Development of Optimized Inhibitor RNAs Allowing Multisite-Targeting of the HCV Genome

Abstract: Engineered multivalent drugs are promising candidates for fighting infection by highly variable viruses, such as HCV. The combination into a single molecule of more than one inhibitory domain, each with its own target specificity and even a different mechanism of action, results in drugs with potentially enhanced therapeutic properties. In the present work, the anti-HCV chimeric inhibitor RNA HH363-10, which has a hammerhead catalytic domain and an aptamer RNA domain, was subjected to an in vitro selection strategy to isolate ten different optimised chimeric inhibitor RNAs. The catalytic domain was preserved while the aptamer RNA domain was evolved to contain two binding sites, one mapping to the highly conserved IIIf domain of the HCV genome’s internal ribosome entry site (IRES), and the other either to IRES domain IV (which contains the translation start codon) or the essential linker region between domains I and II. These chimeric molecules efficiently and specifically interfered with HCV IRES-dependent translation in vitro (with IC50 values in the low µM range). They also inhibited both viral translation and replication in cell culture. These findings highlight the feasibility of using in vitro selection strategies for obtaining improved RNA molecules with potential clinical applications.This work was supported by the Spanish Ministerio de Economía y Competitividad [BFU2015-64359-P]. Work at our laboratory is partially supported by FEDER funds from the EU.Peer reviewe

An engineered inhibitor RNA that efficiently interferes with hepatitis C virus translation and replication

32 páginas, ilustraciones.-- Texto completo borrador de autor.Hepatitis C virus (HCV) translation is mediated by a highly conserved internal ribosome entry site (IRES), mainly located at the 5‟untranslatable region (5‟UTR) of the viral genome. Viral protein synthesis clearly differs from that used by most cellular mRNAs, rendering the IRES an attractive target for novel antiviral compounds. The engineering of RNA compounds is an effective strategy for targeting conserved functional regions in viral RNA genomes. The present work analyses the anti-HCV potential of HH363-24, an in vitro selected molecule composed of a catalytic RNA cleaving domain with an extension at the 3‟ end that acts as aptamer for the viral 5‟UTR. The engineered HH363-24 efficiently cleaved the HCV genome and bound to the essential IIId domain of the IRES region. This action interfered with the proper assembly of the translationally active ribosomal particles 48S and 80S, likely leading to the effective inhibition of IRES function in a hepatic cell line. HH363-24 also efficiently reduced HCV RNA levels up to 70% in a subgenomic replicon system. These findings provide new insights into the development of potential therapeutic strategies based on RNA molecules targeting genomic RNA structural domains and highlight the feasibility of generating novel engineered RNAs as potent antiviral agents.This work was supported by grants BFU2009-08137 and BFU2009-08137 from the Spanish Ministerio de Ciencia e Innovación, CTS-5077 from the Junta de Andalucía to A. B-H. Work at our laboratory is also partially supported by FEDER funds from the EU. C. R-L was funded by grants 2004-20E632 from the Spanish National Research Council (CSIC). B. B-H was funded by grant CTS-5077 from the Junta de Andalucía.Peer reviewe

Bioinformatics analysis of selected aptamer sequences allows the identification of RNA tools for the functional analysis of West Nile virus genomic RNA elements

