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Abstract Hepatitis C virus (HCV) presents a significant global health issue due to its widespread prevalence and the absence of a reliable vaccine for prevention. While significant progress has been achieved in therapeutic interventions since the disease was first identified, its resurgence underscores the need for innovative strategies to combat it. The nonstructural protein NS5A is crucial in the life cycle of the HCV, serving as a significant factor in both viral replication and assembly processes. This significance is highlighted by its inclusion in all existing approved HCV combination therapies. In this study, a quantitative structure–activity relationship (QSAR) was conducted to design new compounds with enhanced inhibitory activity against HCV. In this context, a set of 82 phenylthiazole derivatives was employed to construct a QSAR model using the Monte Carlo optimization technique. This model offers valuable insights into the specific structural characteristics that either enhance or reduce the inhibitory activity. These findings were used to design novel NS5A inhibitors. Moreover, molecular docking was used to predict the binding affinity of the newly designed inhibitors within the NS5A protein, followed by molecular dynamics simulations to investigate the dynamic interactions over time. Additionally, molecular mechanics generalized born surface area calculations were carried out to estimate the binding free energies of the inhibitor candidates, providing additional insights into their binding affinities and stabilities. Finally, the absorption, distribution, metabolism, excretion, and toxicity analysis were performed to assess the pharmacokinetic and toxicity profiles of the inhibitor candidates. This comprehensive approach provides a detailed understanding of the potential efficacy, stability, and safety of the screened drug candidates, offering valuable insights for their further development as potent therapeutic agents against HCV.

Hepatitis C virus (HCV) presents a significant global health issue due to its widespread prevalence and the absence of a reliable vaccine for prevention. While significant progress has been achieved in therapeutic interventions since the disease was first identified, its resurgence underscores the need for innovative strategies to combat it. The nonstructural protein NS5A is crucial in the life cycle of the HCV, serving as a significant factor in both viral replication and assembly processes. This significance is highlighted by its inclusion in all existing approved HCV combination therapies. In this study, a quantitative structure–activity relationship (QSAR) was conducted to design new compounds with enhanced inhibitory activity against HCV. In this context, a set of 82 phenylthiazole derivatives was employed to construct a QSAR model using the Monte Carlo optimization technique. This model offers valuable insights into the specific structural characteristics that either enhance or reduce the inhibitory activity. These findings were used to design novel NS5A inhibitors. Moreover, molecular docking was used to predict the binding affinity of the newly designed inhibitors within the NS5A protein, followed by molecular dynamics simulations to investigate the dynamic interactions over time. Additionally, molecular mechanics generalized born surface area calculations were carried out to estimate the binding free energies of the inhibitor candidates, providing additional insights into their binding affinities and stabilities. Finally, the absorption, distribution, metabolism, excretion, and toxicity analysis were performed to assess the pharmacokinetic and toxicity profiles of the inhibitor candidates. This comprehensive approach provides a detailed understanding of the potential efficacy, stability, and safety of the screened drug candidates, offering valuable insights for their further development as potent therapeutic agents against HCV. [ABSTRACT FROM AUTHOR]

Pakistan has the second highest hepatitis C virus (HCV) prevalence with seroprevalence of 4.5-8.2%, with genotype 3a being most frequently circulating HCV genotype. Current treatment strategies for HCV are based on direct acting antivirals (DAAs), despite high efficacy of antivirals, still therapy failure has been reported in 5-10% cases due to resistance mutations of amino acids. This study was aimed to analyze clinically important resistance associated mutations (RAMs) to daclatasvir (DCV) in HCV GT- 3a NS5A region in treatment naive and DAA treatment experienced patients, to understand their role in treatment failure. Patients samples and data was collected on prescribed questionnaire. Viral nucleic acid was isolated and amplified by gene specific primers followed by sequencing of NS5A region by Sanger method. Amino acid substitutions were identified by using Geno2Pheno tool. Hepatitis c virus genotype 3a was most prevalent genotype in the study group. Successfully sequenced patients were divided into two group based on their treatment history, as treatment naive and experienced groups. Both groups were analyzed for detection of amino acid mutations at positions 28, 30, 31, 58 and 93. A30T was detected in 7.6%, Y93H in 15.6% P58T as 7.6% while S98G was found in 23% treated patients. However, these mutations were not detected in any of treatment naive patients. Some mutations were identified in both treatment naive and experienced groups i.e., A21T, T64A, H85Y, S103P, D172E, H180N, T183A/V, and E137G. Mutations at position 62 was most commonly detected found at the frequency of 83.8% and 84.6% in treatment naive and experienced patients respectively. While mutations at position M28 and L31 were not identified in both groups of current study. Mutations present in both treatment naive as well as in treatment experienced groups suggesting they have no role in DCV resistance, while identification of A30T, Y93H, P58T and S98G in treated patients suggests that DCV RAMs are circulating in Pakistani HCV GT-3a DAAs treated patients, leading to resistance development and treatment failure. Identification of these mutations is important especially in treatment failure patients to design re-treatment strategies. [ABSTRACT FROM AUTHOR]

