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Hepatitis C is a liver illness caused by the hepatitis C virus (HCV). Acute and chronic hepatitis can be caused by the virus. Hepatitis C infection affects an estimated 71 million people worldwide. Two hundred blood samples were collected (128 males and 72 females), ranged in age from 4 to 80 years and diagnosed through a rapid test and the real time-PCR from the teaching medical city hospital in Baghdad from October 2018 until April 2019. The results showed that 89 samples were positive for HCV by serological tests, and other samples are not. Also, the most frequent age of infection of HCV was between 51 and 80 years. In all samples, the partial region of the NS5A gene was successfully amplified and sequenced. PCR-sequencing revealed three subtyping were; subtype 1a, 1b, and 4a. Phylogenetic analysis revealed subtype concordance of 99 % with a bootstrap hepatitis C virus subtype 1b isolate AASN1 Iraq (ID:MT554101.1). The clusters including HCV subtype 1a isolate AASN20 Iraq (ID:MT554100.1) the identical 96 %, and the clusters including HCV subtype 4a isolate AASN35 Iraq (ID:MT554099.1) the identical 99%.

Objectives Due to limited hepatitis C virus (HCV) sequence availability from patients in Russia, the relationship between subtypes and baseline resistance-associated substitutions (RAS) to direct antiretroviral treatment outcome is not fully understood. Methods Deep sequencing of HCV NS3, NS5A, and NS5B sequences was performed on plasma HCV samples from 412 direct-acting antiviral (DAA)-naive patients from Russia. Phylogenetic analysis was performed to investigate sequence similarities between HCV strains from Russia, Asia, Europe, and North America. Pretreatment HCV RAS was assessed with a 15% cutoff. Results HCV genotype GT1b and GT3a sequences in Russia were related to strains in Europe and Asia. The prevalence of GT1a and GT2a was low in Russia. In GT1b, the prevalence of NS5A Y93H was lower in Russia (6%) compared with Asia (15%). The prevalence of NS5B L159F was similar between Russia and Europe (26–39%). GT3a RAS prevalence was similar between Russia and Asia, Europe, and North America. The 2k/1b recombinant strain in Russia was related to strains from Europe. A higher prevalence of the NS5A RAS L31M (10%) was observed in 2k/1b sequences compared to GT1b (1–6%). Conclusions The prevalence of RASs and the phylogenetic analysis showed similarities in HCV strains between Russia, Europe, and North America. This information may be useful for HCV regimens in Russia.

Viral vector-mediated gene therapies have the potential to treat many human diseases; however, host immune responses against the vector and/or the transgene pose a safety risk to the patients and can negatively impact product efficacy. Thus, novel strategies to reduce vector immunogenicity are critical for the advancement of these therapies. T cell activation (TCA) is required for the development of immune responses during gene therapy. We hypothesized that modulation of TCA by incorporating a novel viral immunomodulatory factor into a viral vector may reduce unwanted TCA and immune responses during gene therapy. To test this hypothesis, we identified an immunomodulatory domain of the hepatitis C virus (HCV) NS protein 5A (NS5A) protein and studied the effect of viral vectors expressing NS5A peptide on TCA. Lentiviral vector-mediated expression of a short 20-mer peptide derived from the NS5A protein in human T cells was sufficient to inhibit TCA. Synthetic 20-mer NS5A peptide also inhibited TCA in primary human T cells. Mechanistically, the NS5A protein interacted with Lck and inhibited proximal TCR signaling. Importantly, NS5A peptide expression did not cause global T cell signaling dysfunction as distal T cell signaling was not inhibited. Finally, recombinant adeno-associated virus (AAV) vector expressing the 20-mer NS5A peptide reduced both the recall antigen and the TCR-mediated activation of human T cells and did not cause global T cell signaling dysfunction. Together, these data suggest that expression of a 20-mer NS5A peptide by an AAV vector may reduce unwanted TCA and may contribute to lower vector immunogenicity during gene therapy.

This article was designed to explore the effects and mechanisms of miR-185-5p on the replication of hepatitis C virus (HCV). Quantitative reverse transcription PCR (qRT-PCR) was performed for detecting the abundance of miR-185-5p and HCV RNA in HCV-infected primary hepatocytes and Huh7.5 cells. Dual-luciferase reporter gene assay was used for exploring the interaction between miR-185-5p and GALNT8. Western blot analyzed protein expression of GALNT8, NS3, and NS5A. miR-185-5p was remarkably downregulated in HCV-infected primary hepatocytes and Huh7.5 cells. miR-185-5p upregulation inhibited HCV RNA expression, while its inhibition promoted HCV replication. miR-185-5p induced accumulation of NS3 and NS5A in the cells. Dual-luciferase reporter gene assay verified the targeted relationship between miR-185-5p and GALNT8. In addition, the effects of overexpressing or knocking down miR-185-5p on HCV replication could be correspondingly eliminated by the overexpression or knockdown of GALNT8. miR-185-5p may target GALNT8 in JFH1-infected Huh7.5 cells and then inhibit HCV replication. miR-185-5p may be a potential target for treating HCV.

Background Current approaches of drug repurposing against COVID-19 have not proven overwhelmingly successful and the SARS-CoV-2 pandemic continues to cause major global mortality. SARS-CoV-2 nsp12, its RNA polymerase, shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5B. Besides, HCV enzyme NS5A has pleiotropic activities, such as RNA binding, that are shared with various SARS-CoV-2 proteins. Thus, anti-HCV NS5B and NS5A inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity. Methods SARS-CoV-2-infected Vero cells, HuH-7 cells, Calu-3 cells, neural stem cells and monocytes were used to investigate the effects of daclatasvir and sofosbuvir. In silico and cell-free based assays were performed with SARS-CoV-2 RNA and nsp12 to better comprehend the mechanism of inhibition of the investigated compounds. A physiologically based pharmacokinetic model was generated to estimate daclatasvir’s dose and schedule to maximize the probability of success for COVID-19. Results Daclatasvir inhibited SARS-CoV-2 replication in Vero, HuH-7 and Calu-3 cells, with potencies of 0.8, 0.6 and 1.1 μM, respectively. Although less potent than daclatasvir, sofosbuvir alone and combined with daclatasvir inhibited replication in Calu-3 cells. Sofosbuvir and daclatasvir prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial daclatasvir in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans. Conclusions Daclatasvir, alone or in combination with sofosbuvir, at higher doses than used against HCV, may be further fostered as an anti-COVID-19 therapy.

ObjectiveHCV-genotype 4 infections are a major cause of liver diseases in the Middle East/Africa with certain subtypes associated with increased risk of direct-acting antiviral (DAA) treatment failures. We aimed at developing infectious genotype 4 cell culture systems to understand the evolutionary genetic landscapes of antiviral resistance, which can help preserve the future efficacy of DAA-based therapy.DesignHCV recombinants were tested in liver-derived cells. Long-term coculture with DAAs served to induce antiviral-resistance phenotypes. Next-generation sequencing (NGS) of the entire HCV-coding sequence identified mutation networks. Resistance-associated substitutions (RAS) were studied using reverse-genetics.ResultThe in-vivo infectious ED43(4a) clone was adapted in Huh7.5 cells, using substitutions identified in ED43(Core-NS5A)/JFH1-chimeric viruses combined with selected NS5B-changes. NGS, and linkage analysis, permitted identification of multiple genetic branches emerging during culture adaptation, one of which had 31 substitutions leading to robust replication/propagation. Treatment of culture-adapted ED43 with nine clinically relevant protease-DAA, NS5A-DAA and NS5B-DAA led to complex dynamics of drug-target-specific RAS with coselection of genome-wide substitutions. Approved DAA combinations were efficient against the original virus, but not against variants with RAS in corresponding drug targets. However, retreatment with glecaprevir/pibrentasvir remained efficient against NS5A inhibitor and sofosbuvir resistant variants. Recombinants with specific RAS at NS3-156, NS5A-28, 30, 31 and 93 and NS5B-282 were viable, but NS3-A156M and NS5A-L30Δ (deletion) led to attenuated phenotypes.ConclusionRapidly emerging complex evolutionary landscapes of mutations define the persistence of HCV-RASs conferring resistance levels leading to treatment failure in genotype 4. The high barrier to resistance of glecaprevir/pibrentasvir could prevent persistence and propagation of antiviral resistance.

