Showing papers on "NS5B published in 2020"
TL;DR: Both drugs inhibited independent events during RNA synthesis and this was particularly the case for DCV, which also targeted secondary RNA structures in the SARS-CoV-2 genome, which provide a basis to further explore these agents as COVID-19 antiviral candidates.
Abstract: The infection by the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes major public health concern and economic burden. Although clinically approved drugs have been repurposed to treat individuals with 2019 Coronavirus disease (COVID-19), the lack of safety studies and limited efficiency as well jeopardize clinical benefits. Daclatasvir and sofosbuvir (SFV) are clinically approved direct-acting antivirals (DAA) against hepatitis C virus (HCV), with satisfactory safety profile. In the HCV replicative cycle, daclatasvir and SFV target the viral enzymes NS5A and NS5B, respectively. NS5A is endowed with pleotropic activities, which overlap with several proteins from SARS-CoV-2. HCV NS5B and SARS-CoV-2 nsp12 are RNA polymerases that share homology in the nucleotide uptake channel. These characteristics of the HCV and SARS-CoV-2 motivated us to further study the activity of daclatasvir and SFV against the new coronavirus. Daclatasvir consistently inhibited the production of infectious SARS-CoV-2 virus particles in Vero cells, in the hepatoma cell line HuH-7 and in type II pneumocytes (Calu-3), with potencies of 0.8, 0.6 and 1.1 μM, respectively. Daclatasvir targeted early events during SARS-CoV-2 replication cycle and prevented the induction of IL-6 and TNF-α, inflammatory mediators associated with the cytokine storm typical of SARS-CoV-2 infection. Sofosbuvir, although inactive in Vero cells, displayed EC50 values of 6.2 and 9.5 μM in HuH-7 and Calu-3 cells, respectively. Our data point to additional antiviral candidates, in especial daclatasvir, among drugs overlooked for COVID-19, that could immediately enter clinical trials.
39 citations
TL;DR: To the authors' knowledge, this is the first report of the antiviral activity of sofosbuvir against WNV as well as of selection of mutants in vitro.
25 citations
TL;DR: Results showed that Nb1 could markedly suppress BVDV replication and interact with the BVD V NS5B protein, suggesting that nanobodies have potential for the development of novel antiviral drugs against BVDv infection.
21 citations
TL;DR: Although tremendous scientific progress has been made, the incidence of acute HCV infections has increased by 4-fold since 2005, compounded in the last decade by a surge in opioid and intravenous drug use.
Abstract: In 1989, a collaboration between the Centers for Disease Control (CDC) and a California biotechnology company identified the hepatitis C virus (HCV, formerly known as non-A, non-B hepatitis virus) as the causative agent in the epidemic of silent posttransfusion hepatitis resulting in cirrhosis. We now know that, the HCV genome is a 9.6 kb positive, single-stranded RNA. A single open reading frame encodes a 3011 amino acid residue polyprotein that undergoes proteolysis to yield 10 individual gene products, consisting of 3 structural proteins (core and envelope glycoproteins E1 and E2) and 7 nonstructural (NS) proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B), which participate in posttranslational proteolytic processing and replication of HCV genetic material. Less than 25 years later, a new class of medications, known as direct-acting antivirals (DAAs) which target these proteins, were introduced to treat HCV infection. These highly effective antiviral agents are now approved for use in children as young as 3 years of age and have demonstrated sustained virologic responses exceeding 90% in most genotypes. Although tremendous scientific progress has been made, the incidence of acute HCV infections has increased by 4-fold since 2005, compounded in the last decade by a surge in opioid and intravenous drug use. Unfortunately, awareness of this deadly hepatotropic virus among members of the lay public remains limited. Patient education, advocacy, and counseling must, therefore, complement the availability of curative treatments against HCV infection if this virus is to be eradicated.
20 citations
TL;DR: 19 novel compounds represent good binders to ZIKV RdRp and could be suitable candidates for developing a new and effective anti‐ZIKV polymerase nucleotide inhibitor.
