Hepatitis C Virus RNA Genome Research Articles

Direct Visualization of Hepatitis C Virus RNA in Living Cells and Mice.

RNA virus infection is a global health issue with a significant economic burden. Direct visualization of the viral RNA genome in living cells is crucial for virological research and early clinical diagnosis. Thus, the need to continue research to find imaging toolkits is urgent. The RNA G-quadruplex (G4), a noncanonical secondary structure with stacked planar G-quartets, has recently been demonstrated in the RNA genomes of various viruses, especially the hepatitis C virus (HCV). Recent advancements in small molecular fluorescent probes have paved the way for a novel method of visualizing RNA G4s. Herein, we describe a fluorogenic probe-based RNA G4s light-up system for visualizing the HCV genome in living host cells and HCV RNA-presenting mini-organ-bearing mice without complicated sample pretreatment. Using this approach, we achieved (i) the visualization of HCV RNA genome in living cells, (ii) the investigation of viral RNA subcellular distribution, (iii) the dynamic tracking of native HCV infection and propagation in the host cell, and (iv) the high-contrast HCV RNA imaging in living mice bearing the HCV RNA-presenting mini-organ.

HCV 5-Methylcytosine Enhances Viral RNA Replication through Interaction with m5C Reader YBX1.

Hepatitis C virus (HCV) is a positive-stranded RNA virus that mainly causes chronic hepatitis, cirrhosis and hepatocellular carcinoma. Recently we confirmed m5C modifications within NS5A gene of HCV RNA genome. However, the roles of the m5C modification and its interaction with host proteins in regulating HCV's life cycle, remain unexplored. Here, we demonstrate that HCV infection enhances the expression of the host m5C reader YBX1 through the transcription factor MAX. YBX1 acts as an m5C reader, recognizing the m5C-modified NS5A C7525 site in the HCV RNA genome and significantly enhancing HCV RNA stability. This m5C-modification is also required for YBX1 colocalization with lipid droplets and HCV Core protein. Moreover, YBX1 facilitates HCV RNA replication, as well as viral assembly/budding. The tryptophan residue at position 65 (W65) of YBX1 is critical for these functions. Knockout of YBX1 or the application of YBX1 inhibitor SU056 suppresses HCV RNA replication and viral protein translation. To our knowledge, this is the first report demonstrating that the interaction between host m5C reader YBX1 and HCV RNA m5C methylation facilitates viral replication. Therefore, hepatic-YBX1 knockdown holds promise as a potential host-directed strategy for HCV therapy.

Discovery of a fused bicyclic derivative of 4-hydroxypyrrolidine and imidazolidinone as a new anti-HCV agent

Hepatitis C virus (HCV) infection causes severe liver diseases and remains a major global public health concern. Current direct-acting antiviral (DAA)-based therapies that target viral proteins involving HCV genome replication are effective, however a minority of patients still fail to cure HCV, rendering a window to develop additional antivirals particularly targeting host functions involving in HCV infection. Here, we utilized the HCV infection cell culture system (HCVcc) to screen in-house compounds bearing host-interacting preferred scaffold for the antiviral activity. Compound HXL-10, a novel fused bicyclic derivative of pyrrolidine and imidazolidinone, was identified as a potent anti-HCV agent with a low cytotoxicity and high specificity. Mechanistic studies showed that HXL-10 neither displayed a virucidal effect nor inhibited HCV genomic RNA replication. Instead, HXL-10 might inhibit HCV assembly by targeting host functions. In summary, we developed a novel anti-HCV agent that may potentially offer additive benefits to the current anti-HCV DDA.

Hepatitis C virus core protein: Not just a nucleocapsid building block, but an immunity and inflammation modulator.

