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Tan SL, editor. Hepatitis C Viruses: Genomes and Molecular Biology. Norfolk (UK): Horizon Bioscience; 2006.

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Hepatitis C Viruses: Genomes and Molecular Biology.

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Chapter 2HCV 5′ and 3′UTR: When Translation Meets Replication

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Abstract

Similar to other positive-strand RNA viruses, the non-coding regions of HCV RNA, referred herein as 5′ and 3′ untranslated regions (5′UTR and 3′UTR), contain important sequence and structural elements critical for HCV translation and RNA replication. The 5′UTR harbors an internal ribosome entry site (IRES) that directs viral protein translation via a cap-independent mechanism. As the initiation sites for RNA synthesis, both 5′UTR and 3′UTR contain signals that are indispensable for and regulate viral RNA replication. Additional structural elements involved in translation or RNA replication are also present in both ends of the protein (core and NS5B)-coding regions. These RNA elements interact with each other either directly or through the binding of viral and cellular proteins that are most likely involved in the regulation of translation and RNA replication processes. Since RNA replication and translation occur on the same RNA molecule, mechanisms must exist to regulate and separate these two processes. This chapter details the current understanding of the roles of the UTRs and other structural components in the viral RNA as well as their binding proteins in HCV translation and RNA replication and speculate on the possible mechanisms regulating these two different processes.

Introduction

HCV is a typical flavivirus containing a single-stranded, positive-sense RNA of 9.7 kb in length (Choo et al., 1991). The viral RNA contains a single large open reading frame (ORF) flanked by an untranslated region (UTR) at each end, a genomic organization conserved among members of the Flaviviridae family. One of the most important features of HCV RNA is its high degree of genetic variability as a result of mutations that occur during viral replication. However, the mutation rate varies significantly in the different regions of the HCV genome, of which the 5′UTR and the extreme end of the 3′UTR have the lowest sequence diversity among various genotypes and subtypes (Choo et al., 1991; Miller and Purcell, 1990; Muerhoff et al., 1995). The relatively conserved nature of these regions signifies their functional importance in the viral life cycle.

A combination of phylogenetic analysis, computer modeling, and chemical and enzymatic probing has enabled the identification of structural elements in the 5′ and 3′ UTRs of HCV RNA. The viral RNA elements (internal ribosome entry site, IRES) critically involved in the cap-independent translation of HCV RNA have been analyzed extensively. In contrast, the study of the mechanism of HCV RNA replication was more limited due to the lack of efficient cell culture or small animal models. The generation of consensus cDNA clones that are infectious in chimpanzees provided the first tools for molecular genetic analysis of HCV RNA replication (Beard et al., 1999; Choo et al., 1989; Kolykhalov et al., 1997; Yanagi et al., 1997; Yanagi et al., 1999a; Yanagi et al., 1998). Using this approach, the regions in the 3′UTR that are required for viral replication have been identified (Kolykhalov et al., 1997; Yanagi et al., 1999b). More recently, the development of and advances in the cell-based subgenomic replicon system have identified additional RNA elements of the UTRs and other cis-acting replication elements (CREs) that are involved in RNA replication and translation (Friebe et al., 2005; Friebe et al., 2001; Lee et al., 2004a; You et al., 2004).

A number of viral and cellular proteins have been shown to interact with the essential structural elements in the non-coding and coding regions of HCV RNA and are presumably involved in the regulation of the viral translation and/or RNA replication processes. The precise functional roles of most of these proteins have not been established. The recent development of cell-free HCV RNA replication systems (Ali et al., 2002; Hardy et al., 2003; Lai et al., 2003) provides an additional tool for studying the viral and host proteins involved in the translation and replication of HCV RNA, thus identifying novel targets for the development of more effective antiviral therapies.

Structural and Functional Components of the HCV RNA

The 5′UTR and the extreme end of the 3′UTR are the most conserved regions of HCV RNA in terms of primary sequence and secondary structures. Together with the fact that these structured domains are located at the 5′ and 3′ ends of the genome, it stands to reason that they play important roles in viral RNA translation and/or replication.

5′UTR

The 5′UTR of the HCV genome is 341-nt long in most viral isolates. There is more than 90% sequence identity among different HCV genotypes, with some segments nearly identical among different strains (Bukh et al., 1992). The secondary and tertiary structures of this region are also largely conserved (Brown et al., 1992; Honda et al., 1999a; Honda et al., 1996a). The 5′UTRs of HCV, GBV-B (Muerhoff et al., 1995), and pestiviruses, such as bovine viral diarrhea virus (BVDV) and classical swine fever virus, share extensive homology in primary sequence and secondary structure (Brown et al., 1992; Han et al., 1991; Honda et al., 1996a; Simons et al., 1995), signifying GBV-B and pestiviruses as the closest relatives to HCV (Ohba et al., 1996). A combination of computational, phylogenetic, and mutational analyses of the HCV 5′UTR has identified four major structural domains (domains I–IV) (Fig. 1), most of which are also conserved among HCV genotypes, GBV-B, and pestiviruses (Brown et al., 1992; Honda et al., 1999a; Honda et al., 1996a; Smith et al., 1995). Common features include a large stem-loop III and a pseudoknot (psk). The 5′UTR sequences of HCV and GBV-B have two smaller stem-loops, stem-loop Ia near the extreme 5′ end and stem-loop IV containing the translation initiation codon (Honda et al., 1996a).

Fig. 1. The structures of the 5′UTR (Rijnbrand and Lemon, 2000) and 3′UTR (Ito and Lai, 1997; Kolykhalov et al.

Fig. 1

The structures of the 5′UTR (Rijnbrand and Lemon, 2000) and 3′UTR (Ito and Lai, 1997; Kolykhalov et al., 1996) of HCV RNA (represented by the HCV-H strain). The structural diagram of the 5′UTR was kindly provided by Drs. René (more...)

The first 40 nt of the 5′UTR constitutes domain I, which is involved in RNA replication but not essential for translation; therefore, the function of this region is distinct from the rest of the 5′UTR, which is critical for translation (Friebe et al., 2001; Luo et al., 2003). The remaining domains II–IV constitute an IRES (Fig. 1) (Brown et al., 1992), which mediates the cap-independent translation of the HCV ORF (Tsukiyama-Kohara et al., 1992). Domains II and III are relatively more complex than domain IV and contain multiple stems and loops (Honda et al., 1999a; Lemon and Honda, 1997). Several electron microscopy (Beales et al., 2001; Spahn et al., 2001) and NMR studies (Lukavsky et al., 2000) have provided detailed structural information on the main domains of the IRES. Domains IIIa–IIIc and II extend in opposite directions from a small central domain that includes stem loops and junctions IIIe–IIIf (Spahn et al., 2001). The hairpin loop of the small IIIe subdomain forms a novel tetraloop fold with three exposed Watson–Crick faces that may be involved in 40S ribosome binding (Lukavsky et al., 2000). The stem of subdomain IIId forms a loop E motif similar to those observed in prokaryotic and eukaryotic ribosomal RNA, and a six-nucleotide hairpin loop containing an S-turn motif (Klinck et al., 2000; Lukavsky et al., 2000). The sequences of the hairpin loops of subdomains IIIe and IIId are conserved among all HCV isolates and play an important role in translation initiation.