To research the molecular genetic characteristics of the HCV population circulating among HIV‐infected residents of the Krasnoyarsk Territory, including analysis of resistance‐associated mutations to NS5A inhibitors. Total RNA was isolated from 94 blood plasma samples from HIV/HCVinfected residents of the region and the nucleotide sequences of the Core/E1 gene and NS5A gene regions were obtained and deciphered, which were used for genotyping and searching for resistance mutations.In the HCV samples studied, the circulation of five virus subtypes was recorded: 1b (47,9 %), 3a (37,2 %), 1a (10,6 %), 2a (3,2 %) and 2k (1,1 %). Phylogenetic analysis of HCVs revealed partial clustering within subtypes 1a and 3a on a territorial basis. HCV subtypes 1b, 2a and 2k were grouped with other HCVs previously isolated in Russia, Armenia and Kyrgyzstan. Among the HCVs studied, no clusters were found that were common to HIV‐infected individuals with the same route of infection. Among patients who had no experience of taking DAAs, HCV resistance mutations in the NS5A region were found in 42,3 % of cases for those infected with subtype 3a virus and in 75,6 % of cases for those infected with HCV subtype 1b.The analysis of the diversity and occurrence of mutations of HCV drug resistance to direct antiviral drugs is extremely important for the development of tactics for effective treatment of CHC. The high prevalence of polymorphic mutations that affect sensitivity to DAAs indicates the relevance of introducing HCV resistance analysis into clinical practice.

Hepatitis C virus (HCV) is the most common infection worldwide. The correlation between HCV and renal cell carcinoma (RCC) is still mysterious. Therefore, the relationship between HCV and RCC was investigated. The study included 100 patients with RCC; 32 with HCV infection, and 68 without HCV infection. Expressions of viral proteins (NS3 and NS5A) were tested using an immune electron‐microscope (IEM) and immunohistochemistry (IHC). IHC and quantitative real time‐PCR investigated the presentation of human proteins TP53 and p21 genes. Transmission electron (TEM) detected viral‐like particles in infected RCC tissues. The gene and protein expression of P53 was higher in HCV positive versus HCV negative patients and p21 was lower in HCV positive versus HCV negative in both tumor and normal tissue samples. Viral like particles were observed by TEM in the infected tumor and normal portion of the RCC tissues and the plasma samples. The IEM showed the depositions of NS3 and NS5A in infected renal tissues, while in noninfected samples, were not observed. The study hypothesizes that a correlation between HCV and RCC could exist through successfully detecting HCV‐like particles, HCV proteins, and (p53 and p21) in RCC‐infected patients. [ABSTRACT FROM AUTHOR]

Approximately 71 million people worldwide are supposed to have chronic hepatitis C virus (CHCV). New direct-acting antiviral (DAA) medications have been used, which helped successfully in complete treatment of CHCV and achieved a sustained virological response (SVR). However, some patients may acquire HCV resistance to DAAs, which may result in treatments failure. The aim of the present study was to compare among the patients that were experiencing virologic relapse (VR) and SVR in relation to the patterns of NS5A and NS5B genes resistance associated substitutions (RASs) in the chronic HCV infected Egyptian patients, who received Sofosbuvir (SOF) and Daclatasvir (DCV) combination therapy. All patients that were infected by chronic HCV had completed treatment with SOF and DCV combination therapy and were followed up after the end of this treatment. A total of 10 out of 100 serum specimens were collected from the enrolled patients and analyzed, where two and eight specimens were representatives for VR and SVR, respectively. These samples had undergone reverse transcriptase-polymerase chain reaction amplification (RT-PCR) of NS5A and NS5B genes, partially sequenced by the Sanger method, and then analyzed phylogenetically to determine their genetic subtypes and RASs. Finally, SVR was gained in all but two patients who were experiencing VR that carried natural NS5A-RASs at positions L31M and Y93H. They were considered as significant for DCV resistance as well as natural NS5B-RASs (T282S), which represented the main polymorphism for SOF resistance. In this study, a number of mutational combinations in the analyzed NS5A and NS5B genes were identified, which may increase the risk of treatments failure in the patients administered regimens including multiple DAA, compared to the baseline sequences of those patients that were experiencing SVR.

HCV NS5A is a dimeric phosphoprotein involved in HCV replication. NS5A inhibitors are among direct‐acting antivirals (DAA) for HCV therapy. The Y93H mutant of NS5A is resistant to NS5A inhibitors, but the precise mechanism remains unclear. In this report, we proposed a Ser38‐His93‐Asn91 triad to dissect the mechanism. Using pymol 1.3 software, the homology structure of JFH1 NS5A was determined based on the dimer structure of genotype 1b extracted from the database Protein DataBank (www.ebi.ac.uk/pdbsum) with codes 1ZH1 and 3FQM/3FQQ. FLAG‐NS5A‐WT failed to form dimer in the absence of nonstructural proteins from subgenomic replicon (NS3‐5A); however, FLAG‐NS5A‐Y93H was able to form dimer without the aid of NS3‐5A. The Ser38‐His93‐Asn91 triad in the dimer of the Y93H variant predicts a structural crash of the cleft receiving the NS5A inhibitor daclatasvir. The dimerization assay revealed that the existence of JFH1‐NS5A‐1ZH1 and ‐3FQM homology dimers depended on each other for existence and that both NS5A‐WT 1ZH1 and 3FQM dimers cooperated to facilitate RNA replication. However, NS5A‐Y93H 1ZH1 alone could form dimer and conduct RNA replication in the absence of the 3FQM structure. In conclusion, this study provides novel insight into the functional significance of the Ser38‐His93‐Asn91 triad in resistance of the Y93H variant to NS5A inhibitors. [ABSTRACT FROM AUTHOR]

