Masking the 5' terminal nucleotides of the hepatitis C virus genome by an unconventional microRNA-target RNA complex

Hepatitis C virus subverts liver-specific microRNA, miR-122, to upregulate viral RNA abundance in both infected cultured cells and in the liver of infected chimpanzees. These findings have identified miR-122 as an attractive antiviral target. Thus, it is imperative to know whether a distinct functional complex exists between miR-122 and the viral RNA versus its normal cellular target mRNAs. Toward this goal, effects on viral RNA abundance of mutated miR-122 duplex molecules, bound at each of the two target sites in the viral genome, were compared to effects on microRNA- or siRNA-mediated regulation of reporter target mRNAs. It was found that miR-122 formed an unusual microRNA complex with the viral RNA that is distinct from miR-122 complexes with reporter mRNAs. Notably, miR-122 forms an oligomeric complex in which one miR-122 molecule binds to the 5' terminus of the hepatitis C virus (HCV) RNA with 3' overhanging nucleotides, masking the 5' terminal sequences of the HCV genome. Furthermore, specific internal nucleotides as well as the 3' terminal nucleotides in miR-122 were absolutely required for maintaining HCV RNA abundance but not for microRNA function. Both miR-122 molecules utilize similar internal nucleotides to interact with the viral genome, creating a bulge and tail in the miR-122 molecules, revealing tandemly oriented oligomeric RNA complexes. These findings suggest that miR-122 protects the 5' terminal viral sequences from nucleolytic degradation or from inducing innate immune responses to the RNA terminus. Finally, this remarkable microRNA-mRNA complex could be targeted with compounds that inactivate miR-122 or interfere with this unique RNA structure.     

From the Cover: Competing and noncompeting activities of miR-122 and the 5′ exonuclease Xrn1 in regulation of hepatitis C virus replication

Hepatitis C virus (HCV) replication is dependent on microRNA 122 (miR-122), a liver-specific microRNA that recruits Argonaute 2 to the 5′ end of the viral genome, stabilizing it and slowing its decay both in cell-free reactions and in infected cells. Here we describe the RNA degradation pathways against which miR-122 provides protection. Transfected HCV RNA is degraded by both the 5′ exonuclease Xrn1 and 3′ exonuclease exosome complex, whereas replicating RNA within infected cells is degraded primarily by Xrn1 with no contribution from the exosome. Consistent with this, sequencing of the 5′ and 3′ ends of RNA degradation intermediates in infected cells confirmed that 5′ decay is the primary pathway for HCV RNA degradation. Xrn1 knockdown enhances HCV replication, indicating that Xrn1 decay and the viral replicase compete to set RNA abundance within infected cells. Xrn1 knockdown and miR-122 supplementation have equal, redundant, and nonadditive effects on the rate of viral RNA decay, indicating that miR-122 protects HCV RNA from 5′ decay. Nevertheless, Xrn1 knockdown does not rescue replication of a viral mutant defective in miR-122 binding, indicating that miR-122 has additional yet uncharacterized function(s) in the viral life cycle.     

Targeting microRNA-122 to Treat Hepatitis C Virus Infection

An important host factor for hepatitis C virus (HCV) is microRNA-122 (miR-122). miR-122 is a liver-specific member of a family of small, non-coding RNA molecules known as microRNAs that play major roles in the regulation of gene expression by direct interaction with RNA targets. miR-122 binds directly to two sites in the 5' untranslated region (UTR) of HCV RNA and positively regulates the viral life cycle. The mechanism by which this regulation occurs is still not fully understood. There has been a great deal of interest in potential therapeutics based on small RNAs, and targeting miR-122 to combat HCV is one of the furthest advanced. Chemical inhibitors of miR-122 can be introduced into mammals intravenously and result in potent and specific knockdown of the microRNA, with no detectable adverse effects on liver physiology. This strategy was recently applied to chimpanzees chronically infected with HCV and resulted in a sustained reduction in viral load in the animals. Inhibition of miR-122 therefore presents a very attractive novel approach to treating HCV, a virus for which improved therapeutics are urgently needed.     