Abstract: The Zika virus (ZIKV) outbreak, which started in the year 2015, is considered the fastest and most widely spread outbreak reported for this flavivirus. The polymerase domain of the NS5 protein has been targeted in other viral infections and is recognized as a suitable target in ZIKV infection. Different novel modified compounds against ZIKV NS5 have been tested in silico. A few structures have been solved for ZIKV polymerase and deposited in the protein data bank website. Two of these solved structures (with a resolution of less than 1.9 A) are used in this study to test the binding of 74 novel compounds in silico. Molecular docking is used to quantify the binding affinities of ZIKV polymerase and compare it to the hepatitis C virus NS5B. A total of 19 novel compounds revealed results that are either similar to or better than the physiological molecule, guanosine triphosphate. Water molecules are found to facilitate the binding of the compounds to ZIKV RNA-dependent RNA polymerase (RdRp) structures. The presented 19 novel compounds represent good binders to ZIKV RdRp and could be suitable candidates for developing a new and effective anti-ZIKV polymerase nucleotide inhibitor.
14 citations
TL;DR: Comparisons of the efficacy and barrier to resistance of pangenotypic DAA regimens for HCV genotypes 2 infections, using previously and newly developed efficient cell culture-adapted strains of subtypes 2a, 2b, and 2c suggest the slight superiority of glecaprevir-pibrentasvir against genotype 2b in culture.
Abstract: The introduction of highly efficient therapies with direct-acting antivirals (DAA) for patients with chronic hepatitis C virus (HCV) infection offers exceptional opportunities to globally control this deadly disease. For achieving this ambitious goal, it is essential to prevent antiviral resistance against the most optimal first-line and retreatment DAA choices. We performed independent comparisons of the efficacy and barrier to resistance of pangenotypic DAA regimens for HCV genotype 2 infections, using previously and newly developed efficient cell culture-adapted strains of subtypes 2a, 2b, and 2c. With the applied experimental cell culture conditions, combination treatment with the sofosbuvir-velpatasvir or glecaprevir-pibrentasvir DAA regimen was efficient in eradicating HCV infections; in contrast, single-drug treatments frequently led to viral escape. Sequence analysis of drug targets from recovered viruses revealed known resistance-associated substitutions (RAS) emerging in the NS3 protease or NS5A after treatment failure. These RAS were genetically stable after viral passage, and viruses with these RAS exhibited significant phenotypic resistance. After sofosbuvir treatment failure, only a genotype 2a virus harbored NS5B RAS S282T and thus had decreased susceptibility to nucleotide analogs (nucs). However, in most cases, viral escape from sofosbuvir led to other NS5B substitutions but drug susceptibility was maintained, and in one case, no changes in NS5B were detected. For a genotype 2b virus, after treatment failure with sofosbuvir-velpatasvir, the efficacy of retreatment with glecaprevir-pibrentasvir was maintained due to the high barrier to resistance and low cross-resistance of pibrentasvir. Our findings suggest the slight superiority of glecaprevir-pibrentasvir against genotype 2b in culture, which could have potential therapeutic interest meriting more definitive investigations in the clinic.
14 citations
TL;DR: The current state of knowledge on natural products and their possible activities in the context of HCV infection will be summarised, and many phytochemical constituents have been identified that display considerable inhibition ofHCV at some stage of the life cycle.
Abstract: Chronic hepatitis C virus (HCV) infection is a significant public health problem, with a worldwide prevalence of approximately 170 million. Current therapy for HCV infection includes the pr...
14 citations
TL;DR: The discovery of a novel synthetic compound that harbors the potential of NS5B polymerase inhibition is presented and important information is presented about a novel compound 5b, for the discovery of novel inhibitors that tends to target multiple sites on NS5 B polymerase.
13 citations
TL;DR: Six highly represented amino acid substitutions (HRSs) are identified in NS5A and NS5B of HCV, which are not bona fide resistance-associated substitution (RAS) but have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them.
Abstract: Despite the high virological response rates achieved with current directly-acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of treated patients do not achieve a sustained viral response. Identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultra-deep sequencing (UDS) methods for HCV characterization and patient management. Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV from 220 patients who failed therapy, which are not bona fide resistance-associated substitutions (RAS). They were present frequently in basal and post-treatment virus of patients who failed therapy to different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Coherently, their mutant frequency, measured by the number of deep sequencing reads within the HCV quasispecies that encode the relevant substitutions, ranged between 90% and 100% in most cases. Also, they have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them. Possible mechanisms of HRS origin and dominance, as well as their potential predictive value of treatment response are discussed.
13 citations
TL;DR: This study integrated both immunoinformatic and molecular docking approach to present a multiepitope vaccine against HCV by designating 17 conserved epitopes from eight viral proteins such as Core protein, E1, E2,NS2, NS34A, NS4B, NS5A, and NS5B.