Classical swine fever (CSF) has been eradicated from Western and Central Europe but remains endemic in parts of Central and South America, Asia, and the Caribbean. CSF virus (CSFV) has been endemic in Cuba since 1993, most likely following an escape of the highly virulent Margarita/1958 strain. In recent years, chronic and persistent infections with low-virulent CSFV have been observed. Amino acid substitutions located in immunodominant epitopes of the envelope glycoprotein E2 of the attenuated isolates were attributed to positive selection due to suboptimal vaccination and control. To obtain a complete picture of the mutations involved in attenuation, we applied forward and reverse genetics using the evolutionary-related low-virulent CSFV/Pinar del Rio (CSF1058)/2010 (PdR) and highly virulent Margarita/1958 isolates. Sequence comparison of the two viruses recovered from experimental infections in pigs revealed 40 amino acid differences. Interestingly, the amino acid substitutions clustered in E2 and the NS5A and NS5B proteins. A long poly-uridine sequence was identified previously in the 3′ untranslated region (UTR) of PdR. We constructed functional cDNA clones of the PdR and Margarita strains and generated eight recombinant viruses by introducing single or multiple gene fragments from Margarita into the PdR backbone. All chimeric viruses had comparable replication characteristics in porcine monocyte-derived macrophages. Recombinant PdR viruses carrying either E2 or NS5A/NS5B of Margarita, with 36 or 5 uridines in the 3′UTR, remained low virulent in 3-month-old pigs. The combination of these elements recovered the high-virulent Margarita phenotype. These results show that CSFV evolution towards attenuated variants in the field involved mutations in both structural and non-structural proteins and the UTRs, which act synergistically to determine virulence. [ABSTRACT FROM AUTHOR]

Classical swine fever virus nonstructural protein NS5A is essential for viral genome replication, protein translation, virus assembly, and autophagy. In the present paper, the interaction of cellular PPP2R1A, PP2Ac, and DAPK3 with NS5A has been confirmed. These interactions mediate the dissociation of PP2A from Beclin 1 and its association with DAPK3. Downregulation of DAPK3 or PPP2R1A inhibits CSFV replication. NS5A induced phosphorylation of Ser88 (PK-15 cells) or Ser90 (HEK293T cells) on Beclin 1. Knockdown of Beclin 1 inhibited NS5A-induced autophagy, indicating that NS5A induces Beclin 1-dependent autophagy. Downregulation of DAPK3, PPP2R1A, or PP2Ac by siRNA reduced Beclin 1 phosphorylation and autophagy mediated by NS5A, showing a critical role of PP2A and DAPK3 in Beclin 1 phosphorylation and autophagy triggered by NS5A. In vitro analysis of Beclin 1 phosphorylation revealed PP2A as being essential for DAPK3-mediated phosphorylation of Beclin 1, indicating that PP2A may dephosphorylate DAPK3 to activate its protein kinase activity, with activated DAPK3 phosphorylated Beclin 1, and then triggering autophagy. These data show for the first time that DAPK3 can be activated through dephosphorylation by PP2A. These findings reveal a novel mechanism whereby CSFV NS5A protein activates autophagy via PP2A-DAPK3-Beclin 1 axis to favor viral replication. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is a hepatotropic virus that affects millions of human lives worldwide. Direct-acting antiviral (DAA) regimens are the most effective HCV treatment option. However, amino acid substitution-dependent resistance to DAAs has been a major challenge. This study aimed to determine the increasing risk of DAA resistance due to substitutions in DAA target non-structural proteins (NS3/4A, NS5A, and NS5B). Using a Sequence Retrieval System (SRS) at the virus pathogen resource (ViPR/BV-BRC), n = 32763 target protein sequences were retrieved and analyzed for resistance-associated amino acid substitutions (RAASs) by the Sequence Feature Variant Type (SFVT) antiviral-resistance assessment modeling tool. Reference target protein sequences with 100% identity were retried from UniProt following NCBI BLAST. The types and locations of RAASs were identified and visualized by AlphaFold and PyMol. Linux-r-base/R-studio was used for the data presentation. Multi-drug-resistant variants of NS3/4A in genotype 1 (n = 9) and genotype 5 (n = 5) along with DAA-specific NS3/4A, NS5A, and NS5B variants were identified pan-genotypically. A total of 27 variants (RAASs) of all the targets were identified. Fourteen genotype 1-specific substitutions: V1196A, V1158I, D1194A/T/G, R1181K, T1080S, Q1106R, V1062A, S1148G, A1182V, Y2065N, M2000T, and L2003V were identified. The most frequent substitutions were V1062L and L2003M, followed by Q2002H. L2003V, Q2002H, M2000T, Y2065N, and NL2003M of NS5A and L2003M of NS5B conferred resistance to daclatasvir. S2702T NS5B was the sofosbuvir-resistant variant. D1194A NS3/4A was triple DAA (simeprevir, faldaprevir, and asunaprevir) resistant. The double-drug resistant variants R1181K (faldaprevir and asunaprevir), A1182V and Q1106K/R (faldaprevir and simeprevir), T1080S (faldaprevir and telaprevir), and single drug-resistant variants V1062L (telaprevir), D1194E/T (simeprevir), D1194G (asunaprevir), S1148A/G (simeprevir), and Q1106L (Boceprevir) of NS3/4A were determined. The molecular phenomenon of DAA resistance is paramount in the development of HCV drug candidates. RAASs in NS3, NS5A, and NS5B reduce the susceptibility to DAAs; therefore, continuous RAAS-dependent resistance profiling in HCV is recommended to minimize the probability of DAA therapeutic failure. [ABSTRACT FROM AUTHOR]