Coevolution occurs between viruses and their hosts. The hosts need to evolve means to eliminate pathogenic virus infections, and the viruses, for their own survival and multiplication, have to develop mechanisms to escape clearance by hosts. Hepatitis C virus (HCV) of Flaviviridae is a pathogen which infects human liver and causes hepatitis, a condition of liver inflammation. Unlike most of the other flaviviruses, HCV has an excellent ability to evade host immunity to establish chronic infection. The persistent liver infection leads to chronic hepatitis, liver cirrhosis, hepatocellular carcinoma (HCC), as well as extrahepatic HCV-related diseases. HCV genomic RNA only expresses 10 proteins, many of which bear functions, in addition to those involved in HCV life cycle, for assisting the virus to develop its persistency. HCV core protein is a structural protein which encapsulates HCV genomic RNA and assembles into nucleocapsids. The core protein is also found to exert functions to affect host inflammation and immune responses by altering a variety of host pathways. This paper reviews the studies regarding the HCV core protein-induced alterations of host immunity and inflammatory responses, as well as the involvements of the HCV core protein in pro- and anti-inflammatory cytokine stimulations, host cellular transcription, lipid metabolism, cell apoptosis, cell proliferations, immune cell differentiations, oxidative stress, and hepatocyte steatosis, which leads to liver fibrosis, cirrhosis, and HCC. Implications of roles played by the HCV core protein in therapeutic resistance are also discussed.

Retraction Note: Small interfering RNA targeted to stem-loop II of the 5\u2032 untranslated region effectively inhibits expression of six HCV genotypes

The antiviral action of interferon alpha targets the 5' untranslated region (UTR) used by hepatitis C virus (HCV) to translate protein by an internal ribosome entry site (IRES) mechanism. Although this sequence is highly conserved among different clinical strains, approximately half of chronically infected hepatitis C patients do not respond to interferon therapy. Therefore, development of small interfering RNA (siRNA) targeted to the 5'UTR to inhibit IRES mediated translation may represent an alternative approach that could circumvent the problem of interferon resistance. Four different plasmid constructs were prepared for intracellular delivery of siRNAs targeting the stem loop II-III of HCV 5' UTR. The effect of siRNA production on IRES mediated translation was investigated using chimeric clones between the gene for green fluorescence protein (GFP) and IRES sequences of six different HCV genotypes. The siRNA targeted to stem loop II effectively mediated degradation of HCV IRES mRNA and inhibited GFP expression in the case of six different HCV genotypes, where as siRNAs targeted to stem loop III did not. Furthermore, intracytoplasmic expression of siRNA into transfected Huh-7 cells efficiently degraded HCV genomic RNA and inhibited core protein expression from infectious full-length infectious clones HCV 1a and HCV 1b strains. These in vitro studies suggest that siRNA targeted to stem-loop II is highly effective inhibiting IRES mediated translation of the major genotypes of HCV. Stem-loop II siRNA may be a good target for developing an intracellular immunization strategy based antiviral therapy to inhibit hepatitis C virus strains that are not inhibited by interferon.

Hepatitis C Viral Replication Complex.

The life cycle of the hepatitis C virus (HCV) can be divided into several stages, including viral entry, protein translation, RNA replication, viral assembly, and release. HCV genomic RNA replication occurs in the replication organelles (RO) and is tightly linked to ER membrane alterations containing replication complexes (proteins NS3 to NS5B). The amplification of HCV genomic RNA could be regulated by the RO biogenesis, the viral RNA structure (i.e., cis-acting replication elements), and both viral and cellular proteins. Studies on HCV replication have led to the development of direct-acting antivirals (DAAs) targeting the replication complex. This review article summarizes the viral and cellular factors involved in regulating HCV genomic RNA replication and the DAAs that inhibit HCV replication.

N6-methyladenosine modification of HCV RNA genome regulates cap-independent IRES-mediated translation via YTHDC2 recognition

Hepatitis C virus (HCV) infections are associated with the risk of progression to fibrosis, cirrhosis, and hepatocellular carcinoma. The HCV RNA genome is translated by an internal ribosome entry site (IRES)-dependent mechanism. The structure and function of the HCV IRES have been investigated by both biological and biophysical criteria. Recently, the role of N6-methyladenosine (m6A) in cellular RNA and viral transcripts has been intensely investigated. The HCV RNA genome is m6A-methylated, and this modification regulates the viral life cycle. In this study, we investigated the role of m6A modification of the HCV genome in the IRES-dependent translation function by mutating m6A consensus motifs (DRACH) within the IRES element in stem-loop III and IV regions and studied their effect on translation initiation. There are several DRACH motifs within the IRES element. Of these, the DRACH motif at nucleotide (nt) 329-333, located about 7 nt upstream of initiator AUG (iAUG) codon, regulates IRES-mediated translation initiation. Mutational analysis showed that m6A methylation of the adenosine at nt 331 is essential for the IRES-dependent translation. m6A reader protein YTHDC2, containing the RNA helicase domain, recognizes m6A-methylated adenosine at nt 331 and, in concert with the cellular La antigen, supports HCV IRES-dependent translation. The RNA helicase dead YTHDC2 (E332Q) mutant failed to stimulate HCV translation initiation. This report highlights the functional roles of m6A modification and YTHDC2 in the HCV IRES-dependent translation initiation, thus offering alternative therapeutic avenues to interfere with the infectious process.

