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Showing papers on "NS5B published in 2015"


Journal ArticleDOI
13 Feb 2015-Science
TL;DR: The details of HCV RNA replication are studied by determining crystal structures of stalled polymerase ternary complexes with enzymes, RNA templates, RNA primers, incoming nucleotides, and catalytic metal ions during both primed initiation and elongation of RNA synthesis.
Abstract: Nucleotide analog inhibitors have shown clinical success in the treatment of hepatitis C virus (HCV) infection, despite an incomplete mechanistic understanding of NS5B, the viral RNA-dependent RNA polymerase. Here we study the details of HCV RNA replication by determining crystal structures of stalled polymerase ternary complexes with enzymes, RNA templates, RNA primers, incoming nucleotides, and catalytic metal ions during both primed initiation and elongation of RNA synthesis. Our analysis revealed that highly conserved active-site residues in NS5B position the primer for in-line attack on the incoming nucleotide. A β loop and a C-terminal membrane-anchoring linker occlude the active-site cavity in the apo state, retract in the primed initiation assembly to enforce replication of the HCV genome from the 3' terminus, and vacate the active-site cavity during elongation. We investigated the incorporation of nucleotide analog inhibitors, including the clinically active metabolite formed by sofosbuvir, to elucidate key molecular interactions in the active site.

287 citations


Journal ArticleDOI
TL;DR: Miravirsen is a β-doxy-locked nucleic acid-modified phosphorothioate antisense oligonucleotide targeting the liver-specific microRNA-122 (miR-122).
Abstract: Miravirsen is a β-d-oxy-locked nucleic acid-modified phosphorothioate antisense oligonucleotide targeting the liver-specific microRNA-122 (miR-122). Miravirsen demonstrated antiviral activity against hepatitis C virus (HCV) genotype 1b replicons with a mean 50% effective concentration (EC 50 ) of 0.67 μM. No cytotoxicity was observed up to the highest concentration tested (>320 μM) in different cell culture models, yielding a therapeutic index of ≥297. Combination studies of miravirsen with interferon α2b, ribavirin, and nonnucleoside (VX-222) and nucleoside (2′-methylcytidine) inhibitors of NS5B, NS5A (BMS-790052), or NS3 (telaprevir) indicated additive interactions. Miravirsen demonstrated broad antiviral activity when tested against HCV replicons resistant to NS3, NS5A, and NS5B inhibitors with less than 2-fold reductions in susceptibility. In serial passage studies, an A4C nucleotide change was observed in the HCV 5′ untranslated region (UTR) from cells passaged in the presence of up to 20 μM (40-fold the miravirsen EC 50 concentration) at day 72 of passage but not at earlier time points (up to 39 days of passage). Likewise, a C3U nucleotide change was observed in the HCV 5′UTR from subjects with viral rebound after the completion of therapy in a miravirsen phase 2 clinical trial. An HCV variant constructed to contain the A4C change was fully susceptible to miravirsen. A C3U HCV variant demonstrated overall reductions in susceptibility to miravirsen but was fully susceptible to all other anti-HCV agents tested. In summary, miravirsen has demonstrated broad antiviral activity and a relatively high genetic barrier to resistance. The identification of nucleotide changes associated with miravirsen resistance should help further elucidate the biology of miR-122 interactions with HCV. (The clinical trial study has been registered at ClinicalTrials.gov under registration no. NCT01200420).

174 citations


Journal ArticleDOI
TL;DR: SOF has a high barrier to resistance; however, low‐frequency NS5B substitutions associated with treatment failure were identified that may contribute to resistance of this important drug for chronic HCV infection.

157 citations


Journal ArticleDOI
TL;DR: Dasabuvir retained full activity against replicons known to confer resistance to other polymerase inhibitors, including the S282T variant in the nucleoside binding site and the M423T, P495A, P496S, and V499A single variants in the thumb domain.
Abstract: Dasabuvir (ABT-333) is a nonnucleoside inhibitor of the RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene. Dasabuvir inhibited recombinant NS5B polymerases derived from HCV genotype 1a and 1b clinical isolates, with 50% inhibitory concentration (IC 50 ) values between 2.2 and 10.7 nM, and was at least 7,000-fold selective for the inhibition of HCV genotype 1 polymerases over human/mammalian polymerases. In the HCV subgenomic replicon system, dasabuvir inhibited genotype 1a (strain H77) and 1b (strain Con1) replicons with 50% effective concentration (EC 50 ) values of 7.7 and 1.8 nM, respectively, with a 13-fold decrease in inhibitory activity in the presence of 40% human plasma. This level of activity was retained against a panel of chimeric subgenomic replicons that contained HCV NS5B genes from 22 genotype 1 clinical isolates from treatment-naive patients, with EC 50 s ranging between 0.15 and 8.57 nM. Maintenance of replicon-containing cells in medium containing dasabuvir at concentrations 10-fold or 100-fold greater than the EC 50 resulted in selection of resistant replicon clones. Sequencing of the NS5B coding regions from these clones revealed the presence of variants, including C316Y, M414T, Y448C, Y448H, and S556G, that are consistent with binding to the palm I site of HCV polymerase. Consequently, dasabuvir retained full activity against replicons known to confer resistance to other polymerase inhibitors, including the S282T variant in the nucleoside binding site and the M423T, P495A, P495S, and V499A single variants in the thumb domain. The use of dasabuvir in combination with inhibitors targeting HCV NS3/NS4A protease (ABT-450 with ritonavir) and NS5A (ombitasvir) is in development for the treatment of HCV genotype 1 infections.