The WNV is the etiological agent of recurrent outbreaks of febrile illness and encephalitis worldwide. Since the 1999 outbreak in the US, it is considered a global health threat by the WHO. The West Nile Virus (WNV) shares, with other RNA viruses, a compact RNA genome for storing all the required information for the completion of the infectious cycle. For this purpose, RNA viruses have developed an information storage system based on the use of discrete structural units that accomplish well-defined essential functions. This system complements and overlaps the protein coding one and provides an enormous plasticity to the viral genome. These structural/functional units can be conserved among closely related viruses and even among different isolates or strains. Therefore, they are considered potential therapeutic targets for fighting against viral infection. WNV is an enveloped, single-stranded positive RNA virus belonging to the genus Flavivirus (family Flaviviridae). It is a mosquito-borne flavivirus that naturally cycles between birds and mosquitoes, although it can infect multiple vertebrate hosts including horses and humans. This genus comprises a large number of viruses, including important human pathogens as Dengue, Japanese encephalitis, Yellow fever or Zika virus, among others. The WNV genome is a single-stranded RNA molecule of ~11.000 nts, which contains a single open reading frame (ORF) flanked by essential untranslated regions (UTRs). The UTRs are rich in conserved structural RNA elements, which are functionally indispensable for the consecution of critical viral processes, as replication or translation. Such defined RNA structural elements participate in long-distant RNA-RNA interactions that bring close together both UTRs, thus enabling the acquisition of a closed-loop topology required for the achievement of the viral cycle. The WNV 3¿UTR can be subdivided into three autonomously folded regions, domains I-III, with the presence of duplications of structural cassettes (Figure 1). Domain I is located just downstream of the translation stop codon and appears as a hypervariable sequence followed by two conserved stem-loop domains (SL-I and -II) similar in sequence and structure (figure 1). Domain II is moderately conserved and contains a characteristic duplicated structure known as a dumbbell (DB); this is involved in the formation of a pseudoknot (PK) structural element. Domain III is defined by the highly conserved terminal genomic functional elements sHP (short hairpin) and 3¿SL. The functions of these structural elements have been studied in depth, and are essential for viral replication and the completion of the viral cycle. However, their role in WNV protein synthesis remains elusive. Therefore, determining their functional implications is a requisite to identify new potential targets for anti-WNV agents. In this context, the use of novel molecular tools based on nucleic acids, particularly RNA, for elucidating the role of different viral genomic regions has become a successful tactic recently included into the virologists¿ toolbox. Aptamers are single-stranded DNA or RNA molecules that bind in a very speci¿c and ef¿cient manner to their targets in a structure-dependent way. Aptamers are isolated by SELEX (Systematic Evolution of Ligands by EXponential enrichment), by which a highly heterogeneous initial population of DNA or RNA, usually composed of 1012 to 1015 variants, is subjected to iterative selection rounds against a speci¿c target

Potential of the Other Genetic Information Coded by the Viral RNA Genomes as Antiviral Target

In addition to the protein coding information, viral RNA genomes code functional information in structurally conserved units termed functional RNA domains. These RNA domains play essential roles in the viral cycle (e.g., replication and translation). Understanding the molecular mechanisms behind their function is essential to understanding the viral infective cycle. Further, interfering with the function of the genomic RNA domains offers a potential means of developing antiviral strategies. Aptamers are good candidates for targeting structural RNA domains. Besides its potential as therapeutics, aptamers also provide an excellent tool for investigating the functionality of RNA domains in viral genomes. This review briefly summarizes the work carried out in our laboratory aimed at the structural and functional characterization of the hepatitis C virus (HCV) genomic RNA domains. It also describes the efforts we carried out for the development of antiviral aptamers targeting specific genomic domains of the HCV and the human immunodeficiency virus type-1 (HIV-1).We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)Peer reviewe

The HCV genome domains 5BSL3.1 and 5BSL3.3 act as managers of translation

Peer reviewe

RNA Aptamers as Molecular Tools to Study the Functionality of the Hepatitis C Virus CRE Region

Background: Hepatitis C virus (HCV) contains a (+) ssRNA genome with highly conserved structural, functional RNA domains, many of them with unknown roles for the consecution of the viral cycle. Such genomic domains are candidate therapeutic targets. This study reports the functional characterization of a set of aptamers targeting the cis-acting replication element (CRE) of the HCV genome, an essential partner for viral replication and also involved in the regulation of protein synthesis. Methods: Forty-four aptamers were tested for their ability to interfere with viral RNA synthesis in a subgenomic replicon system. Some of the most efficient inhibitors were further evaluated for their potential to affect the recruitment of the HCV RNA-dependent RNA polymerase (NS5B) and the viral translation in cell culture. Results: Four aptamers emerged as potent inhibitors of HCV replication by direct interaction with functional RNA domains of the CRE, yielding a decrease in the HCV RNA levels higher than 90%. Concomitantly, one of them also induced a significant increase in viral translation (>50%). The three remaining aptamers efficiently competed with the binding of the NS5B protein to the CRE. Conclusions: Present findings confirm the potential of the CRE as an anti-HCV target and support the use of aptamers as molecular tools for investigating the functionality of RNA domains in viral genomes.Spanish Ministry of Economy and Competitiveness, BFU2012-31213 and Junta de Andalucía, CVI-7430, to A.B.-H.; FEDER funds from the EU. We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)Peer reviewe