MicroRNA-122 antagonism against hepatitis C virus genotypes 1-6 and reduced efficacy by host RNA insertion or mutations in the HCV 5' UTR

MicroRNA-122 (miR-122) is believed to stimulate hepatitis C virus (HCV) replication through interaction with two adjacent sites downstream of stem loop I (SLI) within the HCV 5' untranslated region (5' UTR). Recently, it was demonstrated that locked nucleic acid SPC3649-induced miR-122 antagonism suppressed HCV genotype 1a and 1b infection in vivo. However, virus-producing culture systems with 5' UTR of different HCV genotypes have not been available for testing 5' UTR-based treatment approaches. Using JFH1-based Core-NS2 genotype recombinants, we developed 5' UTR-NS2 recombinants of HCV genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, and 6a with efficient growth in Huh7.5 cells. Deletion mutagenesis studies demonstrated that the 5' UTR SLI was essential for genotypes 1-6 infection. However, lack of SLI could be compensated for by insertion of other structured HCV or host RNA sequences, including U3 small nucleolar RNA. We demonstrated that SPC3649-induced miR-122 antagonism had a potent antiviral effect against HCV genotypes 1-6 5' UTR-NS2 viruses. Strikingly, HCV recombinant virus with substitution of SLI and miR-122 binding site 1 (S1) by the U3 RNA sequence was not affected by miR-122 antagonism; this was attributed to the lack of an intact S1 by reverse genetics studies. Therefore, we engineered the corresponding U3 RNA sequences into S1 and demonstrated that HCV recombinants with wild-type SLI and single or combined mutations at four of eight nucleotides of S1 were viable in Huh7.5 cells. These mutations reduced the efficacy of SPC3649 treatment, indicating that escape variants to miR-122 antagonism-based HCV therapy could potentially occur.     

miR-122–based therapies select for three distinct resistance mechanisms based on alterations in RNA structure

Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with a liver-specific microRNA called miR-122. miR-122 binds to two sites in the 5′ untranslated region of the viral genome and promotes HCV RNA accumulation. This interaction is important for viral RNA accumulation in cell culture, and miR-122 inhibitors have been shown to be effective at reducing viral titers in chronic HCV-infected patients. Herein, we analyzed resistance-associated variants that were isolated in cell culture or from patients who underwent miR-122 inhibitor–based therapy and discovered three distinct resistance mechanisms all based on changes to the structure of the viral RNA. Specifically, resistance-associated variants promoted riboswitch activity, genome stability, or positive-strand viral RNA synthesis, all in the absence of miR-122. Taken together, these findings provide insight into the mechanism(s) of miR-122–mediated viral RNA accumulation and provide mechanisms of antiviral resistance mediated by changes in RNA structure.