A machine learning-based treatment prediction model using whole genome variants of hepatitis C virus.

In recent years, the development of diagnostics using artificial intelligence (AI) has been remarkable. AI algorithms can go beyond human reasoning and build diagnostic models from a number of complex combinations. Using next-generation sequencing technology, we identified hepatitis C virus (HCV) variants resistant to directing-acting antivirals (DAA) by whole genome sequencing of full-length HCV genomes, and applied these variants to various machine-learning algorithms to evaluate a preliminary predictive model. HCV genomic RNA was extracted from serum from 173 patients (109 with subsequent sustained virological response [SVR] and 64 without) before DAA treatment. HCV genomes from the 109 SVR and 64 non-SVR patients were randomly divided into a training data set (57 SVR and 29 non-SVR) and a validation-data set (52 SVR and 35 non-SVR). The training data set was subject to nine machine-learning algorithms selected to identify the optimized combination of functional variants in relation to SVR status following DAA therapy. Subsequently, the prediction model was tested by the validation-data set. The most accurate learning method was the support vector machine (SVM) algorithm (validation accuracy, 0.95; kappa statistic, 0.90; F-value, 0.94). The second-most accurate learning algorithm was Multi-layer perceptron. Unfortunately, Decision Tree, and Naive Bayes algorithms could not be fitted with our data set due to low accuracy (< 0.8). Conclusively, with an accuracy rate of 95.4% in the generalization performance evaluation, SVM was identified as the best algorithm. Analytical methods based on genomic analysis and the construction of a predictive model by machine-learning may be applicable to the selection of the optimal treatment for other viral infections and cancer.

Ribosome Pausing at Inefficient Codons at the End of the Replicase Coding Region Is Important for Hepatitis C Virus Genome Replication.

Hepatitis C virus (HCV) infects liver cells and often causes chronic infection, also leading to liver cirrhosis and cancer. In the cytoplasm, the viral structural and non-structural (NS) proteins are directly translated from the plus strand HCV RNA genome. The viral proteins NS3 to NS5B proteins constitute the replication complex that is required for RNA genome replication via a minus strand antigenome. The most C-terminal protein in the genome is the NS5B replicase, which needs to initiate antigenome RNA synthesis at the very 3′-end of the plus strand. Using ribosome profiling of cells replicating full-length infectious HCV genomes, we uncovered that ribosomes accumulate at the HCV stop codon and about 30 nucleotides upstream of it. This pausing is due to the presence of conserved rare, inefficient Wobble codons upstream of the termination site. Synonymous substitution of these inefficient codons to efficient codons has negative consequences for viral RNA replication but not for viral protein synthesis. This pausing may allow the enzymatically active replicase core to find its genuine RNA template in cis, while the protein is still held in place by being stuck with its C-terminus in the exit tunnel of the paused ribosome.

Carbon nanomaterial as platform for electrochemical genosensor: A system for the diagnosis of the hepatitis C in real sample

This work describes the development of a genosensor for the detection of the hepatitis C virus (HCV) in real samples. Gold electrodes were modified with graphene oxide functionalized with ethylenediamine (GO-ETD) for the immobilization of a HCV specific probe. HCV infected sera and viral genomic RNA were evaluated using differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). EIS measurements showed an increase of about twice in charge transfer resistance values, after addition of HCV genomic RNA. In the DPV analyses, the genosensor was able to discriminate HCV positive serum samples from HCV negative serum, with a difference of 3 times between these samples. It was observed an inverse linear relationship between the values of the peak current of the ferrocyanide ion oxidation and the concentration of the samples. The detection limit was 1:483 (v/v) or 1.36nmol·L−1 of RNA. The developed genosensor enables a selective and specific diagnosis of hepatitis C.

