Differential stimulation of hepatitis C virus RNA translation by microRNA-122 in different cell cycle phases

Hepatitis C virus (HCV) replicates preferentially in the liver, and in most cases, the HCV infection becomes chronic and often results in hepatocellular carcinoma. When the HCV plus-strand RNA genome has been delivered to the cytosol of the infected cell, its translation is directed by the internal ribosome entry site (IRES) in the 5′-untranslated region (5′-UTR) of the viral RNA. Thereby, IRES activity is modulated by several host factors. In particular, the liver-specific microRNA-122 (miR-122) interacts with two target sites in the HCV 5′-UTR and stimulates HCV translation, thereby most likely contributing to HCV liver tropism. Here, we show that HCV IRES-dependent translation efficiency in the hepatoma cell line Huh7 is highest during the G₀ and G₁ phases of the cell cycle but significantly drops during S phase and even more in the G₂/M phase. The superimposed stimulation of HCV translation by ectopic miR-122 works best during G₀, G₁ and G₂/M phases but is lower during S phase. However, the levels of Ago2 protein do not substantially change during cell cycle phases, indicating that other cellular factors involved in HCV translation stimulation by miR-122 may be differentially expressed in different cell cycle phases. Moreover, the levels of endogenously expressed miR-122 in Huh7 cells are lowest in S phase, indicating that the predominant G₀/G₁ state of non-dividing hepatocytes in the liver facilitates high expression of the HCV genome and stimulation by miR-122, with yet-unknown factors involved in the differential extent of stimulation by miR-122.Key words: HCV, translation, miR-122, microRNA, miRNA, Ago, Ago2     

微小RNA-122对丙型肝炎病毒复制及感染治疗影响的研究进展

丙型肝炎病毒(HCV)感染后易演变为慢性肝炎,甚至进展为肝硬化、肝癌。目前尚无有效的预防疫苗,抗病毒药物的疗效也较局限。因此,直接靶向抗病毒且无毒副作用的治疗方法是目前研究的重点。微小RNA(miRNA)是一类小分子非编码RNA,主要通过下调宿主基因表达而发挥生物学功能。miRNA-122(miR-122)在HCV感染中的作用受到关注,探讨其影响HCV复制的具体分子机制对将其作为抗病毒治疗的一个靶目标、研发新型靶向抗HCV治疗药物有重要意义。本文主要就miR-122对HCV复制的影响及其成为潜在治疗靶点的研究现状作一综述。     

MicroRNAs and hepatitis C virus: Toward the end of miR-122 supremacy

ABSTRACT: The most common etiologic agents causing chronic hepatitis are hepatitis C and B viruses(HCV and HBV, respectively). Chronic infection caused by HCV is considered one of themajor causative agents of liver cirrhosis and hepatocellular carcinoma worldwide. Incombination with the increasing rate of new HCV infections, the lack of a current vaccineand/or an effective treatment for this virus continues to be a major public health challenge.The development of new treatments requires a better understanding of the virus and itsinteraction with the different components of the host cell. MicroRNAs (miRNAs) are smallnon-coding RNAs functioning as negative regulators of gene expression and represent aninteresting lead to study HCV infection and to identify new therapeutic targets. Until now,microRNA-122 (miR-122) and its implication in HCV infection have been the focus ofdifferent published studies and reviews. Here we will review recent advances in therelationship between HCV infection and miRNAs, showing that some of them emerge inpublications as challengers against the supremacy of miR-122.     

Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides

We report here the results of a systematic high-resolution X-ray crystallographic analysis of complexes of the hepatitis C virus (HCV) RNA polymerase with ribonucleoside triphosphates (rNTPs) and divalent metal ions. An unexpected observation revealed by this study is the existence of a specific rGTP binding site in a shallow pocket at the molecular surface of the enzyme, 30 A away from the catalytic site. This previously unidentified rGTP pocket, which lies at the interface between fingers and thumb, may be an allosteric regulatory site and could play a role in allowing alternative interactions between the two domains during a possible conformational change of the enzyme required for efficient initiation. The electron density map at 1.7-A resolution clearly shows the mode of binding of the guanosine moiety to the enzyme. In the catalytic site, density corresponding to the triphosphates of nucleotides bound to the catalytic metals was apparent in each complex with nucleotides. Moreover, a network of triphosphate densities was detected; these densities superpose to the corresponding moieties of the nucleotides observed in the initiation complex reported for the polymerase of bacteriophage phi6, strengthening the proposal that the two enzymes initiate replication de novo by similar mechanisms. No equivalent of the protein stacking platform observed for the priming nucleotide in the phi6 enzyme is present in HCV polymerase, however, again suggesting that a change in conformation of the thumb domain takes place upon template binding to allow for efficient de novo initiation of RNA synthesis.     

The P body protein LSm1 contributes to stimulation of hepatitis C virus translation,but not replication,by microRNA-122

The P body protein LSm1 stimulates translation and replication of hepatitis C virus (HCV). As the liver-specific microRNA-122 (miR-122) is required for HCV replication and is associated with P bodies, we investigated whether regulation of HCV by LSm1 involves miR-122. Here, we demonstrate that LSm1 contributes to activation of HCV internal ribosome entry site (IRES)-driven translation by miR-122. This role for LSm1 is specialized for miR-122 translation activation, as LSm1 depletion does not affect the repressive function of miR-122 at 3′ untranslated region (UTR) sites, or miR-122–mediated cleavage at a perfectly complementary site. We find that LSm1 does not influence recruitment of the microRNA (miRNA)-induced silencing complex to the HCV 5′UTR, implying that it regulates miR-122 function subsequent to target binding. In contrast to the interplay between miR-122 and LSm1 in translation, we find that LSm1 is not required for miR-122 to stimulate HCV replication, suggesting that miR-122 regulation of HCV translation and replication have different requirements. For the first time, we have identified a protein factor that specifically contributes to activation of HCV IRES-driven translation by miR-122, but not to other activities of the miRNA. Our results enhance understanding of the mechanisms by which miR-122 and LSm1 regulate HCV.     

A genomic approach of the hepatitis C virus generates a protein interaction map

The hepatitis C virus (HCV) causes severe liver disease, including liver cancer. A vaccine preventing HCV infection has not yet been developed, and, given the increasing number of infected people, this virus is now considered a major public-health problem. The HCV genome is a plus-stranded RNA that encodes a single polyprotein processed into at least 10 mature polypeptides. So far, only the interaction between the protease NS3 and its cofactor, NS4A, which is involved in the processing of the non-structural region, has been extensively studied. Our work was aimed at constructing a protein interaction map of HCV. A classical two-hybrid system failed to detect any interactions between mature HCV polypeptides, suggesting incorrect folding, expression or targetting of these proteins. We therefore developed a two-hybrid strategy, based on exhaustive screens of a random genomic HCV library. Using this method, we found known interactions, such as the capsid homodimer and the protease dimer, NS3-NS4A, as well as several novel interactions such as NS4A-NS2. Thus, our results are consistent with the idea that the use of a random genomic HCV library allows the selection of correctly folded viral protein fragments. Interacting domains of the viral polyprotein are identified, opening the possibility of developing specific anti-viral agents, based on their ability to modulate these interactions.     

miR-122 activates hepatitis C virus translation by a specialized mechanism requiring particular RNA components

In animals, microRNAs (miRNAs) generally repress gene expression by binding to sites in the 3'-untranslated region (UTR) of target mRNAs. miRNAs have also been reported to repress or activate gene expression by binding to 5'-UTR sites, but the extent of such regulation and the factors that govern these different responses are unknown. Liver-specific miR-122 binds to sites in the 5'-UTR of hepatitis C virus (HCV) RNA and positively regulates the viral life cycle, in part by stimulating HCV translation. Here, we characterize the features that allow miR-122 to activate translation via the HCV 5'-UTR. We find that this regulation is a highly specialized process that requires uncapped RNA, the HCV internal ribosome entry site (IRES) and the 3' region of miR-122. Translation activation does not involve a previously proposed structural transition in the HCV IRES and is mediated by Argonaute proteins. This study provides an important insight into the requirements for the miR-122-HCV interaction, and the broader consequences of miRNAs binding to 5'-UTR sites.     

