Hepatitis C Virus RNA Genome Research Articles

In vivo inhibition of hepatitis C virus infection by anti‐Claudin‐1 monoclonal antibodies (1062.9)

More than 170 million people worldwide are infected with hepatitis C virus (HCV). Despite concerted research efforts, anti‐HCV agents resistant to the high mutability of the HCV RNA genome have not yet been produced. Claudin 1 (CLDN1) is a co‐receptor for HCV entry into hepatocytes and is an attractive potential target for HCV therapy, because such host factors are genetically stable. Here, we investigated the effects of 4 previously generated mouse anti‐CLDN1 antibodies on HCV infection. All 4 clones attenuated the infection of Huh7 cells by HCV (genotype 2a) and HCV pseudoviral particles (HCVpp, genotypes 1b and 2a). Two of 4 clones also prevented HCV (genotype 1b) infection of human‐liver‐chimeric mice. Injection of mouse anti‐CLDN1 antibodies did not adversely affect serum human albumin levels, apparent character, and motility. For future clinical application, we prepared human‐mouse chimeric anti‐CLDN1 antibodies. The chimeric antibodies prevented HCV and HCVpp infection of Huh7 cells as effectively as did the mouse antibodies. Our findings suggest that mouse and mouse‐human chimeric anti‐CLDN1 antibodies prevent HCV infection and may therefore be promising candidate anti‐HCV agents.Grant Funding Source: supported by the Ministry of Health, Labor, and Welfare of Japan

HCV Genomic RNA Activates the NLRP3 Inflammasome in Human Myeloid Cells

BackgroundElevated plasma levels of IL-1β and IL-18 from patients with hepatitis C virus (HCV) infection indicate a possible activation of inflammasome by HCV.Methodology/Principal FindingsTo demonstrate whether HCV infection activates the inflammasome, we investigated inflammasome activation from HCV infected hepatic Huh7 cells, or monocytic cells and THP-1 derived macrophages challenged with HCV virions, but no any inflammasome activation was detected in these cells. However, when we transfected HCV genomic RNA into monocytes or macrophages, IL-1β was secreted in a dose-dependent manner. We also detected ASC oligomerization and caspase-1 cleavage in HCV RNA transfected macrophages. Using shRNA-mediated gene silencing or specific inhibitors, we found that HCV RNA-induced IL-1β secretion was dependent on the presence of inflammasome components such as NLRP3, ASC and caspase-1. Furthermore, we also found that RIG-I was dispensable for HCV RNA-induced NLRP3 inflammasome activation, while reactive oxygen species (ROS) production was required.ConclusionsOur results indicate that HCV RNA activates the NLRP3 inflammasome in a ROS-dependent manner, and RIG-I is not required for this process.

In silico studies of medicinal compounds against hepatitis C capsid protein from north India.

Hepatitis viral infection is a leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Over one million people are estimated to be persistently infected with hepatitis C virus (HCV) worldwide. As capsid core protein is the key element in spreading HCV; hence, it is considered to be the superlative target of antiviral compounds. Novel drug inhibitors of HCV are in need to complement or replace the current treatments such as pegylated interferon’s and ribavirin as they are partially booming and beset with various side effects. Our study was conducted to predict 3D structure of capsid core protein of HCV from northern part of India. Core, the capsid protein of HCV, handles the assembly and packaging of HCV RNA genome and is the least variable of all the ten HCV proteins among the six HCV genotypes. Therefore, we screened four phytochemicals inhibitors that are known to disrupt the interactions of core and other HCV proteins such as (a) epigallocatechin gallate (EGCG), (b) ladanein, (c) naringenin, and (d) silybin extracted from medicinal plants; targeted against active site of residues of HCV-genotype 3 (G3) (Q68867) and its subtypes 3b (Q68861) and 3g (Q68865) from north India. To study the inhibitory activity of the recruited flavonoids, we conducted a quantitative structure–activity relationship (QSAR). Furthermore, docking interaction suggests that EGCG showed a maximum number of hydrogen bond (H-bond) interactions with all the three modeled capsid proteins with high interaction energy followed by naringenin and silybin. Thus, our results strongly correlate the inhibitory activity of the selected bioflavonoid. Finally, the dynamic predicted capsid protein molecule of HCV virion provides a general avenue to target structure-based antiviral compounds that support the hypothesis that the screened inhibitors for viral capsid might constitute new class of potent agents but further confirmation is necessary using in vitro and in vivo studies.