MiR-27b-mediated suppression of aquaporin-11 expression in hepatocytes reduces HCV genomic RNA levels but not viral titers

BackgroundMicroRNAs (miRNAs) have gained much attention as cellular factors regulating hepatitis C virus (HCV) infection. miR-27b has been shown to regulate HCV infection in the hepatocytes via various mechanisms that have not been fully elucidated. In this study, therefore, we examined the mechanisms of miR-27b-mediated regulation of HCV infection.MethodsIn silico screening analysis, transfection with miR-27b mimic, and a cell-based reporter assay were performed to identify miR-27b target genes. Cell cultured-derived HCV (HCVcc) was added to Huh7.5.1 cells knocked down for aquaporin (AQP) 11 (AQP11) and overexpressing AQP11. HCV replication levels were evaluated by real-time RT-PCR analysis of HCVcc genome.ResultsInfection of Huh7.5.1 cells with HCVcc resulted in significant elevation in miR-27b expression levels. In silico analysis revealed that AQP11, which is an AQP family member and is mainly localized in the endoplasmic reticulum (ER), was a candidate for a target gene of miR-27b. Transfection of a miR-27b mimic significantly reduced AQP11 expression, but a cell-based reporter assay demonstrated that miR-27b did not suppress the expression of a reporter gene containing the 3′-untranslated region of the AQP11 gene, suggesting that miR-27b indirectly suppressed AQP11 expression. AQP11 expression levels were significantly reduced by infection with HCVcc in Huh7.5.1 cells. Knockdown and over-expression of AQP11 significantly reduced and increased HCVcc genome levels in the cells following infection, respectively, however, AQP11 knockdown did not show significant effects on the HCVcc titers in the culture supernatants.ConclusionsThese results indicated that HCV infection induced a miR-27b-mediated reduction in AQP11 expression, leading to a modest reduction in HCV genome levels in the cells, not HCV titers in the culture supernatants.

TNRC6 proteins modulate hepatitis C virus replication by spatially regulating the binding of miR-122/Ago2 complexes to viral RNA.

The liver-specific microRNA, miR-122, is an essential host factor for replication of the hepatitis C virus (HCV). miR-122 stabilizes the positive-strand HCV RNA genome and promotes its synthesis by binding two sites (S1 and S2) near its 5′ end in association with Ago2. Ago2 is essential for both host factor activities, but whether other host proteins are involved is unknown. Using an unbiased quantitative proteomics screen, we identified the TNRC6 protein paralogs, TNRC6B and TNRC6C, as functionally important but redundant components of the miR-122/Ago2 host factor complex. Doubly depleting TNRC6B and TNRC6C proteins reduced HCV replication in human hepatoma cells, dampening miR-122 stimulation of viral RNA synthesis without reducing the stability or translational activity of the viral RNA. TNRC6B/C were required for optimal miR-122 host factor activity only when S1 was able to bind miR-122, and restricted replication when S1 was mutated and only S2 bound by miR-122. TNRC6B/C preferentially associated with S1, and TNRC6B/C depletion enhanced Ago2 association at S2. Collectively, these data suggest a model in which TNRC6B/C regulate the assembly of miR-122/Ago complexes on HCV RNA, preferentially directing miR-122/Ago2 to S1 while restricting its association with S2, thereby fine-tuning the spatial organization of miR-122/Ago2 complexes on the viral genome.

The acidic domain of the hepatitis C virus NS4A protein is required for viral assembly and envelopment through interactions with the viral E1 glycoprotein.

Hepatitis C virus (HCV) assembly and envelopment are coordinated by a complex protein interaction network that includes most of the viral structural and nonstructural proteins. While the nonstructural protein 4A (NS4A) is known to be important for viral particle production, the specific function of NS4A in this process is not well understood. We performed mutagenesis of the C-terminal acidic domain of NS4A and found that mutation of several of these amino acids prevented the formation of the viral envelope, and therefore the production of infectious virions, without affecting viral RNA replication. In an overexpression system, we found that NS4A interacted with several viral proteins known to coordinate envelopment, including the viral E1 glycoprotein. One of the NS4A C-terminal mutations, Y45F, disrupted the interaction of NS4A with E1. Specifically, NS4A interacted with the first hydrophobic region of E1, a region previously described as regulating viral particle production. Indeed, we found that an E1 mutation in this region, D72A, also disrupted the interaction of NS4A with E1. Supernatants from HCV NS4A Y45F transfected cells had significantly reduced levels of HCV RNA, however they contained equivalent levels of Core protein. Interestingly, the Core protein secreted from these cells formed high order oligomers with a density matching the infectious virus secreted from wild-type cells. These results suggest that this Y45F mutation in NS4A causes secretion of low-density Core particles lacking genomic HCV RNA. These results corroborate previous findings showing that the E1 D72A mutation also causes secretion of Core complexes lacking genomic HCV RNA, and therefore suggest that the interaction between NS4A and E1 is involved in the incorporation of viral RNA into infectious HCV particles. Our findings define a new role for NS4A in the HCV lifecycle and help elucidate the protein interactions necessary for production of infectious virus.

