Predicting how RNA virus populations might evolve – a major public health goal – is a challenging task. RNA viruses, such as hepatitis C virus (HCV), replicate by virtue of a relatively low fidelity viral genome-encoded RNA-dependent RNA polymerase. It has been calculated that for each round of replication the mutation rate in HCV reaches a maximum of 1.3 nucleotides (nt) (Bartenschlager and Lohmann, 2000; Cuevas et al., 2009; Ribeiro et al., 2012). The variation introduced generates a dynamic genome pool composed of different but closely related sequences, referred to as a quasispecies (Martell et al., 1992). Quasispecies dynamics involve the constant sampling and selection of mutations that improve viral fitness. Such mutations are critical for escaping the host immune response, for drug resistance, the infection of new host species, and disease emergence. The acquisition of new mutations by viral genomes therefore provides a background for the action of natural selection. The magnitude of the effect of the acquired mutations is dependent on the environment: changes can be favorable for the infection in a given host but may have important fitness costs in a different environment, even within the same infected individual. This phenomenon creates a complex picture for the prediction of the disease severity and impedes the development of novel therapies (Manrubia and Lazaro, 2016; Perales and Domingo, 2016).

The balance between sequence promiscuity and functional conservation in viral genomes is made possible via the use of highly structured genomic regions that can absorb nucleotide variations – as long as these do not alter their active conformation. These are organized as discrete, in cis-functional RNA domains that operate in an interconnected manner to perform functions essential to the execution of the viral cycle (Romero-López and Berzal-Herranz, 2013). Such domains are considered information-carrying units beyond the nucleotide sequence. The study of the cis-acting functional elements, including their localization, sequence and structural conservation, is key to understanding viral infections at the molecular level. In addition, the essential role of genomic functional RNA domains in viral propagation and persistence makes them potential therapeutic targets.

Hepatitis C virus infection, which has a global prevalence of 2.8% (more than 185 million people are infected worldwide; Mohd Hanafiah et al., 2013), causes severe chronic disease that in many patients may eventually make liver transplantation necessary (Hoofnagle, 2002). Currently, the standard of care (SOC) involves the use of pegylated α-interferon combined with the modified nucleosides ribavirin and sofosbuvir (Kowdley et al., 2013; Lawitz et al., 2013). Unfortunately, interferon is not always well tolerated; in addition, the variability of the viral genome prevents a sustained therapeutic response. As mutants resistant to the current SOC arise, new targets and therapeutic agents must be ready. In this context, recent advances in the development of direct antiviral agents (DAAs) have prompted the progress of a new combined therapy consisting in the use of sofosbuvir plus the NS5A inhibitor velpatasvir and the NS3-NS4A inhibitor voxilaprevir (see below) (Bourliere et al., 2017). The two major benefits of this new strategy rely, on one hand, in the lack of α-interferon in this drug cocktail, which significantly reduces the secondary effects of the treatment; on the other, both velpatasvir and voxilaprevir operate as pangenotypic inhibitors, thus simplifying the customization of the therapy.

Hepatitis C virus is a member of the family Flaviviridae and the genus Hepacivirus. Based on the phylogenetic analysis of genomic sequences, up to seven genotypes have been described for HCV showing more than 30% divergence at the nucleotide level. Closely related sets of subtypes and isolates have been identified as well (Smith et al., 2014). The viral genome is a positive RNA molecule of ∼9.6 kb that encodes a single ORF flanked by highly conserved untranslated regions (5′ and 3′UTR; Figure 1) (Choo et al., 1989). Viral proteins are produced as a single polypeptide that is co- and post-translationally processed by viral and cellular proteases to yield structural (core, E1, and E2) and non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B; Figure 1) (Grakoui et al., 1993). It is noteworthy that genetic variation does not occur evenly over the viral genome. On average, the complete sequence genome differs by about 31–33% per position (Smith et al., 2014), but only ∼10% of the entire viral genome is susceptible to positive selection (Patino-Galindo and Gonzalez-Candelas, 2017). The affected regions are intimately related to genotype prevalence (Messina et al., 2015). The 5′ end of the HCV genome and the viral capsid-encoding sequence are the most conserved regions, with around 80–90% sequence identity. The hypervariable region and the envelope protein-encoding sequence show the least sequence identity among isolates (Figure 1) (Le Guillou-Guillemette et al., 2007). These observations agree with the existence of highly conserved structural units throughout the HCV genome (Fricke et al., 2015; Mauger et al., 2015; Pirakitikulr et al., 2016). Some of these operate as essential functional domains – key targets for the development of new therapeutics and diagnostic agents. This review provides a brief overview of the cis-acting signals critical for HCV infection, with special emphasis on the core partner, i.e., the so-called 5BSL3.2 domain at the 3′ end of the viral ORF. It also focuses on the sequences and structural units of the viral genome essential to the preservation of the roles of 5BSL3.2 during the infective cycle.