Drug discovery is a difficult task, and is even more challenging when the target evolves during therapy. Antiviral drug therapy is an excellent example, exemplified by the evolution of therapeutic approaches for treatment of hepatitis C and HIV-1. Nick Meanwell and his colleagues made important contributions leading to molecules for treatment of hepatitis C and HIV-1, each with distinct mechanisms of action. This review summarizes the discovery and impact of these drugs, and will highlight, where applicable, the broader contributions of these discoveries to medicinal chemistry and drug discovery. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductases (RRs or RNRs) catalyze the reduction of the OH group on the 2nd carbon of ribose, reducing four ribonucleotides (NTPs) to the corresponding deoxyribonucleotides (dNTPs) to promote DNA synthesis. Large DNA viruses, such as herpesviruses and poxviruses, could benefit their replication through increasing dNTPs via expression of viral RRs. Little is known regarding the relationship between cellular RRs and RNA viruses. Mammalian RRs contain two subunits of ribonucleotide reductase M1 polypeptide (RRM1) and two subunits of ribonucleotide reductase M2 polypeptide (RRM2). In this study, expression of cellular RRMs, including RRM1 and RRM2, is found to be down-regulated in hepatitis C virus (HCV)-infected Huh7.5 cells and Huh7 cells with HCV subgenomic RNAs (HCVr). As expected, the NTP/dNTP ratio is elevated in HCVr cells. Compared with that of the control Huh7 cells with sh-scramble, the NTP/dNTP ratio of the RRM-knockdown cells is elevated. Knockdown of RRM1 or RRM2 increases HCV replication in HCV replicon cells. Moreover, inhibitors to RRMs, including Didox, Trimidox and hydroxyurea, enhance HCV replication. Among various HCV viral proteins, the NS5A and/or NS3/4A proteins suppress the expression of RRMs. When these are taken together, the results suggest that HCV down-regulates the expression of RRMs in cultured cells to promote its replication. [ABSTRACT FROM AUTHOR]

Many viruses often use a protein degradation system (e.g., the ubiquitin-proteasome pathway or lysosome pathway) to modulate viral propagation and viral pathogenesis. We reported that hepatitis C virus (HCV) infection promotes the lysosomal degradation of hepatocyte nuclear factor-1α (HNF-1α) via chaperone-mediated autophagy (CMA) through an NS5A-mediated association of HNF-1α with cellular chaperone heat shock cognate 70 kDa (HSC70) protein. HSC70 binds to the pentapeptide KFERQ motif (also known as a CMA-targeting motif) on HNF-1α protein and promotes the lysosomal degradation of HNF-1α. The KFERQ motif plays a crucial role in the two lysosomal degradation pathways, CMA and endosomal microautophagy (eMI). Herein, we searched for a novel substrate of HCV-induced lysosomal degradation by examining the NS5A-interacting proteins that carry the KFERQ motif. We identified diacylglycerol O-acyltransferase 1 (DGAT1), which is a key factor for HCV particle formation, as a candidate substrate for HCV-induced lysosomal degradation pathway. The region spanning from amino acids 149–153 of DGAT1 protein matches the rule for the KFERQ motif. DGAT1 protein was co-immunoprecipitated with HSC70, whereas DGAT1 Q149A mutant was not co-immunoprecipitated with HSC70, suggesting that the KFERQ motif is responsible for the interaction between DGAT1 and HSC70. Knockdown of LAMP-2A protein in HCV J6/JFH1-infected cells did not recover DGAT1 protein, whereas knockdown of VPS4B recovered the level of DGAT1 protein, suggesting that DGAT1 is degraded via eMI. These findings lead us to propose that HCV NS5A protein facilitates the recruitment of HSC70 to DGAT1, thereby promoting the lysosomal degradation of DGAT1 via eMI. Abbreviations 3-MA: 3-methyladenine; aa: amino acids; AH: amphipathic helix; BSA: bovine serum albumin; CMA: chaperone-mediated autophagy; DAAs: direct-acting antiviral; DGAT1: diacylglycerol O-acyltransferase 1; DMSO: dimethyl sulfoxide; EL: extracellular lumen; eMI: endosomal microautophagy; ESCRT: endosomal sorting complex required for transport; HA: hemagglutinin; HCV: hepatitis C virus; HNF-1α: hepatocyte nuclear factor-1α; HRP: horseradish peroxidase; HSC70: heat shock cognate 70 kDa protein; IB: immunoblotting; IL: intracellular lumen; IP: immunoprecipitation; LAMP-2A: lysosome-associated membrane protein type 2A; LCS: low-complexity sequences; mAb: monoclonal antibody; MOI: multiplicity of infection; MVB: multivesicular bodies; NS: nonstructural protein; pAb: polyclonal antibody; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PLA: proximity ligation assay; PS: phosphatidylserine; RT: room temperature; TM: transmembrane; TSG: tumor susceptibility gene; VPS4A: vacuolar protein sorting-associated protein 4A; VPS4B: vacuolar protein sorting-associated protein 4B

Hepatitis C virus (HCV) is associated with liver damage and an increased progression rate to cirrhosis and hepatocellular carcinoma. In Portugal, it is prevalent in vulnerable populations such as injection drug users (IDU). HCV is characterized by a high intra-host variability, and the selecting driving forces could select variants containing resistance-associated substitutions (RAS) that reduce treatment effectiveness. The main goal of this study was to analyze the sequence variation of NS5A in treatment-naïve IDU. The epidemiological and clinical status of hepatitis C were analyzed, and samples were sequenced by Sanger and Next-Generation sequencing (NGS) to assess RAS and confirm HCV subtypes. Phylogenetic classification was concordant: 1a, 52.4%; 1b, 10.7%; 3a, 20.2%; 4a, 8.3%; 4d, 7.1%; and one 2k/1b recombinant. A 1a/3a mixed infection was detected by NGS. RAS were found in 34.5% (29/84) of samples using Sanger sequencing, while in 42.9% (36/84) using NGS. In sequences from subtypes 1a and 1b, RAS K24R, M28V, Q30H/R, H58D/P/Q/R, and RAS L31M and P58S were detected, respectively. In subtype 3a, RAS A30S/T, Y93H and polymorphisms in position 62 were identified. Additionally, RAS P58L was detected in genotype 4. The strategy used for the molecular survey of baseline HCV resistance is of particular importance to achieve treatment effectiveness and contribute to the elimination of hepatitis C. [ABSTRACT FROM AUTHOR]