Genomic-Scale Interaction Involving Complementary Sequences in the Hepatitis C Virus 5'UTR Domain IIa and the RNA-Dependent RNA Polymerase Coding Region Promotes Efficient Virus Replication.

The hepatitis C virus (HCV) genome contains structured elements thought to play important regulatory roles in viral RNA translation and replication processes. We used in vitro RNA binding assays to map interactions involving the HCV 5′UTR and distal sequences in NS5B to examine their impact on viral RNA replication. The data revealed that 5′UTR nucleotides (nt) 95–110 in the internal ribosome entry site (IRES) domain IIa and matching nt sequence 8528–8543 located in the RNA-dependent RNA polymerase coding region NS5B, form a high-affinity RNA-RNA complex in vitro. This duplex is composed of both wobble and Watson-Crick base-pairings, with the latter shown to be essential to the formation of the high-affinity duplex. HCV genomic RNA constructs containing mutations in domain IIa nt 95–110 or within the genomic RNA location comprising nt 8528–8543 displayed, on average, 5-fold less intracellular HCV RNA and 6-fold less infectious progeny virus. HCV genomic constructs containing complementary mutations for IRES domain IIa nt 95–110 and NS5B nt 8528–8543 restored intracellular HCV RNA and progeny virus titers to levels obtained for parental virus RNA. We conclude that this long-range duplex interaction between the IRES domain IIa and NS5B nt 8528–8543 is essential for optimal virus replication.

Molecular diagnosis of occult hepatitis C virus infection in Iranian injection drug users.

Occult HCV infection (OCI) has been described as the presence of hepatitis C virus (HCV) genomic RNA in hepatocytes and/or peripheral blood mononuclear cell (PBMC) specimens and the lack of HCV genomic RNA and anti-HCV antibodies (Abs) in plasma samples. Injection drug users (IDUs) are the most important high-risk group for infection with blood-borne viruses, particularly HCV. The purpose of this study was to determine the presence of OCI in IDUs. A prospective cross-sectional study of 126 consecutive Iranian IDUs was performed from March 2017 to January 2018. PBMCs were separated from blood samples from the participants, and after extraction of the viral RNA from the plasma and PBMC specimens, HCV RNA was detected in the samples using RT-nested PCR by amplification of the 5'-NTR of HCV. HCV genotyping was carried out using restriction a fragment length polymorphism (RFLP) assay. The viral RNA was amplified using RT-nested PCR with specific primers for the NS5B gene, and the PCR products were sequenced to confirm the results obtained by HCV RNA detection and HCV genotyping. Out of the 126 IDUs studied, 105 (83.3%) were negative for anti-HCV Abs and HCV RNA in plasma samples, whereas HCV RNA was detected in the PBMC samples of six (5.7%) participants, indicating that these individuals had OCI. Moreover, HCV genomic RNA was detected in PBMC samples from five (23.8%) of the 21 IDUs studied who were positive for anti-HCV Abs and negative for HCV genomic RNA in plasma specimens. These IDUs also had OCI. The HCV genotypes in the PBMC samples from the subjects with OCI were determined. Six (54.5%) subjects were infected with HCV subtype 3a, and five (45.5%) were infected with HCV subtype 1a. This study showed that 8.7% of the Iranian IDUs had OCI, and therefore, a study focusing on the diagnosis of OCI in these individuals can be valuable and informative.