13 citations
Journal Article•
TL;DR: In this article, the potential of 100 phytochemicals against HCV NS5B polymerase was determined Phytochemical structures were retrieved from PubChem database and the phytochemical compounds were docked with the NS 5B active site amino acids, in order to discover their attractions as inhibitors After docking, molecules with top five conformations were selected from 100 molecules by docking scores and RMSD values.
Abstract: Hepatitis C virus (HCV) has major role in spreading of liver diseases worldwide The HCV nonstructural NS5B is a polymerase (RdRp) that is present at the carboxylic-end of the polyprotein chain It is essential and most important for the replication cycle In current study, the potential of 100 phytochemicals against HCV NS5B polymerase was determined Phytochemical structures were retrieved from PubChem database The phytochemicals were docked with the NS5B active site amino acids, in order to discover their attractions as inhibitors After docking, molecules with top five conformations were selected from 100 molecules by docking scores and RMSD values The results demonstrated strong interactions of phytochemicals with the NS5B The selected compounds with best docking scores and RMSD were found to be glycitein, ferulic acid, eugenol, 1-octanol and sebacic acid These were further evaluated through Lipinski's rule of five to explore their molecular properties and drug-likeliness characteristics and all five selected phytochemicals were found to have drug-likeliness characteristics Further, according to ADME analysis, the ferulic acid, 1-octanol and eugenol were found to be nontoxic, non-carcinogenic and have the ability to cross the blood brain barriers Therefore, these phytochemicals could be strong drug candidates for HCV NS5B
TL;DR: In this paper, transient-state kinetics were used to understand the efficiency of inhibition for five nucleoside analogs, including CTP, UMP and sofosbuvir, and they showed that CTP analogs are readily incorporated into a growing primer by NS5B but are also efficiently excised.
TL;DR: The findings show that the antiviral effect of ribavirin on HCV is strain-dependent and is influenced by the specific sequence of multiple HCV nonstructural proteins.
TL;DR: Model the NS5B polymerase of the equine hepacivirus subtypes 1 and 2, TDAV and EPgV, to assess whether current direct-acting antiviral drugs against HCV interact with these proteins and suggest that this compound could be efficient in combating equine pathogens, thus contributing to animal welfare.
TL;DR: Novel nucleosides are designed that incorporate a sterically bulky group at the 5'-carbon of the phosphoester prodrug, which is reasoned would reduce the amounts of non-productive P-O bond cleavage back to the corresponding nucleoside by nucleotidases.
TL;DR: Six highly represented amino acid substitutions (HRSs) are identified from 220 patients who failed therapy to different DAA-based therapies and have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them.
Abstract: Despite the high virological response rates achieved with current directly-acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of treated patients do not achieve a sustained viral response. Identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultra-deep sequencing (UDS) methods for HCV characterization and patient management. Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV from 220 patients who failed therapy, which are not bona fide resistance-associated substitutions (RAS). They were present frequently in basal and post-treatment virus of patients who failed therapy to different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Coherently, their mutant frequency, measured by the number of deep sequencing reads within the HCV quasispecies that encode the relevant substitutions, ranged between 90% and 100% in most cases. Also, they have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them. Possible mechanisms of HRS origin and dominance, as well as their potential predictive value of treatment response are discussed.
TL;DR: The diminished anti-HCV potency of the best compound compared to its 4′-oxo congener is the subject of ongoing research in the lab and is proposed to stem from changes in sugar geometry imparted by the larger sulfur atom.
Abstract: The NS5B RNA-dependent RNA polymerase of the hepatitis C virus (HCV) is a validated target for nucleoside antiviral drug therapy. We endeavored to synthesize and test a series of 4'-thionucleosides with a monophosphate prodrug moiety for their antiviral activity against HCV and other related viruses in the Flaviviridae family. Nucleoside analogs were prepared via the stereoselective Vorbruggen glycosylation of various nucleobases with per-acetylated 2-C-methyl-4-thio-d-ribose built in a 10-step synthetic sequence from the corresponding ribonolactone. Conjugation of the thionucleoside to a ProTide phosphoramidate allowed for evaluation of the prodrugs in the cellular HCV replicon assay with anti-HCV activities ranging from single-digit micromolar (μM) to >200 μM. The diminished anti-HCV potency of our best compound compared to its 4'-oxo congener is the subject of ongoing research in our lab and is proposed to stem from changes in sugar geometry imparted by the larger sulfur atom.