Liver-specific microRNA-122: Biogenesis and function

microRNA-122 (miR-122) was one of the first examples of a tissue-specific miRNA. It is highly expressed in liver, where it constitutes 70% of the total miRNA pool. miR-122 expression is specific to the vertebrate lineage, where the sequence of the mature miRNA is completely conserved. miR-122 is a target for extensive study due to its association with cholesterol metabolism and hepatocellular carcinoma, and its important role in promoting hepatitis C virus (HCV) replication. This review will discuss the biogenesis and function of miR-122.     

Quantitative analysis of the hepatitis C virus replication complex

The hepatitis C virus (HCV) encodes a large polyprotein; therefore, all viral proteins are produced in equimolar amounts regardless of their function. The aim of our study was to determine the ratio of nonstructural proteins to RNA that is required for HCV RNA replication. We analyzed Huh-7 cells harboring full-length HCV genomes or subgenomic replicons and found in all cases a >1,000-fold excess of HCV proteins over positive- and negative-strand RNA. To examine whether all nonstructural protein copies are involved in RNA synthesis, we isolated active HCV replication complexes from replicon cells and examined them for their content of viral RNA and proteins before and after treatment with protease and/or nuclease. In vitro replicase activity, as well as almost the entire negative- and positive-strand RNA, was resistant to nuclease treatment, whereas <5% of the nonstructural proteins were protected from protease digest but accounted for the full in vitro replicase activity. In consequence, only a minor fraction of the HCV nonstructural proteins was actively involved in RNA synthesis at a given time point but, due to the high amounts present in replicon cells, still representing a huge excess compared to the viral RNA. Based on the comparison of nuclease-resistant viral RNA to protease-resistant viral proteins, we estimate that an active HCV replicase complex consists of one negative-strand RNA, two to ten positive-strand RNAs, and several hundred nonstructural protein copies, which might be required as structural components of the vesicular compartments that are the site of HCV replication.     

Liver-specific microRNA miR-122 enhances the replication of hepatitis C virus in nonhepatic cells

The liver-specific microRNA miR-122 has been shown to be required for the replication of hepatitis C virus (HCV) in the hepatoma cell line Huh7. The aim of this study was to test if HCV replication can be modulated by exogenously expressed miR-122 in human embryonic kidney epithelial cells (HEK-293). Our results demonstrate that miR-122 enhances the colony formation efficiency of the HCV replicon and increases the steady-state level of HCV RNA in HEK-293 cells. Therefore, we conclude that although miR-122 is not absolutely required, it greatly enhances HCV replication in nonhepatic cells.     

Structural basis for broad neutralization of hepatitis C virus quasispecies

Monoclonal antibodies directed against hepatitis C virus (HCV) E2 protein can neutralize cell-cultured HCV and pseudoparticles expressing envelopes derived from multiple HCV subtypes. For example, based on antibody blocking experiments and alanine scanning mutagenesis, it was proposed that the AR3B monoclonal antibody recognized a discontinuous conformational epitope comprised of amino acid residues 396–424, 436–447, and 523–540 of HCV E2 envelope protein. Intriguingly, one of these segments (436–447) overlapped with hypervariable region 3 (HVR3), a domain that exhibited significant intrahost and interhost genetic diversity. To reconcile these observations, amino-acid sequence variability was examined and homology-based structural modelling of E2 based on tick-borne encephalitis virus (TBEV) E protein was performed based on 413 HCV sequences derived from 18 subjects with chronic hepatitis C. Here we report that despite a high degree of amino-acid sequence variability, the three-dimensional structure of E2 is remarkably conserved, suggesting broad recognition of structural determinants rather than specific residues. Regions 396–424 and 523–540 were largely exposed and in close spatial proximity at the surface of E2. In contrast, region 436–447, which overlaps with HVR3, was >35 Å away, and estimates of buried surface were inconsistent with HVR3 being part of the AR3B binding interface. High-throughput structural analysis of HCV quasispecies could facilitate the development of novel vaccines that target conserved structural features of HCV envelope and elicit neutralizing antibody responses that are less vulnerable to viral escape.     

