SUMMARY OF RESEARCH

STRUCTURE/FUNCTION OF CONSERVED GENOMIC RNA DOMAINS IN RNA VIRUS.
The research group is co-leaded by Alfredo Berzal-Herranz and Cristina Romero-López.

The lack of knowledge about the functions performed by RNA in living organisms has made that RNA has been long underrated as simply player in the transmission of the genetic information from DNA to proteins. The identification of new classes of RNAs, grouped under the name of non-coding RNAs (ncRNAs), and the elucidation of the functions they perform have placed RNA at the center of biological processes essential for life. The ncRNAs, exert a function by themselves, are functional RNAs, demonstrating the existence of functional genetic information beyond the one that encodes proteins. Their study has also revealed the existence of an additional system of regulation of gene expression that involves different types of non-coding RNAs. The accumulation of information on biological activity of RNA molecules has allowed establishing the molecular basis of different pathological processes, as well as to determine that errors in the RNA metabolism, or the loss of function of RNA molecules are the cause of numerous diseases or pathologies of great sanitary importance.

Viral RNA genomes constitute a particular type of functional RNAs. They are compact genomes that contain all the required information for the completion of the viral infective cycle. To achieve this in a small genome, RNA viruses have developed a system for storing and encoding genetic information beyond the nucleotide sequence that is independent and complementary to the information that encodes proteins. The information is stored in the form of discrete structural elements responsible for essential functions. These structural elements are highly conserved, and distributed throughout the viral genome, despite the existence of genomic regions with a greater concentration of functional RNA elements have been defined, for example non-translatable regions or UTRs. RNA elements exert their function by establishing long-distance RNA-RNA interactions between them and / or by recruiting cellular or viral factors. Thus their functionality is achieved by the establishment of a complex global structure specific of any of the essential functions, e.g., replication, translation, encapsidation.

The hepatitis C virus (HCV) genome is an approximately 9,600 nt-long positive, single-stranded RNA molecule that codes for a single open reading frame flanked by untranslated regions (UTRs). The UTRs are highly conserved in sequence and structure between the different viral isolates, which contrasts with the great genetic variability of the viral genome. In early infection, HCV protein synthesis is initiated by an internal ribosome entry site (IRES)-dependent mechanism different to the cap-dependent method used for the translation of most cellular mRNAs. The IRES element is mostly located at the 5'UTR and spans a short stretch of the core coding sequence. It folds into a well conserved compact structure that contains a subset of well characterized domains and subdomains. These structural elements guide the direct recruitment of the 40S ribosomal subunit and the further binding of eIF3 which aids the incorporation of the ternary complex eIF2-tRNAi Met and the joining of the 60S subunit. This mechanism minimizes protein factor requirements and simplifies the pathway for the assembly of the translationally active 80S ribosome. Further, the presence of domains at the 3' end of the genomic RNA influences translation efficiency, most likely by the acquisition of a closed-loop topology resembling the circular structure adopted by cellular mRNAs. Similarly, the 3'UTR contains several highly conserved functional RNA domains required for replication, translation and viral genome dimerization.

In addition to RNA domains being located in the untranslated regions, numerous cis-acting signals have been identified in the ORFs of RNA viruses. These RNA elements are bifunctional, they simultaneously hold protein coding information and perform specific functions. Thus in the HCV RNA genome a conserved structural region within the 3' end of the viral polymerase (NS5B) coding sequence termed as CRE (cis-acting replication element) has been defined as essential for viral replication. CRE is composed of three stem-loops, being the central one, 5BSL3.2, indispensable for HCV propagation. The 5BSL3.2 is a 48 nt-long RNA domain that folds into a stable stem closed by a 12 nt-long loop. The stem is interrupted by an 8 nt-long bulge, which we described that is involved in a long distance RNA/RNA interaction with the apical loop of the IRES' subdomain IIId at the 5'UTR. This interaction that takes place in the absence of proteins we also demonstrated that is involved in the regulation of the IRES activity. Further studies have showed the existence of a complex network of RNA/RNA interactions involving at least the IRES, the 3'UTR and the CRE regions. Our results have allowed proposing that the 5BSL3.2 domain plays a central role in this network of interactions regulating the switching between the different stages of the viral cycle.

HCV is the prototype of the genus Hepacivirus a member of the family Flaviviridae. The largest genus of this family is the genus Flavivirus, which includes important human pathogens like dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), yellow fever (YFV), among others. The flaviviral genome consists on a positive-sense single-stranded RNA molecule approximately 11,000 nt-long, varying depending on the species. It bears a type 1 cap structure at its 50 end (m7GpppAmp) but it lacks a polyA tail in the 3' end. The RNA genome contains a single ORF flanked by untranslated regions (UTRs). It serves as a messenger for the synthesis of a single polyprotein that is processed by viral and cellular proteases to yield 10 different products. The flanking UTRs are defined by discrete, functionally active structural RNA elements that play important roles in the viral cycle.