Targeting the Hepatitis C Virus with PNAs

The liver specific microRNA-122 (miR-122) has been shown to facilitate the replication and/or translation of the Hepatitis C virus (HCV). Although the exact role played by miR-122 in this process is not fully understood, it has been shown that one of the functions of miR-122 is to stabilize the HCV RNA genome. It has also been shown that miR-122 is a valid antiviral target, as locked nucleic acids (LNAs) developed against miR-122 abolished HCV replication. However, miR-122 has numerous other functions in the hepatic cell, which will also be affected by LNAs. In this study, we adopted a different approach by designing peptide nucleic acids (PNAs) against the highly conserved miR-122 binding sites within the HCV genome, and tested their antiviral properties. We demonstrated the ability of the PNA to bind specifically the 5' UTR of the HCV genome, with a low nanomolar dissociation constant. Moreover, we showed that the PNA is able to invade the HCV 5'-UTR - miR-122 complex displacing miR-122, and thus preventing it from exerting its beneficial function upon HCV replication and/or translation.

A Study of CC-Chemokine Receptor 5 (CCR5) Polymorphism on the Outcome of HCV Therapy in Egyptian Patients

Background:Chronic hepatitis C virus (HCV) infection is a globally serious public health issue.Objectives:In this study, we investigated CC chemokine receptor 5 (CCR5-59029) polymorphism which is considered an important component of the immune system in determining the outcome of HCV infection. Its critical role as a marker in response to interferon therapy of HCV infection is also investigated besides its effect on other clinical patient factors.Patients and Methods:This study was conducted on 82 Egyptian patients with chronic Hepatitis C Virus (HCV) infection who received PEG-INF + Ribavirin treatment for 48 weeks. The study was also conducted on 50 healthy controls (with negative results for HCV antibody and RNA PCR). Full history of patients in this study was recorded. Clinical and histological examinations, qualitative HCV nested RT-PCR, quantitative real –time PCR, and genotyping of HCV RNA genome were performed. CCR5-59029 polymorphism with nucleotide substitution from G to A was amplified. The amplicons were digested with restriction endonuclease Bsp 1286I, and produced RFLPs of the CCR5 genotypes were determined.Results:The present study showed a significant association between the functional SNP of CCR5 gene and the viral response to interferon in chronic HCV Egyptian patients. It was shown that the higher fibrosis stages (F2-F4) had significant association with nonresponse to treatment compared to the lower fibrosis stages (F0-F1) (95% confidence: 5.497 - 55.074, P = 0.0001). In addition, worse liver activity grade (A2-A3) had a very highly significant association with non-responder HCV patients compared to those with better liver activity grade (A1) (95% confidence: 2.242 - 20.974, P = 0.0007). Most importantly HCV patients with G allele had a high significant association with nonresponse to treatment, higher fibrosis stages and worse liver activity grades, while the A allele had a high significant association with sustained response, low fibrosis stages and relatively better liver activity grade (95% confidence: 3.347 - 15.036, P = 0.0001).Conclusions:SNPs within the CCR5 gene should be considered as an important factor used in combination with other host gene SNPs when developing a mathematical model for anticipating response to HCV therapy.