Background: The cellular antiviral responses induced by interferons require some cellular protein kinase for its activation. Evidence indicated that a number of Hepatitis C Virus (HCV) proteins can repress double-stranded (ds) RNA-dependent Protein Kinase (PKR) function and help HCV to escape. However, the reports are controversial, some researchers have suggested that a region in Nonstructural 5A (NS5A) gene called Protein Kinase R-Binding Domain (PKR-BD) is associated with HCV sensitivity to the antiviral effects of Interferon (IFN). In addition, the other factor that might be associated with response to PEGylated-IFNα (Peg-IFNα) and Ribavirin (RBV) combination therapy, is IFNL4 genotypes. Objective: The aim of this study was to investigate the association between amino acid (aa) substitutions in the NS5A region and the IFNL4 genotypes in two Single Nucleotide Polymorphism (SNP) (rs8099917. rs12979860) in patients with HCV genotypes 1a and 3a. We also examined their response to combination therapy and the effect of these mutations on the function and structure of PKR-BD. Methods: Eighty-six patients with hepatitis C were recruited and follow-up for 6 months. Several tests, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), viral load, IFNL4 genotyping, and PKR-BD sequencing were performed. Using several well-known and trustworthy bioinformatics tools, sequences were analyzed to define physio-chemical properties, structural features, immune epitopes and protein-protein interaction. Results: Of the 86 patients, 65.1% had high viral load at baseline, 64% had CT genotype for rs12979860 and 57% had GT genotype for rs8099917. Several aa residues changes were found in the PKR-BD region. We could not find any link between mutations in the PKR-BD region and different genotypes of IFNL4 in response to antiviral therapy. Regardless of pI, PKR-BD 1a and 3a showed similar physio-chemical properties, and 2 phosphorylation sites and one glycosylation site were estimated for both PKR-BD 1a and 3a. Trustworthy software were employed in order to predict B-cell epitopes, 3 regions (6-17, 26-32, 34-41) were found for both proteins, indicating a huge potential of PKR-BD protein to induce humoral immune system. Docking analysis determined non-responder sequences in both 1a and 3a genotypes to have higher energy value and are more compatible with PKR. Conclusion: To sum up, our results could not determine any significant relationship between mutations of PKR-BD and genotypes of IFNL4 with other factors; ALT, AST, viral load. However, docking results showed strengthened interaction between PKR-BD and PKR in non-responders that could have a momentous impact on the illness severity.

Long-term efforts of the international community led to the development of highly efficient direct-acting antivirals (DAAs) that allow treatment of the vast majority of patients with chronic hepatitis C. The proteins encoded in the genome of the hepatitis C virus (HCV) that play a key role in its life cycle (NS3, NS5A, and NS5B) are the targets of this type of drug. There are three classes of DAAs each of which is directed to the inhibition of a specific target protein. Since the HCV has a sufficiently high rate of accumulation of mutations, the development of resistance to these drugs is a big problem. The current recommended treatment regimens with DAAs without the use of interferon and ribavirin are a combination of drugs of different classes providing an increase in the barrier of resistance. Due to the emergence of DAAs, a number of countries (WHO members, with the involvement of Russia) put forward a global strategy to eradicate the HCV. Taking into account the high cost of DAAs and a large number of HCV-infected individuals in Russia, achieving the goals declared by the WHO presents great financial difficulties for our country. However, federal funds allocated for hepatitis C therapy increased significantly over the past 3 years. In addition to increased funding, there is a great potential for reducing the cost of treatment, but its implementation is impossible without the organization of national production of quality generics, issuance of compulsory licenses (given that it is impossible to negotiate with patent holders on licensing), and/or negotiations on price reduction in exchange for volume (for example, the experience of Australia and Portugal). Anyways, Russia faces a very important task to provide therapy for several million patients with hepatitis C in the coming years to get closer to the goal of eradication of the HCV set by the international community.

Hepatitis C virus (HCV) life cycle is highly dependent on cellular proteins for viral propagation. In order to identify the cellular factors involved in HCV propagation, we previously performed a protein microarray assay using the HCV nonstructural 5A (NS5A) protein as a probe. Of ∼9,000 human cellular proteins immobilized in a microarray, adenosylhomocysteinase like 1 (AHCYL1) was among 90 proteins identified as NS5A interactors. Of these candidates, AHCYL1 was selected for further study. In the present study, we verified the physical interaction between NS5A and AHCYL1 by both in vitro pulldown and coimmunoprecipitation assays. Furthermore, HCV NS5A interacted with endogenous AHCYL1 in Jc1-infected cells. Both NS5A and AHCYL1 were colocalized in the cytoplasmic region in HCV-replicating cells. siRNAmediated knockdown of AHCYL1 abrogated HCV propagation. Exogenous expression of the siRNA-resistant AHCYL1 mutant, but not of the wild-type AHCYL1, restored HCV protein expression levels, indicating that AHCYL1 was required specifically for HCV propagation. Importantly, AHCYL1 was involved in the HCV internal ribosome entry site-mediated translation step of the HCV life cycle. Finally, we demonstrated that the proteasomal degradation pathway of AHCYL1 was modulated by persistent HCV infection. Collectively, these data suggest that HCV may modulate the AHCYL1 protein to promote viral propagation.

Background & Aims The efficacy of NS5A inhibitors against several less common subtypes of hepatitis C virus (HCV) is poorly characterised. Some subtypes including 3b, 3g, 6u and 6v commonly harbour amino acid residues in NS5A that may confer resistance to direct-acting antivirals (DAAs) in other common subtypes. Data from patients also suggest that 1l and 4r with amino acid substitutions at positions 28-31 and 93 in NS5A are relatively resistant to DAA therapy. Methods In this study, we tested the efficacy of daclatasvir, elbasvir, ledipasvir, pibrentasvir and velpatasvir against these subtypes using the SGR-JFH1 replicon backbone. Results NS5A inhibitors showed different levels of efficacy with only pibrentasvir effective against all tested subtypes. Daclatasvir and ledipasvir were ineffective against 6u and 6v (half maximal effective concentration [EC50] values of 239–321 nM) while 3b and 3g were only susceptible to pibrentasvir. Analysis of effects of individual mutations indicated that Q30R in 1l increased the EC50 of ledipasvir by 18-fold, conferring intermediate resistance, while those of L31M and Y93H in 4r induced increases in EC50 values of 2,100- and 3,575-fold (high-level resistance). Conclusion The high ledipasvir EC50 values of 1l with the Q30R substitution, 4r L31M and 4r Y93H may explain the treatment failure in patients who were infected with these viruses and treated with ledipasvir + sofosbuvir. This study also shows the ineffectiveness of the first generation NS5A inhibitors against 6u and 6v, and confirms the inherent resistance of 3b and 3g to most NS5A inhibitors. Clinical studies to confirm in vivo sensitivity to NS5A inhibitors are urgently needed so that rational, effective treatment strategies may be developed for unusual subtypes. Lay summary Little is known about the efficacy of NS5A inhibitors against some “unusual” hepatitis C virus (HCV) subtypes including 1l, 3b, 3g, 4r, 6u and 6v. In this study, we manufactured HCV replicons which express the NS5A protein from the unusual HCV subtypes 1l, 3b, 3g, 4r, 6u, 6v. We then tested the effect of the NS5A inhibitors daclatasvir, elbasvir, ledipasvir, pibrentasvir and velpatasvir on blocking replication, using these replicons. We show that these replicons are resistant at some level to all NS5A inhibitors other than pibrentasvir.