In addition to RNA domains being located in the untranslated regions, numerous cis-acting signals have been identified in the ORFs of RNA viruses (for a review, see Romero-López and Berzal-Herranz, 2013). The search for unique, highly conserved structural and functional elements encoded in the HCV ORF has been going on for 15 years. The use of bioinformatic tools and secondary structure mapping, combined with genetic strategies, has finally provided a good overview of the global and local folding of HCV RNA (Tuplin et al., 2002, 2004; Mcmullan et al., 2007; Davis et al., 2008; Diviney et al., 2008; Chu et al., 2013; Fricke et al., 2015; Mauger et al., 2015; Pirakitikulr et al., 2016). The HCV genome is a highly compact molecule, with extensive base-pairing, helping to preserve viral RNA from cellular endonuclease-mediated degradation. Two of these degradation systems, both of which are involved in the innate immune response, include RNase L, which is specific for single-stranded regions, and the double-stranded-specific interference pathway (Li and Lemon, 2013). Importantly, by constraining the length of the helix segments, the viral genome has reached a perfect balance between single- and double-stranded regions with the aim of minimizing the effect of RNase L activity without unleashing the interference mechanism. This is consistent with the presence of alternate, extensive regions of compact folding throughout viral RNA genomes – the so-called GORS (genome-scale ordered RNA structure) elements. The existence of these elements correlates positively with host persistence and cell-to-cell movement (Simmonds et al., 2004; Davis et al., 2008; Witteveldt et al., 2014).

Up to 20 conserved RNA structural elements are scattered throughout the HCV ORF (Figure 1), expanding the functional repertoire of the viral genome via their participation in viral translation, replication, and infectivity (Tuplin et al., 2002, 2004; Mcmullan et al., 2007; Diviney et al., 2008; Chu et al., 2013; Fricke et al., 2015; Mauger et al., 2015; Pirakitikulr et al., 2016). Interestingly, the NS5B coding sequence is specifically enriched in functional RNA domains, with up to 10 distinct conserved stem-loop structural units able to drive HCV RNA and protein synthesis (Figure 1) (Smith and Simmonds, 1997; Walewski et al., 2001; Tuplin et al., 2002, 2004; Hofacker, 2004; Lee et al., 2004; You et al., 2004; Chu et al., 2013; Mauger et al., 2015; Pirakitikulr et al., 2016). One of the best-characterized functional RNA domains in the NS5B coding sequence is the so-called 5BSL3.2 domain (also known as SL9266 following the new standardized nomenclature system according to the genomic nucleotide position of the first 5′ paired residue of the stem-loop). The following sections focus on the molecular and functional features of this domain and its relationships with distant RNA elements in the HCV genome.

Using a combination of phylogenetic comparisons and thermodynamic prediction methods, Tuplin et al. (2002), defined the precise and conserved boundaries within the NS5B coding sequence that mark the limits of a set of genotypically well-conserved secondary structure elements. Five of these had already been proposed by other groups, either in whole or in part (Smith and Simmonds, 1997; Hofacker et al., 1998; Walewski et al., 2001; Tuplin et al., 2002). Nevertheless, it was in 2004 when three independent groups described the existence of a preserved sequence and structural region composed of three stem-loops in the 3′ terminus of the coding sequence. These were named 5BSL3.1, 5BSL3.2, and 5BSL3.3 (or SL9217, SL9266, and SL9324, respectively; Figures 1, 4; Lee et al., 2004; You et al., 2004; Friebe et al., 2005). Reverse genetic analyses in hepatocytes bearing subgenomic replicon constructs, and in full-length viral replication models, showed these stem-loops to operate as a cis-acting replication elements (CRE). The central domain 5BSL3.2 was shown indispensable for HCV propagation (Lee et al., 2004; You et al., 2004; Friebe et al., 2005; Masante et al., 2015; Tuplin et al., 2015).