The role of microRNAs in hepatitis C virus RNA replication

Replication of hepatitis C virus (HCV) RNA is influenced by a variety of microRNAs, with the main player being the liver-specific microRNA-122 (miR-122). Binding of miR-122 to two binding sites near the 5' end of the 5' untranslated region (UTR) of the HCV genomic RNA results in at least two different effects. On the one hand, binding of miR-122 and the resulting recruitment of protein complexes containing Argonaute (Ago) proteins appears to mask the viral RNA's 5' end and stabilizes the viral RNA against nucleolytic degradation. On the other hand, this interaction of miR-122 with the 5'-UTR also stimulates HCV RNA translation directed by the internal ribosome entry site (IRES) located downstream of the miR-122 binding sites. However, it is suspected that additional, yet undefined roles of miR-122 in HCV replication may also contribute to HCV propagation. Accordingly, miR-122 is considered to contribute to the liver tropism of the virus. Besides miR-122, let-7b, miR-196, miR-199a* and miR-448 have also been reported to interact directly with the HCV RNA. However, the latter microRNAs inhibit HCV replication, and it has been speculated that miR-199a* contributes indirectly to HCV tissue tropism, since it is mostly expressed in cells other than hepatocytes. Other microRNAs influence HCV replication indirectly. Some of those are advantageous for HCV propagation, while others suppress HCV replication. Consequently, HCV up-regulates or down-regulates, respectively, the expression of most of these miRNAs.

A Human Proteome Microarray Identifies that the Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) Recognizes the 5′ Terminal Sequence of the Hepatitis C Virus RNA

Stem-loop I (SL1) located in the 5' untranslated region of the hepatitis C virus (HCV) genome initiates binding to miR-122, a microRNA required for hepatitis HCV replication. However, proteins that bind SL1 remain elusive. In this study, we employed a human proteome microarray, comprised of ∼17,000 individually purified human proteins in full-length, and identified 313 proteins that recognize HCV SL1. Eighty-three of the identified proteins were annotated as liver-expressing proteins, and twelve of which were known to be associated with hepatitis virus. siRNA-induced silencing of eight out of 12 candidate genes led to at least 25% decrease in HCV replication efficiency. In particular, knockdown of heterogeneous nuclear ribonucleoprotein K (hnRNP K) reduced HCV replication in a concentration-dependent manner. Ultra-violet-crosslinking assay also showed that hnRNP K, which functions in pre-mRNA processing and transport, showed the strongest binding to the HCV SL1. We observed that hnRNP K, a nuclear protein, is relocated in the cytoplasm in HCV-expressing cells. Immunoprecipitation of the hnRNP K from Huh7.5 cells stably expressing HCV replicon resulted in the co-immunoprecipitation of SL1. This work identifies a cellular protein that could have an important role in the regulation of HCV RNA gene expression and metabolism.

Valine, the branched-chain amino acid, suppresses hepatitis C virus RNA replication but promotes infectious particle formation

Background & aimsConcentrations of the branched-chain amino acid (BCAA) in the serum of patients with liver cirrhosis correlate with their liver function. Oral administration of BCAA can ameliorate hypoalbuminemia and hepatic encephalopathy. In this study, we aim to clarify the role of BCAA in regulating the replication of the hepatitis C virus (HCV). MethodsHCV sub-genomic replicon cells, genome-length replicon cells, and cells infected with cell culture-infectious HCV (HCVcc) were cultured in media supplemented with various concentrations of BCAA, followed by evaluation of the replicon or HCV abundance. ResultsBCAA was capable of suppressing the HCV replicon in a dose-dependent manner and the effect was independent of the mTOR pathway. Of the three BCAAs, valine was identified as being responsible for suppressing the HCV replicon. Surprisingly, an abundance of HJ3-5(YH/QL), an HCVcc, in Huh7 cells was augmented by BCAA supplementation. In contrast, BCAA suppressed an abundance of HJ3-5(wild), an HCVcc that cannot assemble virus particle in Huh7 cells. Internal ribosome entry site of HCV was shown to be a target of BCAA. Single-cycle virus production assays using Huh7-25 cells, which lacked CD81 expression, revealed that BCAA, especially valine, promoted infectious virus particle formation with minimal effect on virus secretion. Thus, BCAA was found to have two opposing effects on HCV production: suppression of the HCV genome RNA replication and promotion of infectious virus formation. ConclusionsBCAA accelerates HCV production through promotion of infectious virus formation in infected cells despite its suppressive effect on HCV genome replication.