The base of domain III forms a highly conserved pseudoknot, which is critical for IRES activity (Wang et al., 1995). Similar pseudoknots with almost identical primary sequences also exist in the pestiviral and GBV-B IRES elements (Lemon and Honda, 1997). The pseudoknot is part of the binding site for the 40S ribosome subunit (Kolupaeva et al., 2000). Another tertiary structural element in domain II, identified by RNA-RNA crosslinking, may also be involved in ribosome binding (Lyons et al., 2001). Domain IV is composed of a small stem-loop (stem-loop IV) in which the initiator codon AUG is located within the single-stranded loop region (Honda et al., 1996a). Stem-loop IV is not required for internal entry of ribosomes. In fact, the stability of this stem-loop structure is negatively correlated with the translation efficiency of the viral RNA (Honda et al., 1996a).

According to a structure-based classification scheme originally designed for picornaviral IRES elements (Wimmer et al., 1993), the HCV IRES, together with the IRES elements of the closely related pestiviruses and GBV-B, is classified into type 3 of four existing types (Lemon and Honda, 1997). The picornaviral and flaviviral IRES elements are significantly different in a number of aspects, suggesting distinct mechanisms of translation initiation for these two virus families (Rijnbrand and Lemon, 2000). The picornaviral IRES elements have been shown to be more efficient than the HCV IRES in directing translation (Borman et al., 1995). In contrast, viruses in the genus Flavivirus (e.g. yellow fever virus) have significantly shorter 5′ UTRs with a cap structure, m7GpppN1mpN2 (Westaway, 1987).

3′UTR

The 3′UTR of HCV varies between 200 and 235 nt long, which typically consists of three distinct regions, in the 5′ to 3′ direction, a variable region, a poly(U/UC) stretch, and a highly conserved 98-nt X region (Blight and Rice, 1997; Kolykhalov et al., 1996; Tanaka et al., 1995; Tanaka et al., 1996; Yamada et al., 1996). The variable region follows immediately the termination codon of the HCV polyprotein, and is variable in length (ranging from 27 to 70 nt) and composition among different genotypes. However, it is highly conserved among viral strains of the same genotype (Kolykhalov et al., 1996; Yanagi et al., 1997; Yanagi et al., 1998). Computer analysis has identified two possible stem-loop structures in the variable region, with the first stem-loop extending into the 3′ end of the NS5B-coding sequence (Han and Houghton, 1992; Kolykhalov et al., 1996). The poly(U/UC) tract consists of a poly(U) stretch and a C(U)n-repeat region (referred to as the transitional region) and varies greatly in length and slightly in sequence among different viral isolates (Tanaka et al., 1996). The transitional regions of genotypes 2a, 3a, and 3b have several conserved A residues, which are not present in genotypes 1b and 2b (Tanaka et al., 1996; Yamada et al., 1996; Yanagi et al., 1999a). The presence of the polypyrimidine tract within the 3′UTR is unique to HCV and GBV-B (Simons et al., 1995) among flaviviruses. The length of this region has been correlated with the replication capability of HCV RNA (Friebe and Bartenschlager, 2002; Kolykhalov et al., 1997; Yanagi et al., 1999b; Yi and Lemon, 2003a). The X region forms three stable stem-loop structures that are highly conserved across all genotypes (Blight and Rice, 1997; Ito and Lai, 1997; Kolykhalov et al., 1996) (Fig. 1). A recent study of the structure of the X region by chemical and enzyme probing has confirmed the presence of SL1 and SL3, but proposed that the region between the two stem-loops folds into two hairpins instead of one and may further form a hypothetical pseudoknot (Dutkiewicz and Ciesiolka, 2005). On the other hand, the complementary sequence of the X region in this region forms a 3-stem-loop structure (Dutkiewicz and Ciesiolka, 2005). There is no poly(A) sequence in the 3′UTR. Instead, the 3′UTR sequence, particularly the X region, is involved in the regulation of translation, much in the same way as the poly(A) sequence in the mRNAs of other RNA viruses. Conceivably, these sequences are involved in the replication, stabilization and also packaging of viral RNA.

As a result of the stem-loop formation in the X region, the HCV genome is predicted to end with a double-stranded stem. Examination of the 3′-terminal sequences of the HCV genome in sera from infected patients revealed that most HCV RNAs contain identical 3′ ends with no extra sequence downstream of the X tail (Tanaka et al., 1996). However, one particular cDNA clone derived from a patient's serum did contain 2 additional nt (UU), thus generating a single-stranded tail (Yamada et al., 1996). The structure of the exact 3′-end will have implications for the initiation of RNA replication.

Other Structural Components in the Protein-Coding Region

Bioinformatic analysis has revealed the possible presence of additional secondary structures in other parts of the HCV genome (Hofacker et al., 1998). These include possible secondary structures in the core- and NS5B-coding regions (Rijnbrand et al., 2001; Smith and Simmonds, 1997; Tuplin et al., 2002). Consistent with the importance of the predicted secondary structures, it has been shown that synonymous nucleotide mutations are suppressed in the core- and NS5B-coding regions and that compensatory mutations are frequently observed within the predicted stems (Ina et al., 1994; Smith and Simmonds, 1997).

The predicted secondary structures within the core-coding region encompass the first 14 nts of the core gene, which form part of the IRES stem-loop IV (Lemon and Honda, 1997). There are two more stem-loops between nt 47 and 167 of the core-coding sequence (nt 391–511 of the genome), which is conserved among all six HCV genotypes (Smith and Simmonds, 1997). This region, corresponding roughly to nt 408–929, has been shown to interact with the 5′UTR, resulting in the reduction of HCV IRES-mediated translation (Honda et al., 1999b). In the NS5B-coding region (Hofacker et al., 1998; Rijnbrand et al., 2001; Smith and Simmonds, 1997; Tuplin et al., 2002; You et al., 2004), six potential stem-loop structures have been predicted based on computer modeling (You et al., 2004). The functional significance of five of these structures in RNA replication has been implicated from mutational analysis and RNA structure probing in the context of the subgenomic replicon. Of particular interest is a cruciform structure (5BSL3) at the 3′ terminus of NS5B, which contains three major stem-loop structures, 5BSL3.1, 5BSL3.2, and 5BSL3.3 (Fig. 2). Its involvement in RNA replication will be discussed in a later section.

Fig. 2. Cis-acting RNA replication regulatory elements in the NS5B-coding region that interact with the 3′UTR (represented by the HCV-Con1 strain).

Fig. 2

Cis-acting RNA replication regulatory elements in the NS5B-coding region that interact with the 3′UTR (represented by the HCV-Con1 strain). (A) The cruciform structure formed at the end of the NS5B-coding sequence contains 5BSL3.1, 5BSL3.2, and (more...)

HCV Translation

Translation of the polyprotein from the HCV RNA genome is the first macromolecular synthetic event after the viral RNA is released into the cytoplasm of host cells. It is carried out by a cap-independent mechanism mediated by the highly structured HCV IRES. The HCV genomic RNA serves as an mRNA for the translation of a single polyprotein, which is processed by cellular and viral proteases into at least 10 structural and nonstructural proteins (De Francesco et al., 2000).

Structural Components Required for Translation

The first 40 nts of the HCV RNA genome, including the first stem-loop domain (domain I), are not required for translation (Honda et al., 1996b; Rijnbrand et al., 1995). Instead, deletion of this domain resulted in a stimulation of translation of a heterologous reporter RNA (Yoo et al., 1992). However, in the context of the HCV subgenomic replicon, deletion of this domain reduced protein expression by 3 to 5 fold (Luo et al., 2003). In addition, a dinucleotide sequence at nt 34–35 has been shown to contribute to the differential translation efficiencies between genotype 1a and 1b isolates (Honda et al., 1999b). It is, therefore, possible that domain I is also involved in the regulation of HCV translation in some fashions. The primary element of the IRES starts at nt 44, which coincides with the 5′ border of domain II (Honda et al., 1999a; Honda et al., 1996b; Reynolds et al., 1995; Rijnbrand et al., 1995). However, the precise 3′ border of the IRES is controversial.