Vimentin (VIM), an indispensable protein, is responsible for the formation of intermediate filament structures within cells and plays a crucial role in viral infections. However, the precise role of VIM in classical swine fever virus (CSFV) infection remains unclear. Herein, we systematically investigated the function of VIM in CSFV replication. We demonstrated that both knockdown and overexpression of VIM affected CSFV replication. Furthermore, we observed by confocal microscopy the rearrangement of cellular VIM into a cage-like structure during CSFV infection. Threedimensional (3D) imaging indicated that the cage-like structures were localized in the endoplasmic reticulum (ER) and ringed around the double-stranded RNA (dsRNA), thereby suggesting that VIM was associated with the formation of the viral replication complex (VRC). Mechanistically, phosphorylation of VIM at serine 72 (Ser72), regulated by the RhoA/ROCK signaling pathway, induced VIM rearrangement upon CSFV infection. Confocal microscopy and coimmunoprecipitation assays revealed that VIM colocalized and interacted with CSFV NS5A. Structurally, it was determined that amino acids 96 to 407 of VIM and amino acids 251 to 416 of NS5A were the respective important domains for this interaction. Importantly, both VIM knockdown and disruption of VIM rearrangement inhibited the localization of NS5A in the ER, implying that VIM rearrangement recruited NS5A to the ER for VRC formation. Collectively, our results suggest that VIM recruits NS5A to form a stable VRC that is protected by the cage-like structure formed by VIM rearrangement, ultimately leading to enhanced virus replication. These findings highlight the critical role of VIM in the formation and stabilization of VRC, which provides alternative strategies for the development of antiviral drugs. IMPORTANCE Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly infectious disease that poses a significant threat to the global pig industry. Therefore, gaining insights into the virus and its interaction with host cells is crucial for developing effective antiviral measures and controlling the spread of CSF. Previous studies have shown that CSFV infection induces rearrangement of the endoplasmic reticulum, leading to the formation of small vesicular organelles containing nonstructural protein and double-stranded RNA of CSFV, as well as some host factors. These organelles then assemble into viral replication complexes (VRCs). In this study, we have discovered that VIM recruited CSFV NS5A to form a stable VRC that was protected by a cage-like structure formed by rearranged VIM. This enhanced viral replication. Our findings not only shed light on the molecular mechanism of CSFV replication but also offer new insights into the development of antiviral strategies for controlling CSFV. [ABSTRACT FROM AUTHOR]

Several direct-acting antivirals (DAAs) are available, providing interferon-free strategies for a hepatitis C cure. In contrast to DAAs, host-targeting agents (HTAs) interfere with host cellular factors that are essential in the viral replication cycle; as host genes, they are less likely to rapidly mutate under drug pressure, thus potentially exhibiting a high barrier to resistance, in addition to distinct mechanisms of action. We compared the effects of cyclosporin A (CsA), a HTA that targets cyclophilin A (CypA), to DAAs, including inhibitors of nonstructural protein 5A (NS5A), NS3/4A, and NS5B, in Huh7.5.1 cells. Our data show that CsA suppressed HCV infection as rapidly as the fastest-acting DAAs. CsA and inhibitors of NS5A and NS3/4A, but not of NS5B, suppressed the production and release of infectious HCV particles. Intriguingly, while CsA rapidly suppressed infectious extracellular virus levels, it had no significant effect on the intracellular infectious virus, suggesting that, unlike the DAAs tested here, it may block a post-assembly step in the viral replication cycle. Hence, our findings shed light on the biological processes involved in HCV replication and the role of CypA. [ABSTRACT FROM AUTHOR]

Introduction and Objectives: In clinical trials, patients with hepatitis C virus (HCV) genotype (GT)1a infection and baseline resistance-associated substitutions (RASs) at amino acid positions 28, 30, 31, or 93 receiving elbasvir/grazoprevir for 12 weeks achieved lower rates of sustained virologic response (SVR) than those without baseline RASs. SVR rates in patients with RASs were improved when elbasvir/grazoprevir treatment duration was extended from 12 to 16 weeks and administered concomitantly with ribavirin. Materials and Methods: This was a retrospective, observational analysis using electronic health record abstraction. Patients with HCV GT1a infection and RASs at positions 28, 30, 31, or 93 who were prescribed 16 weeks of elbasvir/grazoprevir and ≥ 1 prescription for ribavirin were included. SVR was defined as HCV RNA below the lower limit of quantification ≥ 70 days after end of treatment. Results: The primary analysis included patients with baseline RASs at positions 30, 31, or 93 (n = 76); a secondary analysis included patients with RASs at positions 28, 30, 31, or 93 (n = 93). SVR was achieved by 77.6% (59/76) of patients in the primary analysis and 80.6% (75/93) of those in the secondary analysis. Of the 18 (19.4%) patients in the secondary cohort who failed to achieve SVR, 8 relapsed (4 with treatment-emergent NS5A substitutions) and 10 did not have viral sequencing to distinguish relapse from reinfection. Conclusions: This analysis highlights the opportunities in leveraging real-world data to further understand treatment outcomes in smaller, discrete subgroups of patients with HCV infection who cannot be thoroughly evaluated in clinical trials. [ABSTRACT FROM AUTHOR]

