The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Ralf Bartenschlager - One of the best experts on this subject based on the ideXlab platform.
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dimer organization of membrane associated ns5A of hepatitis c virus as determined by highly sensitive 1h detected solid state nmr
Angewandte Chemie, 2021Co-Authors: Vlastimil Jirasko, Ralf Bartenschlager, Alons Lends, Nilsalexander Lakomek, Marielaure Fogeron, Marco E Weber, Alexander A Malar, Susanne Penzel, Beat H Meier, Anja BockmannAbstract:The Hepatitis C virus Nonstructural Protein 5A (NS5A) is a membrane-associated Protein involved in multiple steps of the viral life cycle. Direct-acting antivirals (DAAs) targeting NS5A are a cornerstone of antiviral therapy, but the mode-of-action of these drugs is poorly understood. This is due to the lack of information on the membrane-bound NS5A structure. Herein, we present the structural model of an NS5A AH-linker-D1 Protein reconstituted as proteoliposomes. We use highly sensitive proton-detected solid-state NMR methods suitable to study samples generated through synthetic biology approaches. Spectra analyses disclose that both the AH membrane anchor and the linker are highly flexible. Paramagnetic relaxation enhancements (PRE) reveal that the dimer organization in lipids requires a new type of NS5A self-interaction not reflected in previous crystal structures. In conclusion, we provide the first characterization of NS5A AH-linker-D1 in a lipidic environment shedding light onto the mode-of-action of clinically used NS5A inhibitors.
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overall structural model of ns5A Protein from hepatitis c virus and modulation by mutations confering resistance of virus replication to cyclosporin a
Biochemistry, 2017Co-Authors: Aurelie Badillo, Roland Montserret, Marielaure Fogeron, Anja Bockmann, Veronique Receveurbrechot, Stephane Sarrazin, Francoisxavier Cantrelle, Frederic Delolme, Jennifer Molle, Ralf BartenschlagerAbstract:Hepatitis C virus (HCV) Nonstructural Protein 5A (NS5A) is a RNA-binding phosphoProtein composed of a N-terminal membrane anchor (AH), a structured domain 1 (D1), and two intrinsically disordered domains (D2 and D3). The knowledge of the functional architecture of this multifunctional Protein remains limited. We report here that NS5A-D1D2D3 produced in a wheat germ cell-free system is obtained under a highly phosphorylated state. Its NMR analysis revealed that these phosphorylations do not change the disordered nature of D2 and D3 domains but increase the number of conformers due to partial phosphorylations. By combining NMR and small angle X-ray scattering, we performed a comparative structural characterization of unphosphorylated recombinant D2 domains of JFH1 (genotype 2a) and the Con1 (genotype 1b) strains produced in Escherichia coli. These analyses highlighted a higher intrinsic folding of the latter, revealing the variability of intrinsic conformations in HCV genotypes. We also investigated the eff...
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coordination of hepatitis c virus assembly by distinct regulatory regions in Nonstructural Protein 5A
PLOS Pathogens, 2016Co-Authors: Margarita Zayas, Gang Long, Vanesa Madan, Ralf BartenschlagerAbstract:Hepatitis C virus (HCV) Nonstructural Protein (NS)5A is a RNA-binding Protein composed of a N-terminal membrane anchor, a structured domain I (DI) and two intrinsically disordered domains (DII and DIII) interacting with viral and cellular Proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC) at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC) mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoProteins (ΔE1E2). We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core-RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core Protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i) SC-dependent recruitment of replication complexes to core Protein and (ii) BC-dependent RNA genome delivery to core Protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral Protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles.
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essential role of domain iii of Nonstructural Protein 5A for hepatitis c virus infectious particle assembly
PLOS Pathogens, 2008Co-Authors: Nicole Appel, Margarita Zayas, Sven Miller, Jacomine Krijnselocker, Torsten Schaller, Peter Friebe, Stephanie Kallis, Ulrike Engel, Ralf BartenschlagerAbstract:Persistent infection with the hepatitis C virus (HCV) is a major risk factor for the development of liver cirrhosis and hepatocellular carcinoma. With an estimated about 3% of the world population infected with this virus, the lack of a prophylactic vaccine and a selective therapy, chronic hepatitis C currently is a main indication for liver transplantation. The establishment of cell-based replication and virus production systems has led to first insights into the functions of HCV Proteins. However, the role of Nonstructural Protein 5A (NS5A) in the viral replication cycle is so far not known. NS5A is a membrane-associated RNA-binding Protein assumed to be involved in HCV RNA replication. Its numerous interactions with the host cell suggest that NS5A is also an important determinant for pathogenesis and persistence. In this study we show that NS5A is a key factor for the assembly of infectious HCV particles. We specifically identify the C-terminal domain III as the primary determinant in NS5A for particle formation. We show that both core and NS5A colocalize on the surface of lipid droplets, a proposed site for HCV particle assembly. Deletions in domain III of NS5A disrupting this colocalization abrogate infectious particle formation and lead to an enhanced accumulation of core Protein on the surface of lipid droplets. Finally, we show that mutations in NS5A causing an assembly defect can be rescued by trans-complementation. These data provide novel insights into the production of infectious HCV and identify NS5A as a major determinant for HCV assembly. Since domain III of NS5A is one of the most variable regions in the HCV genome, the results suggest that viral isolates may differ in their level of virion production and thus in their level of fitness and pathogenesis.