TL;DR: Extended exposure of HCV to ribavirin in chronic hepatitis C patients induces NS5B resistance mutations leading to increased polymerase fidelity, which could be one mechanism for Ribavirin resistance.
Abstract: Ribavirin has been used for 25 years to treat patients with chronic hepatitis C virus (HCV) infection; however, its antiviral mechanism of action remains unclear. Here, we studied virus evolution in a subset of samples from a randomized 24-week trial of ribavirin monotherapy versus placebo in chronic HCV patients, as well as the viral resistance mechanisms of the observed ribavirin-associated mutations in cell culture. Thus, we performed next-generation sequencing of the full-length coding sequences of HCV recovered from patients at weeks 0, 12, 20, 32 and 40 and analyzed novel single nucleotide polymorphisms (SNPs), diversity, and mutation-linkage. At week 20, increased genetic diversity was observed in 5 ribavirin-treated compared to 4 placebo-treated HCV patients due to new synonymous SNPs, particularly G-to-A and C-to-U ribavirin-associated transitions. Moreover, emergence of 14 nonsynonymous SNPs in HCV nonstructural 5B (NS5B) occurred in treated patients, but not in placebo controls. Most substitutions located close to the NS5B polymerase nucleotide entry site. Linkage analysis showed that putative resistance mutations were found in the majority of genomes in ribavirin-treated patients. Identified NS5B mutations from genotype 3a patients were further introduced into the genotype 3a cell-culture-adapted DBN strain for studies in Huh7.5 cells. Specific NS5B substitutions, including DBN-D148N+I363V, DBN-A150V+I363V, and DBN-T227S+S183P, conferred resistance to ribavirin in long-term cell culture treatment, possibly by reducing the HCV polymerase error rate. In conclusion, prolonged exposure of HCV to ribavirin in chronic hepatitis C patients induces NS5B resistance mutations leading to increased polymerase fidelity, which could be one mechanism for ribavirin resistance.
TL;DR: Novel methods to analyse and visualise genome-scale ordered RNA structure (GORS) predicted from the increasingly large datasets of complete genome sequences of hepatitis C virus and associations between innate cellular responses with HCV structure and further evolutionary constraints are developed.
Abstract: Mechanisms underlying the ability of hepatitis C virus (HCV) to establish persistent infections and induce progressive liver disease remain poorly understood. HCV is one of several positive-stranded RNA viruses capable of establishing persistence in their immunocompetent vertebrate hosts, an attribute previously associated with formation of large-scale RNA structure in their genomic RNA. We developed novel methods to analyze and visualize genome-scale ordered RNA structure (GORS) predicted from the increasingly large data sets of complete genome sequences of HCV. Structurally conserved RNA secondary structure in coding regions of HCV localized exclusively to polyprotein ends (core, NS5B). Coding regions elsewhere were also intensely structured based on elevated minimum folding energy difference (MFED) values, but the actual stem-loop elements involved in genome folding were structurally poorly conserved, even between subtypes 1a and 1b. Dynamic remodeling was further evident from comparison of HCV strains in different host genetic backgrounds. Significantly higher MFED values, greater suppression of UpA dinucleotide frequencies, and restricted diversification were found in subjects with the TT genotype of the rs12979860 SNP in the IFNL4 gene compared to the CC (nonexpressing) allele. These structural and compositional associations with expression of interferon-λ4 were recapitulated on a larger scale by higher MFED values and greater UpA suppression of genotype 1 compared to genotype 3a, associated with previously reported HCV genotype-associated differences in hepatic interferon-stimulated gene induction. Associations between innate cellular responses with HCV structure and further evolutionary constraints represent an important new element in RNA virus evolution and the adaptive interplay between virus and host.
TL;DR: Examination of genetic diversity and presence of drug resistance mutations within the RNA-dependent RNA polymerase (NS5B) gene in women with HCV genotype 1a demonstrated extensive variation across the NS5B gene.