102 citations


Journal ArticleDOI
29 Sep 2015-Viruses
TL;DR: Direct acting antivirals (DAA) have been identified which bind to one of at least six RdRp inhibitor-binding sites, and are now becoming a mainstay of highly effective and well tolerated antiviral treatment for HCV infection.
Abstract: The hepatitis C virus (HCV) is a pandemic human pathogen posing a substantial health and economic burden in both developing and developed countries. Controlling the spread of HCV through behavioural prevention strategies has met with limited success and vaccine development remains slow. The development of antiviral therapeutic agents has also been challenging, primarily due to the lack of efficient cell culture and animal models for all HCV genotypes, as well as the large genetic diversity between HCV strains. On the other hand, the use of interferon-α-based treatments in combination with the guanosine analogue, ribavirin, achieved limited success, and widespread use of these therapies has been hampered by prevalent side effects. For more than a decade, the HCV RNA-dependent RNA polymerase (RdRp) has been targeted for antiviral development, and direct-acting antivirals (DAA) have been identified which bind to one of at least six RdRp inhibitor-binding sites, and are now becoming a mainstay of highly effective and well tolerated antiviral treatment for HCV infection. Here we review the different classes of RdRp inhibitors and their mode of action against HCV. Furthermore, the mechanism of antiviral resistance to each class is described, including naturally occurring resistance-associated variants (RAVs) in different viral strains and genotypes. Finally, we review the impact of these RAVs on treatment outcomes with the newly developed regimens.

95 citations


Journal ArticleDOI
TL;DR: The antiviral response to a 12‐ to 16‐week course of SOF/RBV treatment in these patients was more similar to responses among genotypes 1 patients than genotype 2 patients, consistent with their genotype 1 NS5B gene.

77 citations


Journal ArticleDOI
18 Dec 2015-Viruses
TL;DR: In this article, a review of hepatitis C virus resistant variants is presented, which depend on the class of direct-acting antiviral drugs used and also vary between hepatitisC virus genotypes and subtypes.
Abstract: There has been a remarkable transformation in the treatment of chronic hepatitis C in recent years with the development of direct acting antiviral agents targeting virus encoded proteins important for viral replication including NS3/4A, NS5A and NS5B. These agents have shown high sustained viral response (SVR) rates of more than 90% in phase 2 and phase 3 clinical trials; however, this is slightly lower in real-life cohorts. Hepatitis C virus resistant variants are seen in most patients who do not achieve SVR due to selection and outgrowth of resistant hepatitis C virus variants within a given host. These resistance associated mutations depend on the class of direct-acting antiviral drugs used and also vary between hepatitis C virus genotypes and subtypes. The understanding of these mutations has a clear clinical implication in terms of choice and combination of drugs used. In this review, we describe mechanism of action of currently available drugs and summarize clinically relevant resistance data.

74 citations


Journal ArticleDOI
16 Sep 2015-Viruses
TL;DR: Despite its large genetic diversity, across all genotypes, codon positions were rarely identified to be positively selected and predominantly found to be under negative selective pressure, suggesting mainly neutral evolution.
Abstract: Treatment with pan-genotypic direct-acting antivirals, targeting different viral proteins, is the best option for clearing hepatitis C virus (HCV) infection in chronically infected patients. However, the diversity of the HCV genome is a major obstacle for the development of antiviral drugs, vaccines, and genotyping assays. In this large-scale analysis, genome-wide diversity and selective pressure was mapped, focusing on positions important for treatment, drug resistance, and resistance testing. A dataset of 1415 full-genome sequences, including genotypes 1–6 from the Los Alamos database, was analyzed. In 44% of all full-genome positions, the consensus amino acid was different for at least one genotype. Focusing on positions sharing the same consensus amino acid in all genotypes revealed that only 15% was defined as pan-genotypic highly conserved (≥99% amino acid identity) and an additional 24% as pan-genotypic conserved (≥95%). Despite its large genetic diversity, across all genotypes, codon positions were rarely identified to be positively selected (0.23%–0.46%) and predominantly found to be under negative selective pressure, suggesting mainly neutral evolution. For NS3, NS5A, and NS5B, respectively, 40% (6/15), 33% (3/9), and 14% (2/14) of the resistance-related positions harbored as consensus the amino acid variant related to resistance, potentially impeding treatment. For example, the NS3 variant 80K, conferring resistance to simeprevir used for treatment of HCV1 infected patients, was present in 39.3% of the HCV1a strains and 0.25% of HCV1b strains. Both NS5A variants 28M and 30S, known to be associated with resistance to the pan-genotypic drug daclatasvir, were found in a significant proportion of HCV4 strains (10.7%). NS5B variant 556G, known to confer resistance to non-nucleoside inhibitor dasabuvir, was observed in 8.4% of the HCV1b strains. Given the large HCV genetic diversity, sequencing efforts for resistance testing purposes may need to be genotype-specific or geographically tailored.

60 citations


Journal ArticleDOI
TL;DR: This cholesterol-conjugated aptamer (chol-aptamer) efficiently entered the cell and inhibited HCV RNA replication, without any alteration in gene expression profiling including innate immune response-related genes.
Abstract: Hepatitis C virus (HCV) is the major cause of progressive liver disease such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Previously, we reported that a 29 nucleotide-long 2'-F pyrimidine modified RNA aptamer against the HCV nonstructural protein 5B efficiently inhibited HCV replication and suppressed HCV infectious virus particle formation in a cell culture system. In this study, we modified this aptamer through conjugation of cholesterol for in vivo availability. This cholesterol-conjugated aptamer (chol-aptamer) efficiently entered the cell and inhibited HCV RNA replication, without any alteration in gene expression profiling including innate immune response-related genes. Moreover, systemic administration of the chol-aptamer was well tolerated without any abnormalities in mice. To evaluate the pharmacokinetics of the chol-aptamer in vivo, dose proportionality, bioavailability, and pharmacokinetic parameters were evaluated by noncompartmental analyses in normal BALB/c mice. Population analysis was performed using nonlinear mixed effects modeling. Moreover, the pharmacokinetics of two different routes (intravenous, IV, versus intraperitoneal, IP) were compared. Cholesterol conjugation showed dose proportionality, extended the time that the aptamer was in the plasma, and enhanced aptamer exposure to the body. Noticeably, the IV route was more suitable than the IP route due to the chol-aptamer remaining in the plasma for a longer period of time.