Introduction and Objectives: In clinical trials, patients with hepatitis C virus (HCV) genotype (GT)1a infection and baseline resistance-associated substitutions (RASs) at amino acid positions 28, 30, 31, or 93 receiving elbasvir/grazoprevir for 12 weeks achieved lower rates of sustained virologic response (SVR) than those without baseline RASs. SVR rates in patients with RASs were improved when elbasvir/grazoprevir treatment duration was extended from 12 to 16 weeks and administered concomitantly with ribavirin. Materials and Methods: This was a retrospective, observational analysis using electronic health record abstraction. Patients with HCV GT1a infection and RASs at positions 28, 30, 31, or 93 who were prescribed 16 weeks of elbasvir/grazoprevir and ≥ 1 prescription for ribavirin were included. SVR was defined as HCV RNA below the lower limit of quantification ≥ 70 days after end of treatment. Results: The primary analysis included patients with baseline RASs at positions 30, 31, or 93 (n = 76); a secondary analysis included patients with RASs at positions 28, 30, 31, or 93 (n = 93). SVR was achieved by 77.6% (59/76) of patients in the primary analysis and 80.6% (75/93) of those in the secondary analysis. Of the 18 (19.4%) patients in the secondary cohort who failed to achieve SVR, 8 relapsed (4 with treatment-emergent NS5A substitutions) and 10 did not have viral sequencing to distinguish relapse from reinfection. Conclusions: This analysis highlights the opportunities in leveraging real-world data to further understand treatment outcomes in smaller, discrete subgroups of patients with HCV infection who cannot be thoroughly evaluated in clinical trials.

Classical swine fever (CSF), caused by the classical swine fever virus (CSFV), is a highly contagious and fatal viral disease, posing a significant threat to the swine industry. Heat shock protein 90 kDa alpha class A member 1 (HSP90AA1) is a very conservative chaperone protein that plays an important role in signal transduction and viral proliferation. However, the role of HSP90AA1 in CSFV infection is unknown. In this study, we found that expression of HSP90AA1 could be promoted in PK-15 and 3D4/2 cells infected by CSFV. Overexpression of HSP90AA1 could inhibit CSFV replication and functional silencing of HSP90AA1 gene promotes CSFV replication. Further exploration revealed that HSP90AA1 interacted with CSFV NS5A protein and reduced the protein levels of NS5A. Since NS5A has an important role in CSFV replication and is closely related to type I IFN and NF-kB response, we further analyzed whether HSP90AA1 affects CSFV replication by regulating type I IFN and NF-kB pathway responses. Our research found HSP90AA1 positively regulated type I IFN response by promoting STAT1 phosphorylation and nuclear translocation processes and promoted the nuclear translocation processes of p-P65. However, CSFV infection antagonizes the activation of HSP90AA1 on JAK/STAT and NF-kB pathway. In conclusion, our study found that HSP90AA1 overexpression significantly inhibited CSFV replication and may inhibit CSFV replication by interacting with NS5A and activating JAK/STAT and NF-kB signaling pathways. These results provide new insights into the mechanism of action of HSP90AA1 in CSFV infection, which abundant the candidate library of anti-CSFV. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is a serious disease that threatens human health. Despite consistent efforts to inhibit the virus, it has infected more than 58 million people, with 300,000 deaths per year. The HCV nonstructural protein NS5A plays a critical role in the viral life cycle, as it is a major contributor to the viral replication and assembly processes. Therefore, its importance is evident in all currently approved HCV combination treatments. The present study identifies new potential compounds for possible medical use against HCV using the quantitative structure–activity relationship (QSAR). In this context, a set of 36 NS5A inhibitors was used to build QSAR models using genetic algorithm multiple linear regression (GA-MLR) and Monte Carlo optimization and were implemented in the software CORAL. The Monte Carlo method was used to build QSAR models using SMILES-based optimal descriptors. Four splits were performed and 24 QSAR models were developed and verified through internal and external validation. The model created for split 3 produced a higher value of the determination coefficients using the validation set (R2 = 0.991 and Q2 = 0.943). In addition, this model provides interesting information about the structural features responsible for the increase and decrease of inhibitory activity, which were used to develop eight novel NS5A inhibitors. The constructed GA-MLR model with satisfactory statistical parameters (R2 = 0.915 and Q2 = 0.941) confirmed the predicted inhibitory activity for these compounds. The Absorption, Distribution, Metabolism, Elimination, and Toxicity (ADMET) predictions showed that the newly designed compounds were nontoxic and exhibited acceptable pharmacological properties. These results could accelerate the process of discovering new drugs against HCV. [ABSTRACT FROM AUTHOR]