The stem-loop IV of the 5′UTR is predicted to extend into the coding region to include the first 10 nts (nt 345–354) of the core-encoding gene. Indeed, several studies have reported the requirement for a short sequence (up to 30 nt) in the core-coding region for optimal IRES function (Honda et al., 1996a; Hwang et al., 1998; Lu and Wimmer, 1996; Reynolds et al., 1996). However, efficient translation has also been observed with certain reporter genes fused immediately after the start codon, without the core protein-coding sequences (Tsukiyama-Kohara et al., 1992; Wang et al., 1993). The differences in the conclusions may have been due to the assay systems and heterologous reporters employed. It has been found that expression of the reporter gene secretory alkaline phosphatase, but not that of chloramphenicol acetyltransferase, depends on the presence of downstream core-coding sequences (Rijnbrand et al., 2001). Conceivably, the core-coding region may contribute to IRES function by preventing undesirable base pairing of the IRES with other inhibitory sequences or by promoting favorable protein binding to the IRES. This core-coding region contains an adenosine-rich stretch, which has been shown to recruit a cellular protein that enhances the HCV IRES activity (Kim et al., 2004a; Reynolds et al., 1995). So far, nt 354 is generally regarded as the consensus 3′ boundary of the IRES (Honda et al., 1999a), but the sequence immediately downstream of the IRES (up to nt 371) may have a stimulating effect on IRES-directed translation. Interestingly, the core-coding sequences further downstream (near the C-terminal portion) have been shown to play a negative-regulatory role in HCV translation (Ito and Lai, 1999; Kim et al., 2003; Wang et al., 2000).

Besides the 5′UTR, the 3′UTR sequences, particularly the X region, may also play a role in HCV RNA translation. It has been shown that HCV RNA containing the X region was translated 3- to 5-fold more efficiently than the corresponding RNAs without this region (Ito et al., 1998). The enhancement of IRES-dependent translation by 3′UTR may be mediated by polypyrimidine tract-binding protein (PTB), which binds to both the 5′ and 3′UTR (Ali and Siddiqui, 1995; Ito and Lai, 1997; Tsuchihara et al., 1997). Since PTB can interact with itself, it can potentially mediate circularization of HCV RNA, thereby enhancing translation. The role of the 3′UTR in translation is reminiscent of the poly(A) tail and the poly(A)-binding protein in the translation of poly(A)-containing mRNAs (Kahvejian et al., 2001). However, a different study reported that deletion of the poly(U/UC) tract or the stem-loop 3 of the X region resulted in an enhancement of translation efficiency; the increase in translation was not mediated by PTB (Murakami et al., 2001). Additional studies are required to understand the role of the 3′UTR in IRES-mediated translation of HCV proteins.

The HCV Translation Machinery

The HCV IRES is responsible for directing the 40S ribosomal subunit in close contact with the start codon for translation initiation (Lemon and Honda, 1997; Wang et al., 1993). Enzymatic and chemical footprinting and domain-deletion experiments have identified domain II and the basal part of domain III, excluding domain IIIb, as the binding site for the 40S ribosome subunits (Kieft et al., 2001; Kolupaeva et al., 2000; Lukavsky et al., 2000; Pestova et al., 1998). Although the HCV IRES with or without domain II recruits the 40S ribosome subunit with comparable efficiency (Otto et al., 2002), interaction of domain II with the 40S subunit induces or stabilizes the conformational changes within the ribosome and facilitates the 3′ end of the coding RNA to thread into the mRNA entry channel (Spahn et al., 2001). The GGG triplet (nt 266–268) of the hexanucleotide (UUGGGU) apical loop of stem-loop IIId and the pseudoknot are essential for ribosome binding (Kolupaeva et al., 2000). Mutagenesis studies have also confirmed that the GGG triplet is essential for IRES activity both in vitro and in vivo (Jubin et al., 2000).

The viral 5′UTR forms a binary complex with the 40S ribosomal subunit in the absence of any canonical or non-canonical initiation factors (Pestova et al., 1998). A ribosomal protein S5, in particular, is important for the efficient translation initiation of HCV RNA (Fukushi et al., 1997; Fukushi et al., 2001b; Pestova et al., 1998). Blocking of the S5 binding to HCV IRES interfered with efficient ribosome assembly at the translation initiation site (Ray and Das, 2004). These features suggest that HCV IRES uses the prokaryotic mode for forming the mRNA-40S ribosome complex (Pestova et al., 1998).

Several basal translation initiation factors have been reported to be involved in the HCV IRES-mediated translation. The eukaryotic initiation factor-3 (eIF3), alone or together with the 40S ribosome subunit and the eIF2-GTP-initiator tRNA complex, can specifically interact with the HCV IRES stem-loop IIIb in the absence of eIF4A, eIF4B and eIF4F, which are required for ribosomal binding during cap- or EMCV IRES-dependent translation (Kieft et al., 2001; Kolupaeva et al., 2000; Pestova et al., 1998; Sizova et al., 1998). eIF3 binding is not necessary for 40S-HCV IRES assembly, but is essential for the joining of 60S subunit to form the active 80S ribosomal complexes (Pestova et al., 1998). These findings suggest that HCV employs a modified mechanism of IRES-dependent translation. Rabbit reticulocyte lysates depleted of certain translation factors, such as eIF4G, cannot support foot-and-mouth-disease virus IRES-, but still can support HCV IRES-dependent translation (Stassinopoulos and Belsham, 2001). eIF2Bγ and eIF2γ have also been identified as cofactors of HCV IRES-mediated translation by a functional genomics approach (Krüger et al., 2000), although their roles in translation have not been established. These findings combined indicate that HCV IRES-dependent translation employs a prokaryotic mode for assembling RNA-ribosome complex and requires only a minimum set of canonical translation factors.

HCV RNA Replication

By analogy with other members of the Flaviviridae, HCV is presumed to replicate its genome through the production of a full-length negative-strand RNA. Positive-strand RNAs are then synthesized from the negative-strand template in five- to ten-fold molar excess over the negative-strand RNA (Lohmann et al., 1999) to be used in translation, replication, and packaging into progeny viruses. Since RNA replication has to initiate from the 3′- end of the RNA template of both strands, the corresponding 5′ and 3′ UTR of HCV RNA genome likely contains the sequences required for the initiation and/or regulation of RNA replication.