Abstract: Viral diversity is an important feature of hepatitis C virus (HCV) infection and an important predictor of disease progression and treatment response. HIV/HCV co-infection is associated with enhanced HCV replication, increased fibrosis, and the development of liver disease. HIV also increases quasispecies diversity of HCV structural genes, although limited data are available regarding the impact of HIV on non-structural genes of HCV, particularly in the absence of direct-acting therapies. The genetic diversity and presence of drug resistance mutations within the RNA-dependent RNA polymerase (NS5B) gene were examined in 3 groups of women with HCV genotype 1a infection, including those with HCV mono-infection, antiretroviral (ART)-naive women with HIV/HCV co-infection and CD4 cell count <350 cells/mm3, and ART-naive women with HIV/HCV co-infection and CD4 cell count ≥350 cells/mm3. None had ever been treated for HCV infection. There was evidence of significant diversity across the entire NS5B gene in all women. There were several nucleotides and amino acids with distinct distributions across the three study groups, although no obvious clustering of NS5B sequences was observed based on HIV co-infection or CD4 cell count. Polymorphisms at amino acid positions associated with resistance to dasabuvir and sofosbuvir were limited, although the Q309R variant associated with ribavirin resistance was present in 12 individuals with HCV mono-infection, 8 HIV/HCV co-infected individuals with CD4 <350 cells/mm3, and 12 HIV/HCV co-infected individuals with CD4 ≥350 cells/mm3. Previously reported fitness altering mutations were rare. CD8+ T cell responses against the human leukocyte antigen (HLA) B57-restricted epitopes NS5B2629-2637 and NS5B2936-2944 are critical for HCV control and were completely conserved in 44 (51.8%) and 70 (82.4%) study participants. These data demonstrate extensive variation across the NS5B gene. Genotypic variation may have a profound impact on HCV replication and pathogenesis and deserves careful evaluation.
TL;DR: AIBEE prodrug 18c provided liver triphosphate concentrations that were 6-fold higher than sofosbuvir at both biopsy time points, suggesting that 18c could be a highly effective agent for treating HCV infected patients in the clinic.
TL;DR: Describing HCV genotypes circulating among Egyptian HCV patients, dissecting the full sequences of HCV NS3-4A and NS5B regions, and demonstrating potential NS3 epitopes that could be used in engineering T cells against HCV epitopes are demonstrated.
Abstract: Recently, treatment of HCV infection has been improved after the development of direct acting antivirals (DAAs) which target different viral proteins (NS3-4A, NS5A and NS5B) The activity and effectiveness of these DAAs are affected by the presence of resistance associated substitutions (RASs) This study aimed to characterize HCV genotypes circulating among Egyptian HCV patients, to dissect the full sequences of HCV NS3-4A and NS5B regions, and to characterize RASs associated with NS3-4A and NS5B inhibitors in HCV treatment-naive patients Genotyping of 80 HCV samples from treatment-naive patients was done using restriction fragment length polymorphism and phylogenetic analysis based on some full NS5B sequences Results showed the prevalence of HCV subtype 4a Twenty four new full sequences of NS3-4A and NS5B regions of subtype 4a were deposited in the GenBank database In general, the substitutions associated with NS3-4A-targeting drugs were absent predicting possible responsiveness of Egyptian HCV patients to these drugs In addition, the absence of amino acid substitutions associated with resistance to Sofosbuvir may predict good response to treatment with Sofosbuvir Some amino acid substitutions associated with resistance to different classes of non-nucleoside inhibitors were detected Further investigations on treated Egyptian HCV patients may evaluate the effectiveness of the massively used drugs Many predicted T-cell-binding epitopes in NS3-4A and NS5B regions were found to be highly conserved in the currently studied isolates; a finding that might be important for HCV vaccine development We demonstrated potential NS3 epitopes that could be used in engineering T cells against HCV epitopes
TL;DR: This study not only provides an effective approach to predict the biological activity of RNA replication's inhibitors, but also extends the QSAR modeling technique.
Abstract: Non-structural viral protein 5B (NS5B) is a viral protein in hepatitis C virus. Although various inhibitors against NS5B have been found, the activity prediction of similar untested inhibitors is still highly desirable. In this respect, the Tchebichef moments (TMs) calculated from the images of molecular structures were regarded as the independent variables while the inhibitory activity (pIC50 ) was the dependent variable, and the predictive model was established by means of stepwise regression. The R-squared of leave-one-out cross-validation (Q2 ) for the training set and the R-squared of prediction ( Rp2 ) for external independent test set were 0.919 and 0.927, respectively. The obtained model was also evaluated strictly. Compared with the multivariate curve resolution with alternating least squares (MCR-ALS) and the QSAR approaches derived from the literature, the proposed method is more accurate and reliable. This study not only provides an effective approach to predict the biological activity of RNA replication's inhibitors, but also extends the QSAR modeling technique.