Background & Aims: Direct-acting antiviral (DAA) regimens provide a cure in >95% of patients with chronic HCV infection. However, in some patients in whom therapy fails, resistance-associated substitutions (RASs) can develop, limiting retreatment options and risking onward resistant virus transmission. In this study, we evaluated RAS prevalence and distribution, including novel NS5A RASs and clinical factors associated with RAS selection, among patients who experienced DAA treatment failure. Methods: SHARED is an international consortium of clinicians and scientists studying HCV drug resistance. HCV sequence linked metadata from 3,355 patients were collected from 22 countries. NS3, NS5A, and NS5B RASs in virologic failures, including novel NS5A substitutions, were examined. Associations of clinical and demographic characteristics with RAS selection were investigated. Results: The frequency of RASs increased from its natural prevalence following DAA exposure: 37% to 60% in NS3, 29% to 80% in NS5A, 15% to 22% in NS5B for sofosbuvir, and 24% to 37% in NS5B for dasabuvir. Among 730 virologic failures, most were treated with first-generation DAAs, 94% had drug resistance in ≥1 DAA class: 31% single-class resistance, 42% dual-class resistance (predominantly against protease and NS5A inhibitors), and 21% triple-class resistance. Distinct patterns containing ≥2 highly resistant RASs were common. New potential NS5A RASs and adaptive changes were identified in genotypes 1a, 3, and 4. Following DAA failure, RAS selection was more frequent in older people with cirrhosis and those infected with genotypes 1b and 4. Conclusions: Drug resistance in HCV is frequent after DAA treatment failure. Previously unrecognized substitutions continue to emerge and remain uncharacterized. Lay summary: Although direct-acting antiviral medications effectively cure hepatitis C in most patients, sometimes treatment selects for resistant viruses, causing antiviral drugs to be either ineffective or only partially effective. Multidrug resistance is common in patients for whom DAA treatment fails. Older patients and patients with advanced liver diseases are more likely to select drug-resistant viruses. Collective efforts from international communities and governments are needed to develop an optimal approach to managing drug resistance and preventing the transmission of resistant viruses.

Hepatitis C virus (HCV) is associated with liver damage and an increased progression rate to cirrhosis and hepatocellular carcinoma. In Portugal, it is prevalent in vulnerable populations such as injection drug users (IDU). HCV is characterized by a high intra-host variability, and the selecting driving forces could select variants containing resistance-associated substitutions (RAS) that reduce treatment effectiveness. The main goal of this study was to analyze the sequence variation of NS5A in treatment-naïve IDU. The epidemiological and clinical status of hepatitis C were analyzed, and samples were sequenced by Sanger and Next-Generation sequencing (NGS) to assess RAS and confirm HCV subtypes. Phylogenetic classification was concordant: 1a, 52.4%; 1b, 10.7%; 3a, 20.2%; 4a, 8.3%; 4d, 7.1%; and one 2k/1b recombinant. A 1a/3a mixed infection was detected by NGS. RAS were found in 34.5% (29/84) of samples using Sanger sequencing, while in 42.9% (36/84) using NGS. In sequences from subtypes 1a and 1b, RAS K24R, M28V, Q30H/R, H58D/P/Q/R, and RAS L31M and P58S were detected, respectively. In subtype 3a, RAS A30S/T, Y93H and polymorphisms in position 62 were identified. Additionally, RAS P58L was detected in genotype 4. The strategy used for the molecular survey of baseline HCV resistance is of particular importance to achieve treatment effectiveness and contribute to the elimination of hepatitis C.

The human myxovirus resistance B (MxB) protein is an interferon-induced restriction factor that fights a wide range of viruses. We previously demonstrated that MxB binds to hepatitis C virus (HCV)-encoded non-structural protein 5A (NS5A) and inhibits HCV infection by impairing the formation of cyclophilin A (CypA)–NS5A complex. However, the molecular details about how the presence of MxB diminishes the binding of NS5A to CypA remain uncovered. In this study, through molecular dynamic simulations and biochemical assays, we characterized that MxB binds to NS5A domain I through its N-terminal and GTPase domains. Specifically, amino acids (aa.) 189–191 and aa. 330–334 within MxB, together with NS5A residues aa. 71–73, are crucial for MxB–NS5A interaction. Furthermore, we predicted the CypA:NS5A and CypA:NS5A:MxB complexes and calculated the per-residue energy decomposition for identified key residues of the CypA–NS5A interface. A 28% decrease in CypA–NS5A binding affinity was observed in the presence of MxB, suggesting a weakened CypA–NS5A association upon binding of MxB to NS5A, which may contribute to the MxB-mediated inhibitory effect on the formation of CypA–NS5A complex. This work provides information for the antiviral mechanism of MxB and may facilitate the discovery of new strategies to combat CypA-dependent viruses.

The human myxovirus resistance B (MxB) protein is an interferon-induced restriction factor that fights a wide range of viruses. We previously demonstrated that MxB binds to hepatitis C virus (HCV)-encoded non-structural protein 5A (NS5A) and inhibits HCV infection by impairing the formation of cyclophilin A (CypA)–NS5A complex. However, the molecular details about how the presence of MxB diminishes the binding of NS5A to CypA remain uncovered. In this study, through molecular dynamic simulations and biochemical assays, we characterized that MxB binds to NS5A domain I through its N-terminal and GTPase domains. Specifically, amino acids (aa.) 189–191 and aa. 330–334 within MxB, together with NS5A residues aa. 71–73, are crucial for MxB–NS5A interaction. Furthermore, we predicted the CypA:NS5A and CypA:NS5A:MxB complexes and calculated the per-residue energy decomposition for identified key residues of the CypA–NS5A interface. A 28% decrease in CypA–NS5A binding affinity was observed in the presence of MxB, suggesting a weakened CypA–NS5A association upon binding of MxB to NS5A, which may contribute to the MxB-mediated inhibitory effect on the formation of CypA–NS5A complex. This work provides information for the antiviral mechanism of MxB and may facilitate the discovery of new strategies to combat CypA-dependent viruses. [ABSTRACT FROM AUTHOR]