Hepatitis C virus (HCV) is a positive-sense RNA virus of the Flaviviridae family. The ∼9.6 kb HCV genomic RNA consists of a single open reading frame, which gives rise to the viral polyprotein that is processed into the 10 mature viral proteins flanked by highly structured 5′ and 3′ untranslated regions (UTRs) (1, 2). As a positive-sense RNA virus, the viral genome itself must serve as a template for the different stages of the viral life cycle, including viral translation, replication, and packaging (2). To this end, the viral 5′ and 3′ UTRs contain several cis-acting RNA elements that play important roles in directing the various stages of the viral life cycle (2–4). Specifically, the 5′ UTR contains the viral internal ribosomal entry site (IRES) made up of stem–loops (SL) II-IV, which is required for viral translation, while sequences and SL structures in both the 5′ (SLI-II) and 3′ UTRs (variable region, polyU/UC-tract, and 96-nt X-tail) are required for viral RNA replication (2, 5–8). Additionally, the 5′ terminus of the HCV genome interacts with the highly abundant, liver-specific human microRNA (miRNA), miR-122 (9–12).miR-122 is a highly expressed miRNA in the liver with ∼135,000 copies per hepatocyte (9, 13). While miRNAs typically interact with the 3′ UTRs of their target messenger RNAs (mRNAs) to dampen gene expression, miR-122 interacts to two sites in the 5′ UTR (site 1 and site 2) of the viral genome, and these interactions promote viral RNA accumulation (9, 10, 12). Several recent studies have led to a new model for miR-122:HCV RNA interactions that suggest that miR-122 plays at least three roles in the HCV life cycle (Fig. 1) (14–17). Firstly, the HCV 5′ UTR is thought to initially adopt an energetically favorable conformation (termed SLII^alt), which results in the recruitment of an Ago:miR-122 complex to site 2 of the HCV genome. This results in an RNA chaperone-like switch in conformation, akin to a bacterial riboswitch (18), resulting in the formation of SLII and allowing the viral IRES (SLII-IV) to form (15–17). Secondly, this change in conformation allows the recruitment of Ago:miR-122 to site 1, which protects the 5′ terminus from pyrophosphatase activity and subsequent exoribonuclease-mediated decay (12, 14, 19–21). Finally, the Ago protein bound to site 2 makes direct contact with the viral IRES, promoting HCV IRES-mediated translation (16).Open in a separate windowFig. 1.Model of miR-122 interactions with the HCV genome. The HCV genomic RNA is thought to enter the cell in an energetically stable conformation termed SLII^alt. The recruitment of the first Ago:miR-122 molecule to the accessible (unpaired) site 2 serves as an RNA chaperone, akin to a bacterial riboswitch, which refolds the RNA into the functional SLII conformation and allows the viral IRES (SLII-IV) to form (1). Subsequent recruitment of a second Ago:miR-122 molecule to site 1 promotes genome stability by protecting the 5′ terminus from cellular pyrophosphatases and exoribonuclease-mediated decay (2). In order to accommodate the Ago:miR-122 complex at site 1, the Ago:miR-122 complex at site 2 releases its auxiliary interactions but is likely stabilized by interactions between the Ago protein and SLII of the HCV IRES (3). Collectively, these interactions promote HCV IRES-mediated translation. miR-122 seed and auxiliary binding sequences are indicated (red).Due to the importance of miR-122 in the HCV life cycle, two miR-122 inhibitors (antisense oligonucleotides), the first miRNA-based drugs to enter clinical trials, have been developed and used to treat chronic HCV infection in the clinic (22, 23). Both Miravirsen (Santaris Pharma, a/s) and RG-101 (Regulus Therapeutics) miR-122 inhibitors have completed Phase II or Ib clinical trials, respectively, to investigate their clinical efficacy in chronic HCV infection (22, 23). Excitingly, both treatments led to dose-dependent and sustained reductions in viral loads; and, in the latter study, two patients achieved a sustained virological response (at least up to 76 wk posttreatment) after receiving a single dose of RG-101 (23). Neither treatment was associated with significant adverse events or long-term safety issues, suggesting that antisense targeting of miR-122 may be an effective treatment that could be used in future combination therapies. Interestingly, while no resistance was apparent during treatment, when viral RNA rebounded after the cessation of the inhibitor, several resistance-associated variants (RAVs) were identified in the 5′ UTR of the HCV genome (23). Specifically, C3U (genotype 1) was identified as a RAV in both the Miravirsen and RG-101 trials, while the C2GC3U (genotype 3/4) RAV was identified in the RG-101 trial alone, with both RAVs identified in multiple patients (22, 23). Additional RAVs were identified in cell culture, including studies performed with genotype 1 (A4C) and genotype 2 (U4C, G28A, and C37U), which were identified alone (A4C and G28A) or in combination with other RAVs (i.e., G28A+C37U, U4C+G28A+C37U) (24–26). As the cell culture–based studies identifying RAVs were performed with genotype 2 and the majority of the RAV nucleotide identities are present in genotype 2 (save for A4C, although U4C was deemed an equivalent RAV observed in this genotype), herein, we explored the mechanism of action of these collective RAVs (C2GC3U, C3U, U4C, G28A, and C37U) using genotype 2a (J6/JFH-1) reporter RNAs (Fig. 2A) (22–26). Previous work suggests that the G28A mutation is “riboswitched” and promotes the formation of the functional SLII structure even in the absence of miR-122 (16, 17). Similar to G28A, we hypothesized that the other RAVs also alter the structure of the viral RNA in a manner that negates the requirement for one or more miR-122 activities. Thus, we sought to provide insight into the mechanism(s) of action of the RAVs using RNA structure analysis and assays for viral RNA accumulation and decay. Our analyses suggest that each of the RAVs alter the structure of the viral RNA, and we identify three distinct resistance mechanisms based on unique changes in viral RNA structure.Open in a separate windowFig. 2.RAV accumulation in cell culture. (A) The positions of the RAVs on the 5′ UTR of the HCV RNA. Nucleotides 1 to 45 of the HCV genome (black) and the binding topology of the two miR-122 molecules (teal) are shown. Full-length RLuc HCV genomic reporter RNAs (WT and RAVs) were coelectroporated with a capped Firefly fuciferase (FLuc) mRNA into (B) Huh-7.5 or (C) miR-122 KO cells. Full-length RLuc HCV genomic reporter RNAs containing mutations at (D) site 1 (S1:p3A) or (E) site 2 (S2:p3A) were coelectroporated with a capped FLuc mRNA and compensatory miR-122p3U molecules into miR-122 KO cells. Luciferase activity was measured at the indicated time points post-electroporation. The limit of detection is indicated, and all data are representative of at least three independent replicates. Error bars represent the SD of the mean.     