Structural Components Required for RNA Replication

Since the 5′UTR is involved in the initiation of both translation and RNA replication, any possible effects of this region on translation will impact RNA replication indirectly and vice versa. Therefore, the direct role of 5′UTR in RNA replication is difficult to assess. Separation of RNA replication and translation was initially achieved by inserting the IRES elements of poliovirus or classical swine fever virus between the serially deleted 5′UTR of HCV and the ORF (Friebe et al., 2001; Kim et al., 2002b; Reusken et al., 2003). The deletions introduced into the 5′-terminal 40 nt upstream of the IRES region abolished RNA replication but only moderately affected translation. The first 125 nt of the HCV genome, which includes domain I and II of the 5′UTR, was shown to be essential and sufficient for RNA replication (Friebe et al., 2001; Kim et al., 2002b; Reusken et al., 2003). This region overlaps with the 5′end of the IRES. The replication efficiency of RNA was tremendously increased by the inclusion of the complete 5′UTR (Friebe et al., 2001). Compared with its close relative BVDV, the requirements for RNA sequences or structures within the 5′UTR of HCV appear to be more complex because much longer sequences or particular structures within the IRES are necessary for efficient RNA replication (Frolov et al., 1998; Wilhelm Grassmann et al., 2005). However, further studies are required to show whether the sequences downstream are directly involved in RNA replication or merely contribute to the preservation of the structural and functional integrity of the minimal replication signal.

Consistent with a role for the 3′ terminal nt of the viral RNA in the initiation of negative-strand RNA, the 3′UTR sequences have been shown to play an essential role in HCV RNA replication in vitro (Friebe and Bartenschlager, 2002; Yi and Lemon, 2003a) and in vivo (Kolykhalov et al., 2000; Yanagi et al., 1999b). The 3′UTR sequences were first shown to be required for the replication of HCV RNA when deletion of the 3′ terminal sequences destroyed the ability of otherwise infectious synthetic genome-length HCV RNA to initiate infection in intrahepatically inoculated chimpanzees (Kolykhalov et al., 2000; Yanagi et al., 1999b). Using a subgenomic HCV replicon, the 3′ terminal RNA signals required for HCV RNA replication were determined to be approximately 225 nt from the 3′ end of the genome (Yi and Lemon, 2003a). The 3′-most 150 nt of the genome, which includes the 98-nt X region and the 52 nt of the poly(U/UC) tract, are essential for replication of HCV RNA, while the remaining upstream region of the 3′UTR plays a facilitating role (Friebe and Bartenschlager, 2002; Ito and Lai, 1997; Yi and Lemon, 2003a; Yi and Lemon, 2003b). These results suggest an interesting symmetry in the 5′- and 3′- terminal RNA replication signals since the 5′-most domains I and II of the 5′UTR are essential for replication, while sequences lying further downstream within the 5′UTR help to facilitate replication but are not absolutely required (Friebe et al., 2001; Kim et al., 2002b). The X region interacts with the recombinant HCV RNA polymerase (Cheng et al., 1999; Oh et al., 2000), although other parts of the 3′ end of HCV genome may contain additional NS5B-binding sites (Cheng et al., 1999). The NS5B-binding domain within the X region has been mapped to stem II and the single-stranded region connecting stem-loops I and II (Oh et al., 2000). Truncation of 40 nts or more from the 3′ end of the X region abolished its template activity in vitro (Oh et al., 1999; Oh et al., 2000). A more extensive mutational analysis of the 3′-end 46 nt that form the terminal hairpin (stem-loop I) in the HCV replicon provided strong functional evidence for the existence of the structure and for an essential role of the structure in the replication of HCV RNA (Yi and Lemon, 2003b). It is interesting that the X region is also necessary for efficient translation of HCV protein (Ito et al., 1998); thus, the same set of sequences are involved in both RNA replication and translation.

The poly(U/UC) tract is required for HCV RNA replication (Friebe and Bartenschlager, 2002; Kolykhalov et al., 1997; Yanagi et al., 1999b; Yi and Lemon, 2003a). It is possible that this region assists in circularizing the viral genome, which has been shown to be important for efficient RNA replication of other flaviviruses (Khromykh et al., 2001). This sequence binds several cellular proteins (e.g. PTB), which may mediate RNA-RNA interaction (Ito and Lai, 1999) and/or the binding of the replicase complex to RNA. The length of the poly(U/UC) region may influence viral replication as HCV RNA with a longer poly(U/UC) region had a replicative advantage in chimpanzees (Kolykhalov et al., 1997; Yanagi et al., 1999b) than the one with a shorter poly(U/UC). Similar observation was made in the subgenomic replicon RNAs (Friebe and Bartenschlager, 2002; Yi and Lemon, 2003a). Conversely, the poly(U/UC)-rich sequence may serve as a modulator of RNA replication under some conditions, as shown in an in vitro RNA polymerase reaction, in which HCV RNA polymerase stutters at this region (Oh et al., 1999).

The sequences within the variable region of the 3′UTR are not essential for RNA replication (Friebe and Bartenschlager, 2002; Yanagi et al., 1999b; Yi and Lemon, 2003a), a finding similar to those of other flaviviruses (Khromykh and Westaway, 1997; Mandl et al., 1998; Men et al., 1996). Interruption of sequence integrity within this region by insertion of the extraneous sequences in this region did not interfere with the replication of the HCV RNA or replicons (Friebe and Bartenschlager, 2002; Yanagi et al., 1999b). Nevertheless, deletions in this region impaired the efficiency of amplification of subgenomic replicons (Yi and Lemon, 2003a).

Some of the conserved RNA elements identified in the NS5B-coding region may serve as recognition sites for the HCV replicase complex since partially purified NS5B specifically binds to the coding sequences of NS5B RNA (Cheng et al., 1999), but their involvement in RNA replication has not been established until recently. The NS5B-coding region contains a predicted cruciform structure (5BSL3) consisting of three stem-loops, 5BSL3.1, 5BSL3.2, and 5BSL3.3 (Fig. 2). Mutations disrupting the 5BSL3.2 blocked RNA replication, whereas 5BSL3.1 and 5BSL3.3 were shown not to be required for RNA replication (Friebe et al., 2005; You et al., 2004). Insertion of 5BSL3.2 alone into the variable region of the 3′UTR was sufficient to rescue RNA replication of a replicon in which all three 5BSLs in the NS5B-coding region were disrupted, indicating that 5BSL3.2 can act as a cis-acting RNA replication element. This insertion allowed the analysis of individual elements within 5BSL3.2 in more detail without the complication of introducing amino acid changes in the NS5B-coding region (Friebe et al., 2005).

5BSL3.2 consists of an 8-bp lower helix, a 6-bp upper helix, a 12-base terminal loop, and an 8-base internal loop; the stem structures, but not their primary sequences, are required for RNA replication (You et al., 2004). In addition, a kissing-loop interaction between a 7-nt-long complementary sequence in 5BSL3.2 and SL2 in the X region has been proven essential for RNA replication (Friebe et al., 2005). In the upper loop of 5BSL3.2, a CACAGC sequence motif is found to be virtually invariant among HCV genotypes and is also present in cis-acting RNA sequences of distantly related flaviviruses, such as Kunjin virus, West Nile virus, or Dengue virus (Markoff, 2003). Given the high genetic conservation in this particular region of the genome, it may be speculated that certain ubiquitously expressed and evolutionarily conserved host cell proteins are involved in the formation of a replication complex that interacts with the 3′ end of the flavivirus genome.

