The biological function of RNA has, for long time, been considered to be restricted to its role in the transmission of genetic information from DNA to proteins. The discovery in the early 1980′s that the RNA is capable of catalyzing chemical reactions [1
] led to the reconsideration of this dogma and aroused the interest in unraveling the molecular mechanisms of the catalytic activity of RNA molecules and, later, in deciphering the unknown functional roles of RNA within cells. Currently, it is widely accepted that RNA plays a central role in many biological processes in all living organisms. An increasing number of functional RNA molecules, so-called “noncoding RNAs”, has been identified. Although they do not encode proteins, they encode information that is also essential for life. In fact, errors in RNA metabolism or the loss of function of RNA molecules are the cause of pathological processes of sanitary importance. Therefore, determining the function of certain RNA molecules has made it possible to clarify the molecular mechanisms of different biological processes. The genomes of RNA viruses constitute a particular class of functional RNA molecules. Viral RNA genomes are compact entities that carry all the information that the virus requires to complete the infectious cycle. Besides acting as a replication and translation template, viral RNA genomes play several essential functions for the completion of the viral cycle and their regulation. To achieve all these functions, they have developed an information coding system that complements and overlaps the protein coding one. This information coding system uses discrete RNA units that fold into their specific structures for storing information. These structural units are, in fact, functional genomic RNA domains and are highly conserved among the viral population. They can be grouped in complex folded RNA regions that interact among them to achieve the proper functioning. Thus, the structure of the entire genome has to be preserved for the efficient viral propagation. This structural conservation contrasts with the great sequence variability of the viral RNA genomes, which helps the viral populations to adapt to novel molecular and cellular contexts and to escape host defenses. It also contributes toward the development of resistance to antiviral drugs. The genome variability is, therefore, limited by the preservation of the genome structure. The precise equilibrium between the sequence variability and structure preservation determines the success of the RNA viral populations.
Deciphering the mechanisms that underlie each of the functions performed by functional genomic RNA domains and understanding the molecular strategies used by RNA genomes to contain all the information necessary for each of them are scientific problems of great relevance and interest. Elucidating these mechanisms would provide information of enormous importance to address the control of infections caused by RNA viruses. The current information indicates that structural RNA domains play out their different biological roles (e.g., in replication, translation, or encapsidation) by directly recruiting viral and/or cellular factors and/or by forming high-order structures via the establishment of long-range RNA–RNA interaction networks (for a review, see Reference [3
The essentiality of these genomic RNA domains for the virus makes them excellent candidates as targets for the development of antiviral strategies. Drugs aimed at interfering with their function either by blocking their interactions with viral or cellular factors or by impeding their proper folding may seriously compromise the viral viability. Therefore, they should be considered promising antiviral agents. Aptamers specifically recognize and efficiently bind to structural elements within a target molecule, being among the most promising molecules for interfering with the function of structural domains of viral RNA genomes.
Members of the family Flaviviridae bear a positive single-stranded RNA genome. They are responsible of worldwide public health problems. The family comprises three genera: Flavivirus, that includes important human pathogens like Dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), or Zika virus (ZIKV), among many others; Pestivirus, represented by the virus responsible of the Bovine viral diarrhea (BVDV) or the classic swine fever (CSFV); and Hepacivirus, with the hepatitis C virus (HCV) as the most significant member. Their genome contains multiple highly conserved structural RNA domains, which store essential information for the completion of the viral cycle. This review focuses on the HCV genome, paying special attention to the work carried out in our laboratory aimed at the structural and functional characterization of genomic RNA domains. The second part of the review summarizes the main strategies we have developed seeking aptamers targeted against specific genomic RNA domains of the HCV and HIV genomes.
2. HCV Genomic Functional RNA Domains
HCV is likely the member of the family Flaviviridae
that has attracted major efforts to understand the molecular mechanisms that govern its infective cycle. Its genome is a 9600-nt-long single-stranded RNA molecule, which codes for a single open reading frame (ORF) flanked by highly conserved untranslated regions (UTRs). Although the UTRs comprise numerous conserved structural/functional RNA elements that play essential roles for the consecution of the viral cycle, these RNA elements are also distributed throughout the coding region (Figure 1
). The 5′ UTR is a highly conserved and complexly folded region that is mainly occupied by an internal ribosome entry site (IRES) that spans around 30 nt within the viral capsid coding region [4
]. The IRES directs the recruitment of the 40S ribosomal subunit in the absence of a cap structure and initiates the viral protein synthesis [6
]. It is folded into four structural domains that comprise a set of highly conserved subdomains, each with essential roles in the ribosome recruitment and translation initiation. In addition, 5′ UTR structural domains are essential for viral replication and infectivity [6
]. The initiation of replication takes places at the 3′ UTR [8
]. It is a highly conserved 200–250-nt-long region, which contains several functional RNA elements grouped into highly conserved domains required for efficient viral replication. The 3′ UTR also plays an important role in the viral translation, regulating the IRES activity [13
The use of complementary experimental approaches (bioinformatics tools, secondary structure mapping, and genetic strategies) has provided a good overview of the HCV genome folding [15