Oxidative stress (OS) and insulin resistance (IR) induced by hepatitis C virus (HCV) infection, are involved in the development of chronic hepatitis C (CHC) complications and progression to hepatocellular carcinoma. The aim of this study was to investigate the effect of pegylated interferon alpha (IFNα) + ribavirin (PegIFNα+RVB) or sofosbuvir + NS5A inhibitor (SOF+InNS5A) on IR and the components of OS. HCV was genotyped in 20 CHC patients grouped by treatment with either PegIFNα+RVB (n = 10) or SOF+InNS5A (n = 10). The treatment’s effect on OS-induced damage to lipids (HNE-HDL), proteins (advanced glycation end products [AGEs]), and DNA (8-OHdG) as well as the concentrations of proinflammatory cytokines (IL-2, TNFα, IFNγ), ALT, AST, GSH and platelets was determined. Superoxide dismutase (SOD) and catalase activity as well as IR, determined by the HOMA1-IR index, was evaluated. The HCV genotypes (GT) found were GT1b (45%), GT1a (30%), GT2b (20%), and GT2a (5%). Viral RNA became undetectable by week 12 with SOF+InNS5A in 100% of the cases and with PegIFNα+RVB in 70% of the cases. After viral RNA became undetectable, regardless of treatment and GT, a significant increase in the platelet concentration and SOD activity was observed, whereas ALT, insulin, and IR decreased (p

Hepatitis C virus (HCV) is a life-threatening virus that causes liver infection. If it is not detected in an early phase, the virus can lead to severe liver damages, including hepatic fibrosis, liver cirrhosis, and hepatocellular carcinoma. Today, computational design of the HCV diagnostic kit is employed to increase the specificity and sensitivity of the ELISA (enzyme-linked immunosorbent assay) diagnosis method according to the specific genotypes of the virus in each geographical region as well as to reduce costs in developing and low-income countries. The aim of this study was to design a multi-epitope protein from common HCV genotypes in Iran (1a, 1b, and 3a). For this purpose, potential immunodominant epitopes and highly antigenic regions were identified for six antigenic proteins and all of the segments were joined using a proper linker. The physico-chemical characteristics of the designed multi-epitope protein were evaluated and tertiary structures of the construct were modeled. Then, the models were evaluated and the best one was determined. Finally, the sequence of the protein was reverse-translated and optimized for high expression in E. coli expression host. The findings of the present study indicated that the designed construct could detect the common HCV genotypes in Iran with high sensitivity and specificity.

Patients with thalassemia major are at high risk of hepatitis C through blood transfusion from donors infected by hepatitis C virus (HCV). The use of direct-acting antiviral (DAA) therapy against such HCV infections has increased in different populations. However, resistant viral variants can affect treatment outcomes, and therefore improved surveillance strategies are needed. Accordingly, we aimed to evaluate resistance-associated substitutions (RASs) to HCV DAAs at the baseline of treatment in thalassemia patients in a referral center. Out of 89 thalassemia patients who suffered from HCV infection and were referred to our center between 2016 and 2017, 43 underwent further analysis of the HCV nonstructural proteins NS5A and NS5B using polymerase chain reaction (PCR) sequencing methods. Unique primers were designed using bioinformatics software for separate detection of HCV subtypes 1a, 3a, and 1b. Detection of RASs was performed based on previously published literature. Statistical analysis was carried out using SPSS version 19. The participants, 60.4% (26/43) of whom were male, had a mean age ± standard deviation (SD) of 33.0 ± 5.0 years. HCV subtype 1a was found in 27 cases, 3a in 13, and 1b in three. In HCV subtype 1a there were 163 mutations in NS5A and 212 mutations in NS5B. The frequency of RASs was 20.9% (8 RASs in 9 patients), including M28V and H58P in subtype 1a, L28M, R30Q, C316N, and C316S in subtype 1b, and S24F in subtype 3a. Statistically, the subtype 1b and a higher mutation rate in NS5A were associated with RASs (p-value

Previously, it was found that the hepatitis C virus NS5A interacted with ACBD3 in a genotype-dependent manner. However, the region in NS5A responsible for association with ACBD3 is not clear. Domain I of NS5A was identified as critical for ACBD3 binding. By comparing the differences of amino acids in domain I from different genotypes of NS5A, it was found that key amino acids potentially corresponded to the affinity of the NS5A-ACBD3 interaction. The findings not only revealed that domain I of NS5A associates with ACBD3 but they also shed mechanistic light on how NS5A is associated with ACBD3 in a genotype-dependent manner.

Background Many long noncoding RNAs (lncRNAs) have important roles in biological processes. The lncRNA HULC was found to be upregulated in human hepatoma tissues. HULC is thought to be involved in multiple steps of hepatoma development and progression; however, the relationship between HULC and hepatitis C virus (HCV) infection, which is a leading cause of hepatoma, remains unclear. Methods We examined the effect of HCV replication on HULC expression and the underlying mechanism using cell culture systems. Subsequently, we tested the effect of HULC suppression and overexpression on HCV replication. Finally, we examined the impact of HCV eradication on HULC expression using human liver tissue and blood samples. Results HCV replication increased HULC expression in cell cultures. A promoter assay showed that an HCV nonstructural protein, NS5A, increased HULC transcription. HULC suppression inhibited HCV replication; conversely, its overexpression enhanced HCV replication. These effects on HCV replication seemed to occur by the modification of HCV translation. Measurements from human liver and blood samples showed that HCV eradication significantly reduced HULC levels in the liver and blood. Conclusions HCV infection increases HULC expression in vitro and in vivo. HULC modulates HCV replication through an HCV internal ribosome entry site–directed translation step.

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-naïve patients. Genotyping of 80 HCV samples from treatment-naïve 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. Electronic supplementary material The online version of this article (10.1007/s11262-020-01776-y) contains supplementary material, which is available to authorized users.

Interferon lambda was discovered in recent years to be an antiviral agent, and research on different aspects of this antiviral factor in viral infection and investigations of its effectiveness are also progressing. The immunological effects of interferon lambda on different cell populations is not precisely known, which may be due to its use of a heterodimeric receptor consisting of IL-10R2 and IFN-λR1, which are not broadly expressed in all types of cells. In the present study, signaling by interferon lambda and its effect on the expression of hepatitis C virus (HCV) proteins were measured, and the expression pattern of some antiviral proteins and IL-10 levels were investigated in peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from 50 patients with chronic genotype 1a HCV infection and 10 healthy individuals as controls. The PBMCs were treated with various doses of interferon lambda at different times of cultivation. Real-time PCR was used for relative quantification of Mxa, PKR, OAS, ISG15 and HCV core mRNAs. Expression of the NS5A protein was measured by flow cytometry, and IL-10 production was assessed by ELISA. A significant increase in the expression of mRNA encoding antiviral proteins and a decrease in the expression of mRNAs encoding the HCV core protein were observed when cells were treated with interferon lambda in an intermittent manner. The expression of HCV NS5A protein and interleukin 10 levels were also lower than in the control group. It was shown that the maximum antiviral effect of interferon lambda in PBMCs is dependent on the dose and treatment time.

Ribavirin remains relevant for successful treatment of chronic hepatitis C virus (HCV) infections in low-income settings, as well as for therapy of difficult-to-treat HCV patients. We studied the effect of ribavirin against cell-culture adapted HCV of genotypes 1, 2 and 3, representing ~80% of global infections. TNcc(1a) was the most sensitive to ribavirin, while J6/JFH1(2a) was the most resistant. EC50s ranged from 21 μM (95%CI: 20–22 μM) to 189 μM (95%CI: 173–207 μM). Substitutions at position 415 of NS5B resulted in little or no change to ribavirin sensitivity (0.7–0.9 fold) but conferred moderate drug resistance during extended treatment of genotype 1 (1.8-fold). NS5A and NS5B sequences could alter ribavirin sensitivity 2-4-fold, although their contribution was not simply additive. Finally, we detected limited accumulation of mutations associated with ribavirin treatment. Our 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.