Initiation of Negative- and Positive-Strand RNA Synthesis

Recombinant NS5B proteins are capable of primer-independent initiation of RNA synthesis on a variety of virus-specific and nonspecific RNA templates in vitro (Ferrari et al., 1999; Lohmann et al., 1998; Oh et al., 1999). However, there are conflicting descriptions of the precise initiation site of negative-strand RNA transcription on the HCV-specific templates (Hong et al., 2001; Kim et al., 2002a; Oh et al., 2000; Shim et al., 2002). Oh et al. reported that the transcription of the negative-strand RNA was initiated within the loop sequence of the 3′X stem-loop I, at approximately 21 nt from the 3′ end of the RNA (Oh et al., 2000). Kim et al. reported that transcription initiated further downstream, within the 3′ stem sequence of SL1 (Kim et al., 2002a). Shim et al. has shown that transcription can be initiated by a recombinant NS5B polymerase in vitro at the 3′ end of short oligonucleotide templates representing the 3′ terminus of the positive-strand genomic RNA (Shim et al., 2002). The terminal U is preferred as the initiation nt (Shim et al., 2002), which is confirmed by the study of a subgenomic replicon (Yi and Lemon, 2003b). Hong et al. proposed that a unique β-hairpin within the thumb domain of the NS5B polymerase positions the terminal sequences of the genome so as to initiate de novo transcription from the 3′ terminal nucleotides (Hong et al., 2001). It was proposed that the β-hairpin ensures the initiation of de novo RNA synthesis at the 3′ terminus by preventing movement of the 3′ end of the single-stranded RNA template into the active site of the enzyme. Conceivably, the presence of other viral and cellular proteins may affect the selection of the initiation point of RNA replication in vivo.

The initiation of the positive-strand RNA synthesis has not been as well studied. Conceivably, the 3′ terminus of the negative-strand RNA is essential for positive-strand RNA replication. In vitro replication studies using recombinant NS5B showed that the minimal RNA fragment required for efficient replication of the negative-strand RNA spans nt −239 to −1 (Oh et al., 1999), which is complementary to domains I to III of the 5′UTR. Various site-specific mutation studies on the 5′UTR of the HCV replicons have revealed the importance of these regions on HCV genome replication. However, these studies did not distinguish their effects on either positive- or negative-strand RNA synthesis (Friebe et al., 2001; Kim et al., 2002b). The predicted secondary structures of positive- or negative-strand RNA of 5′UTR are slightly different (Schuster et al., 2002). In in vitro RNA synthesis using the full-length HCV RNA as the template, the NS5B polymerase is capable of positive-strand RNA synthesis, continuing from the 3′ end of the full-length negative-strand RNA product, resulting in a dimeric hairpin HCV RNA (Oh et al., 1999). The significance of such a product is not clear.

The HCV RNA Replication Machinery

HCV RNA replication is believed to occur in the cytoplasm of virus-infected cells based on the cytoplasmic localization of viral RNA (Gowans, 2000) and polymerase (Hwang et al., 1997; Selby et al., 1993). RNA is synthesized by a membrane-associated replication complex that includes the HCV RNA-dependent RNA polymerase (RdRP) NS5B, most of the other viral NS proteins (NS3, NS4A, NS4B, and NS5A), and possibly cellular proteins (Asabe et al., 1997; Bartenschlager et al., 1995; Ishido et al., 1998; Lin et al., 1997; Tu et al., 1999). Among the viral NS proteins, NS4B protein by itself induces membranous alterations that morphologically resemble the membranous webs found in replicon cells where viral RNA replication takes place (Egger et al., 2002; Gosert et al., 2003; Shi et al., 2003). A variety of biochemical evidence suggests that NS4B anchors the formation of the RNA replication complex (Gao et al., 2004), which is formed on the detergent-resistant membrane structures containing cholesterol-rich lipid rafts (Aizaki et al., 2004; Shi et al., 2003). Interestingly, all the nonstructural proteins, except NS5A, have to be translated in cis from the ORF of the very RNA molecule in order for RNA replication to occur (Appel et al., 2005). This finding suggests that the viral proteins are assembled in an ordered and sequential way into the replication complex soon after translation. The only trans-acting protein NS5A may enter the replication complex by binding to a cellular protein, VAP-33 (see below).

From the replicon studies, it appears that the RNA replication requires all the HCV nonstructural proteins except NS2. The NS3 is directly involved in RNA synthesis probably through its helicase function. The RNA helicase function is presumed to be necessary for unwinding the secondary structures of RNA template and to separate the positive- and negative-strand HCV RNA during replication. The HCV helicase lies within the C-terminal half of NS3, which has been shown to possess NTPase, single-stranded (ss) polynucleotide binding, and duplex-unwinding activities (Kim et al., 1995; Tai et al., 1996). NS3 alone has only a weak RNA unwinding activity, which can be significantly enhanced by the presence of NS4A (Pang et al., 2002). The resolution of the crystal structure of NS3 either alone or complexed with deoxyuridine octamer has provided additional insights into the mechanism of the HCV NS3 helicase function (Cho et al., 1998; Kim et al., 1998; Yao et al., 1999).

NS5B is a membrane-associated phosphoprotein (Hwang et al., 1997), which contains signature motifs, such as the GDD, shared by other viral RdRps (Koonin, 1991). The C-terminal 21 aa of NS5B plays a role in anchoring the protein to the membrane (Yamashita et al., 1998) but also plays a direct role in RNA synthesis (Lee et al., 2004b; Vo et al., 2004). NS5B also interacts with a SNARE-like cellular membrane protein, human vesicle-associated membrane protein (VAMP)-associated protein of 33 kDa (hVAP-33), which may directly or indirectly target the polymerase to the RNA replication site (Gao et al., 2004; Tu et al., 1999). Reduction of hVAP-33 expression either by dominant-negative mutants or small interfering RNA (siRNA) of hVAP-33 blocked the association of NS5B with detergent-resistant membranes and led to an inhibition of HCV RNA replication (Gao et al., 2004; Zhang et al., 2004).

Although multiple potential phosphorylation sites exist within the NS5B aa sequence, no site is conserved among all HCV isolates examined (Altschul et al., 1997), suggesting that phosphorylation of NS5B may vary among different isolates. Screening of a phage-display library with HCV NS5B protein as bait has identified one peptide with amino acid sequences homologous to protein kinase C-related kinase 2 (PRK2) (Kim et al., 2004b). In vitro analysis has revealed that PRK2 binds and phosphorylates the N-terminal region of NS5B. Further studies in the subgenomic replicon system have indicated that phosphorylation of NS5B by PRK2 is involved in the regulation of HCV RNA replication. It is not clear whether this phosphorylation has an effect on the NS5B polymerase activity and whether it is conserved among different isolates.

The crystal structure of NS5B shares significant similarity to those of other polymerases, but also displays certain striking differences (Ago et al., 1999; Bressanelli et al., 1999; Lesburg et al., 1999). The domain organization in NS5B can be subdivided into the fingers, palm and thumb, similar to other polymerases. However, as other polymerases, such as the poliovirus 3D polymerase, are distinctly U-shaped, the fingers and the thumb domains of NS5B exhibit extensive contacts between each other, resulting in a globular-shaped molecule. The encircled active site is relatively inflexible and can accommodate only a template:primer duplex without global conformational changes. The C terminus of NS5B (excluding the hydrophobic tail) is present in the active site of the protein and has been hypothesized to play a role in the regulation of RdRp activity and template discrimination (Ago et al., 1999).