Structural biology of human CD81, a receptor for hepatitis C virus

Human CD81, which is belonged to tetraspanin family, has been previously identified as a receptor for the hepatitis C virus envelope E 2 glycoprotein. The crystal structure of the human CD81 long extracellular domain, binding site for E 2 glycoprotein, is presented here at 1.6 A resolution. The tertiary structure of CD81-LEL, which is composed of five alpha-helices, is resemble for a mushroom-shaped molecules (stalk and head subdomains) and forms a dimer in the crystallographic asymmetric unit. The two disulfide bridges, which are conserved all the tetraspanin and are necessary for CD 81-HCV interaction, are stabilizing the conformation of the head domain. This head domain is solvent exposed surface region and is locating the amino acid residues which are essential for the E 2 binding. The hydrophobic cluster in this head domain may suggest that the presence of a docking site for a low complementary surface cavity in the partner E 2 glycoprotein. We proposed that the dimer structure may be important in the interactions of HCV E 2 glycoprotein and also the viral protein may occur in dimeric aggregation on the HCV envelope. This common structural motif of the tetraspanin provides the first insight onto the mechanism of HCV binding to human cell and may be targets for structure-based antiviral drug.     

Two-step affinity purification of the hepatitis C virus ribonucleoprotein complex

Positive-strand RNA viruses replicate their RNA genome within a ribonucleoprotein (RNP) complex that is associated with cellular membranes. We used a two-step method of purification to isolate hepatitis C virus (HCV) RNP complexes from human hepatoma cell line Huh7, which stably expresses HCV subgenomic replicons. The procedure involved hybridization of replicon-expressing cellular lysates with oligonucleotides tagged with biotin and digoxigenin at their respective termini complementary to subgenomic replicon RNA followed by avidin-agarose enrichment of the mixture and subsequent immunoprecipitation of biotin-eluted material with anti-digoxigenin antibody. The immunoprecipitates were immunoblotted with antisera against HCV nonstructural (NS) proteins. The analysis revealed the association of all the HCV NS proteins (NS3, NS4a, NS4b, NS5a, and NS5b) that are encoded by the subgenomic replicon RNA. The HCV RNP complex migrated in a native polyacrylamide gel with an approximate molecular mass of 450 kD. The association of these viral proteins in the RNP complex reinforces the widely acknowledged notion that RNA viruses accomplish replication within a membranous RNP complex.     

Position-dependent function for a tandem microRNA miR-122-binding site located in the hepatitis C virus RNA genome

MicroRNAs usually interact with 3' noncoding regions (3'NCRs) of target mRNAs leading to downregulation of mRNA expression. In contrast, liver-specific microRNA miR-122 interacts with the 5' end of the hepatitis C virus RNA genome, resulting in increased viral RNA abundance. We find that inserting the viral miR-122 binding site into the 3' noncoding region of a reporter mRNA leads to downregulation of mRNA expression, indicating that the location of the miR-122 binding site dictates its effect on gene regulation. Furthermore, we discovered an adjacent, second miR-122 binding site, separated from the first by a highly conserved 14-nucleotide sequence. Mutational analysis demonstrates that both miR-122 binding sites in a single viral genome are occupied by the microRNA and function cooperatively to regulate target gene expression. These findings set a paradigm for dual, position-dependent functions of tandem microRNA-binding sites. Targeting an oligomeric microRNA complex offers potential as an antiviral-intervention strategy.     