Affinity Capture and Identification of Host Cell Factors Associated with Hepatitis C Virus (+) Strand Subgenomic RNA

Hepatitis C virus (HCV) infection leading to chronic hepatitis is a major factor in the causation of liver cirrhosis, hepatocellular carcinoma, and liver failure. This process may involve the interplay of various host cell factors, as well as the interaction of these factors with viral RNA and proteins. We report a novel strategy using a sequence-specific biotinylated peptide nucleic acid (PNA)-neamine conjugate targeted to HCV RNA for the in situ capture of subgenomic HCV (+) RNA, along with cellular and viral factors associated with it in MH14 host cells. Using this affinity capture system in conjunction with LC/MS/MS, we have identified 83 cellular factors and three viral proteins (NS5B, NS5A, and NS3-4a protease-helicase) associated with the viral genome. The capture was highly specific. These proteins were not scored with cured MH14 cells devoid of HCV replicons because of the absence of the target sequence in cells for the PNA-neamine probe and also because, unlike oligomeric DNA, cellular proteins have no affinity for PNA. The identified cellular factors belong to different functional groups, including signaling, oncogenic, chaperonin, transcriptional regulators, and RNA helicases as well as DEAD box proteins, ribosomal proteins, translational regulators/factors, and metabolic enzymes, that represent a diverse set of cellular factors associated with the HCV RNA genome. Small interfering RNA-mediated silencing of a diverse class of selected proteins in an HCV replicon cell line either enhanced or inhibited HCV replication/translation, suggesting that these cellular factors have regulatory roles in HCV replication.

Sequence and Structural Analysis of 3' Untranslated Region of Hepatitis C Virus, Genotype 3a, From Pakistani Isolates

BackgroundHepatitis C virus (HCV) is the cause of high morbidity and mortality worldwide, inflicting around one million people in Pakistan alone. The HCV genomic RNA harbors conserved structural elements that are indispensable for its replication. The 3’ untranslated region (UTR) contains several of these elements essentially involved in regulating the major steps of the viral life cycle.ObjectivesDifferences in regulatory elements of HCV may contribute towards differential infectivity of local isolates. The present study explicates sequence analysis and secondary structure prediction of HCV 3'UTR region of subtype 3a from Pakistan to characterize this particular region.Patients and MethodsHCV 3'UTR region was amplified, cloned and sequenced from five different patients. Sequence and structural analysis was performed and phylogenetic analysis was carried out using the 3'UTR sequence reported in NCBI nucleotide data base (http://www.ncbi.nlm.nih.gov/nuccore) by other studies.ResultsSequence analysis of the amplified fragment from five patients indicated that the 3'UTR is composed of 214-235 nts. Its sequence contains a type-specific variable region followed by a poly U/UC region and a highly conserved X-tail of 98 nts. The variable region reported here has 26 nts and one stem loop at the secondary structure that differentiate it from HCV genotype 1a ( GT1a) 3'UTR which contains additional 14 nts and two stem loops. The poly U/UC region varied in length (100-79 nts) and nucleotide sequence within the Pakistani isolates, and among different genotypes. Some substitutions found in the X-tail do not affect secondary structure of this element suggesting that this region might play an important role in replication, stabilization and packaging of HCV genome. Additionally, U residues are not present at the end of the X-tail in Pakistani 3a isolates as otherwise reported for the variants of genotype 1b.ConclusionsSequence and structural diversity of the 3'UTR variable region and Poly U/UC region found in the local isolates indicate specificity in the regulating elements of 3'UTR that might be associated with differential replication efficacy of the HCV Pakistani isolates. The study necessitates functional characterization of these regulating elements to elucidate variable viral efficiency and pathogenicity associated with inter-geographical isolates.

