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

Structure-function relationship in viral RNA genomes: The case of hepatitis C virus

The acquisition of a storage information system beyond the nucleotide sequence has been a crucial issue for the propagation and dispersion of RNA viruses. This system is composed by highly conserved, complex structural units in the genomic RNA, termed functional RNA domains. These elements interact with other regions of the viral genome and/or proteins to direct viral translation, replication and encapsidation. The genomic RNA of the hepatitis C virus (HCV) is a good model for investigating about conserved structural units. It contains functional domains, defined by highly conserved structural RNA motifs, mostly located in the 5’-untranslatable regions (5’UTRs) and 3’UTR, but also occupying long stretches of the coding sequence. Viral translation initiation is mediated by an internal ribosome entry site located at the 5’ terminus of the viral genome and regulated by distal functional RNA domains placed at the 3’ end. Subsequent RNA replication strongly depends on the 3’UTR folding and is also influenced by the 5’ end of the HCV RNA. Further increase in the genome copy number unleashes the formation of homodimers by direct interaction of two genomic RNA molecules, which are finally packed and released to the extracellular medium. All these processes, as well as transitions between them, are controlled by structural RNA elements that establish a complex, direct and long-distance RNARNA interaction network. This review summarizes current knowledge about functional RNA domains within the HCV RNA genome and provides an overview of the control exerted by direct, long-range RNA-RNA contacts for the execution of the viral cycle.Spanish Ministry of Economy and Competitiveness, No. BFU2012-31213; Junta de Andalucía, No. CVI-7430; and FEDER funds from the EUPeer reviewe

The role of the RNA-RNA interactome in the hepatitis C virus life cycle

RNA virus genomes are multifunctional entities endowed with conserved structural elements that control translation, replication and encapsidation, among other processes. The preservation of these structural RNA elements constraints the genomic sequence variability. The hepatitis C virus (HCV) genome is a positive, single-stranded RNA molecule with numerous conserved structural elements that manage different steps during the infection cycle. Their function is ensured by the association of protein factors, but also by the establishment of complex, active, long-range RNA-RNA interaction networks-the so-called HCV RNA interactome. This review describes the RNA genome functions mediated via RNA-RNA contacts, and revisits some canonical ideas regarding the role of functional high-order structures during the HCV infective cycle. By outlining the roles of long-range RNA-RNA interactions from translation to virion budding, and the functional domains involved, this work provides an overview of the HCV genome as a dynamic device that manages the course of viral infection.This work was supported by the Spanish Ministerio de Economía y Competitividad (BFU2015-64359-P) grant to A.B.-H.; work at our laboratory is partially supported by FEDER funds from the E

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

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 either 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.This work was supported by grant BFU2006-02568 from the Spanish Ministerio de Educación y Ciencia and CTS-233 from the Junta de Andalucía to A. B-H C. R-L was funder by grant BMC2003-669. R. D-G was the recipient of a fellowship from the Spanish Ministerio de Educación y CienciaPeer reviewe

Selection of RNA aptamers targeting the 3¿ untranslated region of the West Nile Virus genome

West Nile Virus (WNV) is a positive polarity, single-stranded RNA virus that causes West Nile fever, for which no cure has been found to date. WNV, like other RNA viruses, needs to compact all the information to complete the viral cycle into a very small genome. Beyond the information that is stored in the primary structure, the genome of RNA viruses bear functional structural domains that perform multiple essential functions for the viral cycle. In WNV, several of these functional domains are found in the 3'UTR region. Based on the importance of these functional domains, in this work, RNA aptamers have been studied as a possible therapeutic agent. Aptamers are oligonucleotides with the ability to efficiently bind to a molecule, not taking into account only the sequence of the target but also its structural motifs. In this work, various aptamers directed against the 3'UTR region of WNV, which could potentially inhibit processes of the WNV viral cycle, have been analysed and selected by in silico analysis. We have also studied certain characteristics of the SL-I structural element of the WNV 3¿UTR, which shows a high chance of interacting with host molecules. This work will lead further studies towards the generation of antiviral aptamers against WNV and a deeper understanding of WNV interaction with the host cell

Aptamers: Future Pharmaceutical Drugs

In vitro selection strategies are powerful tools for the identification of nucleic acids with unsuspected activities. Their successful application during last two decades has positioned these technologies among those most promising for the development of therapeutic and diagnostic agents. Aptamers are DNA or RNA molecules with special conformational features, able to efficiently bind to a target molecule. They are obtained by SELEX, a particular in vitro selection strategy, and have found utility during last years as a novel class of pharmaceuticals compounds. This review summarizes the progress made in the use of aptamers as therapeutic agents, paying special attention to those examples that have concluded in pharmacological formulations currently included in preclinical and clinical trials.The work in the group of A.B.-H. is funded by grant BFU2009-08137 from the Spanish Ministry of Science and Innovation; grant CTS-5077 from the Junta de Andalucía; and by FEDER funds from the EU.Peer Reviewe

Functional Information Stored in the Conserved Structural RNA Domains of Flavivirus Genomes