An efficient strategy to deepen the characterization of the functional RNA domains is the use of nucleic acids as tools. In particular RNA molecules have shown to efficiently interfere with the structure of RNA motifs and to compete out RNA/RNA interactions, therefore to interfere with the functionality of RNA domains. Among these tools stand out for their great potential the aptamers. Aptamers are RNA or DNA oligonucleotides that specifically and efficiently bind to a ligand molecule. We have isolated collections of aptamers selected against RNA domains of the HCV and WNV genomes. Their characterization has allowed us identifying genomic sequences and structural domains as potential antiviral targets.

Our research line is the study or the structure/function of viral RNA domains. Currently we are focused in the characterization of these domains in the genomes of the HCV and WNV. We also want to explore its potential as antiviral or therapeutic targets. In parallel, we address the development of general-purpose strategies for the design and application of RNA molecules as efficient tools in biotechnology and basic and biomedical research.

FUNDING AGENCIES LAST 5 YEARS

- IMPLICACIONES FUNCIONALES DE INTERACCIONES RNA/RNA EN GENOMAS DE FLAVIVIRUS. PROYECTO, PN2019 - Proyectos I+D+i «Retos Investigación», Ref: PID2019-104018RB-I00, (2020 - 2023).

PUBLICATIONS LAST 5 YEARS

-Berzal-Herranz, A.; Berzal-Herranz, B.; Ramos-Lorente, S.E.; Romero-López, C., The Genomic 3′ UTR of Flaviviruses Is a Translation Initiation Enhancer, International Journal of Molecular Sciences, 2022, Vol. 23: 15-8604, ARTÍCULO, Id:903474

-Cristina Romero-López; Sara Esther Ramos-Lorente; Alfredo Berzal-Herranz, In vitro methods to decipher the structure of viral RNA genomes, Pharmaceuticals, 2021, Vol. 14: 11-1192, ARTÍCULO, Id:862847

-Romero-lópez, C.; Berzal-herranz, A.; Martínez-guitarte, J.L.; de la Fuente, M., Criter¿a: A novel temperature¿dependent noncoding rna switch in the telomeric transcriptome of chironomus riparius, International Journal of Molecular Sciences, 2021, Vol. 22: 19-10310, ARTÍCULO, Id:854536

-Ramos-Lorente, S.; Romero-López, C.; Berzal-Herranz, A., Information encoded by the flavivirus genomes beyond the nucleotide sequence, International Journal of Molecular Sciences, 2021, Vol. 22: 7-3738, ARTÍCULO DE REVISIÓN, Id:846358

-Berzal-Herranz, A.; Romero-López, C., Two examples of RNA aptamers with antiviral activity. Are aptamers the wished antiviral drugs?, Pharmaceuticals, 2020, Vol. 13: 1-10, ARTÍCULO, Id:812178

-Romero-López, C.; Berzal-Herranz, A., The role of the RNA-RNA interactome in the hepatitis C virus life cycle, International Journal of Molecular Sciences, 2020, Vol. 21: 4-1479, ARTÍCULO DE REVISIÓN, Id:805178

-Castillo-Martínez, J.; Ovejero, T.; Romero-López, C.; Sanmartín, I.; Berzal-Herranz, B.; Oltra, E.; Berzal-Herranz, A.; Gallego, J., Structure and function analysis of the essential 3′X domain of hepatitis C virus, RNA, 2020, Vol. 26: 186-198, ARTÍCULO, Id:804552

-Berzal-Herranz, A.; Romero-López, C.; Berzal-Herranz, B.; Ramos-Lorente, S., Potential of the other genetic information coded by the viral RNA genomes as antiviral target, Pharmaceuticals, 2019, Vol. 12: 1-38, ARTÍCULO DE REVISIÓN, Id:762931

-Miras, M.; Rodríguez-Hernández, A.M.; Romero-López, C.; Berzal-Herranz, A.; Colchero, J.; Aranda M.A.; Truniger, V., A dual interaction between the 5¿- and 3¿-ends of the Melon necrotic spot virus (MNSV) RNA genome is required for efficient cap-independent translation, Frontiers in Plant Science, 2018, Vol. 9: 1-625, ARTÍCULO, Id:748167

-Romero-López, C.; Ríos-Marco, P.; Berzal-Herranz, B.; Berzal-Herranz, A., The HCV genome domains 5BSL3.1 and 5BSL3.3 act as managers of translation, Scientific Reports, 2018, Vol. 8: 1-16101, ARTÍCULO, Id:735054

DOCTORAL THESES LAST 5 YEARS