Bioinformatic prediction, biochemical structural mapping and NMR studies have all been undertaken to try to decipher the three-dimensional folding of 5BSL3.2 (Lee et al., 2004; You et al., 2004; Friebe et al., 2005; Tuplin et al., 2012), which was found to be a 48 nt-long imperfect hairpin with a 12 nt-long apical loop (Figure 4). The stem is interrupted at its 3′ end by an 8 nt-long bulge. Interestingly, both unpaired regions are phylogenetically conserved across different genotypes and show low synonymous site sequence variation (Figure 4) (You et al., 2004) that cannot be explained only by the need to preserve the NS5B coding sequence. Such a high degree of conservation undoubtedly points to a role in the functional control of the infective cycle, mediated by the apical loop and the bulge of the 5BSL3.2 domain (Lee et al., 2004; You et al., 2004; Friebe et al., 2005; Tuplin et al., 2012, 2015). Interestingly, the 5′-CACAGC-3′ sequence motif in the apical loop is found in other conserved elements of distantly related flaviviruses, such as Kunjin virus, West Nile virus or Dengue virus, where it operates as a single CRE (Markoff, 2003; Friebe et al., 2005). This observation points to the existence of common molecular mechanisms for viral RNA synthesis across different members of the family Flaviviridae.

Co-variation data confirm the existence in the basal part of the stem-loop of three out of the eight base pairs. In the upper stem, three of the six base pairs are invariable and two of the other remaining three can be predicted through compensatory base pair changes (Figure 4) (You et al., 2004). These phylogenetic data provide a convincing clue about the existence of the 5BSL3.2 hairpin in vivo.

From a structural point of view, the molecular context surrounding the 5BSL3.2 domain is striking. It is embedded between two other stem-loops, 5BSL3.1 and 5BSL3.3, to yield a high-order structure that can be depicted as a cruciform element (Figure 5) (Lee et al., 2004; You et al., 2004; Friebe et al., 2005). Domains 5BSL3.1 and 5BSL3.3 were initially identified as evolutionarily conserved elements (Smith and Simmonds, 1997; Tuplin et al., 2004) that fold into stable stem-loop structures (Figure 4). RNase and chemical mapping have confirmed this secondary structure and support the proposed large cruciform structure (You et al., 2004). However, neither mutagenesis nor biophysical methods have yet confirmed the existence of this high-order structure in vivo. This might be due to the dynamic and relatively unstable nature of long-distance RNA–RNA contacts, which can promote the switch between different metastable structural states in the RNA molecule, depending on the presence of specific ligands or external stimuli. Thus, such contacts suggest a regulatory operation mode that might rely on the versatility and efficiency of the HCV CRE region.

The remarkable structural features of the 5BSL3.2 domain, as well as its strong conservation, point to its critical involvement in the HCV infective cycle. As mentioned above, the observation that both the apical loop and the bulge of the 5BSL3.2 domain are highly preserved among different genotypes and viral isolates suggests their participation in interactions with other viral RNA elements. Several reports have provided substantial proof of the existence of long-distance RNA–RNA contacts, together forming a complex network of interactions that appears to be governed by 5BSL3.2 (Friebe et al., 2005; Romero-López and Berzal-Herranz, 2009; Romero-López et al., 2012, 2014, 2017; Tuplin et al., 2012, 2015; Shetty et al., 2013; Fricke et al., 2015). Such a network would organize the three-dimensional folding of the viral genome into a compact conformation that correlates with virulence and the persistence of infection (Davis et al., 2008). To date, two conformational rearrangements of the HCV genome are known to be mediated by the 5BSL3.2 domain: viral genome circularization and the structural tuning of the 3′ end of the HCV genome.

The recruitment of both host and viral components is important in the regulatory activities of the CRE region (Lourenco et al., 2008). The apical loop of the 5BSL3.2 domain binds to the viral polymerase NS5B protein (Zhang et al., 2005), favoring the positioning of the polymerase in the 3′X tail and thus the initiation of replication. The interaction of the NS5B protein with the apical loop of 5BSL3.2 might compete with the contact 5BSL3.2-3′X. Swapping from NS5B recruitment to 3′X interaction could therefore be used by the 5BSL3.2 domain as a regulatory mechanism for promoting transitions between steps during the infective cycle.

