplex Conversion for Trinucleotide Repeat Sequences

Direct evidence for RNA–RNA interactions at the 3′ end of the Hepatitis C virus genome using surface plasmon resonance

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