RNA Structural Elements of Hepatitis C Virus Controlling Viral RNA Translation and the Implications for Viral Pathogenesis

Hepatitis C virus (HCV) genome multiplication requires the concerted action of the viral RNA, host factors and viral proteins. Recent studies have provided information about the requirement of specific viral RNA motifs that play an active role in the viral life cycle. RNA regulatory motifs controlling translation and replication of the viral RNA are mostly found at the 5'' and 3'' untranslated regions (UTRs). In particular, viral protein synthesis is under the control of the internal ribosome entry site (IRES) element, a complex RNA structure located at the 5''UTR that recruits the ribosomal subunits to the initiator codon. Accordingly, interfering with this RNA structural motif causes the abrogation of the viral cycle. In addition, RNA translation initiation is modulated by cellular factors, including miRNAs and RNA-binding proteins. Interestingly, a RNA structural motif located at the 3''end controls viral replication and establishes long-range RNA-RNA interactions with the 5''UTR, generating functional bridges between both ends on the viral genome. In this article, we review recent advances on virus-host interaction and translation control modulating viral gene expression in infected cells.     

Inhibition of hepatitis C virus (HCV)-RNA-dependent translation and replication of a chimeric HCV poliovirus using synthetic stabilized ribozymes

Ribozymes are catalytic RNA molecules that can be designed to cleave specific RNA sequences. To investigate the potential use of synthetic stabilized ribozymes for the treatment of chronic hepatitis C virus (HCV) infection, we designed and synthesized hammerhead ribozymes targeting 15 conserved sites in the 5' untranslated region (UTR) of HCV RNA. This region forms an internal ribosome entry site that allows for efficient translation of the HCV polyprotein. The 15 synthetic ribozymes contained modified nucleotides and linkages that stabilize the molecules against nuclease degradation. All 15 ribozymes were tested for their ability to reduce expression in an HCV 5' UTR/luciferase reporter system and for their ability to inhibit replication of an HCV-poliovirus (HCV-PV) chimera. Treatment with several ribozymes resulted in significant down-regulation of HCV 5' UTR/luciferase reporter expression (range 40% to 80% inhibition, P <.05). Moreover, several ribozymes showed significant inhibition (>90%, P <.001) of chimeric HCV-PV replication. We further show that the inhibitory activity of ribozymes targeting site 195 of HCV RNA exhibits a sequence-specific dose response, requires an active catalytic ribozyme core, and is dependent on the presence of the HCV 5' UTR. Treatment with synthetic stabilized anti-HCV ribozymes has the potential to aid patients who are infected with HCV by reducing the viral burden through specific targeting and cleavage of the viral genome.     

Translation and replication of hepatitis C virus genomic RNA depends on ancient cellular proteins that control mRNA fates

Inevitably, viruses depend on host factors for their multiplication. Here, we show that hepatitis C virus (HCV) RNA translation and replication depends on Rck/p54, LSm1, and PatL1, which regulate the fate of cellular mRNAs from translation to degradation in the 5′-3′-deadenylation-dependent mRNA decay pathway. The requirement of these proteins for efficient HCV RNA translation was linked to the 5′ and 3′ untranslated regions (UTRs) of the viral genome. Furthermore, LSm1–7 complexes specifically interacted with essential cis-acting HCV RNA elements located in the UTRs. These results bridge HCV life cycle requirements and highly conserved host proteins of cellular mRNA decay. The previously described role of these proteins in the replication of 2 other positive-strand RNA viruses, the plant brome mosaic virus and the bacteriophage Qß, pinpoint a weak spot that may be exploited to generate broad-spectrum antiviral drugs.     

Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5' UTR as in the 3' UTR

In animals, microRNAs (miRNAs) bind to the 3' UTRs of their target mRNAs and interfere with translation, although the exact mechanism of inhibition of protein synthesis remains unclear. Functional miRNA-binding sites in the coding regions or 5' UTRs of endogenous mRNAs have not been identified. We studied the effect of introducing miRNA target sites into the 5' UTR of luciferase reporter mRNAs containing internal ribosome entry sites (IRESs), so that potential steric hindrance by a microribonucleoprotein complex would not interfere with the initiation of translation. In human HeLa cells, which express endogenous let-7a miRNA, the translational efficiency of these IRES-containing reporters with 5' let-7 complementary sites from the Caenorhabditis elegans lin-41 3' UTR was repressed. Similarly, the IRES-containing reporters were translationally repressed when human Ago2 was tethered to either the 5' or 3' UTR. Interestingly, the method of DNA transfection affected our ability to observe miRNA-mediated repression. Our results suggest that association with any position on a target mRNA is mechanistically sufficient for a microribonucleoprotein to exert repression of translation at some step downstream of initiation.     

Interferons alpha, beta, gamma each inhibit hepatitis C virus replication at the level of internal ribosome entry site-mediated translation.

Interferon (IFN)-alpha is the standard therapy for the treatment of chronic hepatitis C, but the mechanisms underlying its antiviral action are not well understood. In this report, we demonstrated that IFN-alpha, -beta and -gamma inhibit replication of the hepatitis C virus (HCV) in a cell culture model at concentrations between 10 and 100 IU/ml. We demonstrated that the antiviral actions each of each these IFNs are targeted to the highly conserved 5' untranslated region of the HCV genome, and that they directly inhibit translation from a chimeric clone between full-length HCV genome and green fluorescent protein (GFP). This effect is not limited to HCV internal ribosome entry site (IRES), since these IFNs also inhibit translation of the encephalomyocardititis virus (EMCV) chimeric mRNA in which GFP is expressed by IRES-dependent mechanisms (pCITE-GFP). These IFNs had minimal effects on the expression of mRNAs from clones in which translation is not IRES dependent. We conclude that IFN-alpha, -beta and -gamma inhibit replication of sub-genomic HCV RNA in a cell culture model by directly inhibiting two internal translation initiation sites of HCV- and EMCV-IRES sequences present in the dicistronic HCV sub-genomic RNA. Results of this in vitro study suggest that selective inhibition of IRES-mediated translation of viral polyprotein is a general mechanism by which IFNs inhibits HCV replication.     

Regulation of eukaryotic protein synthesis: Selective influenza viral mRNA translation is mediated by the cellular RNA-binding protein GRSF-1

To better understand regulation of eukaryotic protein synthesis, we studied cellular and viral mRNA translation in influenza virus-infected cells. Influenza virus infection results in a dramatic shut-off of cellular protein synthesis that is concomitant with selective viral mRNA translation. Earlier work showed that these events are mediated by viral and/or cellular factors binding to the 5' untranslated region (5' UTR) of viral mRNAs. To identify trans-acting cellular proteins responsible for selective viral protein synthesis, we employed the yeast three-hybrid system. Using the 5' UTR of the influenza virus nucleocapsid protein (NP) mRNA as bait, we identified the cellular RNA-recognition motif containing RNA-binding protein G-rich sequence factor 1 (GRSF-1) as a positive-acting translational regulatory factor. The in vivo yeast assay revealed GRSF-1 specifically bound to the NP 5' UTR but not select NP 5' UTR mutants or cellular RNA 5' UTRs. These data were confirmed by gel shift assays using recombinant GRSF-1. Importantly, recombinant GRSF-1 specifically stimulated translation of a NP 5' UTR-driven template in cell-free translation systems. Furthermore, translation efficiency of NP 5' UTR-driven templates was reduced markedly in GRSF-1-depleted HeLa cell extracts, but restored in GRSF-1-reconstituted extracts. GRSF-1 also stimulated translation of an NP 5' UTR-driven template in HeLa cell extracts that were depleted of essential factors by addition of RNA oligonucleotides representing the viral 5' UTR RNA. Taken together, these data document the functional demonstration of a cellular protein binding to influenza virus RNAs and, importantly, suggest that influenza virus may recruit GRSF-1 to the 5' UTR to ensure preferential translation of viral mRNAs in infected cells.     