In recent years, exhaustive lists of cellular proteins susceptible to recruitment by the CRE region have been produced (Oakland et al., 2013; Ríos-Marco et al., 2016). Interestingly, some have been shown to influence viral translation, replication or both (Oakland et al., 2013; Ríos-Marco et al., 2016). Oakland et al. (2013) showed Ewing’s Sarcoma binding protein 1 (EWSR1) to interact with the CRE region; EWSR1 is a nuclear factor that regulates RNA synthesis and processing as well as the transport of pre-mRNAs involved in cell cycle progression and the response to DNA damage (Zinszner et al., 1994; Paronetto et al., 2011). It also binds other factors related to splicing, such as those belonging to the heterogeneous ribonuclear protein family (hnRNPs). Its regulatory role during mitosis has also been reported (Wang et al., 2016). The involvement of ESWR1 in tumorigenesis has attracted much attention since HCV infection can lead to the development of hepatocellular carcinoma. Though EWSR1 is preferentially located in the nucleus, it can be translocated to the cytosol of HCV-infected hepatocytes (Oakland et al., 2013). Here it binds to the viral genome in a manner dependent on the structure resulting from the interaction 5BSL3.2-3′X, promoting efficient HCV RNA replication, at least in cell culture (Oakland et al., 2013).

Subsequent proteomic analyses have identified a collection of RNA-binding proteins with highly conserved RNA recognition motifs able to bind to the CRE region (Ríos-Marco et al., 2016). hnRNPA1 is a very abundant nuclear and cytosolic protein involved in the packaging of pre-mRNAs into spliceosomal particles, and in the translocation of processed poly(A) mRNAs from the nucleus to the cytoplasm (Dreyfuss et al., 2002). During HCV infection, hnRNPA1 operates as an IRES-dependent translation initiation enhancer via its interaction with the IRES region (Lu et al., 2004), and as a negative regulatory partner of viral RNA synthesis, most likely by competing with NS5B for a common interacting site in the 5BSL3.2 domain (Ríos-Marco et al., 2016). Additionally, hnRNPA1 plays an important role as a splicing factor in the maturation of IFR3 (interferon regulatory factor 3), which is involved in interferon-mediated immunity (Guo et al., 2013). Sequestering hnRNPA1 by 5BSL3.2 would, therefore, influence interferon production, helping HCV escape the cellular immune response. HMGB1 (high mobility group box 1 protein) also interferes with HCV replication by binding to the CRE (Jung et al., 2011; Ríos-Marco et al., 2016), and is considered an antiviral factor (Jung et al., 2011). Finally, host proteins with helicase activity, such as DDX3, DDX5, and DDX17, might recognize the 5BSL3.2 domain and promote HCV replication and/or translation (Ríos-Marco et al., 2016). This last observation confirms the role of the CRE as a regulator of viral protein and RNA synthesis (Ariumi et al., 2007; Randall et al., 2007; Oakland et al., 2013; Ríos-Marco et al., 2016).

MicroRNAs (miRNAs) produced by the host cell have been identified as agents that regulate viral infection (Bruscella et al., 2017). Via a little-understood mechanism, miRNAs influence HCV infection at different stages. For example, miR-122, a highly abundant miRNA in hepatocytes, promotes viral translation and replication via its interaction with the IRES region at different target sites (Jopling et al., 2005; Jangra et al., 2010; Roberts et al., 2011), while miR-199a^∗ represses RNA replication (Murakami et al., 2009). Let-7b also acts as a negative regulatory agent of HCV replication via its direct interaction with the region connecting domains 5BSL3.2 and 5BSL3.3 (Cheng et al., 2012). Let-7b is involved in cell differentiation and has been intimately associated with the development of cancer (Takamizawa et al., 2004; Yu et al., 2007). This, along with the observation that cell cycle regulatory proteins bind to the CRE, provides additional evidence of the potential role of the CRE region in HCV-associated hepatocellular carcinoma.

