The great plasticity of RNA virus genomes allows them to perform different functions during the infectious cycle, helping viral populations adapt to novel molecular and cellular contexts, and to escape host defenses. It also contributes toward the development of resistance to antiviral drugs. These feats are achieved by the genome preserving a degree of variability while avoiding challenges to viral fitness. Genome variability can become a threat to viral survival if it reaches the error catastrophe limit (Schuster, 1993; Eigen, 2002), but RNA viruses have overcome this by storing information required for essential functions in discrete, highly conserved, genomic RNA structural domains. These complexly folded regions may overlap the nucleotide sequence coding for viral proteins. They play out their different biological roles (e.g., in replication, translation, or encapsidation) by directly recruiting viral and/or cellular factors, or by forming high-order regulatory structures via the establishment of long-range RNA–RNA interaction networks resulting in the formation of the complex global structures required for correct viral functioning. By means of this dynamic folding, the RNA genome can perform functions during the viral cycle other than simply coding for proteins (Romero-López and Berzal-Herranz, 2013).

Flavivirus spp. (from now on flaviviruses) belong to the family Flaviviridae. They are small (40–65 nm diameter), enveloped (icosahedral nucleocapsid) viruses with a positive single-stranded RNA genome. The genus includes important human pathogens responsible for ongoing/recurrent outbreaks of disease in areas where such diseases are not traditionally endemic; West Nile virus (WNV), dengue virus (DENV, perhaps the most important human pathogen of the genus) and Zika virus (ZIKV), for example, are all dramatically expanding their original geographic distribution. Other well-known flaviviruses include the causal agents of yellow fever (YFV), Japanese encephalitis (JEV), St. Louis encephalitis (SLEV), Murray Valley encephalitis (MVEV), or tick-borne encephalitis (TBEV) among of over 70 flaviviruses that have been identified. Some authors believe there could be over 2,000 left to discover (Pybus et al., 2002).

Most flaviviruses are transmitted to vertebrate hosts by the bite of haematophagous arthropods (thus classifying them within the heterogeneous group of arboviruses). Flaviviruses have traditionally been assigned to one of three clusters according to their arthropod vectors (Kuno et al., 1998; Cook and Holmes, 2006; Cook et al., 2012): mosquito-borne (MBFV), tick-borne (TBFV), and no-known-vector (NKV) flaviviruses (Table 1). These clusters can be further divided into clades and species. The members of the MBFV and TBFV clusters replicate in vertebrates and arthropods, while the NKV flaviviruses can be subdivided into two clades infecting solely bats or rodents, with no arthropod vector involved in the infective cycle. A fourth cluster, gathers together the insect-specific flaviviruses (ISFV), has recently been defined and characterized (Cook et al., 2012). It is the most divergent group and can be subdivided according to the mosquito host involved (mainly Aedes spp. and Culex spp.). ISFVs do not infect any vertebrate host (Table 1). Finally, Tamana bat virus (TABV), which infects exclusively mammalian cells, shows no serological relationship with any other flavivirus, and has only very distant phylogenetic relationships with them. Its taxonomic position, therefore, is not well defined (de Lamballerie et al., 2002; Roby et al., 2014).

Certainly, flaviviruses pose health problems for humans (and some other vertebrates) that may be associated with enormous social and economic costs. Over the last decade, the number of outbreaks of flavivirus-induced disease has increased all over the world. The main causes include the geographic expansion of their mosquito vectors, and increasing human travel to the areas of highest infection risk. They cause non-specific symptoms in the initial phase of infection in humans, which hinders their control, and as for other RNA viruses, no efficient therapeutic or immunoprophylactic strategies have been developed. The World Health Organization¹ and the Centers for Disease Control² therefore both cite flaviviruses as a global health threat.

The functional importance of the highly conserved structural genomic RNA domains in different RNA viruses (Romero-López and Berzal-Herranz, 2013) renders them potential therapeutic targets for new antiviral drugs. This review focuses on the role of the functionally active structural RNA domains identified in the flavivirus genome. Their mechanisms of action in the regulation of essential functions of the viral cycle are discussed, and a short overview is provided of the flavivirus subgenomic RNAs (sfRNAs). Recent advances in the development of novel therapeutic strategies entailing the use of nucleic acid-based agents to target RNA molecules are also described.