ATPase coupling in the processive RNA helicase NS3 from hepatitis C virus

Hepatitis C virus (HCV) NS3 motor protein contains both RNA helicase and serine protease activities that are vital to HCV replication in infected cells. NS3 belongs to the DExD/H‐box family of RNA helicases, which recognize short structured stretches of RNA and utilize ATP to remodel RNA and RNA‐protein complex structures. In contrast, NS3 is hypothesized to processively unwind the + and ‐ strands of the entire HCV RNA genome during viral replication, which likely requires that NS3 ATPase activity is tightly coupled to helicase translocation. To characterize the coupling of NS3 ATPase and translocation activities, we utilized equilibrium binding, steady‐state and transient kinetic biophysical methods. We compared a series of RNA substrates and found that RNA length affects both the concentration‐dependence and maximal rate of the RNA‐stimulated ATPase activity. Using transient kinetics, we demonstrated that the steady‐state rate of ATPase cycling is partially limited by a nucleotide‐dependent isomerization of the NS3‐RNA complex. On shorter RNAs, translocating NS3 protomers fall off the 5′ end of the nucleic acid track, and undergo this isomerization upon rebinding the ssRNA in order to restart productive translocation and rapid RNA‐stimulated ATPase. NS3 with an intact protease domain engages more productively with the RNA lattice, which increases apparent processivity of the ssRNA translocase.

Hepatitis C virus NS4B blocks the interaction of STING and TBK1 to evade host innate immunity

Hepatitis C virus (HCV) is a major human viral pathogen that causes chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. In most cases, acute HCV infection becomes persistent, at least in part due to viral evasion of host innate immune response. Although HCV genomic RNA contains pathogen-associated molecular pattern (PAMP) that is able to induce host interferon responses, HCV can shut down the responses by using the viral NS3/4A protease to cleave MAVS/VISA and TRIF, two key adaptor molecules essential for the interferon signaling activation. The aim of this study was to explore a novel NS3/4A-independent mechanism HCV utilizes to evade host innate immune responses. We used the interferon promoter-reporter system to screen HCV encoded proteins for their activities to suppress the interferon signaling and to determine the molecular targets of viral proteins. Co-immunoprecipitation, confocal microscopy, and siRNA-based gene silencing were used to investigate the molecular mechanism. We found that, in addition to NS3/4A, NS4B can suppress double-stranded RNA or RNA virus induced interferon activation. NS4B interacts with STING/MITA, an important molecule that mediates the HCV PAMP induced interferon signaling. Mechanistic studies indicated that NS4B disrupts the interactions between STING/MITA and TBK1. In conclusion, we reported an additional mechanism for HCV evasion of host interferon responses in which viral NS4B protein targets STING/MITA to suppress the interferon signaling. Our results present important evidence for the control of interferon response by HCV, and shed more light on the molecular mechanisms underlying the persistence of HCV infection.

A novel strategy to inhibit the reproduction and translation of hepatitis C virus

Hepatitis C virus (HCV), a positive single-stranded RNA virus, is a major cause of liver disease in humans. Herein we report a novel strategy to inhibit the reproduction and translation of HCV using a short RNA, named an Additional RNA, to activate the endonuclease activity of Argonaute 2 (Ago2). In the presence of the Additional RNA, the HCV genome RNA has the requisite 12 nucleotides of base-pairing with microRNA-122. This activates the endonuclease activity of Ago2, resulting in cleavage and release of the HCV genome RNA from Ago2 and microRNA-122. The free HCV genome RNA would be susceptible to intracellular degradation, effectively inhibiting its reproduction and translation. This study presents a new method to inhibit HCV that may hold great potential for HCV treatment in the future.

MiR-122: An Antiviral Target against Hepatitis C Virus

microRNA-122 (miR-122), a liver specific microRNA, has been shown to facilitate the Hepatitis C virus (HCV) replication and/or translation. Although the exact role played by miR-122 in this process is not fully understood, it has been shown that one of the functions of miR-122 is to stabilize the HCV RNA genome upon binding. There are two miR-122 binding sites within the HCV genome 5'-untranslated region (5'-UTR), named the S1 and S2 sites, both containing the miR-122 seed sequence. It has been shown that miR-122 is a valid antiviral target, as locked nucleic acids developed against miR-122 abolished the HCV replication. However, miR-122 has numerous other functions in the hepatic cell, which will also be affected by these LNAs. In this study, we adopted a different approach, namely to design peptide nucleic acids (PNAs) against the miR-122 binding sites within the HCV genome, sites conserved in all HCV genotypes and to test their antiviral properties. Our results indicate that one such PNA designed against the site S2 binds to the 5'-UTR of the HCV genome. And moreover, has the ability to invade the miR-122/HCV 5'UTR complex, releasing miR-122.