]. It has allowed the identification of up to 20 highly conserved structural elements among different viral isolates throughout the ORF of the HCV genome (Figure 1
). This high structural conservation within a genome with a high rate of sequence variability indicates that each structural unit codes important information for the efficiency of the virus infective cycle. In contrast to the genomic UTRs, information about the structure and function of the ORF structural elements is scarce. Among them, the so-called CRE, cis-replication element, is probably the one that has attracted more attention (Figure 1
). The CRE is defined by three stable stem-loops known as 5BSL3.1, 5BSL3.2, and 5BSL3.3 located at the 3′ end of the protein coding region [24
]. The central domain, 5BSL3.2, is absolutely indispensable for HCV propagation, acting as an essential element for viral replication. Further, it has been shown that CRE is a negative regulator of the HCV IRES-dependent translation [26
3. Long-Range Genomic RNA–RNA Interactions
Specific structural elements of the 5′ and 3′ ends of the HCV genome are involved in the viral replication and translation. This implies the existence of a communication between the two genomic ends. It was commonly accepted that the acquisition of a genomic circular conformation was mediated by the recruitment at both ends of the cellular and viral proteins, as it was demonstrated for the recruitment of IRES-activity stimulating proteins by the genomic HCV 3′ UTR [13
]. We questioned whether the conserved structural RNA elements located in the two ends of the HCV genome were directly involved in the formation of the circular conformation. We first tested the ability of two independent RNA fragments comprising the 5′ or the 3′ genomic ends to bind with each other. Interestingly both RNA genomic ends form a stable complex in in vitro assays [31
]. Further characterization allowed us to demonstrate that this interaction takes place between the two essential structural RNA elements IIId within domain III of the IRES and the 5BSL3.2 within the CRE (Figure 2
). It responds to a kissing ALIL (apical loop–internal loop) interaction. This result represented the first evidence of a long-range RNA–RNA interaction involving the two genomic ends, supporting a protein-independent circularization of the HCV RNA genome. The functional characterization of this interaction allowed us to conclude that it actually occurs in vivo, being the CRE a negative regulator of the HCV IRES activity [26
] (Figure 2
). The use of CRE mutants depleted of specific structural units demonstrated that this translation regulation activity is mainly restricted to the central structural unit 5BSL3.2 [26
The 5BSL3.2 domain is also involved in an Apical loop–Apical loop (ALAL) long-distance interaction with the highly conserved 3′SLII domain included in the 3′X tail region at the 3′ UTR [33
] (Figure 2
). This interaction promotes viral replication. A third functional long-range interaction has been described, which involves the internal loop of the 5BSL3.2 domain and the upstream Alt region [17
] (Figure 2
A functional RNA–RNA interaction seems to take place between domain VI, at the 5′ end of the protein coding region, and the highly conserved linker sequence between domains I and II within the 5′ UTR. This interaction has been proposed to participate in the regulation of both the viral replication and translation processes [20
]. Interestingly, the interacting sequence within the interdomain region overlaps with the interaction site of the liver-specific micro RNA miR-122, which has been shown to be involved in viral replication and translation [41
]. It has been proposed that the interaction of miR-122 would induce a conformational switch of the 5′ genomic end encompassing the IRES, which would be responsible for the regulation of the activity of this region [39
All together, these results probe the existence of a network of long-range RNA–RNA interactions. This network involves essential structural genomic RNA domains of at least the IRES, CRE, and the genomic 3′ UTR X-tail region and governs the regulation of the essential viral processes (reviewed in References [27
]; Figure 2
), in which the CRE 5BSL3.2 domain seems to be the conductor of the orchestra directing the regulatory interactions of all other structural elements.
To understand the molecular mechanism by which long-distance interactions may regulate viral processes, a detailed structural analysis at the nucleotide level of the HCV genome was carried out. For this purpose, a combination of chemical probing methods, SHAPE (Selective 2′-hydroxyl acylation and primer extension)-based technology analysis and accessibility using oligonucleotide microarrays, was used. The results of this analysis allowed us to conclude that interactions between elements of both ends of the viral genomes promote to each other their structural tuning at the secondary and tertiary levels, which undoubtedly leads to the mutual modulation of their functionality [32
] (Figure 3
). Each one tunes the structural conformation of essential domains of the other end of the genome. These structural modifications correlate well with the functional regulation observed. Thus, modulation of the translation efficiency at the 5′ genomic end by the 3′ end is achieved by promoting the conformational fine-tuning of the IRES-essential domains III and IV, while the 5′ genomic end promotes significant structural changes at the 3′ genomic end, mainly at the 3′X tail region. The latest could account for variations in the translational and replicational efficiencies.
Besides intramolecular genomic RNA–RNA interactions, intermolecular ones have also been described. Thus, the dimerization of the HCV genome is initiated at a highly conserved palindromic sequence named DLS (Dimerization Linkage Sequence) located at the 3′SLII element of the 3′X tail region [45
] (Figure 2
). The 3′X tail region folds into two alternative conformations [46
]: a three stem-loop (3′SLI-3′SLII-3′SLIII) [48
] and a two stem-loop (3′SLI-3′SLII’) conformers, being the latest dimerization competent one, which exposes the DLS in the apical portion of the stem-loop SLII’. A functional analysis of the 3′X tail in the presence of the IRES and/or the CRE allowed us to demonstrate that genomic dimerization is modulated by long-distance elements [49
]. This confirms the functionality of the described long-distance RNA–RNA interactions involving the CRE-3′X tail and IRES-3′X tail.
All together, these data demonstrate the existence of a dynamic network of RNA–RNA contacts that tune the structural conformation of essential distal RNA elements, resulting in the regulation of the different stages of the viral cycle. Our data support that the 5BSL3.2 domain operates at the core of this regulatory system.