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 5′ end (m⁷GpppAmp) (Ray et al., 2006; Zhou et al., 2007; Saeedi and Geiss, 2013) but it lacks a polyA tail in the 3′ end (Wengler, 1981; Brinton et al., 1986). The RNA genome contains a single ORF flanked by untranslated regions (UTRs) (Castle et al., 1985; Wengler et al., 1985; Castle and Wengler, 1987). It serves as a messenger for the synthesis of a single polyprotein that is processed by viral and cellular proteases (Brinton, 2002) to yield 10 different products (Rice et al., 1985) (Figure 1B). The flanking UTRs are defined by discrete, functionally active structural RNA elements that play important roles in the viral cycle. These can be divided into essential partners in the infection process (e.g., promoters) and other elements not essential for viral RNA propagation but which help to regulate the processes involved. The functional RNA elements of all flaviviruses appear as highly conserved, complex folding regions, despite the lack of extensive sequence conservation across the Flavivirus genus (Brinton, 2014).

In addition to the accumulation of genomic RNA during flavivirus infection, subgenomic, non-coding flavivirus RNA molecules (sfRNAs) ranging from 300 to 500 nt-long accumulate in the cytoplasm (Urosevic et al., 1997; Lin et al., 2004; Pijlman et al., 2008). These molecules are the result of incomplete digestion of the viral genome by the host cell 5′–3′ exoribonuclease Xrn1, which cleaves the viral RNA but stalls at defined locations in the highly folded 3′UTR (Pijlman et al., 2008) (Figure 4). This resistance to Xrn1 activity is dependent on specific residues; these have been elucidated for WNV (Pijlman et al., 2008; Funk et al., 2010), YFV (Silva et al., 2010), DENV-2 (Chapman et al., 2014b), and MVEV (Chapman et al., 2014a), and are confirmed to be conserved across flaviviruses (Chapman et al., 2014a). Such residues share a common structural environment defined by a three-way junction and a characteristic and conserved pseudoknot, PK1 (Pijlman et al., 2008; Chapman et al., 2014b) (Figures 3, 4), which is essential for sfRNA generation. In the DENV and JEV groups (Table 1), this structure has been located within the SL-I and SL-II of Domain I of the 3′UTR, respectively (Pijlman et al., 2008; Funk et al., 2010) (Figures 4A,B), while in YFV the single SL (SL-E) provides the stalling point (Silva et al., 2010) (Figure 4C). In MBFVs, it has been reported that the abrogation of PK1 leads to the production of shorter species of sfRNAs derived from the Xrn1 stalling at the downstream pseudoknot structures PK2 [SL-II in DENV-2 (Figure 4B), SL-IV in JEV (Figure 4A) and ψ-DB in YFV (Figure 4C)] and/or PK3 [5′DB in JEV and DENV and DB in YFV (Figure 4)] (Pijlman et al., 2008; Funk et al., 2010; Chapman et al., 2014a). Consecutive pseudoknots therefore appear to act as security or check points to assess the production of sfRNAs. The three-way junction organizes the three-dimensional folding by bringing the basal stem and the 3′ apical loop of the structure close together, yielding a ring-like topology with the free 5′ end inside it, as determined by X-ray crystallography (Chapman et al., 2014a). Thus, rather than providing a simple unfolding mechanism, Xrn1 turns the ring inside-out to provide access to the susceptible residues at the 5′ end. This architecture may also be responsible for the selection of directionality during extension by viral polymerase (Chapman et al., 2014a).