59 citations


Journal ArticleDOI
11 May 2015-RNA
TL;DR: It is proposed that the 5'-3' UTR base-pairing in the HCV genome might play an important role in viral RNA replication, and several previously unknown long-range interactions are predicted, most importantly a possible circularization interaction between distinct elements in the 5' and 3' U TR, reminiscent of the cyclization elements of the related flaviviruses.
Abstract: Hepatitis C virus (HCV) is a hepatotropic virus with a plus-strand RNA genome of ∼9.600 nt. Due to error-prone replication by its RNA-dependent RNA polymerase (RdRp) residing in nonstructural protein 5B (NS5B), HCV isolates are grouped into seven genotypes with several subtypes. By using whole-genome sequences of 106 HCV isolates and secondary structure alignments of the plus-strand genome and its minus-strand replication intermediate, we established refined secondary structures of the 5' untranslated region (UTR), the cis-acting replication element (CRE) in NS5B, and the 3' UTR. We propose an alternative structure in the 5' UTR, conserved secondary structures of 5B stem-loop (SL)1 and 5BSL2, and four possible structures of the X-tail at the very 3' end of the HCV genome. We predict several previously unknown long-range interactions, most importantly a possible circularization interaction between distinct elements in the 5' and 3' UTR, reminiscent of the cyclization elements of the related flaviviruses. Based on analogy to these viruses, we propose that the 5'-3' UTR base-pairing in the HCV genome might play an important role in viral RNA replication. These results may have important implications for our understanding of the nature of the cis-acting RNA elements in the HCV genome and their possible role in regulating the mutually exclusive processes of viral RNA translation and replication.

54 citations


Journal ArticleDOI
TL;DR: In vitro infectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations are developed and open new avenues for the culture adaptation of HCV isolates of different genotypes.
Abstract: The first discovered and sequenced hepatitis C virus (HCV) genome and the first in vivo infectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77C in vivo infectious clones. We initially adapted a genome with the HCV-1 5′UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3′UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 10 4.0 focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 10 3.5 and 10 4.4 FFU/ml, respectively. IMPORTANCE Hepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but not in vitro . HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developed in vitro infectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, the in vitro infectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes.

Journal ArticleDOI
17 Jul 2015-Viruses
TL;DR: The structural and functional properties of the HCV RNA-dependent RNA polymerase (NS5B) are reviewed in order to understand the fundamental processes underlying the replication of viral genomes.
Abstract: Viral polymerases replicate and transcribe the genomes of several viruses of global health concern such as Hepatitis C virus (HCV), human immunodeficiency virus (HIV) and Ebola virus. For this reason they are key targets for therapies to treat viral infections. Although there is little sequence similarity across the different types of viral polymerases, all of them present a right-hand shape and certain structural motifs that are highly conserved. These features allow their functional properties to be compared, with the goal of broadly applying the knowledge acquired from studying specific viral polymerases to other viral polymerases about which less is known. Here we review the structural and functional properties of the HCV RNA-dependent RNA polymerase (NS5B) in order to understand the fundamental processes underlying the replication of viral genomes. We discuss recent insights into the process by which RNA replication occurs in NS5B as well as the role that conformational changes play in this process.

Journal ArticleDOI
04 Dec 2015-Viruses
TL;DR: In this paper, a review of tools available to investigate drug resistance in preclinical studies, clinical trials and clinical practice is presented, which includes tools for drug resistance detection in pre-clinical and clinical settings.
Abstract: Recent advances in molecular biology have led to the development of new antiviral drugs that target specific steps of the Hepatitis C Virus (HCV) lifecycle. These drugs, collectively termed direct-acting antivirals (DAAs), include non-structural (NS) HCV protein inhibitors, NS3/4A protease inhibitors, NS5B RNA-dependent RNA polymerase inhibitors (nucleotide analogues and non-nucleoside inhibitors), and NS5A inhibitors. Due to the high genetic variability of HCV, the outcome of DAA-based therapies may be altered by the selection of amino-acid substitutions located within the targeted proteins, which affect viral susceptibility to the administered compounds. At the drug developmental stage, preclinical and clinical characterization of HCV resistance to new drugs in development is mandatory. In the clinical setting, accurate diagnostic tools have become available to monitor drug resistance in patients who receive treatment with DAAs. In this review, we describe tools available to investigate drug resistance in preclinical studies, clinical trials and clinical practice.