Lysosome incorporate and degrade proteins in a process known as autophagy. There are three types of autophagy; macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Although autophagy is considered a nonselective degradation process, CMA is known as a selective degradation pathway. All proteins internalized in the lysosome via CMA contain a pentapeptide KFERQ-motif, also known as a CMA-targeting motif, which is necessary for selectivity. CMA directly delivers a substrate protein into the lysosome lumen using the cytosolic chaperone HSC70 and the lysosomal receptor LAMP-2A for degradation. Hepatitis C virus (HCV) NS5A protein interacts with hepatocyte-nuclear factor 1α (HNF-1α) together with HSC70 and promotes the lysosomal degradation of HNF-1α via CMA, resulting in HCV-induced pathogenesis. HCV NS5A promotes recruitment of HSC70 to the substrate protein HNF-1α. HCV NS5A plays a crucial role in HCV-induced CMA. Further investigations of HCV NS5A-interacting proteins containing CMA-targeting motifs may help to elucidate HCV-induced pathogenesis.

Zika virus (ZIKV) is a mosquito-borne pathogen classified by the World Health Organization (WHO) as a public health emergency of international concern in 2016, and it is still identified as a priority disease. Although most infected individuals are asymptomatic or show mild symptoms, a risk of neurologic complications is associated with infection in adults. Additionally, infection during pregnancy is directly linked to microcephaly and other congenital malformations. Since there are no currently available vaccines or approved therapeutics for this virus, there is a critical unmet need in developing treatments to prevent future ZIKV outbreaks. Towards this end, we performed a large-scale cell-based high-content screen of 51,520 chemical compounds to identify potential antiviral drug candidates. The compound (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) was found to inhibit replication of multiple ZIKV strains and in different cell systems. SBI-0090799 did not affect viral entry or RNA translation but suppressed RNA replication by preventing the formation of the membranous replication compartment. Selection of drug-resistant viruses identified single amino acid substitions in the N-terminal region of non-structural protein NS4A arguing this to be the likely drug target. These resistance mutations rescued viral RNA replication and restored the formation of the membranous replication compartment. This mechanism of action is similar to clinically-approved NS5A inhibitors for hepatitis C virus (HCV). Taken together, SBI-0090799 represents a promising lead candidate for the development of an antiviral treatment against ZIKV infection for the mitigation of severe complications and potential resurgent outbreaks of the virus. IMPORTANCE This study describes the elucidation of (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) as selective and potent inhibitor of Zika virus (ZIKV) replication using a high throughput screening approach. Mapping and resistance studies, supported by electron microscopy observations, indicate that the small molecule is functioning through inhibition of NS4A-mediated formation of ZIKV replication compartments in the endoplasmic reticulum (ER). Intriguingly, this defines a novel non-enzymatic target and chemical matter for the development of a new class of ZIKV antivirals. Moreover, chemical modulation affecting this non-structural protein mirrors the identification and development of hepatitis C virus (HCV) NS5A inhibitor daclatasvir and its derivatives, similarly interfering with the formation of the viral replication compartment and also targeting a protein with no enzymatic activity, which have been part of a curative strategy for HCV.

Despite the effectiveness of direct-acting antiviral agents in treating hepatitis C virus (HCV), cases of treatment failure have been associated with the emergence of resistance-associated substitutions. To better guide clinical decision-making, we developed and validated a near-whole-genome HCV genotype-independent next-generation sequencing strategy. HCV genotype 1–6 samples from direct-acting antiviral agent treatment-naïve and -treated HCV-infected individuals were included. Viral RNA was extracted using a NucliSens easyMAG and amplified using nested reverse transcription-polymerase chain reaction. Libraries were prepared using Nextera XT and sequenced on the Illumina MiSeq sequencing platform. Data were processed by an in-house pipeline (MiCall). Nucleotide consensus sequences were aligned to reference strain sequences for resistance-associated substitution identification and compared to NS3, NS5a, and NS5b sequence data obtained from a validated in-house assay optimized for HCV genotype 1. Sequencing success rates (defined as achieving &gt, 100-fold read coverage) approaching 90% were observed for most genotypes in samples with a viral load &gt, 5 log10 IU/mL. This genotype-independent sequencing method resulted in &gt, 99.8% nucleotide concordance with the genotype 1-optimized method, and 100% agreement in genotype assignment with paired line probe assay-based genotypes. The assay demonstrated high intra-run repeatability and inter-run reproducibility at detecting substitutions above 2% prevalence. This study highlights the performance of a freely available laboratory and bioinformatic approach for reliable HCV genotyping and resistance-associated substitution detection regardless of genotype.

Resistance-associated substitutions (RASs) may exist prior to treatment and contribute to the failure of treatment with direct-acting antivirals (DAAs). As the major site of HCV replication, naturally occurring variants with RASs may segregate into the liver. In the present study, we performed viral population sequencing to retrospectively investigate the NS3 and NS5A RAS profiles in 34 HIV/HCV coinfected patients naïve to anti-HCV treatment who underwent diagnostic liver biopsy between 2000 and 2006 and had liver and plasma samples available. Sixteen were infected by HCV genotype (GT) 1a, 11 by GT3a, and 7 by GT4d. The analysis of the NS3 domain in GT1a showed a difference in strain between the liver and plasma in three cases, with a preponderance of specific RASs in the liver compartment. In GT4d samples, 6/7 coupled liver and plasma samples were concordant with no RASs. Sequence analysis of the NS5A domain showed the presence of RASs in the livers of 2/16 patients harboring GT1a but not in the corresponding plasma. In GT4d, NS5A RASs were detected in 7/7 liver tissues and 5/7 plasma samples. NS3 domain and NS5A domain were found to be conserved in plasma and livers of patients infected with GT3a. Thus, RASs within GT1a and GT4d more likely segregate into the liver and may explain the emergence of resistant strains during DAA treatment.

Molecular diagnosis of viral genotyping devoid of polymerase chain reaction (PCR) amplification in clinical cohorts has hitherto been challenging. Here we present a simplified molecular diagnostic strategy for direct genotyping of hepatitis C virus (HCV) 1 and 3 (prevalent worldwide) using a combination of rationally designed genotype-specific antisense oligonucleotides (ASOs) and plasmonic gold nanoparticles. The ASOs specific to genotypes 1 and 3 have been designed from the nonstructural region 5A (NS5A) of the viral genome using the ClustalW multiple sequence alignment tool. A total of 79 clinical samples including 18 HCV genotype 1, 18 HCV genotype 3, one HIV positive, one HBV positive, and 41 healthy controls have been tested against both the designed ASOs. The study reveals 100% specificity and sensitivity with the employed samples and thereby opens up new avenues for PCR-free direct genotyping of other viruses as well, through the rational design of ASOs.

SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.

Background Steatosis is an important clinical manifestation associated with chronic hepatitis C virus (HCV) infection. AMP-activated protein kinase (AMPK), a major mediator of lipid metabolism, regulates HCV-associated hepatic steatosis, but the underlying mechanisms remain obscure. Here we investigated the mechanism of HCV nonstructural protein 5A (NS5A)-induced lipid accumulation by the AMPK/SREBP-1c pathway. Methods We generated model mice by injecting recombinant lentiviral particles expressing the NS5A protein (genotype 3a) via the tail vein. The serum levels of alanine aminotransferase (ALT), free fatty acids (FFAs) and triglycerides (TG) were examined. H&E and Oil Red O staining were used to examine lipid droplets. Immunohistochemistry staining, quantitative real-time PCR and Western blotting were used to determine the expression of lipogenic genes. Results Our results showed that the serum levels of ALT, FFAs and TG, as well as the accumulation of hepatic lipid droplets, were increased significantly in mice infected with NS5A-expressing lentiviral particles. NS5A inhibited AMPK phosphorylation and increased the expression levels of sterol regulatory element binding protein-1c (SREBP-1c), acetyl-coenzyme A carboxylase 1 (ACC1) and fatty acid synthase (FASN) in vivo and in vitro. Further investigation revealed that pharmacological activation or ectopic expression of AMPK neutralized the upregulation of SREBP-1c, ACC1 and FASN, and ameliorated hepatic lipid accumulation induced by NS5A. Ectopic expression of SREBP-1c enhanced NS5A-induced hepatic lipid accumulation, which was dramatically reversed by pharmacological activation of AMPK. Conclusions Collectively, we demonstrate that NS5A induces hepatic lipid accumulation via the AMPK/SREBP-1c pathway.