HepG2 cells expressing microRNA miR-122 support the entire hepatitis C virus life cycle

The liver-specific microRNA miR-122 is required for efficient hepatitis C virus (HCV) RNA replication both in cell culture and in vivo. In addition, nonhepatic cells have been rendered more efficient at supporting this stage of the HCV life cycle by miR-122 expression. This study investigated how miR-122 influences HCV replication in the miR-122-deficient HepG2 cell line. Expression of this microRNA in HepG2 cells permitted efficient HCV RNA replication and infectious virion production. When a missing HCV receptor is also expressed, these cells efficiently support viral entry and thus the entire HCV life cycle.     

Visualization of the structures of the hepatitis C virus replication complex

Hepatitis C viral RNA synthesis has been demonstrated to occur on a lipid raft membrane structure. Lipid raft membrane fraction purified by membrane flotation analysis was observed using transmission electron microscopy and atomic force microscopy. Particles around 0.7 um in size were found in lipid raft membrane fraction purified from hepatitis C virus (HCV) replicon but not their parental HuH7 cells. HCV NS5A protein was associated with these specialized particles. After several cycles of freezing-thawing, these particles would fuse into larger sizes up to 10 um. Knockdown of seven proteins associated with lipid raft (VAPA, COPG, RAB18, COMT, CDC42, DPP4, and KDELR2) of HCV replicon cells reduced the observed number of these particles and suppressed the HCV replication. Results in this study indicated that HCV replication complexes with associated lipid raft membrane form distinct particle structures of around 0.7 um as observed from transmission electron microscopy and atomic force microscopy.     

microRNA-122可调节线粒体的生物发生

目的探讨microRNA-122在代谢中的作用机制。方法首先通过生物信息学预测和双荧光素酶报告基因实验去验证14个代谢相关的microRNA-122靶基因,接着分别利用瞬时转染microRNA-122前体的293A细胞、HeLa细胞和HepG2细胞去检测维持线粒体形态功能的3种标志蛋白Immt、AIF、Cox IV的含量以及通过NRF-1/2及ERR-α途径直接调控线粒体生物生成的PGC-1α的表达,并同时采用线粒体DNA定量实验和活体细胞线粒体损伤/氧化(NAO)荧光测定实验检测这些相应的细胞稳定克隆中线粒体的拷贝数。结果发现其中一些预测的线粒体生物发生相关的靶基因能被microRNA-122显著影响,并且在过表达了microRNA-122前体的不同种类的细胞中Immt、AIF和Cox IV的蛋白水平,PGC-1α的RNA水平和线粒体的拷贝数都呈现较为显著的下降。结论 mi-croRNA-122可通过调节线粒体的生物发生来影响代谢。     

Structure of hepatitis C virus polymerase in complex with primer-template RNA

The replication of the hepatitis C viral (HCV) genome is accomplished by the NS5B RNA-dependent RNA polymerase (RdRp), for which mechanistic understanding and structure-guided drug design efforts have been hampered by its propensity to crystallize in a closed, polymerization-incompetent state. The removal of an autoinhibitory β-hairpin loop from genotype 2a HCV NS5B increases de novo RNA synthesis by >100-fold, promotes RNA binding, and facilitated the determination of the first crystallographic structures of HCV polymerase in complex with RNA primer-template pairs. These crystal structures demonstrate the structural realignment required for primer-template recognition and elongation, provide new insights into HCV RNA synthesis at the molecular level, and may prove useful in the structure-based design of novel antiviral compounds. Additionally, our approach for obtaining the RNA primer-template-bound structure of HCV polymerase may be generally applicable to solving RNA-bound complexes for other viral RdRps that contain similar regulatory β-hairpin loops, including bovine viral diarrhea virus, dengue virus, and West Nile virus.