Journal ArticleDOI
TL;DR: The first investigation to examine the effect of a large panel of anti-HCV agents on DMV formation reveals that CypI and NS5Ai act at the same membranous web biogenesis step of HCV RNA replication, thus indicating a new therapeutic target of chronic hepatitis C.
Abstract: Although the mechanisms of action (MoA) of nonstructural protein 3 inhibitors (NS3i) and NS5B inhibitors (NS5Bi) are well understood, the MoA of cyclophilin inhibitors (CypI) and NS5A inhibitors (NS5Ai) are not fully defined. In this study, we examined whether CypI and NS5Ai interfere with hepatitis C virus (HCV) RNA synthesis of replication complexes (RCs) or with an earlier step of HCV RNA replication, the creation of double-membrane vesicles (DMVs) essential for HCV RNA replication. In contrast to NS5Bi, both CypI and NS5Ai do not block HCV RNA synthesis by way of RCs, suggesting that they exert their antiviral activity prior to the establishment of enzymatically active RCs. We found that viral replication is not a precondition for DMV formation, since the NS3-NS5B polyprotein or NS5A suffices to create DMVs. Importantly, only CypI and NS5Ai, but not NS5Bi, mir-122, or phosphatidylinositol-4 kinase IIIα (PI4KIIIα) inhibitors, prevent NS3-NS5B-mediated DMV formation. NS3-NS5B was unable to create DMVs in cyclophilin A (CypA) knockdown (KD) cells. We also found that the isomerase activity of CypA is absolutely required for DMV formation. This not only suggests that NS5A and CypA act in concert to build membranous viral factories but that CypI and NS5Ai mediate their early anti-HCV effects by preventing the formation of organelles, where HCV replication is normally initiated. This is the first investigation to examine the effect of a large panel of anti-HCV agents on DMV formation, and the results reveal that CypI and NS5Ai act at the same membranous web biogenesis step of HCV RNA replication, thus indicating a new therapeutic target of chronic hepatitis C.

Journal ArticleDOI
TL;DR: A novel role for HuR, as a regulator of proteins assembling at the 3′ untranslated region (UTR) of viral RNA in the context of hepatitis C virus (HCV) infection is reported and it is suggested that HuR influences the formation of a cellular/viral ribonucleoprotein complex, which is important for efficient initiation of viralRNA replication.
Abstract: HuR is a ubiquitous, RNA binding protein that influences the stability and translation of several cellular mRNAs. Here, we report a novel role for HuR, as a regulator of proteins assembling at the 3′ untranslated region (UTR) of viral RNA in the context of hepatitis C virus (HCV) infection. HuR relocalizes from the nucleus to the cytoplasm upon HCV infection, interacts with the viral polymerase (NS5B), and gets redistributed into compartments of viral RNA synthesis. Depletion in HuR levels leads to a significant reduction in viral RNA synthesis. We further demonstrate that the interaction of HuR with the 3′ UTR of the viral RNA affects the interaction of two host proteins, La and polypyrimidine tract binding protein (PTB), at this site. HuR interacts with La and facilitates La binding to the 3′ UTR, enhancing La-mediated circularization of the HCV genome and thus viral replication. In addition, it competes with PTB for association with the 3′ UTR, which might stimulate viral replication. Results suggest that HuR influences the formation of a cellular/viral ribonucleoprotein complex, which is important for efficient initiation of viral RNA replication. Our study unravels a novel strategy of regulation of HCV replication through an interplay of host and viral proteins, orchestrated by HuR. IMPORTANCE Hepatitis C virus (HCV) is highly dependent on various host factors for efficient replication of the viral RNA. Here, we have shown how a host factor (HuR) migrates from the nucleus to the cytoplasm and gets recruited in the protein complex assembling at the 3′ untranslated region (UTR) of HCV RNA. At the 3′ UTR, it facilitates circularization of the viral genome through interaction with another host factor, La, which is critical for replication. Also, it competes with the host protein PTB, which is a negative regulator of viral replication. Results demonstrate a unique strategy of regulation of HCV replication by a host protein through alteration of its subcellular localization and interacting partners. The study has advanced our knowledge of the molecular mechanism of HCV replication and unraveled the complex interplay between the host factors and viral RNA that could be targeted for therapeutic interventions.

Journal ArticleDOI
TL;DR: A relevant proportion of treatment naïve genotype 1b infected patients evaluated in this study harboured N316 polymorphism and might poorly respond to sofosbuvir treatment, which has been approved for treatment of HCV chronic infection in USA and Europe including Italy.
Abstract: The detection of baseline resistance mutations to new direct-acting antivirals (DAAs) in HCV chronically infected treatment-naive patients could be important for their management and outcome prevision. In this study, we investigated the presence of mutations, which have been previously reported to be associated with resistance to DAAs in HCV polymerase (NS5B) and HCV protease (NS3) regions, in sera of treatment-naive patients. HCV RNA from 152 naive patients (84 % Italian and 16 % immigrants from various countries) infected with different HCV genotypes (21,1a; 21, 1b; 2, 2a; 60, 2c; 22, 3a; 25, 4d and 1, 4k) was evaluated for sequence analysis. Amplification and sequencing of fragments in the NS5B (nt 8256–8640) and NS3 (nt 3420–3960) regions of HCV genome were carried out for 152 and 28 patients, respectively. The polymorphism C316N/H in NS5B region, associated with resistance to sofosbuvir, was detected in 9 of the 21 (43 %) analysed sequences from genotype 1b-infected patients. Naturally occurring mutations V36L, and M175L in the NS3 protease region were observed in 100 % of patients infected with subtype 2c and 4. A relevant proportion of treatment naive genotype 1b infected patients evaluated in this study harboured N316 polymorphism and might poorly respond to sofosbuvir treatment. As sofosbuvir has been approved for treatment of HCV chronic infection in USA and Europe including Italy, pre-treatment testing for N316 polymorphism on genotype 1b naive patients should be considered for this drug.