Using direct-acting antiviral agents (DAAs) against hepatitis C virus (HCV) infection results in a high treatment response rate. However, several factors can significantly alter this outcome such as resistance-associated substitutions (RASs) in HCV NS5A gene. This study aimed to evaluate the prevalence of naturally occurring RASs of NS5A in HCV genotype 3 (HCV-3) sequences isolated from individuals with chronic HCV-3 infection. All the registered sequences in the GenBank under "NS5A" AND "Hepacivirus C" query were evaluated and screened, those which followed our inclusion criteria were enrolled in our pooled analysis. The retrieved sequences of included studies were evaluated for substitutions, RASs, and RASs conferring100 resistance fold change (RASs100 × ) in NS5A amino acid positions 24, 28, 30, 31, 62, 92, and 93. From 7 enrolled studies, a total of 370 HCV-3a isolates were retrieved and investigated. Forty-eight (13.0%, 95% CI = 9.9-16.8%) isolates harbored NS5A RASs. Moreover, Y93H was the only NS5A RAS100 × observed in 13 (3.5%, 95% CI = 2.0-5.9%) retrieved sequences. The low frequency of naturally occurring NS5A RASs, especially those with clinical relevance (RASs100 × ), among individuals with HCV-3 infection and the high rate of treatment response to DAAs suggest not to investigate every individual with HCV-3 infection for NS5A RASs before treatment.

Background and objective Direct-acting antivirals (DAA) facing resistance continue to be used in some areas worldwide. Thus, identifying hepatitis C virus (HCV) genotypes/subtypes and loci with certain prevalent resistance-associated substitutions (RASs) deserves attention. We investigated the global and regional frequencies of naturally occurring RASs among all confirmed HCV subtypes (n=86) and explored co-occurring and mutually exclusive RAS pairs within and between genes NS3, NS5A, and NS5B. Methods A total of 213,908 HCV sequences available as of July 10, 2019 were retrieved from the NCBI nucleotide database. After curation, 17,312 NS3, 8,478 NS5A, and 25,991 NS5B sequence fragments from DAA-naive patients were screened for RASs. MEGA 6.0 was used to translate aligned nucleotide sequences into amino acid sequences, and RAS pairs were identified by hypergeometric analysis. Results RAS prevalence varied significantly among HCV subtypes. For example, D168E, highly resistanct to all protease inhibitors except voxilaprevir, was nearly absent in all subtypes except in 43.48% of GT5a sequences. RASs in NS3 exhibiting significantly different global distribution included Q80K in GT1a with the highest frequency in North America (54.49%), followed by in Europe (22.66%), Asia (6.98%), Oceania (6.62%), and South America (1.03%). The prevalence of NS3 S122G in GT1b was highest in Asia (26.6%) and lowest in Europe (2.64%). NS5A L28M, R30Q, and Y93H in GT1b, L31M in GT2b, and NS5B C316N in GT1b was most prevalent in Asia. A150V in GT3a, associated with sofosbuvir treatment failure, was most prevalent in Asia (44.09%), followed by Europe (31.19%), Oceania (24.29%), and North America (19.05%). Multiple mutually exclusive or co-occurring RAS pairs were identified, including Q80K+R155K and R155K+D168G in GT1a and L159F+C316N and R30Q (NS5A)+C316N (NS5B) in GT1b. Conclusion Our data may be of special relevance for those countries where highly effective antivirals might not be available. Considering the specific RASs prevalence will help the clinicians to make optimal treatment choices. The RASs pairs would benefit anti-HCV drug development.

AIMS Development of direct-acting antivirals (DAAs) has made antihepatitis C virus (HCV) treatment highly safe and effective. However, the emergence of resistant-associated variants (RAVs) after failure of DAA therapy affects retreatment outcomes. In particular, genotype 1 HCV with P32 deletion has been reported to be highly resistant to all approved non-structural protein (NS)5A inhibitors. However, analysis of RAVs in genotype 2 HCV has been limited. Accordingly, in this study, we evaluated the roles of genotype 2 HCV variants in antiviral drug efficacy. METHODS We utilized HCV-2b/2a (JFH-1) chimeric virus (genotype 2a), which replicates more robustly than JFH-1. We constructed various genotype 2a JFH-1-based HCV cell culture systems with NS3 (D168E), NS5A (F28S, F28S/M31I, P32 deletion, and Y93H), and NS5B (S282 T) RAVs and analyzed the replication ability and sensitivity to various anti-HCV reagents. RESULTS Genotype 2a-based HCV with NS5A-P32 deletion could not replicate even in long-term cultures. Genotype 2a-based HCV with NS5A-F28S/M31I showed significantly higher replication ability than the wild-type strain, and replication could not be suppressed, even with high concentrations of NS5A inhibitors, including pibrentasvir and velpatasvir (

Combination therapy with glecaprevir (GLE) and pibrentasvir (PIB) has high efficacy for pan-genotypic hepatitis C virus (HCV)-infected patients. However, the efficacy for patients who acquired potent NS5A inhibitor resistance-associated variants (RAVs) as a result of failure to respond to previous direct-acting antiviral (DAA) therapies is unclear. We investigated the efficacy of GLE/PIB treatment for genotype 1b HCV strains containing RAVs using subgenomic replicon systems and human hepatocyte transplanted mice. Mice were injected with serum samples obtained from a DAA-naïve patient or daclatasvir plus asunaprevir (DCV/ASV) treatment failures including NS5A-L31M/Y93H, -P58S/A92K or -P32 deletion (P32del) RAVs, then treated with GLE/PIB. HCV was eliminated by GLE/PIB treatment in mice with wild-type and NS5A-L31M/Y93H but relapsed in mice with NS5A-P58S/A92K, followed by emergence of additional NS5A mutations after cessation of the treatment. In NS5A-P32del-infected mice, serum HCV RNA remained positive during the GLE/PIB treatment. NS5A-P58S/A92K showed 1.5-fold resistance to PIB relative to wild-type based on analysis using HCV subgenomic replicon systems. When mice were administered various proportions of HCV wild-type and P32del strains and treated with GLE/PIB, serum HCV RNA remained positive in mice with high frequencies of P32del. In these mice, the P32del was undetectable by deep sequencing before GLE/PIB treatment, but P32del strains relapsed after cessation of the GLE/PIB treatment. GLE/PIB is effective for wild-type and NS5A-L31M/Y93H HCV strains, but the effect seems to be low for P58S/A92K and NS5A-P32del RAVs. Although NS5A-P32del was not detected, the mutation may be present at low frequency in DCV/ASV treatment failures.

Bicyclol is a synthetic drug for hepatoprotection in clinic since 2004. Preliminary clinical observations suggest that bicyclol might be active against hepatitis C virus (HCV) with unknown mechanism. Here, we showed that bicyclol significantly inhibited HCV replication in vitro and in hepatitis C patients. Using bicyclol as a probe, we identified glycolipid transfer protein (GLTP) to be a novel restrictive factor for HCV replication. The GLTP preferentially bound host vesicle-associated membrane protein-associated protein-A (VAP-A) in competition with the HCV NS5A, causing an interruption of the complex formation between VAP-A and HCV NS5A. As the formation of VAP-A/NS5A complex is essential for viral RNA replication, up-regulation of GLTP by bicyclol reduced the level of VAP-A/NS5A complex and thus inhibited HCV replication. Bicyclol also exhibited an inhibition on HCV variants resistant to direct-acting antiviral agents (DAAs) with an efficacy identical to that on wild type HCV. In combination with bicyclol, DAAs inhibited HCV replication in a synergistic fashion. GLTP appears to be a newly discovered host restrictive factor for HCV replication, Up-regulation of GLTP causes spontaneous restriction of HCV replication., Graphical abstract Treatment of the HCV-infected Huh7.5 cells with bicyclol decreased the level of miR-449b and thus induced the expression of glycolipid transfer protein (GLTP). The up-regulated GLTP preferentially bound host cofactor vesicle-associated membrane protein-associated protein-A (VAP-A) in competition with the HCV NS5A, causing a decrease of the HCV replicative complex level of VAP-A/NS5A, and thus inhibited HCV replication.fx1

A 42-year-old Chinese man with chronic hepatitis C virus (HCV) infection visited our hospital for antiviral therapy. The subgenotype could not be determined using the HCV GENOTYPE Primer Kit (Institute of Immunology, Tokyo, Japan), which can identify genotype 3a HCV exclusively among genotype 3 HCV. Thus, the whole-genome sequence of HCV was analyzed using the MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK), a third-generation single-molecule sequencing platform. Consequently, a total of 9442 bases with a 73.6 mean depth, corresponding to the sequences between nt25 and PolyU/UC were determined (LC414155.2). The similarity analysis revealed that the obtained sequence was classified into genotype 3b HCV and showed nucleotide identities from 87.6% to 93.9% with those of 12 previously reported strains. Furthermore, possible resistance-associated substitutions in non-structural protein (NS)3, NS5A, and NS5B based on consensus sequences of 12 genotype 3b HCV strains, including NS5A-Y93H and NS5B-S282 T substitutions, were absent. In conclusion, the MinION nanopore sequencer is useful for analyzing the HCV genome, especially the genomes of genotype 3 HCV strains for which standardized real- time PCR methods for all subgenotypes have not been established.