From a functional point of view, full-length sfRNAs play important roles in regulating the switch between translation and replication during the infectious cycle (Lin et al., 2004). They promote cytopathic effects and pathogenicity in mice (Pijlman et al., 2008; Funk et al., 2010; Chapman et al., 2014a; Liu et al., 2014) and they disrupt the generation of a proper immune response at different levels, while shortened sfRNA species lead to attenuated viral forms. In particular, full-length sfRNAs inhibit the antiviral activity of IFN-α/β by an unknown mechanism (Schuessler et al., 2012), as well as that of the antiviral RNAi pathway, probably by acting as Dicer decoy substrates (Schnettler et al., 2012). Intrinsic to sfRNA formation, Xrn1 function is inhibited and, thus, endogenous mRNAs are accumulated (Moon et al., 2012). Moreover, DENV-2 sfRNA has been shown to interact with stress granules (Bidet and Garcia-Blanco, 2014). Detailed information on the roles of sfRNAs is provided in recent reviews (Roby et al., 2014; Clarke et al., 2015; Charley and Wilusz, 2016).

The acquisition of a circular conformation in viral RNA genomes is a successful strategy that provides important advantages in the completion of the infective cycle. First of all, it efficiently ensures the propagation of undamaged, full-length genomes (Hahn et al., 1987). Further, the initiation of protein synthesis and the replication process is governed by the establishment of a closed loop topology. Transitions between different steps of the viral cycles are directly dependent on the existence of complex networks of RNA–RNA contacts (Romero-López and Berzal-Herranz, 2013).

The acquisition of the circular topology is mediated by direct, long distance RNA–RNA interactions between different complementary sequence motifs at the 5′ and 3′ ends of the genome (Figure 4). Such interactions have been probed by psoralen/UV crosslinking assays (You et al., 2001), electrophoretic mobility shift assays (Alvarez et al., 2005; Zhang et al., 2008a), atomic force microscopy (Alvarez et al., 2005), and structure probing (Dong et al., 2008; Polacek et al., 2009a). Though some of the complementary sequence motifs involved in genome cyclization show low conservation rates across the flaviviruses, the circularization mechanism is ubiquitous and required for flaviviral propagation (Khromykh et al., 2001; Song et al., 2008).

In MBFV, at least three pairs of sequence motifs have been shown to participate in the cyclization process (Figures 4, 5). These include:

Data derived from structural, phylogenetic and functional analyses have allowed a theoretical model of the genomic cyclization process to be proposed (Friebe et al., 2011). Accordingly, the latter is initiated via the interaction between the 5′ and the 3′CYC motifs (Polacek et al., 2009a). The duplex then further extends via the DAR contacts which “open” the sHP element (Friebe and Harris, 2010; Friebe et al., 2011). Additional UAR-mediated interactions help to unwind the basal portion of the 3′SL domain to further promote conformational rearrangements within the 3′ end of the viral genome. Recently, a cis-acting element present in the capsid coding sequence of DENV was found to interact with 5′DB at the 3′UTR, forming a PK structural element. This interaction was proven to have a different effect on viral RNA replication in mosquito and mammalian cells (de Borba et al., 2015).

Tick-borne genomic cyclization occurs by the formation of at least the two long-distance interactions 5′–3′CSA and 5′–3′CSB (Mandl et al., 1993; Khromykh et al., 2001). The sequence motifs involved in these interactions are unrelated to those in MDFVs. The 5′–3′CSA interaction is the equivalent of the 5′–3′UAR interaction in MBFV, despite being located at different positions (Mandl et al., 1993) and is also crucial for RNA synthesis (Khromykh et al., 2001). The 5′CSB and 3′CSB motifs are located at genomic positions similar to 5′CYC and 3′CYC in MBFVs, but their interaction is not essential in TBFV replication (Kofler et al., 2006). Genome circularization in TBFVs is also enhanced by a kissing-loop contact involving two stem-loops, 5′SL6 and 3′SL3, located in the capsid coding region at the 5′ and 3′ ends, respectively (Tsetsarkin et al., 2016). The 5′SL6 domain was previously shown to be required for efficient replication (Tuplin et al., 2011).

In NKV flaviviruses, two interactions have been predicted involved in genome cyclization. The first involves a sequence motif located upstream of the AUG start codon and a complementary one within the 3′SL; the second is established between a motif within the capsid coding region and the corresponding counterpart upstream of the 3′SL (Leyssen et al., 2002).