Journal ArticleDOI
TL;DR: Cell-based assays show that PIs can directly inhibit viral RNA synthesis and also block a late stage in virus assembly/maturation at clinically relevant concentrations.
Abstract: Hepatitis C virus (HCV) NS3 is a multifunctional protein composed of a protease domain and a helicase domain linked by a flexible linker. Protease activity is required to generate viral nonstructural (NS) proteins involved in RNA replication. Helicase activity is required for RNA replication, and genetic evidence implicates the helicase domain in virus assembly. Binding of protease inhibitors (PIs) to the protease active site blocks NS3-dependent polyprotein processing but might impact other steps of the virus life cycle. Kinetic analyses of antiviral suppression of cell culture-infectious genotype 1a strain H77S.3 were performed using assays that measure different readouts of the viral life cycle. In addition to the active-site PI telaprevir, we examined an allosteric protease-helicase inhibitor (APHI) that binds a site in the interdomain interface. By measuring nucleotide incorporation into HCV genomes, we found that telaprevir inhibits RNA synthesis as early as 12 h at high but clinically relevant concentrations. Immunoblot analyses showed that NS5B abundance was not reduced until after 12 h, suggesting that telaprevir exerts a direct effect on RNA synthesis. In contrast, the APHI could partially inhibit RNA synthesis, suggesting that the allosteric site is not always available during RNA synthesis. The APHI and active-site PI were both able to block virus assembly soon (<12 h) after drug treatment, suggesting that they rapidly engage with and block a pool of NS3 involved in assembly. In conclusion, PIs and APHIs can block NS3 functions in RNA synthesis and virus assembly, in addition to inhibiting polyprotein processing. IMPORTANCE The NS3/4A protease of hepatitis C virus (HCV) is an important antiviral target. Currently, three PIs have been approved for therapy of chronic hepatitis C, and several others are in development. NS3-dependent cleavage of the HCV polyprotein is required to generate the mature nonstructural proteins that form the viral replicase. Inhibition of protease activity can block RNA replication by preventing expression of mature replicase components. Like many viral proteins, NS3 is multifunctional, but how PIs affect stages of the HCV life cycle beyond polyprotein processing has not been well studied. Using cell-based assays, we show here that PIs can directly inhibit viral RNA synthesis and also block a late stage in virus assembly/maturation at clinically relevant concentrations.

Journal ArticleDOI
TL;DR: Using a pan-genotypic population-sequencing method with degenerated primers targeting the NS5A region, baseline resistance can be examined and the data could have a potential role in selecting the optimal and cost-efficient treatment for the patient.
Abstract: Background: The future treatment of hepatitis C virus (HCV) infection will be combinations of direct-acting antivirals (DAAs) that not only target multiple viral targets, but are also effective against different HCV genotypes. Of the many drug targets in HCV, one promising target is the non-structural 5A protein (NS5A), against which inhibitors, namely daclatasvir, ledipasvir and ombitasvir, have shown potent efficacy. However, since HCV is known to have very high sequence diversity, development of resistance is a problem against but not limited to NS5A inhibitors (i.e. resistance also found against NS3-protease and NS5B non-nucleoside inhibitors), when used in suboptimal combinations. Furthermore, it has been shown that natural resistance against DAAs is present in treatment-naive patients and such baseline resistance will potentially complicate future treatment strategies. Methods: A pan-genotypic population-sequencing method with degenerated primers targeting the NS5A region was developed. We h...

Journal ArticleDOI
TL;DR: It is found that HCV infection and replication inhibited the activation of NF-κB by the inflammatory cytokine tumor necrosis factor–α (TNF-α), which was mediated by the viral protein NS3 and, to a lesser extent, NS5B.
Abstract: The transcription factor nuclear factor κB (NF-κB) is crucial for innate immune defense against viral infections, and its activation requires the ubiquitylation of upstream proteins, including the adaptor protein NEMO (NF-κB essential modulator). Many infectious pathogens, including hepatitis C virus (HCV), inhibit NF-κB signaling in host cells, which promotes pathogen survival. Frequently, HCV-infected individuals develop a chronic infection, which suggests that HCV can subvert host antiviral responses. We found that HCV infection and replication inhibited the activation of NF-κB by the inflammatory cytokine tumor necrosis factor-α (TNF-α), which was mediated by the viral protein NS3 and, to a lesser extent, NS5B. NS3 directly interacted with linear ubiquitin chain assembly complex (LUBAC), competed with NEMO for binding to LUBAC, and inhibited the LUBAC-mediated linear ubiquitylation of NEMO and the subsequent activation of NF-κB. Together, our results highlight an immune evasion strategy adopted by HCV to modulate host antiviral responses and enhance virus survival and persistence.

Journal ArticleDOI
TL;DR: A new quantitative system to measure the cis- and trans-requirements for HCV NS gene function in RNA replication is described and several lethal mutations in the NS3, NS4A, NS 4B, NS5A, and NS5B genes that can be complemented in trans, alone or in combination are identified.
Abstract: Many positive-strand RNA viruses encode genes that can function in trans, whereas other genes are required in cis for genome replication The mechanisms underlying trans- and cis-preferences are not fully understood Here, we evaluate this concept for hepatitis C virus (HCV), an important cause of chronic liver disease and member of the Flaviviridae family HCV encodes five nonstructural (NS) genes that are required for RNA replication To date, only two of these genes, NS4B and NS5A, have been trans-complemented, leading to suggestions that other replicase genes work only in cis We describe a new quantitative system to measure the cis- and trans-requirements for HCV NS gene function in RNA replication and identify several lethal mutations in the NS3, NS4A, NS4B, NS5A, and NS5B genes that can be complemented in trans, alone or in combination, by expressing the NS3–5B polyprotein from a synthetic mRNA Although NS5B RNA binding and polymerase activities can be supplied in trans, NS5B protein expression was required in cis, indicating that NS5B has a cis-acting role in replicase assembly distinct from its known enzymatic activity Furthermore, the RNA binding and NTPase activities of the NS3 helicase domain were required in cis, suggesting that these activities play an essential role in RNA template selection A comprehensive complementation group analysis revealed functional linkages between NS3-4A and NS4B and between NS5B and the upstream NS3–5A genes Finally, NS5B polymerase activity segregated with a daclatasvir-sensitive NS5A activity, which could explain the synergy of this antiviral compound with nucleoside analogs in patients Together, these studies define several new aspects of HCV replicase structure-function, help to explain the potency of HCV-specific combination therapies, and provide an experimental framework for the study of cis- and trans-acting activities in positive-strand RNA virus replication more generally