Poly(rC)-Binding Protein 1 Limits Hepatitis C Virus Virion Assembly and Secretion

The hepatitis C virus (HCV) co-opts numerous cellular elements, including proteins, lipids, and microRNAs, to complete its viral life cycle. The cellular RNA-binding protein, poly(rC)-binding protein 1 (PCBP1), was previously reported to bind to the 5′ untranslated region (UTR) of the HCV genome; however, its importance in the viral life cycle has remained unclear. Herein, we sought to clarify the role of PCBP1 in the HCV life cycle. Using the HCV cell culture (HCVcc) system, we found that knockdown of endogenous PCBP1 resulted in an overall decrease in viral RNA accumulation, yet resulted in an increase in extracellular viral titers. To dissect PCBP1’s specific role in the HCV life cycle, we carried out assays for viral entry, translation, genome stability, RNA replication, as well as virion assembly and secretion. We found that PCBP1 knockdown did not directly affect viral entry, translation, RNA stability, or RNA replication, but resulted in an overall increase in infectious particle secretion. This increase in virion secretion was evident even when viral RNA synthesis was inhibited, and blocking virus secretion could partially restore the viral RNA accumulation decreased by PCBP1 knockdown. We therefore propose a model where endogenous PCBP1 normally limits virion assembly and secretion, which increases viral RNA accumulation in infected cells by preventing the departure of viral genomes packaged into virions. Overall, our findings improve our understanding of how cellular RNA-binding proteins influence viral genomic RNA utilization during the HCV life cycle.     

Reciprocal effects of micro-RNA-122 on expression of heme oxygenase-1 and hepatitis C virus genes in human hepatocytes

BACKGROUND & AIMS: Heme oxygenase-1 (HO-1) is an antioxidant defense and key cytoprotective enzyme, which is repressed by Bach1. Micro-RNA-122 (miR-122) is specifically expressed and highly abundant in human liver and required for replication of hepatitis C virus (HCV) RNA. This study was to assess whether a specific miR-122 antagomir down-regulates HCV protein replication and up-regulates HO-1. METHODS: We transfected antagomir of miR-122, 2'-O-methyl-mimic miR-122, or nonspecific control antagomir, into wild-type (WT) Huh-7 cells or Huh-7 stably replicating HCV subgenomic protein core through nonstructural protein 3 of HCV (NS3) (CNS3 replicon cells) or NS3-5B (9-13 replicon cells). RESULTS: Antagomir of miR-122 reduced the abundance of HCV RNA by 64% in CNS3 and by 84% in 9-13 cells. Transfection with 2'-O-methlyl-mimic miR-122 increased HCV levels up to 2.5-fold. Antagomir of miR-122 also decreased Bach1 and increased HO-1 mRNA levels in CNS3, 9-13, and WT Huh-7 cells. Increasing HO-1 by silencing Bach1 with 50 nmol/L Bach1-short interfering RNA or by treatment with 5 mumol/L cobalt protoporphyrin or heme (known inducers of HO-1) decreased HCV RNA and protein by 50% in HCV replicon cells. CONCLUSIONS: Down-regulation of HCV replication using an antagomir targeted to miR-122 is effective, specific, and selective. Increasing HO-1, by silencing the Bach1 gene or by treatment with cobalt protoporphyrin or heme, decreases HCV replication. Thus, miR-122 plays an important role in the regulation of HCV replication and HO-1/Bach1 expression in hepatocytes. Down-regulation of miR-122 and up-regulation of HO-1 may be new strategies for anti-HCV intervention and cytoprotection.     