In in vitro RdRP assays, NS5B often uses the 3′ end of the template RNA or an artificial oligonucleotide as a primer. (Al et al., 1998; Behrens et al., 1996; De Francesco et al., 1996; Ferrari et al., 1999; Lohmann et al., 1997; Yamashita et al., 1998; Yuan et al., 1997). However, it can also initiate de novo RNA synthesis in a primer-independent manner (Luo et al., 2000; Oh et al., 1999; Sun et al., 2000; Zhong et al., 2000). NS5B binds in vitro preferentially to several regions in the 3′-end of HCV RNA, including the 3′-coding region of NS5B, the U/UC-rich sequence, and part of the X region (in the stem I and II) (Fig. 1) (Cheng et al., 1999; Oh et al., 2000). Partial deletion of the 3′UTR of HCV RNA abolished the template activity of the RNA (Cheng et al., 1999; Oh et al., 2000). Thus, it appears that NS5B recognizes some specific sequence or structural elements at the 3′ end of HCV RNA (Cheng et al., 1999; Oh et al., 2000). Once it binds the stem structure of the 3′UTR, however, NS5B initiates RNA synthesis only from the single-stranded RNA region closest to the 3′ end of the template (Oh et al., 2000). This conclusion is supported by another study showing that the RdRp reaction mediated by NS5B requires a stable secondary structure and a single-stranded sequence with at least one 3′-end cytidylate in the RNA template (Kao et al., 2000).

Since the 3′ end of HCV RNA ends with a near-perfect double-stranded stem (stem I) (Fig. 1), then how does HCV RNA synthesis initiate in vivo, if the in vitro mechanism reflects the mechanism of RNA synthesis in vivo? There are several potential mechanisms whereby the 3′ end sequence of the viral RNA is retained during RNA replication: (1) The 3′ end of HCV RNA may be extended by a terminal transferase so that there is a single-stranded tail at the 3′ end to allow NS5B to initiate from the precise 3′-end. Indeed, an HCV cDNA clone containing two additional nt (UU) at the 3′-end of HCV RNA has been detected (Yamada et al., 1996). (2) RNA helicase or unwinding proteins may be present in the HCV replicative complex to unwind the 3′-end stem structure into the single-stranded region. (3) RNA synthesis may initiate internally in the single-stranded region within the 3′UTR; the 3′-end sequence may be recovered during the positive-strand RNA synthesis since the complementary sequence can be made by fold-back RNA synthesis. (4) The presence of other viral or cellular proteins may alter the choice of the initiation site of RNA replication.

HCV RdRp activity has been detected in the crude replication complexes prepared from lysates of cells carrying HCV replicons. This lysate can synthesize RNA from the endogenous template, but not exogenously added templates, and requires both NS5B and NS3 (Ali et al., 2002; Hardy et al., 2003; Lai et al., 2003). The whole complex is localized on the detergent-resistant membrane and contains all the nonstructural proteins of HCV. The viral RNA is enclosed within the membrane complex and shielded from outside. All the nonstructural proteins are probably anchored on the membrane structures by a series of protein-protein interactions between them and with a cellular protein hVAP-33 (Tu et al., 1999). It has been shown that most of the HCV NS proteins, including NS3, NS4A, NS4B, NS5A, and NS5B, can interact with each other either directly or indirectly (Asabe et al., 1997; Bartenschlager et al., 1995; Gao et al., 2004; Ishido et al., 1998; Lin et al., 1997; Tu et al., 1999). Interestingly, while NS5B interacts with the N terminus of hVAP-33, NS5A binds the C terminus of hVAP-33. The importance of NS5A in HCV replication has been further suggested by the detection of a number of adaptive mutations clustered in a defined region of NS5A in a subgenomic HCV replicon (Blight et al., 2000). It is conceivable that this region may mediate the interaction of NS5A with a cellular protein that inhibits HCV replication. Further evidence supporting the existence of a replication complex consisting of multiple HCV NS proteins came from an analysis of the adaptive mutations derived from a subgenomic HCV replicon (Lohmann et al., 2001). An adaptive mutation in NS5B was found incompatible with those in NS5A or NS4B when introduced back into the same replicon. These mutations may affect contact sites between these proteins in the replication complex, resulting in a dramatic reduction in replication efficiency.

Regulation of HCV Translation and Replication

The 5′ and 3′ UTRs are clearly the sites of important events leading to the onset of translation and replication of HCV RNA. The binding of viral or cellular proteins to the UTRs may modulate the secondary and/or tertiary structure of the viral RNA to facilitate its recognition by the translation machinery and/or the replicase complex. These proteins may recruit additional cellular factors and mediate long-range cross-talks between the ends of HCV RNA.

Regulation of Translation by Viral and Cellular Proteins

The HCV IRES-mediated translation is relatively inefficient as compared to that of other viruses (Borman et al., 1995). It has been suggested that HCV has a self-modulating mechanism to maintain a low level of replication and translation that may promote viral persistence. In this regard, it was speculated that domain IV of the IRES may be stabilized by interaction with the viral core protein, resulting in translation inhibition (Honda et al., 1996a). It has indeed been shown that the core protein binds to several sites within HCV IRES, thereby inhibiting translation (Li et al., 2003; Shimoike et al., 1999; Zhang et al., 2002). However, other studies have suggested that the core-coding sequence, rather than the core protein itself, is responsible for the suppression of IRES-mediated translation, possibly through long-range RNA-RNA interactions with the 5′UTR (Kim et al., 2003; Wang et al., 2000). The sites of RNA-RNA interaction have been mapped to nt 24–38 within the 5′UTR and nt 428–442 of the core-coding sequence (Kim et al., 2003), which is part of a stem-loop structure (Wang et al., 2000). The stem-loop IV of the IRES may be one of the candidates for feedback control, since the stabilization of this structure can reduce IRES activity and the primary sequence within this stem-loop is conserved in nearly all HCV strains (Honda et al., 1996a). However, these conflicting reports may have been due to the different reporter RNA constructs used in the different studies since the stable RNA structure assumed by some heterologous sequences fused directly at the initiation codon may be detrimental to translation directed by IRES (Rijnbrand et al., 2001). Furthermore, a cellular protein PTB binds to the 3′-end of the core-coding region and negatively regulates HCV translation (Ito and Lai, 1999). Thus, translation can be regulated by multiple RNA segments and viral proteins.

Several other HCV proteins, E2 (Taylor et al., 1999) and NS5A (Gale et al., 1997; He et al., 2003), may have an indirect effect on HCV translation by inhibiting PKR, but the biological significance of this effect is not clear.

Besides the canonical translation factors, such as the 40S ribosomal subunit and eIF3, the HCV IRES also recruits noncanonical cellular translation factors, such as La autoantigen (Ali and Siddiqui, 1997) and PTB (Ali and Siddiqui, 1995), which may regulate translation (Fig. 3). The La antigen is an RNA-binding protein belonging to the RNA recognition motif (RRM) superfamily (Gottlieb and Steitz, 1989). It has been implicated in various cellular processes (Ford et al., 2001; Gottlieb and Steitz, 1989) and the translation initiation of picornaviruses and flaviviruses (Ray and Das, 2002; Wolin and Cedervall, 2002). The La antigen recognizes the intact HCV IRES structure and significantly augments the IRES-directed translation in vitro (Ali and Siddiqui, 1997; Costa-Mattioli et al., 2004; Pudi et al., 2003; Pudi et al., 2004). Inhibition of HCV IRES activity caused by sequestration of La protein can be rescued by the addition of purified La protein (Das et al., 1998; Izumi et al., 2004). La protein binds to the GCAC motif near the initiator AUG within stem-loop IV (Pudi et al., 2003). Mutations in the GCAC, which alter the primary sequence while retaining the overall secondary structure, affect the binding of La protein to HCV IRES and significantly inhibit IRES-mediated translation both in vitro and in vivo (Pudi et al., 2004). It has been suggested that the nucleic acid-dependent ATPase activity of La may promote the transformation of stem-loop IV into single-stranded conformation, which is favorable for 40S ribosome binding and the formation of active initiation complex (Lemon and Honda, 1997; Pudi et al., 2004). In addition, La protein may enhance the binding of the ribosomal protein S5 to HCV IRES, which, in turn, facilitates the formation of the IRES-40S complex (Pudi et al., 2004). A recent study suggests that La antigen may also be involved in HCV RNA replication (Domitrovich et al., 2005).