Hepatitis C virus (HCV) is a highly variable infectious agent, classified into 8 genotypes and 86 subtypes. Our laboratory has implemented an in-house developed high-resolution HCV subtyping method based on next-generation sequencing (NGS) for error-free classification of the virus using phylogenetic analysis and analysis of genetic distances in sequences from patient samples compared to reference sequences. During routine diagnostic, a sample from an Equatorial Guinea patient could not be classified into any of the existing subtypes. The whole genome was analyzed to confirm that the new isolate could be classified as a new HCV subtype. In addition, naturally occurring resistance-associated substitutions (RAS) were analyzed by NGS. Whole-genome analysis based on p-distances suggests that the sample belongs to a new HCV genotype 1 subtype. Several RAS in the NS3 (S122T, D168E and I170V) and NS5A protein (Q(1b)24K, R(1b)30Q and Y93L+Y93F) were found, which could limit the use of some inhibitors for treating this subtype. RAS studies of new subtypes are of great interest for tailoring treatment, as no data on treatment efficacy are reported. In our case, the patient has not yet been treated, and the RAS report will be used to design the most effective treatment.

Resistance-associated substitutions (RASs) of hepatitis C virus (HCV) in the NS5A region impair the efficacy of NS5A inhibitors. In this study, we evaluated the characteristics of the novel RASs observed in treatment-failure patients, A92K and a deletion at P32 (P32del), and the susceptibility of viruses with these RASs to various anti-HCV reagents by using JFH-1 based recombinant HCV with NS5A from a genotype 1b Con1 strain (JFH1/5ACon1). We introduced A92K or P32del solely or in combination with Q24K, L28M, R30Q or L31F into the NS5A of JFH1/5ACon1. Viruses harboring R30Q/A92K showed high extracellular core antigens and infectivity titers, whereas the other viruses with RASs showed low replication levels and infectivity titers. All the viruses with A92K or P32del were markedly resistant to ledipasvir, velpatasvir and elbasvir. Interestingly, viruses with R30Q/A92K were more susceptible to grazoprevir than viruses without RAS. All the viruses had a similar susceptibility to ribavirin and sofosbuvir. In conclusion, combination RASs R30Q/A92K enhanced virus production whereas other RASs impaired virus replication. Both A92K and P32del conferred severe resistance even to second generation NS5A inhibitors. However, these viruses were susceptible to grazoprevir, ribavirin and sofosbuvir. Thus, combination regimens with these reagents may eradicate viruses harboring A92K or P32del.

Daclatasvir (DCV) is a highly potent direct-acting antiviral that targets the non-structural protein 5A (NS5A) of hepatitis C virus (HCV) and has achieved great clinical successes. Previous studies demonstrate its impact on the viral replication complex assembly. However the precise mechanism by which DCV impairs the replication complex assembly remains elusive. In this study, by using HCV subgenomic replicons and a viral replicase assembly surrogate system that expresses the HCV NS3-5B polyprotein to mimic the viral replicase assembly, we dissected the impacts of DCV on aggregation and tertiary structure of NS5A, the protein-protein interactions within the viral replicase and the quaternary structure of the viral replicase. We found that DCV didn’t affect aggregation and tertiary structure of NS5A. DCV induced a quaternary structural change of the viral replicase, evidenced by selectively increasing of the NS4B’s sensitivity to proteinase K digestion. Mechanically, DCV impaired the NS4B-involved protein-protein interactions within the viral replicase. The DCV-resistant mutant Y93H was refractory to the DCV-induced reduction of the NS4B-invoved protein interactions and the quaternary structural change of the viral replicase. In addition, Y93H reduced NS4B-involed protein-protein interactions within the viral replicase and attenuated viral replication. We propose that DCV may induce a position change of NS5A, which allosterically affects the protein interactions within the replicase components and disrupts the replicase assembly.ImportanceThe development of the direct-acting antivirals (DAA) has resulted in great clinical achievements for Hepatitis C Virus (HCV) treatment. Daclatasvir (DCV) is an inhibitor targeting the non-enzymatic NS5A, with the 50% effective concentration values in the picomolar range. Accumulated data suggest that DCV blocks the biogenesis of the HCV replication complex. However the mechanistic actions of DCV are still largely unknown. Insights into the action mechanism of DCV on the viral replication complex assembly of HCV may enlighten the development of next generation of DAAs and new anti-viral strategies for other positive-strand RNA viruses for which there are a scarcity of DAAs. Herein, using HCV subgenomic replicons and a viral replicase assembly surrogate system, we dissected the mechanistic actions of DCV on the viral replicase assembly. We found that DCV allosterically impairs NS4B-involved protein-protein interactions within the viral replicase and disrupts the quaternary structure of the viral replicase.

The NS5A non-structural protein of classical swine fever virus (CSFV) is a multifunctional protein involved in viral genomic replication, protein translation and regulation of cellular signaling pathways, and assembly of infectious virus particles. Previous report showed that NS5A inhibited nuclear factor kappa B (NF-κB) signaling induced by poly(I:C); however, the mechanism was not elucidated. Here, we report that NS5A interacts with NF-κB essential modulator (NEMO),a regulatory subunit of the IκB kinase (IKK) complex, and that the zinc finger domain of NEMO essential for NEMO ubiquitination and IKK activation is required for the interaction of NEMO with NS5A. Viral infection or NS5A expression by itself reduced the protein level of NEMO. Mechanistic analysis revealed that NS5A mediated proteasomal degradation of NEMO. Ubiquitination assay showed that NS5A induced K27-but not K48-linked polyubiquitination of NEMO. In addition, NS5A blocked k63-linked polyubiquitination of NEMO, thereby inhibiting activation of IKK and NF-κB. These findings indicate that NS5A inhibits NF-κB signaling by mediating proteasomal degradation of NEMO and blocking k63-linked polyubiquitination of NEMO, thereby revealing a novel mechanism by which CSFV inhibits host innate immunity.ImportanceClassical swine fever (CSF) is a economically important swine viral disease leading to hemorrhagic fever and immuno-suppression. In order to successfully infect and replicate in a host cell, viruses have evolved various strategies to antagonize host innate immunity. It is known that CSFV non-structural protein Npro interacts with interferon regulatory factor 3 (IRF3) and mediates its proteasomal degradation, thereby inhibiting the production of type I interferon. However, no other mechanism by which CSFV antagonizes host innate immunity has so far been reported. Here, we show that NS5A inhibits NF-κB signaling by mediating proteasomal degradation of NEMO and by blocking k63-linked polyubiquitination of NEMO, thereby revealing a novel mechanism by which CSFV antagonizes host innate immunity.