The genus Flavivirus comprises a large number of small, positive-sense single-stranded, RNA viruses able to replicate in the cytoplasm of certain arthropod and/or vertebrate host cells. The genus, which has some 70 member species, includes a number of emerging and re-emerging pathogens responsible for outbreaks of human disease around the world, such as the West Nile, dengue, Zika, yellow fever, Japanese encephalitis, St. Louis encephalitis, and tick-borne encephalitis viruses. Like other RNA viruses, flaviviruses have a compact RNA genome that efficiently stores all the information required for the completion of the infectious cycle. The efficiency of this storage system is attributable to supracoding elements, i.e., discrete, structural units with essential functions. This information storage system overlaps and complements the protein coding sequence and is highly conserved across the genus. It therefore offers interesting potential targets for novel therapeutic strategies. This review summarizes our knowledge of the features of flavivirus genome functional RNA domains. It also provides a brief overview of the main achievements reported in the design of antiviral nucleic acid-based drugs targeting functional genomic RNA elements.Work in our laboratory is supported by the Spanish Ministerio de Economía y Competitividad (BFU2012-31213 and BFU2015-64359-P) and the Consejería de Economía Innovación, Ciencia y Empleo, Junta de Andalucía (CVI-7430). It is also partially funded by FEDER funds from the EU.The authors also acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer reviewedPeer Reviewe

Ribozymes, a new therapeutic strategy?

Las ribozimas son moléculas de RNA con actividad catalítica, capaces de catalizar reacciones químicas en el interior celular. En su contexto natural la gran mayoría de las ribozimas descritas llevan a cabo el procesamiento de otras moléculas de RNA. A diferencia de las ribonucleasas proteicas, las ribozimas presentan una gran especificidad de substrato, esta es la característica en la que se fundamenta la idea de que las ribozimas puedan ser utilizadas como supresores génicos específicos y servir de base para el desarrollo de nuevos agentes terapéuticos. La necesidad de disponer de nuevas estrategias terapéuticas se hace más evidente cuando surgen enfermedades contra las cuales los fármacos y terapias empleados no son eficientes. La repercusión social de determinadas enfermedades de difícil o escasa probabilidad de curación (SIDA, Cáncer, etc.), ha agudizado la necesidad de buscar tratamientos alternativos. La actuación directa sobre la información genética responsable de la dolencia, es una de las aproximaciones experimentales en desarrollo. En este sentido, la utilización de ácidos nucleicos como agentes supresores constituye, desde hace algunos años, una línea de investigación importante. Como resultado están surgiendo un conjunto de nuevas tecnologías en tomo a conceptos tales como oligonucleótidos antisense, ribozimas y triples hélices. Las ribozimas actuarían bloqueando la transmisión de información genética a nivel del RNA mediante la destrucción de genomas RNA (virus RNA) o de mRNAs. El diseño y la aplicación efectiva de ribozimas con nuevas especificidades requiere un profundo conocimiento acerca de estas moléculas así como de sus mecanismos de reconocimiento e interacción con los RNAs substrato. En este trabajo se describen los RNAs catalíticos mejor caracterizados, así como las diferentes estrategias desarrolladas hasta el momento para la utilización de las ribozimas como inactivadores génicos.Ribozymes are RNA molecules endowed with catalytic activity, they are able to cataIyze chemical reactions inside the cells. In their natural environment, most of the described ribozymes carry out processing of other RNA molecule. In contrast to the protein ribonucleases, ribozymes exhibit a high-substrate specificity. This feature is the base for the potential use of ribozymes as specific gene suppressors and therefore to develop new therapeutic agents. The need of new therapeutic strategies is even more significant with the rising of diseases against which the existent drugs and therapies do not show the desirable efficiency. The social repercussions of some diseases of difficult or low probability of control (AIDS, Cancer, etc.), has made more acute the need of looking for altemative treatments. Among the experimental approaches that are being carried out is the direct action on the genetic information responsible of such diseases. On this line, several strategies have emerged in the past few years, mainly based on the use of nucleic acids as specific gene suppressors, e.g. antisense oligonucleotides, ribozymes and triplex helix. The ribozymes would act blocking the transmission of genetic information at the RNA level, mediating destruction of RNA genomes (RNA virus) or mRNAs. A deep understanding of the mechanisms used by these molecules for the recognition and interaction with their RNA substrates as well as of these molecules themselves is necessary for the design and effective application of ribozymes with new specificity. In this work we describe the best characterized catalytic RNAs, as well as the different strategies developed for their use as gene tic suppressors

The 3¿UTR of the West Nile Virus genomic RNA is a potential antiviral target site

The protein coding-information only represents a small portion of the genetic load of a living organism. It is well established that essential information codes functional RNAs, called non-coding RNAs (ncRNAs), which play key roles in the essential biological processes of the cell life. Many mRNAs also act as truly ncRNAs besides being translated into proteins. Therefore, the repertoire of potential drug targets to fight diseases goes beyond proteins. Viral RNA genomes encode all the information for completion of the infectious cycle. They are multifunctional molecules, which act as replication templates and mRNAs. Further, defined structural domains in viral RNA genomes play key functions for the completion of the viral cycle and the regulation of the essential processes; these domains have also been involved in virulence. The West Nile Virus (WNV) genome consists in a single stranded RNA molecule, which contains a single ORF flanked by untranslated regions (UTRs). The 3¿UTR is required for efficient translation, but the mechanisms involved in this regulation are still obscure. In this work, we show evidences that the WNV-3¿UTR specifically recruits the 40S ribosomal subunit. We have localized two potential binding sites of the 40S. Binding of the 40S induced conformational changes in highly conserved structural domains within the WNV-3¿UTR. Functional assays support the hypothesis that recruitment of the 40S particle by the 3¿UTR is required for an efficient translation. Interfering with the 40S recruitment, by targeting the WNV-3¿UTR binding sites, constitutes a potential antiviral strategy by the development of new therapeutic compounds