A chimeric GB virus B with 5' nontranslated RNA sequence from hepatitis C virus causes hepatitis in tamarins

Only humans and chimpanzees are fully permissive for replication of hepatitis C virus (HCV), an important cause of liver cirrhosis and cancer worldwide. The absence of suitable animal models limits opportunities for in vivo evaluation of candidate hepatitis C therapeutics and slows progress in the field. Here, we describe a chimeric virus derived from GB virus B (GBV-B), an unclassified hepatotropic member of the family Flaviviridae that is closely related to HCV and infects tamarins (Saguinus sp.), in which a functionally important HCV regulatory sequence replaced an analogous sequence in the 5' nontranslated region (5'NTR) of the GBV-B genome. The transplanted sequence comprised domain III of the internal ribosome entry site (IRES), which directly binds the 40S ribosome subunit and is a target for candidate therapeutics. The chimeric 5'NTR retained ribosome binding activity and was competent in directing protein translation both in cell-free translation reactions and in transfected primary tamarin hepatocyte cultures. Virus rescued from the chimeric RNA replicated in the liver of tamarins, causing biochemical and histopathological changes typical of viral hepatitis. However, adaptive mutations were required elsewhere in the genome for efficient replication. Virus was not rescued from other, translationally competent, chimeric RNAs in which domain II of the IRES was exchanged. Thus, the 5'NTR appears to contain virus-specific replication signals that interact with other sites within the viral genome or with viral proteins. In conclusion, such novel chimeric flaviviruses offer opportunities for new insights into HCV replication mechanisms, while potentially facilitating the evaluation of candidate therapeutics in vivo.     

The effects of the conserved extreme 3' end sequence of hepatitis C virus (HCV) RNA on the in vitro stabilization and translation of the HCV RNA genome

BACKGROUND/AIMS: The discovery of an additional 98-base in the extreme 3' end of the hepatitis C virus (HCV) has fueled much speculation as to the role of this sequence on the behavior of the virus. It is now known that this additional 98-base sequence is present and conserved amongst HCV genotypes. This sequence is capable of forming complex and stable high-order structures that may be important in stabilizing the RNA to degradation, facilitating translation and regulating replication of the virus. We have examined the possible role of the HCV extreme 3' end sequence in stabilizing the HCV RNA genome and regulating translation in vitro. METHODS: The extreme 3' end sequence was cloned to downstream of two pre-existing two HCV clones: HCV1 (genotype 1a) and HCV-BK (genotype 1b). The reconstructed full-length clones were then tested in vitro for their stability and translation efficiency. RESULTS: We showed that the addition of the conserved 3' end sequence greatly enhanced the stability of HCV1 RNA but had only minimal effect on HCV-BK RNA in mammalian cytoplasmic extracts, suggesting that the requirements for HCV RNA stability vary amongst isolates. Following the optimization of in vitro translation conditions, it was demonstrated that the addition of this 3' end sequence did not affect the translation level from either HCV clone. CONCLUSIONS: The conserved 3' end of the HCV genome confers differential stabilizing effects on two HCV genotype 1 isolates and has no obvious role in the in vitro translation of either clone.     

MicroRNAs,Hepatitis C Virus,and HCV/HIV-1 Co-Infection: New Insights in Pathogenesis and Therapy

MicroRNAs (miRNAs) can exert a profound effect on Hepatitis C virus (HCV) replication. The interaction of HCV with the highly liver-enriched miRNA, miR-122 represents one such unique example of viruses having evolved mechanism(s) to usurp the host miRNA machinery to support viral life cycle. Furthermore, HCV infection can also trigger changes in the cellular miRNA profile, which may ultimately contribute to the outcome of viral infection. Accumulating knowledge on HCV-host miRNA interactions has ultimately influenced the design of therapeutic interventions against chronic HCV infection. The importance of microRNA modulation in Human Immunodeficiency Virus (HIV-1) replication has been reported, albeit only in the context of HIV-1 mono-infection. The development of HCV infection is dramatically influenced during co-infection with HIV-1. Here, we review the current knowledge on miRNAs in HCV mono-infection. In addition, we discuss the potential role of some miRNAs, identified from the analyses of public data, in HCV/HIV-1 co-infection.