The Genomic 3′ UTR of Flaviviruses Is a Translation Initiation Enhancer

Viruses rely on the cellular machinery of host cells to synthesize their proteins, and have developed different mechanisms enabling them to compete with cellular mRNAs for access to it. The genus Flavivirus is a large group of positive, single-stranded RNA viruses that includes several important human pathogens, such as West Nile, Dengue and Zika virus. The genome of flaviviruses bears a type 1 cap structure at its 5′ end, needed for the main translation initiation mechanism. Several members of the genus also use a cap-independent translation mechanism. The present work provides evidence that the WNV 5′ end also promotes a cap-independent translation initiation mechanism in mammalian and insect cells, reinforcing the hypothesis that this might be a general strategy of flaviviruses. In agreement with previous reports, we show that this mechanism depends on the presence of the viral genomic 3′ UTR. The results also show that the 3′ UTR of the WNV genome enhances translation of the cap-dependent mechanism. Interestingly, WNV 3′ UTR can be replaced by the 3′ UTR of other flaviviruses and the translation enhancing effect is maintained, suggesting a molecular mechanism that does not involve direct RNA-RNA interactions to be at work. In addition, the deletion of specific structural elements of the WNV 3′ UTR leads to increased cap-dependent and cap-independent translation. These findings suggest the 3′ UTR to be involved in a fine-tuned translation regulation mechanism

Structure and function analysis of the essential 3′X domain of hepatitis C virus

The 3X domain of hepatitis C virus has been reported to control viral replication and translation by modulating the exposure of a nucleotide segment involved in a distal base-pairing interaction with an upstream 5BSL3.2 domain. To study the mechanism of this molecular switch, we have analyzed the structure of 3X mutants that favor one of the two previously proposed conformations comprising either two or three stem–loops. Only the two-stem conformation was found to be stable and to allow the establishment of the distal contact with 5BSL3.2, and also the formation of 3X domain homodimers by means of a universally conserved palindromic sequence. Nucleotide changes disturbing the two-stem conformation resulted in poorer replication and translation levels, explaining the high degree of conservation detected for this sequence. The switch function attributed to the 3X domain does not occur as a result of a transition between two- and three-stem conformations, but likely through the sequestration of the 5BSL3.2-binding sequence by formation of 3X homodimers.We thank R. Bartenschlager (University of Heidelberg, Germany) for providing the plasmid coding for HCV replicon pFK-I389-Fluc-NS3-3′ET, and Angel Cantero-Camacho for his contribution toward the design of mutant 3′X sequences. This work was supported by Ministerio de Economía y Competitividad of Spain (BFU-2012-30770 and BFU2015-65103-R to J.G., and BFU2015-64359-P to A.B.-H.) and Universidad Católica de Valencia of Spain (PRUCV/2015/629 and 2017-114-001 to J.G., and EDUCV contract E-46-2017-0118740 to J.C.-M.)

Inter- and Intramolecular RNA–RNA Interactions Modulate the Regulation of Translation Mediated by the 3′ UTR in West Nile Virus

RNA viruses rely on genomic structural elements to accomplish the functions necessary to complete the viral cycle. These elements participate in a dynamic network of RNA–RNA interactions that determine the overall folding of the RNA genome and may be responsible for the fine regulation of viral replication and translation as well as the transition between them. The genomes of members of the genus Flavivirus are characterized by a complexly folded 3′ UTR with a number of RNA structural elements that are conserved across isolates of each species. The present work provides evidence of intra- and intermolecular RNA–RNA interactions involving RNA structural elements in the 3′ UTR of the West Nile virus genome. The intermolecular interactions can be visualized in vitro by the formation of molecular dimers involving the participation of at least the SLI and 3′DB elements. Certainly, the 3′ UTR of dengue virus, which lacks the SLI element, forms molecular dimers in lower quantities via a single interaction site, probably 3′DB. The functional analysis of sequence or deletion mutants revealed an inverse relationship between 3′ UTR dimerization and viral translation efficiency in cell cultures. A network of RNA–RNA interactions involving 3′ UTR structural elements might therefore exist, helping to regulate viral translation