Journal ArticleDOI
TL;DR: This work proposes that SL9266/PK functions as a temporal switch, modulating the mutually incompatible processes of translation and replication, and demonstrates that LNAs which block formation of the closed conformation inhibit genome translation.
Abstract: A phylogenetically conserved RNA structure within the NS5B coding region of hepatitis C virus functions as a cis-replicating element (CRE). Integrity of this CRE, designated SL9266 (alternatively 5BSL3.2), is critical for genome replication. SL9266 forms the core of an extended pseudoknot, designated SL9266/PK, involving long distance RNA–RNA interactions between unpaired loops of SL9266 and distal regions of the genome. Previous studies demonstrated that SL9266/PK is dynamic, with ‘open’ and ‘closed’ conformations predicted to have distinct functions during virus replication. Using a combination of site-directed mutagenesis and locked nucleic acids (LNA) complementary to defined domains of SL9266 and its interacting regions, we have explored the influence of this structure on genome translation and replication. We demonstrate that LNAs which block formation of the closed conformation inhibit genome translation. Inhibition was at least partly independent of the initiation mechanism, whether driven by homologous or heterologous internal ribosome entry sites or from a capped message. Provision of SL9266/PK in trans relieved translational inhibition, and mutational analysis implied a mechanism in which the closed conformation recruits a cellular factor that would otherwise suppresses translation. We propose that SL9266/PK functions as a temporal switch, modulating the mutually incompatible processes of translation and replication.

Journal ArticleDOI
TL;DR: It is demonstrated that the viral p7 and NS5B proteins cooperate to promote virion infectivity by decreasing sphingomyelin content in the virion.
Abstract: The molecular mechanisms that govern hepatitis C virus (HCV) assembly, release, and infectivity are still not yet fully understood. In the present study, we sequenced a genotype 2A strain of HCV (JFH-1) that had been cell culture adapted in Huh-7.5 cells to produce nearly 100-fold-higher viral titers than the parental strain. Sequence analysis identified nine mutations in the genome, present within both the structural and nonstructural genes. The infectious clone of this virus containing all nine culture-adapted mutations had 10-fold-higher levels of RNA replication and RNA release into the supernatant but had nearly 1,000-fold-higher viral titers, resulting in an increased specific infectivity compared to wild-type JFH-1. Two mutations, identified in the p7 polypeptide and NS5B RNA-dependent RNA polymerase, were sufficient to increase the specific infectivity of JFH-1. We found that the culture-adapted mutation in p7 promoted an increase in the size of cellular lipid droplets following transfection of viral RNA. In addition, we found that the culture-adaptive mutations in p7 and NS5B acted synergistically to enhance the specific viral infectivity of JFH-1 by decreasing the level of sphingomyelin in the virion. Overall, these results reveal a genetic interaction between p7 and NS5B that contributes to virion specific infectivity. Furthermore, our results demonstrate a novel role for the RNA-dependent RNA polymerase NS5B in HCV assembly. Importance Hepatitis C virus assembly and release depend on viral interactions with host lipid metabolic pathways. Here, we demonstrate that the viral p7 and NS5B proteins cooperate to promote virion infectivity by decreasing sphingomyelin content in the virion. Our data uncover a new role for the viral RNA-dependent RNA polymerase NS5B and p7 proteins in contributing to virion morphogenesis. Overall, these findings are significant because they reveal a genetic interaction between p7 and NS5B, as well as an interaction with sphingomyelin that regulates virion infectivity. Our data provide new strategies for targeting host lipid-virus interactions as potential targets for therapies against HCV infection.

Journal ArticleDOI
TL;DR: Coupling data mining of sequence databases with spin glass models from statistical physics, a computational approach is developed to translate clinical sequence databases into empirical fitness landscapes quantifying the replicative capacity of the virus as a function of its amino acid sequence.
Abstract: Hepatitis C virus (HCV) afflicts 170 million people worldwide, 2%–3% of the global population, and kills 350 000 each year. Prophylactic vaccination offers the most realistic and cost effective hope of controlling this epidemic in the developing world where expensive drug therapies are not available. Despite 20 years of research, the high mutability of the virus and lack of knowledge of what constitutes effective immune responses have impeded development of an effective vaccine. Coupling data mining of sequence databases with spin glass models from statistical physics, we have developed a computational approach to translate clinical sequence databases into empirical fitness landscapes quantifying the replicative capacity of the virus as a function of its amino acid sequence. These landscapes explicitly connect viral genotype to phenotypic fitness, and reveal vulnerable immunological targets within the viral proteome that can be exploited to rationally design vaccine immunogens. We have recovered the empirical fitness landscape for the HCV RNA-dependent RNA polymerase (protein NS5B) responsible for viral genome replication, and validated the predictions of our model by demonstrating excellent accord with experimental measurements and clinical observations. We have used our landscapes to perform exhaustive in silico screening of 16.8 million T-cell immunogen candidates to identify 86 optimal formulations. By reducing the search space of immunogen candidates by over five orders of magnitude, our approach can offer valuable savings in time, expense, and labor for experimental vaccine development and accelerate the search for a HCV vaccine. Abbreviations: HCV—hepatitis C virus, HLA—human leukocyte antigen, CTL—cytotoxic T lymphocyte, NS5B—nonstructural protein 5B, MSA—multiple sequence alignment, PEG-IFN—pegylated interferon.