TL;DR: The data demonstrate that while mutation of NS5B L2 loop affects replication, individual IFNL4-associated variants have modest but consistent effects on replication both in the presence and absence of IFNλ4, and suggest a nuanced effect of each individual position on viral replication.
Abstract: Host IFNL4 haplotype status contributes to the development of chronic hepatitis C virus infection in individuals who are acutely infected with the virus. In silico studies revealed that specific amino acid variants at multiple sites on the HCV polyprotein correlate with functional single nucleotide polymorphisms (SNPs) in the IFNL4 locus. Thus, SNPs at the IFNL4 locus may select variants that influence virus replication and thereby outcome of infection. Here, we examine the most significantly IFNL4-associated amino acid variants that lie in the Lambda (L) 2 loop of the HCV NS5B RNA polymerase. L2 loop variants were introduced into both sub-genomic replicon and full-length infectious clones of HCV and viral replication examined in the presence and absence of exogenous IFN{lambda}4. Our data demonstrate that while mutation of NS5B L2 loop affects replication, individual IFNL4-associated variants have modest but consistent effects on replication both in the presence and absence of IFN{lambda}4. Given the strong genetic association between these variants and IFNL4, these data suggest a nuanced effect of each individual position on viral replication, the combined effect of which might mediate resistance to the effects of IFN{lambda}4.
TL;DR: In this article, a molecular docking approach was used to screen phytochemicals with the best binding and spatial affinity with NS5B at the Palm I region, which provided seven hit compounds including Betanin, 3,5'- dihydroxythalifaboramine, Diarctigenin, 6'-desmethylthalifaboramines, Cephalotaxine, 5alpha-O-(3'-dimethylamino-3'- phenylpropionyl) taxinine M and IsoTetrandrine with minimum binding score compared to
Abstract: BACKGROUND Hepatitis C Virus (HCV) is one of the serious health issues affecting one-third of the world's population. The high variations of the HCV genome are ascribed to quick replication by NS5B Polymerase and are thus the most attractive target for developing anti-HCV agents. OBJECTIVE The current study aimed to discover novel phytochemicals that harbor the potential of NS5B polymerase inhibition. METHOD In this computational study, a molecular docking approach was used to screen phytochemicals with the best binding and spatial affinity with NS5B at the Palm I region. The top-ranked compounds were then subjected to in-silico pharmacokinetic and toxicological study. RESULTS AND CONCLUSION The virtual screening provided seven 'hit compounds' including Betanin, 3,5'- dihydroxythalifaboramine, Diarctigenin, 6'-desmethylthalifaboramine, Cephalotaxine, 5alpha-O-(3'-dimethylamino-3'- phenylpropionyl) taxinine M and IsoTetrandrine with minimum binding score compared to the reference drug, Sofosbuvir (-14.7 kcal/mol). The absorption, distribution, metabolism, excretion, and toxicity (ADMET) and thorough toxicological analysis revealed a favorable and the safety profile of these compounds. The study would demonstrate the phytochemicals identified might serve as potential antiviral compounds that can potentially an alternative approach for amelioration of HCV.
TL;DR: Using ribosome profiling of cells replicating full-length infectious HCV genomes, it is uncovered that ribosomes accumulate at the HCV stop codon and about 30 nucleotides upstream of it, which 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.
Abstract: 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.
TL;DR: Fluorene compounds showing strong inhibitory effects on GT 1b and 3a of HCV are reported and some compounds were effective against resistance-associated variants to DAAs.
TL;DR: The HCV prevalence found among crack users is almost threefold that observed in the general population in Brazil supporting that this population is at higher risk for HCV.