Fig. 3. Cellular proteins that interact with HCV RNA.

Fig. 3

Cellular proteins that interact with HCV RNA. The 5′UTR interacts with a basal translation factor (eIF3), noncanonical translation factors (PTB and La), and other cellular proteins that may regulate translation (hnRNP L and PCBP). The numbers (more...)

PTB interacts with three distinct pyrimidine-rich sequences within the HCV IRES (Ali and Siddiqui, 1995) (Fig. 3). The interaction of PTB with domain III of the IRES has been confirmed by electron microscopy analysis (Beales et al., 2001). Immunodepletion of PTB results in the loss of IRES-directed translation, which, however, cannot be restored by the addition of purified PTB, suggesting that additional factors tightly associated with PTB are also required to enhance IRES activity (Ali and Siddiqui, 1995). In addition to the IRES, PTB has also been shown to interact with the 3′ X region (Ito and Lai, 1997; Tsuchihara et al., 1997) and to enhance HCV IRES-mediated translation (Ito et al., 1998). This long-range effect suggests that the HCV 5′ and 3′UTR may interact with each other through PTB or other viral or cellular proteins. Furthermore, the presence of RNA aptamers of PTB inhibited HCV IRES translation (Anwar et al., 2000). In contrast, results obtained in a study of the subgenomic replicon system do not support a significant role of PTB in HCV replication (Tischendorf et al., 2004). However, PTB has been found in the detergent-resistant membrane complex in cells harboring the HCV subgenomic replicon, while it is in the detergent-sensitive membrane in the control cells, indicating the recruitment of PTB to the HCV RNA replication complex; knockdown of PTB inhibited HCV RNA replication (Domitrovich et al., 2005)(Aizaki and Lai, unpublished).

Besides PTB, also known as heterogeneous nuclear ribonucleoprotein I (hnRNP I), several other proteins of the hnRNP family have been shown to interact with HCV IRES. hnRNP L specifically interact with the 3′ border of the HCV IRES in the core-coding sequence; the binding correlates with the translation efficiency from the IRES (Hahm et al., 1998). The mouse minute virus nonstructural protein NS1-associated protein 1 (NSAP1) (Harris et al., 1999), a homolog of hnRNP R, also known as SYNCRIP (Synaptotagmin-binding cytoplasmic RNA-interacting protein) (Hassfeld et al., 1998), was recently shown to enhance IRES-dependent translation through the interaction with an adenosine-rich region in the 5′-proximal region of the core-coding sequence (Kim et al., 2004a; Reynolds et al., 1995). This protein appears to be involved in RNA replication as well (Choi and Lai, unpublished observation). Poly(rC)-binding protein (PCBP), which is also known as hnRNP E and involved in the expression regulation of numerous cellular and viral RNAs (Ostareck-Lederer et al., 1998), interacts with the HCV 5′UTR (Fukushi et al., 2001a; Spångberg and Schwartz, 1999). PCBP has been implicated in the regulation of poliovirus IRES activity by binding to the 5′UTR of the viral genome (Gamarnik and Andino, 1998; Gamarnik and Andino, 2000). However, the specific interaction of PCBP-2 with the 5′ terminal domain I of HCV RNA has no effect on IRES-mediated translation (Fukushi et al., 2001a). Consistent with the role of domain I in RNA replication (Friebe et al., 2001; Kim et al., 2002b; Reusken et al., 2003), PCBP-2 may be involved in the replication rather than translation of HCV RNA.

Using a functional genomics approach, the proteasome α-subunit PSMA7 has been shown to be involved in IRES-mediated translation, but it is unknown whether the protein acts directly on IRES or indirectly through the regulation of other cellular proteins (Kruger et al., 2001). In summary, multiple cellular proteins binding to the 5′ or 3′UTR can regulate HCV translation; some of them regulate both translation and RNA replication.

Regulation of RNA Replication by Viral and Cellular Proteins

All the viral nonstructural proteins except NS2 are required for replication, but the modes of their participation are not clear. Adaptive mutations in the HCV replicons that allowed the replicons to enhance replication efficiencies have been detected in all of the viral NS proteins, particularly NS3 and NS5A, indicating that every viral nonstructural protein (except NS2) contributes to RNA replication. The purified recombinant HCV NS3 protein or its helicase domain alone can interact efficiently and specifically with the 3′-terminal sequences of both positive- and negative-strand RNA but not with the corresponding complementary 5′-terminal RNA sequences (Banerjee and Dasgupta, 2001). Specific interaction of NS3 with the 3′-terminal sequences of the positive-strand RNA appears to require the entire 3′UTR. A predicted stem-loop structure present at the 3′ terminus (nt 5 to 20 from the 3′ end) of the negative-strand RNA, particularly the three G-C pairs within the stem, appears to be important for NS3 binding to the negative-strand UTR. This interaction may anchor RNA-protein complexes to the cytoplasmic membrane where viral replication complexes are formed.

The poly(U/UC)-rich region of the 3′UTR is a hot spot in the HCV genome for binding cellular proteins (Fig. 3), two of which are the Drosophila melanogaster embryonic lethal, abnormal visual system (ELAV)-like RNA-binding protein, HuR, and hnRNP C (Gontarek et al., 1999; Spångberg et al., 2000). Both HuR and hnRNP C interact with the 3′ ends of both the positive- and negative-strand HCV RNA. Due to its pyrimidine-rich nature, it is not surprising that the poly(U/UC)-rich region has been identified to interact with PTB (Gontarek et al., 1999; Luo, 1999). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) also interacts with the poly(U/UC) tract (Petrik et al., 1999), but the functional relevance of this interaction has yet to be determined. Based on studies of hepatitis A virus (HAV), the binding of GAPDH to the 5′UTR of HAV may directly influence IRES-dependent translation and/or replication of viral RNA by destabilizing the folded structure of the stem-loop IIIa of HAV IRES and competing with PTB for the binding to this structure (Schultz et al., 1996; Yi et al., 2000). The 3′UTR has also been shown to bind La autoantigen, which protects the HCV RNA from rapid degradation (Spångberg et al., 2001). Although the role of these proteins in HCV RNA replication has not be characterized, a group of host factors that bind to the 3′UTR of the closely related pestivirus BVDV has been shown to be required for viral RNA replication (Isken et al., 2003). It is conceivable that these cellular proteins are involved in not only RNA replication but translation as well, possibly through the 5′ and 3′ UTR interaction, causing the circularization of the viral RNA.

In addition to viral and cellular proteins, a liver-specific cellular microRNA, miR-122, was suggested by a recent study to regulate HCV RNA replication by directly interacting with the 5′UTR (Jopling et al., 2005). A 7-nt sequence (ACACUCC) complementary to the seed sequence of miR-122 was found in both the 5′ and 3′ UTRs and predicted to be potential binding sites for miR-122. Disruption of sequence complementarity between the 5′UTR, but not the 3′UTR, and miR-122 reduced HCV RNA replication without affecting RNA stability or translation. It is speculated that miR-122 may aid in RNA folding or RNA sequestration in replication complexes. Since miR-122 is expressed in Huh7 but not HepG2 cells, it may also play a role in host-range determination.