Journal ArticleDOI
TL;DR: This study offers important functional data on how cell culture-adaptive mutations identified in genotype 5a JFH1-based HCVcc permit high-titer culture by affecting HCV genesis through increasing virus assembly and HCV fitness by enhancing the virus specific infectivity and cell-to-cell transmission ability, without influencing the biophysical particle properties.
Abstract: Recombinant hepatitis C virus (HCV) clones propagated in human hepatoma cell cultures yield relatively low infectivity titers. Here, we adapted the JFH1-based Core-NS2 recombinant SA13/JFH1 C3405G,A3696G (termed SA13/JFH1 orig ), of the poorly characterized genotype 5a, to Huh7.5 cells, yielding a virus with greatly improved spread kinetics and an infectivity titer of 6.7 log 10 focus-forming units (FFU)/ml. We identified several putative adaptive amino acid changes. In head-to-head infections at fixed multiplicities of infection, one SA13/JFH1 orig mutant termed SA13/JFH1 Core-NS5B , containing 13 amino acid changes (R114W and V187A [Core]; V235L [E1]; T385P [E2]; L782V [p7]; Y900C [NS2]; N2034D, E2238G, V2252A, L2266P, and I2340T [NS5A]; A2500S and V2841A [NS5B]), displayed fitness comparable to that of the polyclonal high-titer adapted virus. Single-cycle virus production assays in CD81-deficient Huh7-derived cells demonstrated that these changes did not affect replication but increased HCV assembly and specific infectivity as early as 24 h posttransfection. Infectious coculture assays in Huh7.5 cells showed a significant increase in cell-to-cell transmission for SA13/JFH1 Core-NS5B viruses as well as viruses with only p7 and nonstructural protein mutations. Interestingly, the E2 hypervariable region 1 (HVR1) mutation T385P caused (i) increased sensitivity to neutralizing patient IgG and human monoclonal antibodies AR3A and AR4A and (ii) increased accessibility of the CD81 binding site without affecting the usage of CD81 and SR-BI. We finally demonstrated that SA13/JFH1 orig and SA13/JFH1 Core-NS5B , with and without the E2 mutation T385P, displayed similar biophysical properties following iodixanol gradient ultracentrifugation. This study has implications for investigations requiring high virus concentrations, such as studies of HCV particle composition and development of whole-virus vaccine antigens. IMPORTANCE Hepatitis C virus (HCV) is a major global health care burden, affecting more than 150 million people worldwide. These individuals are at high risk of developing severe end-stage liver diseases. No vaccine exists. While it is possible to produce HCV particles resembling isolates of all HCV genotypes in human hepatoma cells (HCVcc), production efficacy varies. Thus, for several important studies, including vaccine development, in vitro systems enabling high-titer production of diverse HCV strains would be advantageous. Our study offers important functional data on how cell culture-adaptive mutations identified in genotype 5a JFH1-based HCVcc permit high-titer culture by affecting HCV genesis through increasing virus assembly and HCV fitness by enhancing the virus specific infectivity and cell-to-cell transmission ability, without influencing the biophysical particle properties. High-titer HCVcc like the one described in this study may be pivotal in future vaccine-related studies where large quantities of infectious HCV particles are necessary.

Journal ArticleDOI
TL;DR: Compound 33, an arylidene derivative, was found to be the most active compound in this series with an IC50 value of 25.3 µM, and molecular docking studies were performed on the thumb pocket‐II of NS5B to postulate the binding mode for these compounds.
Abstract: In continuation of our efforts to develop new derivatives as hepatitis C virus (HCV) NS5B inhibitors, we synthesized novel 5-arylidene-4-thiazolidinones. The novel compounds 29–42, together with their synthetic precursors 22–28, were tested for HCV NS5B inhibitory activity; 12 of these compounds displayed IC50 values between 25.3 and 54.1 µM. Compound 33, an arylidene derivative, was found to be the most active compound in this series with an IC50 value of 25.3 µM. Molecular docking studies were performed on the thumb pocket-II of NS5B to postulate the binding mode for these compounds.

Journal ArticleDOI
TL;DR: This review summarizes and updates knowledge on molecular mechanisms ofHCV proteins involved in anti-IFN activity as well as examining amino acid variations and mutations in several regions of HCV proteins associated with the response to IFN-based therapy and pattern of resistance associated amino acid variants (RAV) to antiviral agents.
Abstract: For many years, the standard of treatment for hepatitis C virus (HCV) infection was a combination of pegylated interferon alpha (Peg-IFN-α) and ribavirin for 24–48 weeks. This treatment regimen results in a sustained virologic response (SVR) rate in about 50 % of cases. The failure of IFN-α-based therapy to eliminate HCV is a result of multiple factors including a suboptimal treatment regimen, severity of HCV-related diseases, host factors and viral factors. In recent years, advances in HCV cell culture have contributed to a better understanding of the viral life cycle, which has led to the development of a number of direct-acting antiviral agents (DAAs) that target specific key components of viral replication, such as HCV NS3/4A, HCV NS5A, and HCV NS5B proteins. To date, several new drugs have been approved for the treatment of HCV infection. Application of DAAs with IFN-based or IFN-free regimens has increased the SVR rate up to >90 % and has allowed treatment duration to be shortened to 12–24 weeks. The impact of HCV proteins in response to IFN-based and IFN-free therapies has been described in many reports. This review summarizes and updates knowledge on molecular mechanisms of HCV proteins involved in anti-IFN activity as well as examining amino acid variations and mutations in several regions of HCV proteins associated with the response to IFN-based therapy and pattern of resistance associated amino acid variants (RAV) to antiviral agents.