Abstract: The aim is to investigate the prevalence, risk factors, and hepatitis C virus (HCV) genotypes/subtypes among crack users in-treatment in Central Brazil. A cross-sectional survey in which 600 in-treatment crack users were interviewed and tested for anti-HCV Ab by enzyme-linked immunosorbent assay was conducted between August 2012 and April 2013. Anti-HCV-positive samples were also submitted for HCV RNA detection by polymerase chain reaction. Positive HCV RNA samples were genotyped by direct sequencing analysis of the NS5B region of the viral genome, followed by phylogenetic analysis. Of the total, 3.7% (95.0% CI, 2.4%-5.6%) were anti-HCV positive. Age over 40 years and history of injecting drugs were risk factors for HCV, while snorting cocaine was a protector variable. HCV RNA was detected in 14 of 22 anti-HCV-positive samples, and the genotypes 1 (n = 10) and 3 (n = 2), subtypes 1a (n = 7), 1b (n = 3), and 3a (n = 2) were identified. The HCV prevalence found among crack users is almost threefold that observed in the general population in Brazil supporting that this population is at higher risk for HCV. The findings of cocaine insufflation as a protective behavior for HCV infection in this population should be explored.
TL;DR: Novel methods to analyse and visualise genome-scale ordered RNA structure (GORS) predicted from the increasingly large datasets of complete genome sequences of hepatitis C virus and associations between innate cellular responses with HCV structure and further evolutionary constraints are developed.
Abstract: Mechanisms underlying the ability of hepatitis C virus (HCV) to establish persistent infections and induce progressive liver disease remain poorly understood. HCV is one of several positive-stranded RNA viruses capable of establishing persistence in their immunocompetent vertebrate hosts, an attribute associated with formation of large scale RNA structure in their genomic RNA. We developed novel methods to analyse and visualise genome-scale ordered RNA structure (GORS) predicted from the increasingly large datasets of complete genome sequences of HCV. Structurally conserved RNA secondary structure in coding regions of HCV localised exclusively to polyprotein ends (core, NS5B). Coding regions elsewhere were also intensely structured based on elevated minimum folding energy difference (MFED) values, but the actual stem-loop elements involved in genome folding were structurally entirely distinct, even between subtypes 1a and 1b. Dynamic remodelling was further evident from comparison of HCV strains in different host genetic background. Significantly higher MFED values, greater suppression of UpA dinucleotide frequencies and restricted diversification were found in subjects with the TT genotype of the rs12979860 SNP in the IFNL4 gene compared to the CC (non-expressing) allele. These structural and compositional associations with expression of interferon-λ4 were recapitulated on a larger scale by higher MFED values and greater UpA suppression of genotype 1 compared to genotype 3a, associated with previously reported HCV genotype-associated differences in hepatic interferon-stimulated gene induction. Associations between innate cellular responses with HCV structure and further evolutionary constraints represents an important new element in RNA virus evolution and the adaptive interplay between virus and host.
TL;DR: This study is the first to analyze the drug resistance sites and to evaluate genetic barriers in NS5B sequences that could affect the responsiveness of Chinese HCV patients to DAA therapy, and the results provide a valuable basis for drug development and introduction of foreign-origin antiviral drugs in China that targeting the HCV NS 5B region.
Abstract: The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase that is required for viral genome replication and constitutes the most important target region for drugs being developed as direct-acting antivirals (DAAs) against HCV genotype 1 However, the extreme genetic variability leading to drug resistance mutations and genetic barriers has dramatically compromised the effectiveness of DAA therapy The purpose of this study was to analyze the genetic variability of NS5B polymerase in HCV patients from different provinces of China to identify the impact of these resistance sites on genetic barriers We analyzed 3489 NS5B sequences of HCV strains circulating in different regions of China, obtained from the GenBank database, 153 of which were from three cities in Sichuan Province (Yibin, Zigong and Zhangzhou) Sequence alignment was conducted using MEGA 60, the genetic information was translated into amino acids, and the percentage of polymorphic amino acid sites was calculated The Vijver method was used to evaluate the occurrence of genetic barriers in HCV NS5B sequences Blood samples were collected from 153 HCV patients from Sichuan for NS5B sequence analysis using real-time PCR and the Sanger method Of the 17 antiviral drug resistance sites summarized from the published literature, nine were found in Chinese NS5B sequences, and C316Y was identified as the dominant mutation Analysis of genetic barriers revealed that the probability of mutation to a drug-resistance-associated amino acid, in response to selective pressure from antiviral drugs was 100% at site 96 and 997% at site 282 Our study is the first to analyze the drug resistance sites and to evaluate genetic barriers in NS5B sequences that could affect the responsiveness of Chinese HCV patients to DAA therapy The results provide a valuable basis for drug development and introduction of foreign-origin antiviral drugs in China that targeting the HCV NS5B region