In Guangdong Province, hepatitis C virus (HCV) had been found to confer resistance to direct-acting antivirals (DAAs). There were few studies of HCV subtypes and resistance-associated substitutions (RASs) of HCV in different high-risk populations. In this study, we aimed to determine the subtype distribution and the RASs in high-risk population groups, including drug users (DU), men who have sex with men (MSM), female sex workers (FSW), and male patients with sexually transmitted diseases (STD) in Guangdong Province (a highly developed province with a large population). Using a city-based sampling strategy,1356 samples were obtained from different population groups. Phylogenetic analyses determined subtypes based on Core, NS5B, or NS5A sequences. HCV subtype distribution and RASs in various risk groups and regions were analyzed. Ten subtypes, of which 6 h and 6 k were novel in Guangdong, were identified. The primary subtype among all risk groups was 6a. RASs in 1b and 3a were different from those observed in other studie s. Subtype 3b in western Guangdong was higher than the other three regions. No RASs were found in 6a or any other genotype 6. The HCV subtypes are expanding in high-risk populations in Guangdong. Drug use by other risk groups and commercial sex by DU may bridge the dissemination of 6a from DU to other populations. The RAS profiles of 1b and 3a differed from those reported in studies conducted in southwestern China. Further research is required to determine the reason for this discrepancy. Moreover, the combination of RASs was high in subtype 3b. To guide HCV treatment of subtype 3b, pretreatment subtyping of HCV genotype 3 should be considered in western cities in the near future. • Ten HCV subtypes were detected among high-risk population groups in Guangdong, China. • Commercial sex and drug use may bridge the epidemic of 6a. • RAS profiles of 1b and 3a in Guangdong differed from those in southwestern China. • RAS combination of A30K + L31M was high among study participants with 3b. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) infects millions of people worldwide and is a leading cause of liver disease. Despite recent advances in antiviral therapies, viral resistance can limit drug efficacy and understanding the mechanisms that confer viral escape is important. We employ an unbiased interactome analysis to discover host binding partners of the HCV non-structural protein 5A (NS5A), a key player in viral replication and assembly. We identify ASPP2, apoptosis-stimulating protein of p53, as a new host co-factor that binds NS5A via its SH3 domain. Importantly, silencing ASPP2 reduces viral replication and spread. Our study uncovers a previously unknown role for ASPP2 to potentiate HCV RNA replication.

• The cellular mitophagy induced by CSFV NS5A protein. • CSFV NS5A induce mitophagy correlate with ROS production. • Mitophagy promote CSFV replication. The classical swine fever virus (CSFV) is one of the most harmful pathogens of swine and causes considerable economic loss. Mitophagy is a selective form of autophagy that degrades damaged mitochondria by combining with lysosomes. Previous studies have been reported that CSFV infection can induce mitophagy, but which effector protein is responsible for this process remains unclear. Herein, we revealed here that the CSFV nonstructural protein 5A (NS5A) plays a critical role in inducing cellular mitophagy. Specifically, the expression of CSFV NS5A in the PK-15 cells induces membrane potential loss and mitochondrial fission, and the quantities of mitophagosomes, the expression of Parkin and PINK1 were significantly increased compared with mock cells. Intriguingly, we found that Parkin-overexpression promotes CSFV propagation. Furthermore, the expression level of reactive oxygen species (ROS) was increased by CSFV NS5A protein, while NS5A-induced mitophagy correlated with the quantity of ROS production. In summary, our results reveal a new function of NS5A in inducing cellular mitophagy and broaden our understanding of the mechanism of CSFV-induced mitophagy, which may provide a new way to develop an antiviral strategy. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) infection can lead to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV employs diverse strategies to evade host antiviral innate immune responses to mediate a persistent infection. In the present study, we show that nonstructural protein 5A (NS5A) interacts with an NF-κB inhibitor immunomodulatory kinase, IKKε, and subsequently downregulats beta interferon (IFN-β) promoter activity. We further demonstrate that NS5A inhibits DDX3-mediated IKKε and interferon regulatory factor 3 (IRF3) phosphorylation. We also note that hyperphosphorylation of NS5A mediats protein interplay between NS5A and IKKε, thereby contributing to NS5A-mediated modulation of IFN-β signaling. Lastly, NS5A inhibits IKKε-dependent p65 phosphorylation and NF-κB activation. Based on these findings, we propose NS5A as a novel regulator of IFN signaling events, specifically by inhibiting IKKε downstream signaling cascades through its interaction with IKKε. Taken together, these data suggest an additional mechanistic means by which HCV modulates host antiviral innate immune responses to promote persistent viral infection.

Hepatitis C virus (HCV) is a major cause of chronic liver disease and is highly dependent on cellular proteins for viral propagation. Using protein microarray analysis, we identified 90 cellular proteins as HCV nonstructural 5A (NS5A) interacting partners, and selected telomere length regulation protein (TEN1) for further study. TEN1 forms a heterotrimeric complex with CTC and STN1, which is essential for telomere protection and maintenance. Telomere length decreases in patients with active HCV, chronic liver disease, and hepatocellular carcinoma. However, the molecular mechanism of telomere length shortening in HCV-associated disease is largely unknown. In the present study, protein interactions between NS5A and TEN1 were confirmed by immunoprecipitation assays. Silencing of TEN1 reduced both viral RNA and protein expression levels of HCV, while ectopic expression of the siRNA-resistant TEN1 recovered the viral protein level, suggesting that TEN1 was specifically required for HCV propagation. Importantly, we found that TEN1 is re-localized from the nucleus to the cytoplasm in HCV-infected cells. These data suggest that HCV exploits TEN1 to promote viral propagation and that telomere protection is compromised in HCV-infected cells. Overall, our findings provide mechanistic insight into the telomere shortening in HCV-infected cells.