Roles of phosphoinositides and phosphoinositides kinases in hepatitis C virus RNA replication

Phosphoinositides (PIs) play an essential role in mediating key signaling pathways on biological membranes. Hepatitis C virus (HCV) replicates its RNA genome by establishing a viral replication complex (RC) on host cell membranes. Recently, an increasing body of literature reported that not only PIs themselves but also several PIs-specific kinases are required for efficient replication of HCV RNA genome. Especially, PI 4-kinases type III alpha, beta as well as their enzymatic products including phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) are consistently identified to be host factors essential for HCV replication. In this article, the current state of our knowledge of PIs and PIs-specific kinases together with their roles in modulating HCV replication is reviewed. The effects of various PIs-specific kinases inhibitors on HCV replication are also highlighted, proposing them as promising candidate targets to which a new class of anti-HCV therapeutics can be envisaged.

High plasma level of nucleocapsid-free envelope glycoprotein-positive lipoproteins in hepatitis C patients

Hepatitis C virus (HCV) particles associate viral and lipoprotein moieties to form hybrid lipoviral particles (LVPs). Cell culture-produced HCV (HCVcc) and ex vivo-characterized LVPs primarily differ by their apolipoprotein (apo) B content, which is low for HCVcc, but high for LVPs. Recombinant nucleocapsid-free subviral LVPs are assembled and secreted by apoB-producing cell lines. To determine whether such subviral particles circulate in HCV-infected individuals, LVPs complexed with immunoglobulin were precipitated with protein A from low-density plasma fractions of 36 hepatitis C patients, and their lipid content, apolipoprotein profile, and viral composition were determined. HCV RNA in LVPs was quantified and molar ratios of apoB and HCV genome copy number were calculated. LVPs lipidome from four patients was determined via electrospray ionization/tandem mass spectrometry. Protein A-purified LVPs contained at least the envelope glycoprotein E2 and E2-specific antibodies. LVPs were present in every patient and were characterized by high lipid content, presence of apolipoproteins characteristic of triglyceride-rich lipoproteins (TRLs), HCV RNA, and viral glycoprotein. Importantly, save for four patients, LVPs fractions contained large amounts of apoB, with on average more than 1 × 10(6) apoB molecules per HCV RNA genome. Because there is one apoB molecule per TRL, this ratio suggested that most LVPs are nucleocapsid-free, envelope glycoprotein-containing subviral particles. LVPs and TRLs had similar composition of triacylglycerol and phospholipid classes. LVPs are a mixed population of particles, comprising predominantly subviral particles that represent a distinct class of modified lipoproteins within the TRL family.

Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Hepatitis C virus

Hepatitis C virus (HCV) is a major public health problem and a leading cause of chronic liver disease. An estimated 180 million people are infected worldwide. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay for rapid detection of HCV genomic RNA and compared the sensitivity of LAMP with nested-PCR. A total of 30 blood samples from HCV-infected patients were analyzed with six primers targeting conserved sequences of the HCV 5'UTR within 70min, under isothermal conditions at 62°C. Then, visualized by gel electrophoresis with ethidium bromide staining and detected by the naked-eye after adding SYBR Green I. All samples positive for HCV by nested PCR were confirmed by LAMP method. When visualized by gel electrophoresis and ethidium bromide staining, the HCV LAMP assay products appeared in a ladder pattern, with many bands of different sizes. The HCV LAMP product could also be detected by the naked-eye after adding SYBR Green I to the reaction tube and observing a color change from orange to green in positive samples. The HCV LAMP had the same sensitivity as a nested-PCR assay, the detection limit for the both systems were found to be 10copies/mL of HCV RNA. The LAMP assay reported here is superior for rapid amplification, simple operation, and easy detection and will be useful for rapid and reliable clinical diagnosis of HCV in areas with limited resources, such as developing countries.