Simple Summary According to the last estimate by the World Health Organization (WHO), more than 71 million individuals have chronic hepatitis C worldwide. The persistence of HCV infection leads to chronic hepatitis, which can evolve into liver cirrhosis and ultimately into hepatocellular carcinoma (HCC). Although the pathogenic mechanisms are not fully understood, it is well established that an interplay between host cell factors, including microRNAs (miRNA), and viral components exist in all the phases of the viral infection and replication. Those interactions establish a complex equilibrium between host cells and HCV and participate in multiple mechanisms characterizing hepatitis C pathogenesis. The present review aims to describe the role of HCV structural and non-structural proteins in the modulation of cellular miRNA during HCV infection and pathogenesis. Abstract Hepatitis C virus (HCV) genome encodes for one long polyprotein that is processed by cellular and viral proteases to generate 10 polypeptides. The viral structural proteins include the core protein, and the envelope glycoproteins E1 and E2, present at the surface of HCV particles. Non-structural (NS) proteins consist of NS1, NS2, NS3, NS4A, NS4B, NS5a, and NS5b and have a variable function in HCV RNA replication and particle assembly. Recent findings evidenced the capacity of HCV virus to modulate host cell factors to create a favorable environment for replication. Indeed, increasing evidence has indicated that the presence of HCV is significantly associated with aberrant miRNA expression in host cells, and HCV structural and non-structural proteins may be responsible for these alterations. In this review, we summarize the recent findings on the role of HCV structural and non-structural proteins in the modulation of host cell miRNAs, with a focus on the molecular mechanisms responsible for the cell re-programming involved in viral replication, immune system escape, as well as the oncogenic process. In this regard, structural and non-structural proteins have been shown to modulate the expression of several onco-miRNAs or tumor suppressor miRNAs.

RNA viruses, such as hepatitis C virus (HCV), influenza virus, and SARS-CoV-2, are notorious for their ability to evolve rapidly under selection in novel environments. It is known that the high mutation rate of RNA viruses can generate huge genetic diversity to facilitate viral adaptation. However, less attention has been paid to the underlying fitness landscape that represents the selection forces on viral genomes, especially under different selection conditions. Here, we systematically quantified the distribution of fitness effects of about 1,600 single amino acid substitutions in the drug-targeted region of NS5A protein of HCV. We found that the majority of nonsynonymous substitutions incur large fitness costs, suggesting that NS5A protein is highly optimized. The replication fitness of viruses is correlated with the pattern of sequence conservation in nature, and viral evolution is constrained by the need to maintain protein stability. We characterized the adaptive potential of HCV by subjecting the mutant viruses to selection by the antiviral drug daclatasvir at multiple concentrations. Both the relative fitness values and the number of beneficial mutations were found to increase with the increasing concentrations of daclatasvir. The changes in the spectrum of beneficial mutations in NS5A protein can be explained by a pharmacodynamics model describing viral fitness as a function of drug concentration. Overall, our results show that the distribution of fitness effects of mutations is modulated by both the constraints on the biophysical properties of proteins (i.e., selection pressure for protein stability) and the level of environmental stress (i.e., selection pressure for drug resistance). IMPORTANCE Many viruses adapt rapidly to novel selection pressures, such as antiviral drugs. Understanding how pathogens evolve under drug selection is critical for the success of antiviral therapy against human pathogens. By combining deep sequencing with selection experiments in cell culture, we have quantified the distribution of fitness effects of mutations in hepatitis C virus (HCV) NS5A protein. Our results indicate that the majority of single amino acid substitutions in NS5A protein incur large fitness costs. Simulation of protein stability suggests viral evolution is constrained by the need to maintain protein stability. By subjecting the mutant viruses to selection under an antiviral drug, we find that the adaptive potential of viral proteins in a novel environment is modulated by the level of environmental stress, which can be explained by a pharmacodynamics model. Our comprehensive characterization of the fitness landscapes of NS5A can potentially guide the design of effective strategies to limit viral evolution.

Hepatitis C virus (HCV) nonstructural protein NS4B is necessary for HCV replication. Our previous research found that NS4B-associated cellular proteins PREB and Surfeit 4 are involved in HCV replication. However, the molecular mechanism of HCV replication is not fully understood. Here we identified cellular ovarian cancer immunoreactive antigen domain containing 2 (OCIAD2) protein as a novel NS4B-associated HCV host cofactor by screening with small interfering RNA. Knockdown of OCIAD2 reduced significantly the HCV replication in a dose-dependent and genotype-independent manner. Further research showed that OCIAD2 was recruited into the HCV RNA replication complex by the interaction with NS4B. Interestingly, HCV replication induced OCIAD2 expression. In turn, overexpression of wild OCIAD2 also promoted virus replication whereas that of OCIAD2 mutant lacking the ability to bind NS4B exerted no effect on HCV replication. We also examined whether OCIAD2 interacted with other proteins participating in the HCV RNA replication complex including viral proteins NS5A, NS5B, and cellular proteins PREB, Surfeit 4. The results showed that OCIAD2 interacted with PREB and NS5A, but not NS5B or Surfeit 4. Our findings provide new insights into the function of OCIAD2 and HCV replication mechanism.

Currently, direct-acting antiviral drugs (DAAs) are widely used as therapeutic methods for hepatitis C virus (HCV)-positive patients, however, patients may experience treatment failure, and the dynamic changes of HCV genomes in these patients are unknown. In this study, three real-world DAAs cohorts were enrolled to observe clinical efficacy. In addition, serum samples from treatment failure patients at baseline and relapse were used to analyze changes of the HCV genomes at near full-length genome level, including resistance-associated variants (RAVs), viral quasispecies diversity and selection analysis. Next-generation sequencing was used as the detection method. The overall sustained virological response at 12 w after the end of treatment was achieved in 91.9% (57/62) of HCV patients, and 3 paired samples obtained from relapsed patients. All the 3 patients harbored baseline NS5A RAVs, the frequency of NS5A RAVs increased in 2 patients and a new NS5A RAV emerged in 1 patient at relapse, and almost all the viral strains existed with NS5A RAVs at relapse. The results of the viral quasispecies diversity analysis revealed that viral quasispecies diversity decreased at relapse compared to baseline, and the results of selection analysis indicated that the virus population experienced a bottleneck phenomenon, recent selective sweep and population expansion or was under purification selection after DAAs treatment. This study indicated that the clinical efficacy was excellent in real-world DAAs cohorts, and the viral strains existed at relapse were selective by DAAs therapy.

Porcine LFBKαVβ6 cells have been successfully used for diagnostics and propagation of all FMDV serotypes/subtypes. Unfortunately, after initial characterization, these cells showed contamination with bovine viral diarrhea virus (BVDV), a non-cytopathic adventitious agent. Persistent infection with BVDV could interfere with diagnostic tests and, also prevent consideration for other uses, i.e., vaccine production. In this study, we developed a three-prong methodology to completely remove BVDV from LFBKαVβ6 cells. Combined treatment with siRNA against BVDV NS5A, porcine interferon alpha and ribavirin resulted in the elimination of BVDV, as determined by immunohistochemistry analysis, quantitative RT-PCR and RNA sequencing. Importantly, elimination of BVDV from LFBKαVβ6 did not affect FMDV growth and plaque phenotype from different serotypes isolated and propagated in the clean cell line, newly named MGPK αVβ6-C5. Additionally, isolation of FMDV from field oro-pharyngeal samples, was successful at the same sensitivity as in BVDV-contaminated LFBKαVβ6 cells. Our results identified a direct method to efficiently eliminate BVDV from porcine cells without altering FMDV permissiveness, diagnostic value, or potential for use in vaccine production. Furthermore, these cells may provide an improved platform for diagnostics and propagation of other viruses of interest in the veterinary field and the virology community at large.

Hepatitis C virus (HCV) genotype 3 presents a high level of both baseline and acquired resistance to direct-acting antivirals (DAAs), particularly those targeting the NS5A protein. To understand this resistance we studied a cohort of Brazilian patients treated with the NS5A DAA, daclatasvir and the nucleoside analogue, sofosbuvir. We observed a novel substitution at NS5A amino acid residue 98 [serine to glycine (S98G)] in patients who relapsed post-treatment. The effect of this substitution on both replication fitness and resistance to DAAs was evaluated using two genotype 3 subgenomic replicons. S98G had a modest effect on replication, but in combination with the previously characterized resistance-associated substitution (RAS), Y93H, resulted in a significant increase in daclatasvir resistance. This result suggests that combinations of substitutions may drive a high level of DAA resistance and provide some clues to the mechanism of action of the NS5A-targeting DAAs.