In addition to the RNA–RNA interactions, genomic cyclization might be stabilized by viral and host protein factors recruited by different genomic RNA structural domains (Blackwell and Brinton, 1997; Ta and Vrati, 2000; De Nova-Ocampo et al., 2002; Garcia-Montalvo et al., 2004). These factors include the La protein (De Nova-Ocampo et al., 2002; Vashist et al., 2009), polypyrimidine-tract binding protein (PTB) (De Nova-Ocampo et al., 2002; Kim and Jeong, 2006) or translation elongation factor 1α (eEF-1α) (De Nova-Ocampo et al., 2002). Interestingly, such proteins are involved, at different extent, with the progression of the translation process, which points to cyclization as a feasible strategy to control viral protein synthesis. Different RNA helicases as FBP1 (far upstream element-binding protein), DDX3, DDX5, and DDX6 have also been proved to bind to both the 5′ and the 3′UTRs of the flavivirus genome, and affect replication in opposite ways (Chien et al., 2011; Ward et al., 2011; Li et al., 2013, 2014). These findings demonstrate that the control of the cyclization event is mediated by the RNA recruitment of host factors. They also show that flaviviruses can use the genome cyclation for regulating transitions between different steps of the infective cycle. Finally, host proteins related to mRNA splicing such as hnRNPA2 (Katoh et al., 2011) or Lsm1 (Dong et al., 2015) interact with the cyclization sequence motifs and/or with functional RNA domains located in the untranslated regions. The recruitment of these proteins is required for and efficient viral replication process though their molecular mechanism is still unknown.

A proper balance between linear and circular forms of the genome is required to ensure the initiation of plus-strand RNA synthesis, encapsidation, and even the switch from translation to replication. This is because sequences involved in the cyclization process overlap with essential structural domains that cannot be formed in the circular topology. In this context, the thermodynamic stability of the single structural domains is critical for efficient transition from one conformation to another. Thus, mutations that stabilize the circular or the linear form spontaneously revert to “less stable” architectures (Clyde et al., 2008; Villordo et al., 2010; Iglesias et al., 2011). The genome cyclization operates as a control system regulating the progression of the flavivirus infective cycle.

The information and functions encoded in structural genomic RNA domains render interference with the proper folding of these elements a candidate means of interfering with viral propagation. In this context, the use of nucleic acids as therapeutic agents is of growing interest. The development of any such therapy, however, must overcome a number of challenges, including the maintenance of the stability of the nucleic acid agents and efficient delivery to the target cell. These problems have been largely addressed by combinatorial chemistry, and a range of chemical nucleotide substitutions are now available. The use of chemically modified oligonucleotides has resulted in the improvement of the pharmacodynamic and pharmacokinetic properties of these antiviral nucleic acids-based antiviral agents (Haasnoot and Berkhout, 2009).

In recent decades, pioneering work into antisense oligonucleotide-based inhibitors has laid the ground for the design of thus-based antiviral compounds (Haasnoot and Berkhout, 2009). Antisense oligonucleotides are short nucleic acids with sequences complementary to those of their targets. They interfere with the function of essential regions within RNA molecules by different mechanisms. The first attempt to design antisense oligonucleotides against a flavivirus RNA genome used the DENV genome as a model (Raviprakash et al., 1995). A set of propynil-phosphorothioate-modified antisense oligonucleotides targeting five regions throughout the viral RNA showed that interfering with the sequence motif surrounding the translation initiation codon and the SL-IV domain within the 3′UTR was an effective antiviral strategy in cell culture.

These preliminary but promising results in DENV prompted further efforts to develop other antisense oligonucleotides against other flaviviruses, such as WNV. The use of phosphorodiamidate morpholino oligomers (PMOs) as potential anti-WNV drugs has been reported (Deas et al., 2005). Two oligonucleotides targeting the extreme 5′ end of the viral genome and the 3′CYC motif were found to efficiently interfere with viral translation and replication. Further, the conjugation of these PMOs at their 5′ end with an arginine-rich peptide (PPMO) improved uptake by cells, yielding an agent capable of strongly suppressing the viral cycle. It was suggested that the high conservation rate of the targeted regions allowed the design of sets of PPMOs targeting a spectrum of related flaviviruses belonging to the JEV group (Deas et al., 2007). This could lead to important advances in the use of nucleic acid-based compounds, not only as inhibitory molecules but also as biotechnological tools for the detection of different viruses in biological samples. In addition, modified PMOs could help us understand the molecular mechanisms underlying the function of the targeted structural RNA domains.