Stabilization of hepatitis C virus RNA by an Ago2–miR-122 complex

MicroRNAs (miRNAs) are small noncoding RNAs that regulate eukaryotic gene expression by binding to regions of imperfect complementarity in mRNAs, typically in the 3' UTR, recruiting an Argonaute (Ago) protein complex that usually results in translational repression or destabilization of the target RNA. The translation and decay of mRNAs are closely linked, competing processes, and whether the miRNA-induced silencing complex (RISC) acts primarily to reduce translation or stability of the mRNA remains controversial. miR-122 is an abundant, liver-specific miRNA that is an unusual host factor for hepatitis C virus (HCV), an important cause of liver disease in humans. Prior studies show that it binds the 5' UTR of the messenger-sense HCV RNA genome, stimulating translation and promoting genome replication by an unknown mechanism. Here we show that miR-122 binds HCV RNA in association with Ago2 and that this slows decay of the viral genome in infected cells. The stabilizing action of miR-122 does not require the viral RNA to be translationally active nor engaged in replication, and can be functionally substituted by a nonmethylated 5' cap. Our data demonstrate that a RISC-like complex mediates the stability of HCV RNA and suggest that Ago2 and miR-122 act coordinately to protect the viral genome from 5' exonuclease activity of the host mRNA decay machinery. miR-122 thus acts in an unconventional fashion to stabilize HCV RNA and slow its decay, expanding the repertoire of mechanisms by which miRNAs modulate gene expression.

Eradication of hepatitis C virus subgenomic replicon by interferon results in aberrant retinol-related protein expression.

Hepatitis C virus (HCV) infection induces several changes in hepatocytes, such as oxidative stress, steatosis, and hepatocarcinogenesis. Although considerable progress has been made during recent years, the mechanisms underlying these functions remain unclear. We employed proteomic techniques in HCV replicon-harboring cells to determine the effects of HCV replication on host-cell protein expression. We examined two-dimensional electrophoresis (2-DE) and mass spectrometry to compare and identify differentially expressed proteins between HCV subgenomic replicon-harboring cells and their "cured" cells. One of the identified proteins was confirmed using enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Full-length HCV genome RNA replicating and cured cells were also assessed using ELISA. Replicon-harboring cells showed higher expression of retinal dehydrogenase 1 (RALDH-1), which converts retinol to retinoic acid, and the cured cells showed higher expression of retinol-binding protein (RBP), which transports retinol from the liver to target tissues. The alteration in RBP expression was also confirmed by ELISA and Western blot analysis. We conclude that protein expression profiling demonstrated that HCV replicon eradication affected retinol-related protein expression.

Analysis of the RNA chaperoning activity of the hepatitis C virus core protein on the conserved 3′X region of the viral genome

The core protein of hepatitis c virus (HCV) is a structural protein with potent RNA chaperoning activities mediated by its hydrophilic N-terminal domain D1, which is thought to play a key role in HCV replication. To further characterize the core chaperoning properties, we studied the interactions between core D1 and the conserved HCV 3′X genomic region required for genome replication. To this end, we monitored the real-time annealing kinetics of native and mutated fluorescently labelled 16-nt palindromic sequence (DLS) and 27-nt Stem Loop II (SL2) from X with their respective complementary sequences. Core D1 and peptides consisting of the core basic domains were found to promote both annealing reactions and partly switch the loop–loop interaction pathway, which predominates in the absence of peptide, towards a pathway involving the stem termini. The chaperone properties of the core D1 peptides were found to be mediated through interaction of their basic clusters with the oligonucleotide phosphate groups, in line with the absence of high affinity site for core on HCV genomic RNA. The core ability to facilitate the interconversion between different RNA structures may explain how this protein regulates RNA structural transitions during HCV replication.