Genetic Analysis of Serum-Derived Defective Hepatitis C Virus Genomes Revealed Novel Viral cis Elements for Virus Replication and Assembly

ABSTRACT Defective viral genomes (DVGs) of hepatitis C virus (HCV) exist, but their biological significances have not been thoroughly investigated. Here, we analyzed HCV DVGs circulating in patient sera that possess deletions in the structural protein-encoding region. About 30% of 41 HCV clinical isolates possess DVGs that originated from the full-length genome in the same patients. No correlation between DVGs, viremia, and alanine aminotransferase (ALT) levels was found. Sequencing analysis of DVGs revealed the existence of deletion hot spots, with upstream sites in E1 and downstream sites in E2 and NS2. Interestingly, the coding sequences for the core protein and the C-terminal protease domain of NS2 were always intact in DVGs despite the fact that both proteins are dispensable for HCV genome replication. Mechanistic studies showed that transmembrane segment 3 (TMS3) of NS2, located immediately upstream of its protease domain, was required for the cleavage of NS2-NS3 and the replication of DVGs. Moreover, we identified a highly conserved secondary structure (SL750) within the core domain 2-coding region that is critical for HCV genome packaging. In summary, our analysis of serum-derived HCV DVGs revealed novel viral cis elements that play important roles in virus replication and assembly. IMPORTANCE HCV DVGs have been identified in vivo and in vitro , but their biogenesis and physiological significances remain elusive. In addition, a conventional packaging signal has not yet been identified on the HCV RNA genome, and mechanisms underlying the specificity in the encapsidation of the HCV genome into infectious particles remain to be uncovered. Here, we identified new viral cis elements critical for the HCV life cycle by determining genetic constraints that define the boundary of serum-derived HCV DVGs. We found that transmembrane segment 3 of NS2, located immediately upstream of its protease domain, was required for the cleavage of NS2-NS3 and the replication of DVGs. We identified a highly conserved secondary structure (SL750) within the core-coding region that is critical for HCV genome packaging. In summary, our analysis of serum-derived HCV DVGs revealed previously unexpected novel cis elements critical for HCV replication and morphogenesis.

Signals Involved in Regulation of Hepatitis C Virus RNA Genome Translation and Replication.

Hepatitis C virus (HCV) preferentially replicates in the human liver and frequently causes chronic infection, often leading to cirrhosis and liver cancer. HCV is an enveloped virus classified in the genus Hepacivirus in the family Flaviviridae and has a single-stranded RNA genome of positive orientation. The HCV RNA genome is translated and replicated in the cytoplasm. Translation is controlled by the Internal Ribosome Entry Site (IRES) in the 5′ untranslated region (5′ UTR), while also downstream elements like the cis-replication element (CRE) in the coding region and the 3′ UTR are involved in translation regulation. The cis-elements controlling replication of the viral RNA genome are located mainly in the 5′- and 3′-UTRs at the genome ends but also in the protein coding region, and in part these signals overlap with the signals controlling RNA translation. Many long-range RNA–RNA interactions (LRIs) are predicted between different regions of the HCV RNA genome, and several such LRIs are actually involved in HCV translation and replication regulation. A number of RNA cis-elements recruit cellular RNA-binding proteins that are involved in the regulation of HCV translation and replication. In addition, the liver-specific microRNA-122 (miR-122) binds to two target sites at the 5′ end of the viral RNA genome as well as to at least three additional target sites in the coding region and the 3′ UTR. It is involved in the regulation of HCV RNA stability, translation and replication, thereby largely contributing to the hepatotropism of HCV. However, we are still far from completely understanding all interactions that regulate HCV RNA genome translation, stability, replication and encapsidation. In particular, many conclusions on the function of cis-elements in HCV replication have been obtained using full-length HCV genomes or near-full-length replicon systems. These include both genome ends, making it difficult to decide if a cis-element in question acts on HCV replication when physically present in the plus strand genome or in the minus strand antigenome. Therefore, it may be required to use reduced systems that selectively focus on the analysis of HCV minus strand initiation and/or plus strand initiation.

Theory Meets Experiment: Metal Ion Effects in HCV Genomic RNA Kissing Complex Formation.

The long-range base pairing between the 5BSL3. 2 and 3′X domains in hepatitis C virus (HCV) genomic RNA is essential for viral replication. Experimental evidence points to the critical role of metal ions, especially Mg2+ ions, in the formation of the 5BSL3.2:3′X kissing complex. Furthermore, NMR studies suggested an important ion-dependent conformational switch in the kissing process. However, for a long time, mechanistic understanding of the ion effects for the process has been unclear. Recently, computational modeling based on the Vfold RNA folding model and the partial charge-based tightly bound ion (PCTBI) model, in combination with the NMR data, revealed novel physical insights into the role of metal ions in the 5BSL3.2-3′X system. The use of the PCTBI model, which accounts for the ion correlation and fluctuation, gives reliable predictions for the ion-dependent electrostatic free energy landscape and ion-induced population shift of the 5BSL3.2:3′X kissing complex. Furthermore, the predicted ion binding sites offer insights about how ion-RNA interactions shift the conformational equilibrium. The integrated theory-experiment study shows that Mg2+ ions may be essential for HCV viral replication. Moreover, the observed Mg2+-dependent conformational equilibrium may be an adaptive property of the HCV genomic RNA such that the equilibrium is optimized to the intracellular Mg2+ concentration in liver cells for efficient viral replication.