Journal ArticleDOI
TL;DR: This zebrafish model of HCV may prove to be a novel and simple in vivo model for the study of the mechanisms ofHCV replication and may also prove useful in the disovery of new anti-HCV drugs.
Abstract: Persistent infection with hepatitis C virus (HCV) is a major risk factor in the development of hepatocellular carcinoma The elucidation of the pathogenesis of HCV-associated liver disease is hampered by the absence of an appropriate small animal model Zebrafish exhibits high genetic homology to mammals, and is easily manipulated experimentally In this study, we describe the use of a zebrafish model for the analysis of HCV replication mechanisms As the 5' untranslated region (UTR), the core protein, the non-structural protein 5B (NS5B) and the 3'UTR are essential for HCV replication, we constructed a HCV sub-replicon gene construct including the 4 gene sequences and the enhanced green fluorescent protein (EGFP) reporter gene; these genes were transcribed through the mouse hepatocyte nuclear factor 4 (mHNF4) promoter By microinjection of the subgenomic replicon vector into zebrafish larvae, the virus was easily detected by observing EGFP fluorescence in the liver The positive core and NS5B signals showed positive expression of the HCV gene construct in zebrafish by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis Importantly, the negative strand sequence of the HCV subgenomic RNA was detected by RT-PCR and hybridization in situ, demonstrating that the HCV sub-replicon has positive replication activity Furthermore, the hybridization signal mainly appeared in the liver region of larvae, as detected by the sense probe of the core protein or NS5B, which confirmed that the sub-replicon amplification occurred in the zebrafish liver The amplification of the sub-replicon caused alterations in the expression of certain genes, which is similar to HCV infection in human liver cells To verify the use of this zebrafish model in drug evaluation, two drugs against HCV used in clinical practice, ribavirin and oxymatrine, were tested and these drugs showed significant inhibition of replication of the HCV sub-replicon in the larvae In conclusion, this zebrafish model of HCV may prove to be a novel and simple in vivo model for the study of the mechanisms of HCV replication and may also prove useful in the disovery of new anti-HCV drugs

Journal ArticleDOI
TL;DR: A pharmacoinformatics-based approach was applied for the identification of active plant-derived compounds against nonstructural protein 5B that can be developed into commercial drugs having high binding energy and promising ADMET properties.
Abstract: Hepatitis C virus (HCV) is one of the major viruses affecting the world today. It is a highly variable virus, having a rapid reproduction and evolution rate. The variability of genomes is due to hasty replication catalyzed by nonstructural protein 5B (NS5B) which is also a potential target site for the development of anti-HCV agents. Recently, the US Food and Drug Administration approved sofosbuvir as a novel oral NS5B inhibitor for the treatment of HCV. Unfortunately, it is much highlighted for its pricing issues. Hence, there is an urgent need to scrutinize alternate therapies against HCV that are available at affordable price and do not have associated side effects. Such a need is crucial especially in underdeveloped countries. The search for various new bioactive compounds from plants is a key part of pharmaceutical research. In the current study, we applied a pharmacoinformatics-based approach for the identification of active plant-derived compounds against NS5B. The results were compared to docking results of sofosbuvir. The lead compounds with high-binding ligands were further analyzed for pharmacokinetic and pharmacodynamic parameters based on in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) profile. The results showed the potential alternative lead compounds that can be developed into commercial drugs having high binding energy and promising ADMET properties.

Journal ArticleDOI
23 Feb 2015-PLOS ONE
TL;DR: DsiRNA molecules are as potent as 21 nt siRNAs for the inhibition of HCV replication and may provide future approaches for HCV therapy if the emergence of resistant mutants can be addressed.
Abstract: Hepatitis C virus (HCV) frequently establishes persistent infections in the liver, leading to the development of chronic hepatitis and, potentially, to liver cirrhosis and hepatocellular carcinoma at later stages. The objective of this study was to test the ability of five Dicer substrate siRNAs (DsiRNA) to inhibit HCV replication and to compare these molecules to canonical 21 nt siRNA. DsiRNA molecules were designed to target five distinct regions of the HCV genome – the 5’ UTR and the coding regions for NS3, NS4B, NS5A or NS5B. These molecules were transfected into Huh7.5 cells that stably harboured an HCV subgenomic replicon expressing a firefly luciferase/neoR reporter (SGR-Feo-JFH-1) and were also tested on HCVcc-infected cells. All of the DsiRNAs inhibited HCV replication in both the subgenomic system and HCVcc-infected cells. When DsiRNAs were transfected prior to infection with HCVcc, the inhibition levels reached 99.5%. When directly compared, canonical siRNA and DsiRNA exhibited similar potency of virus inhibition. Furthermore, both types of molecules exhibited similar dynamics of inhibition and frequencies of resistant mutants after 21 days of treatment. Thus, DsiRNA molecules are as potent as 21 nt siRNAs for the inhibition of HCV replication and may provide future approaches for HCV therapy if the emergence of resistant mutants can be addressed.

Journal ArticleDOI
Fabao Zhao1, Na Liu1, Peng Zhan1, Xuemei Jiang, Xinyong Liu1 
TL;DR: Indole analogues are the most important series of NS5B thumb site I inhibitors with considerable antiviral activity, and five fundamental principles, the general structure-activity relationships (SARs) model of indole scaffold, were summarized, which could pave the way for further structural optimization of indoles based anti-HCV agents.