The Evidence for Direct or Indirect 5′-3′ Interaction

Translation of vast majority of eukaryotic mRNAs, which are capped at the 5′ end and polyadenylated at the 3′ end, has been shown to adopt a closed-loop mechanism, in which the mRNAs are circularized via a 5′-3′ interaction mediated by the cap-binding proteins, eIF4F and eIF4G, and the poly(A)-binding protein, PABP. The eIF4G-PABP interaction has also been shown to be required for poly(A)-mediated stimulation of picornaviral IRES-dependent translation, indicating that the 5′-3′ crosstalk is mechanistically conserved between classical eukaryotic mRNAs and picornaviral RNA (Herold and Andino, 2001; Michel et al., 2001; Michel et al., 2000). Circularization has been shown to be important for efficient RNA replication of other flaviviruses (Khromykh et al., 2001). Even in the absence of a poly(A) tail in HCV RNA, the closed-loop model may still be preserved in HCV IRES-mediated translation by the presence of RNA sequences and proteins that can functionally replace the poly(A) tail and PABP (Ito and Lai, 1999). Indeed, the X region of the 3′UTR has been shown to bind PTB and enhance translation of HCV RNA (Ito et al., 1998), suggesting that the functions of the X region may be similar to that of poly(A) in eukaryotic mRNA translation (Kahvejian et al., 2001). Since PTB also interacts with the 5′UTR, it may mediate crosstalk between the 5′- and 3′-ends of HCV RNA. Thus, the mechanism of translation enhancement by PTB may be similar to that of eIF4G-PABP in the translation of cellular and viral RNAs that contain a poly(A) tail.

Switch between Translation and RNA Replication

Since RNA replication and translation occur on the same RNA molecules, the question arises how these two processes are coordinated. For some RNA viruses, there is evidence of coupling between RNA replication and translation. For example, poliovirus defective-interfering RNA without a translatable ORF can not replicate; the nature of the protein product is not critical, but the translatability is essential (Collis et al., 1992; Hagino-Yamagishi and Nomoto, 1989; Novak and Kirkegaard, 1994). This requirement has been demonstrated for several other viral RNAs, such as clover yellow mosaic virus RNA (White et al., 1992), Kunjin virus (Khromykh et al., 2000), and rubella virus (Liang and Gillam, 2001). In coronavirus, the cis-acting protein appears to confer a replication advantage to the RNA; the longer the ORF, the more robust the RNA replication is (de Groot et al., 1992; Kim et al., 1993; Liao and Lai, 1995). The mechanism of the coupling of these two processes is not yet clear. However, there are also viral RNAs (e.g., vesicular stomatitis virus, influenza virus, Sindbis virus) whose replication does not depend on translation of the ORF on the same RNA. In any case, translation and replication must be separated since translation goes in the 5′ to 3′ direction, whereas negative-strand RNA synthesis goes from 3′ to 5′ on the same positive-strand RNA template. When the translation machinery meets the replication complex in opposite direction, there must be a mechanism to prevent confrontation. The situation is akin to the separation of transcription and replication of cellular DNA.

In HCV, the 5′ and 3′UTR sequences are involved in the regulation of both translation and RNA replication. There is substantial overlap in the UTR regions required for translation and RNA replication. Nevertheless, the structural and sequence requirement for these two processes may be different. It is conceivable that the structural changes involved in translation and RNA replication may be effected by the viral or cellular proteins binding to these regions. Indeed, several cellular proteins binding to the 5′ and 3′UTR of HCV have been shown to affect both translation and replication.

In poliovirus, a switch between translation and RNA replication has been proposed to be controlled by PCBP, which enhances translation by binding to the 5′-terminal cloverleaf structure of the poliovirus RNA, and the viral 3CD polymerase, which promotes negative-strand RNA synthesis by binding to the same RNA structure, possibly by altering the structure of this region (Gamarnik and Andino, 1998; Gamarnik and Andino, 2000). Interestingly, PCBP-1 and 2 have also been shown to interact with the HCV 5′UTR, with PCBP-2 binding particularly to stem-loop I, suggesting a possibly similar role of these proteins in regulating a switch between HCV RNA replication and translation (Fukushi et al., 2001a; Spångberg and Schwartz, 1999). In addition, the HCV core protein may also be involved in the switch by down-regulating IRES-dependent translation as a regulatory mechanism required for the initiation of RNA replication (Li et al., 2003; Shimoike et al., 1999; Zhang et al., 2002). Since many of the cellular proteins binding to the 5′ and 3′UTR of HCV have been reported to regulate both translation and replication, it is conceivable that the relative ratios of the different proteins may control the switch between translation and replication. Furthermore, the HCV RNA elements required for translation and those for replication partially overlap. So, the key question in this regard is how the structures of these elements are altered by RNA-RNA or protein-RNA interactions so that the RNA can be properly directed to be used for translation or replication.

Alternatively, an entirely different mechanism may operate to regulate translation and RNA replication of HCV. The RNA replication complex has been shown to reside in the cholesterol-rich, detergent-resistant membrane complex (Aizaki et al., 2004; Shi et al., 2003), whereas translation occurs on the detergent-sensitive, endoplasmic reticulum membrane. Thus, there may be separate machineries in different subcellular compartments for these two processes. The different viral and cellular proteins may bind to RNA molecules differentially in these two different compartments. The key question in this regard is how the RNA is transported from the replication complex to the site of translation or vice versa so that these two functions can be separated.

Perspectives

The 5′ and 3′UTR are the most conserved regions of HCV RNA and play key roles in regulating translation and RNA replication. The knowledge on these two processes is still rudimentary, but the development of subgenomic and genomic replicons and the infectious culture systems (Lohmann et al., 1999; Wakita et al., 2005) provides promises for the unraveling of these two processes in the near future. These two regions also offer promising targets for developing antiviral agents.

Within the past two years, small molecule inhibitors of the NS3 protease and the RNA-dependent RNA polymerase have been shown in early clinical studies to be efficacious in both treatment-naïve patients and patients who failed interferon therapy. However, the extensive genetic heterogeneity of HCV RNA and the rapid evolution of quasispecies present a substantial challenge for these inhibitors to broad-spectrum activity. The high degree of sequence conservation in the 5′UTR and 3′UTR among different HCV genotypes makes these regions attractive targets for antiviral therapies, such as antisense oligonucleotides (Soler et al., 2004), ribozymes (Welch et al., 1996; Welch et al., 1998), and siRNAs (Kronke et al., 2004; Randall and Rice, 2004). The inhibition of HCV RNA translation or replication has been observed with these inhibitors that target the 5′UTR alone or together with the core-coding sequence of HCV (Hanecak et al., 1996; Kronke et al., 2004; Macejak et al., 2000; McCaffrey et al., 2003; Ohkawa et al., 1997; Sakamoto et al., 1996). Universal siRNAs targeting similar regions have been generated and proven to be effective against all known genotypes (Kronke et al., 2004; Yokota et al., 2003). Encouragingly, early clinical trials have demonstrated efficacy of some of these inhibitors in HCV-infected patients despite the limitations associated with RNA-based therapies and the inherent structures of the UTR sequences (Branch, 1998; Crooke and Bennett, 1996; Gomez et al., 2004). The interventions directing against conserved domains of viral RNAs may provide valuable alternatives to small molecule inhibitors that target HCV proteins.

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