The HCV cycle is extensively compartmentalized, both spatially and temporally: viral protein synthesis, replication, and encapsidation occur in different cellular localizations and do not overlap in time (Shulla and Randall, 2015). The switch between one stage and the next must therefore be carefully controlled. The machinery required for this is not well-understood, but it is widely assumed that functional genomic domains, along with host and viral factors, together control the progression of the infective cycle.

During early infection, HCV virions are endocytosed and their genomes released into the cytosol by a little-understood mechanism (Figure 6; for a review see Scheel and Rice, 2013). Endoplasmic reticulum (ER)-associated translation is then initiated by the IRES region. During this stage, subdomain IIId of the IRES is preferentially occupied by the translational machinery, thus favoring the contacts 5BSL3.2-Alt and 5BSL3.2-3′X (Figure 6). The emerging HCV polyprotein is co- and post-translationally cleaved by cellular proteases and viral NS2-NS3 and NS3-NS4A proteases to release 10 HCV proteins (Grakoui et al., 1993). The accumulation of non-structural viral proteins promotes significant rearrangements in the ER membrane to create optimized microenvironments for RNA replication (as seen for other positive-strand RNA viruses) (Egger et al., 2002; Moradpour et al., 2003; Meyers et al., 2016; Falcon et al., 2017). The binding of the replicase complex to the 3′X tail of the viral genome, and the concomitant recruitment of cellular components at the CRE region (Oakland et al., 2013; Ríos-Marco et al., 2016), releases the 5BSL3.2 domain for new interactions with distant genomic RNA elements such as subdomain IIId within the IRES (Figure 6). This contact promotes the viral genome’s acquisition of a circular form. In addition, the interaction IIId-5BSL3.2 blocks the recruitment of 40S ribosomal particles by the IRES, contributing to the creation of a translationally repressed-state(Romero-López and Berzal-Herranz, 2012). 5BSL3.2 is thus considered a specific inhibitor of HCV IRES function (Romero-López and Berzal-Herranz, 2012). The IIId-5BSL3.2 interaction also controls the structural switch at the 3′X tail and partially interferes with the formation of dimeric genomes (Romero-López et al., 2017). From a thermodynamic point of view, the interaction IIId-5BSL3.2 can swap with the contact Alt-5BSL3.2 (Shetty et al., 2013) to create a favorable replicative environment (Figure 6) (Diviney et al., 2008; Tuplin et al., 2012). Viral RNA synthesis is then initiated using the positive genome strand as a template, yielding the complementary, negative strand, which is used for the generation of new progeny RNA genomes (for a review, see Kim and Chang, 2013). In a later stage of the cycle, viral genomes accumulate in the cytosol, where they serve as mRNAs for the production of HCV proteins, thus initiating new rounds of translation-replication (Figure 6). At this time, the IRES region is occluded by the translational machinery, favoring the interaction Alt-5BSL3.2. Because of the high concentration of viral RNAs, genomic dimerization is thermodynamically favored (Figure 6). This results in viral RNA storage, but HCV dimeric genomes are also excellent templates for the initiation of replication (Cantero-Camacho and Gallego, 2015; Masante et al., 2015; Cantero-Camacho et al., 2017). According to this model, HCV genomic dimerization may play a replication enhancing role during late infection. Finally, a fraction of the newly synthesized RNA molecules is shuttled for encapsidation and released to the extracellular medium (Figure 6).

The model proposed above recapitulates many of the findings made regarding the molecular biology of HCV. More importantly, it provides an overview of the critical role of long-distance RNA–RNA contacts in the progression of the infective cycle, and points to 5BSL3.2 as a multifunctional component encoded in the viral genome.

The contrast between the high structural conservation and sequence variability of the HCV RNA genome reflects an efficient mechanism for retaining essential functionalities in certain structural elements while favoring the acquisition of novel capabilities. Whereas many genomic domains govern essential steps in infection, the control exerted by 5BSL3.2 at multiple levels reflects the functional versatility and fine regulation that can be achieved by a single stem-loop. This domain is thus an interesting target for novel therapeutic agents. Exploring the structural dynamics of functional RNA domains offers a powerful, informative methodology for future drug design. Extending the knowledge acquired in HCV to related viruses, such as flaviviruses, will allow conformational maps of common structural domains to be produced, help reveal similar viral mechanisms involved in the infective cycle, and perhaps contribute to the development of novel therapeutic and diagnostic strategies.

CR-L and AB-H designed and wrote the paper.

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.