International audience; The hepatitis C virus (HCV) life cycle is a tightly regulated process, during which structural and non-structural proteins cooperate. However, the interplay between HCV proteins during genomic RNA replication and progeny virion assembly is not completely understood. Here, we studied the dynamics and intracellular localization of non-structural 5A protein (NS5A), which is a protein involved both in genome replication and encapsidation. An NS5A-eGFP (enhanced green fluorescent protein) tagged version of the strain JFH-1-derived wild-type HCV was compared to the corresponding assembly-deficient viruses Δcore, NS5A basic cluster 352–533 mutant (BCM), and serine cluster 451 + 454 + 457 mutant (SC). These analyses highlighted an increase of NS5A motility when the viral protein core was lacking. Although to a lesser extent, NS5A motility was also increased in the BCM virus, which is characterized by a lack of interaction of NS5A with the viral RNA, impairing HCV genome encapsidation. This observation suggests that the more static NS5A population is mainly involved in viral assembly rather than in RNA replication. Finally, NS4B exhibited a reduced co-localization with NS5A and lipid droplets for both Δcore and SC mutants, which is characterized by the absence of interaction of NS5A with core. This observation strongly suggests that NS5A is involved in targeting NS4B to lipid droplets (LDs). In summary, this work contributes to a better understanding of the interplay between HCV proteins during the viral life cycle.

Positive-strand RNA viruses such as hepatitis C virus (HCV), flaviviruses, and coronaviruses are medically important. Assembly of replicase on host membranes is a conserved replication strategy and an attractive antiviral target. The mechanisms of replicase assembly are largely unknown, due to the technical difficulties in purifying the replicase and carrying out structural studies. Here, with an HCV replicase assembly surrogate system, we employed a bioorthogonal system to introduce the photolabile unnatural amino into each residue in the cytosolic regions of NS4B and the amphipathic helix (AH) of NS5A. Photocrosslinking enabled visualization of NS4B oligomerization and NS5A dimerization at pinpointed interacting residues and identifying contacting sites among the replicase components. Characterization of the interacting sites revealed hub elements in replicase assembly by docking replicase components to prompt protein-protein interactions. The results provide information about the molecular architecture of the replicase, advancing understanding of the mechanism of replicase assembly., Graphical abstract, Zhang et al. use a polyprotein expression model to introduce a photoactivatable unnatural amino acids at individual residues to characterize the interaction between HCV replicase components NS4B, NS5A, and NS3. The detailed landscape of protein-protein interactions provides a model for replicase assembly.

BackgroundHepatitis C virus (HCV), non-structural 5A (NS5A), and non-structural 5B (NS5B) resistance-associated substitutions (RASs) are the main causes of failure to direct-acting antiviral agents (DAAs). NS5A and NS5B RASs can occur in patients with HCV infection naturally and before exposure to DAAs.ObjectivesThis study aimed to evaluate naturally-occurring NS5A and NS5B RASs in Iranian patients with HCV genotype 1a (HCV-1a) and -3a infections.MethodsIn this cross-sectional study, viral RNA was extracted from serum specimens. NS5A and NS5B regions were amplified using RT-PCR followed by DNA sequencing. The results of nucleotide sequences were aligned against reference sequences of HCV-1a and -3a and the amino acid substitutions were analyzed using geno2pheno [hcv] web application.ResultsAmong 135 patients with hepatitis C, NS5A amino acid substitutions/RASs were identified in 26.4% and 15.9% of patients with HCV-1a and -3a infections, respectively. The identified amino acid substitutions/RASs in the NS5A region of patients with HCV-1a infection were M28T/V/I 11.1%, Q30R/H 4.2%, L31M 1.4%, and H58Y/P/C/D/Q/S/T 16.7%. Y93H substitution was not found in HCV-1a sequences. In patients with HCV-3a infection, NS5A amino acid substitutions/RASs were A30T/K 9.5%, L31F 1.6%, P58S/T/C 3.2%, Y93H 3.2%, and Y93N 3.2%. No resistance substitutions were identified in NS5B sequences from patients with HCV-1a and -3a infections.ConclusionIn this study, baseline amino acid substitutions/RASs were only identified in the NS5A region in Iranian patients with HCV-1a and -3a infections, and the prevalence of these amino acid substitutions/RASs were in accordance with similar studies. There were no RASs in the HCV-1a and -3a NS5B region.

Jasminum multiflorum Burm. f. (J. multiflorum) is an ornamental plant with traditional medicinal importance. This study aims to evaluate the activity of J. multiflorum isolated compounds against hepatocellular carcinoma cells infected with hepatitis C virus (HCV) in vitro. The in vitro anti-viral and anti-oncogenic-related activity were validated by anchorage-independent assay plus transwell migration/invasion and spreading assay. In addition to chromatographic isolation of the active metabolites. The flower extract demonstrated a significant antiviral potential through reducing active viral replication by more than 90%. Study results credit this to specific reduction of viral NS5A and cellular EphA2 protein levels. Molecular docking analysis proved the role of the isolated compounds especially multifloroside, jasfloroside A and jasfloroside B as possible anti HCV molecules.

Classical swine fever virus (CSFV), an enveloped virus belonging to the genus Pestivirus of the Flaviviridae family, utilizes cell host factors for its own replication. ARFGAP1, GTPase activating protein of ADP-ribosylation factor 1, regulates COP I vesicle formation and function in cells and is involved in the life cycle of several viruses. However, the effect of ARFGAP1 on the infection of CSFV has not been illustrated. Here we showed that inhibition of ARFGAP1 either by QS11 or by lentivirus-mediated silencing repressed CSFV replication. While, subsequent experiments revealed that CSFV production were increased in cells with sufficient ARFGAP1 expression. However, ARFGAP1 was not involved in CSFV binding, entry, access to cell vesicles, and RNA replication during the early stages of infection. Then, we showed that ARFGAP1 interacted with the viral protein of NS5A, measured by immunoprecipitation, GST-pulldown, and confocal microscopy assays. Furthermore, we revealed that ARFGAP1 could alleviated CSFV NS5A-induced endoplasmic reticulum stress (ERS). Altogether, these results demonstrate that ARFGAP1, a NS5A binding protein, is involved in CSFV replication.

Made available in DSpace on 2021-06-26T02:36:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 MRC China Scholarship Council Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Hepatitis C virus (HCV) genotype 3 presents a high level of both baseline and acquired resistance to direct-acting antivirals (DAAs), particularly those targeting the NS5A protein. To understand this resistance we studied a cohort of Brazilian patients treated with the NS5A DAA, daclatasvir and the nucleoside analogue, sofosbuvir. We observed a novel substitution at NS5A amino acid residue 98 [serine to glycine (S98G)] in patients who relapsed post-treatment. The effect of this substitution on both replication fitness and resistance to DAAs was evaluated using two genotype 3 subgenomic replicons. S98G had a modest effect on replication, but in combination with the previously characterized resistance-associated substitution (RAS), Y93H, resulted in a significant increase in daclatasvir resistance. This result suggests that combinations of substitutions may drive a high level of DAA resistance and provide some clues to the mechanism of action of the NS5A-targeting DAAs. Sao Paulo State Univ, Inst Biosci Languages & Exact Sci, BR-15054000 Sao Jose Do Rio Preto, SP, Brazil Univ Leeds, Fac Biol Sci, Sch Mol & Cellular Biol, Leeds LS2 9JT, W Yorkshire, England Univ Leeds, Astbury Ctr Struct Mol Biol, Leeds LS2 9JT, W Yorkshire, England Univ Sao Paulo, Ribeirao Preto Sch Med, BR-14049900 Ribeirao Preto, SP, Brazil Univ Sao Paulo, Ribeirao Preto Fac Pharmaceut Sci, BR-14040903 Ribeirao Preto, SP, Brazil Sao Paulo State Univ, Inst Biosci Languages & Exact Sci, BR-15054000 Sao Jose Do Rio Preto, SP, Brazil MRC: MR/S001026/1 FAPESP: 2016/03807-0 FAPESP: 2018/04678-5