Cellular RNA interference (RNAi) has also been widely examined in recent years as a means of generating novel antiviral RNA molecules. This strategy is based on the design of short, double-stranded RNA molecules (the so-called small interfering RNAs or siRNAs), which are loaded into the RNA-induced silencing complex (RISC). The sense strand of the duplex then guides the complex to the target region, where it base-pairs fully to induce degradation of the target RNA molecule. As antisense oligonucleotides, siRNAs can be chemically produced or endogenously synthesized from appropriate expression vectors. Numerous authors have reported the use of siRNAs – both in cell culture and in infected mice – against the coding region of the WNV genome (Mccown et al., 2003; Bai et al., 2005; Geiss et al., 2005; Kumar et al., 2006; Ong et al., 2006, 2008; Yang et al., 2008) and the conserved functional domains within the 3′UTR (Zhang et al., 2008b; Anthony et al., 2009). The results confirm the potential of this strategy in the development of new antiviral compounds.

The use of RNA or DNA aptamers (short oligonucleotides that efficiently and specifically bind to a target molecule) represents another promising strategy for developing antiviral agents against flaviviruses. They also provide an interesting means of developing molecular tools for deciphering the functional role of genomic structural elements, and therefore the identification of potential therapeutic targets. This has already been shown for other, closely related viruses such as HCV (Marton et al., 2011, 2013; Fernández-Sanlés et al., 2015) as well as non-related viruses such as HIV (Sánchez-Luque et al., 2014). Aptamers can be chemically modified quite easily to increase their stability and improve their efficiency.

The successful clinical use of any of the above strategies is conditioned by the appearance of resistant mutants. In fact, WNV particles resistant to PMOs targeting the conserved 3′UAR sequence motif have already been isolated (Zhang et al., 2008b). They contained a single nucleotide mutation in the target sequence that impaired or weakened the PMO interaction, while the 5′UAR–3′UAR base-pairing was restored by the selection of a compensatory mutation. Novel strategies are therefore required, based on combining antiviral compounds with different specificities, including recognition of specific structural features, and even different mechanisms of action. In this context, the use of antisense oligonucleotides, siRNAs and other nucleic acid molecules (e.g., aptamers) in combination with other drugs, such as interferon or neutralizing antibodies, may provide effective and potent antiviral cocktails.

The acquisition of compact genomes was an important evolutionary achievement of RNA viruses; these genomes can store all the information required for the completion of the infectious cycle in reduced packages. This is possible due to the existence of a supracoding system beyond the nucleotide sequence, defined by discrete, folded domains, and higher-order structures. These elements operate both alone and in combination to create complex networks of contacts that regulate multiple steps of the viral cycle, and to recruit host and viral factors. Understanding how host–virus interactions shape viral evolution will help to elucidate the factors that govern the emergence of new viruses and the expansion of already known RNA viral pathogens. The lack of technics or experimental approaches to determine the RNA structure and to analyze the kinetics of RNA–RNA interactions in cell culture, together with the lack of experimental strategies to specifically interfere with the folding of the RNA genomic elements, represent an important limitation for understanding their function in the viral cycle. Importantly, the phylogenetic conservation of the genomic RNA structural domains and their interactions across members of Flavivirus, provide alternative and complementary potential targets to the viral proteins for novel antiviral compounds. Advances made in the field of nucleic acid synthesis have provided excellent candidate molecules for fighting RNA viruses by interfering with the essential functions performed by their genomic functional domains. Different pharmaceutical companies are now investigating the potential of nucleic acid therapeutic strategies, assessing long-term antiviral responses and trying to minimize secondary effects.

All authors participated in writing the manuscript (led by CR-L and AB-H), commented upon, and approved its final version. AF-S and PR-M prepared the figures.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.