Identification and Analysis of Novel Inhibitors against NS3 Helicase and NS5B RNA-Dependent RNA Polymerase from Hepatitis C Virus 1b (Con1).

Hepatitis C virus (HCV) leads to severe liver diseases, including liver fibrosis, cirrhosis and hepatocellular carcinoma. Non-structural protein 3 helicase (NS3h) and non-structural protein 5B RNA-dependent RNA polymerase (NS5B) are involved in the replication of HCV RNA genome, and have been proved to be excellent targets for discovery of direct-acting antivirals. In this study, two high-throughput screening systems, fluorescence polarization (FP)-based ssDNA binding assay and fluorescence intensity (FI)-based dsRNA formation assay, were constructed to identify candidate NS3h and NS5B inhibitors, respectively. A library of approximately 800 small molecules and crude extracts, derived from marine microorganisms or purchased from the National Compound Resource Center, China, were screened, with three hits selected for further study. Natural compound No.3A5, isolated from marine fungi, inhibited NS3h activity with an IC50 value of 2.8 μM. We further demonstrated that compound No.3A5 inhibited the abilities of NS3h to bind ssDNA in electrophoretic mobility shift assay and to hydrolyze ATP. The NS3h-inhibitory activity of compound No.3A5 was reversible in our dilution assay, which indicated there was no stable NS3h-No.3A5 complex formed. Additionally, compound No.3A5 exhibited no binding selectivity on NS3h or single strand binding protein of Escherichia coli. In NS5B assays, commercial compounds No.39 and No.94 previously reported as kinase inhibitors were found to disrupt dsRNA formation, and their IC50 values were 62.9 and 18.8 μM, respectively. These results highlight how identifying new uses for existing drugs is an effective method for discovering novel HCV inhibitors. To our knowledge, all inhibitors reported in this study were originally discovered with HCV anti-non-structural protein activities in vitro.

Hepatitis C Virus Infection Increases c-Jun N-Terminal Kinase (JNK) Phosphorylation and Accentuates Hepatocyte Lipoapoptosis

BackgroundHepatitis C virus (HCV) infection and metabolic diseases including nonalcoholic steatohepatitis (NASH) exhibit a complex interplay. Although free fatty acid-mediated apoptosis is a prominent feature of NASH, the impact of HCV infection on hepatocyte lipotoxicity has remained largely unexplored. The study aimed at identifying whether infection by HCV affected the apoptotic pathway in hepatocytes during fatty acid assault.Material/MethodsOR6 cells, which are derived from human hepatocellular carcinoma Huh-7 cells and harbor a full-length HCV RNA genome replication system, were treated with palmitate. Apoptosis was examined by 4′,6-diamidino-2-phenylindole staining. Activation and expression of JNK, Bim, cIAP-1, and Mcl-1 were examined by immunoblotting. mRNA expression of CHOP, a major player in endoplasmic reticulum stress-mediated apoptosis, was assessed by real-time PCR.ResultsPalmitate-induced hepatocyte apoptosis was significantly enhanced in OR6 cells compared to cured cells, in which the HCV genome had been eradicated by treatment with interferon-α. Although basal expression of CHOP mRNA was enhanced in OR6 cells compared to cured cells, it was similarly upregulated in both cell lines following palmitate treatment. Notably, palmitate-induced JNK phosphorylation was accentuated in OR6 cells compared to cured cells. Inhibition of JNK with SP600125 attenuated palmitate-induced apoptosis. Palmitate-mediated upregulation of BH3-only protein Bim, which acts downstream of JNK, was also enhanced in OR6 cells compared to cured cells. In contrast, Mcl-1 and cIAP-1 were equally reduced in OR6 cells and cured cells following palmitate treatment.ConclusionsThese findings suggest that during lipoapoptosis, HCV infection may enhance hepatocyte toxicity by increasing JNK phosphorylation.