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mutational analysis of hepatitis c virus Nonstructural Protein 5A potential role of differential phosphorylation in rna replication and identification of a genetically flexible domain
Journal of Virology, 2005Co-Authors: Nicole Appel, Thomas Pietschmann, Ralf BartenschlagerAbstract:Nonstructural Protein 5A of the hepatitis C virus (HCV) is a highly phosphorylated molecule implicated in multiple interactions with the host cell and most likely involved in RNA replication. Two phosphorylated variants of NS5A have been described, designated according to their apparent molecular masses (in kilodaltons) as p56 and p58, which correspond to the basal and hyperphosphorylated forms, respectively. With the aim of identifying a possible role of NS5A phosphorylation for RNA replication, we performed an extensive mutation analysis of three serine clusters that are involved in phosphorylation and hyperphosphorylation of NS5A. In most cases, alanine substitutions for serine residues in the central cluster 1 that enhanced RNA replication to the highest levels led to a reduction of NS5A hyperphosphorylation. Likewise, several highly adaptive mutations in NS4B, which is also part of the replication complex, resulted in a reduction of NS5A hyperphosphorylation too, arguing that alterations of the NS5A phosphorylation pattern play an important role for RNA replication. On the other hand, a deletion encompassing all highly conserved serine residues in the C-terminal region of NS5A that are involved in basal phosphorylation did not significantly affect RNA replication but reduced formation of p56. This region was found to tolerate even large insertions with only a moderate effect on replication. Based on these results, we propose a model of the role of NS5A phosphorylation in the viral life cycle.
Michael M C Lai - One of the best experts on this subject based on the ideXlab platform.
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Nonstructural Protein 5A is incorporated into hepatitis c virus low density particle through interaction with core Protein and microtubules during intracellular transport
PLOS ONE, 2014Co-Authors: Chaokuen Lai, Vikas Saxena, Chunghsin Tseng, Kingsong Jeng, Michinori Kohara, Michael M C LaiAbstract:Nonstructural Protein 5A (NS5A) of hepatitis C virus (HCV) serves dual functions in viral RNA replication and virus assembly. Here, we demonstrate that HCV replication complex along with NS5A and Core Protein was transported to the lipid droplet (LD) through microtubules, and NS5A-Core complexes were then transported from LD through early-to-late endosomes to the plasma membrane via microtubules. Further studies by cofractionation analysis and immunoelectron microscopy of the released particles showed that NS5A-Core complexes, but not NS4B, were present in the low-density fractions, but not in the high-density fractions, of the HCV RNA-containing virions and associated with the internal virion core. Furthermore, exosomal markers CD63 and CD81 were also detected in the low-density fractions, but not in the high-density fractions. Overall, our results suggest that HCV NS5A is associated with the core of the low-density virus particles which exit the cell through a preexisting endosome/exosome pathway and may contribute to HCV natural infection.
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Nonstructural 5A Protein of hepatitis c virus modulates tumor necrosis factor α stimulated nuclear factor κb activation
Journal of Biological Chemistry, 2002Co-Authors: Kyujin Park, Soon B Hwang, Sooho Choi, Soo Young Lee, Michael M C LaiAbstract:The hepatitis C virus Nonstructural Protein 5A (NS5A) is a multifunctional phosphoProtein that leads to pleiotropic responses, in part by regulating cell growth and cellular signaling pathways. Here we show that overexpression of NS5A inhibits tumor necrosis factor (TNF)-alpha-induced nuclear factor kappaB (NF-kappaB) activation in HEK293 cells, as determined by luciferase reporter gene expression and by electrophoretic mobility shift assay. When overexpressed, NS5A cannot inhibit the recruitment of TNF receptor-associated factor 2 (TRAF2) and IkappaB kinase (IKK)beta into the TNF receptor 1-TNF receptor-associated death domain complex. In contrast, NS5A is a part of the TNF receptor 1 signaling complex. NF-kappaB activation by TNF receptor-associated death domain and TRAF2 was inhibited by NS5A, whereas MEKK1 and IKKbeta-dependent NF-kappaB activation was not affected, suggesting that NS5A may inhibit NF-kappaB activation signaled by TRAF2. Coimmunoprecipitation and colocalization of NS5A and TRAF2 expressed in vivo provide compelling evidence that NS5A directly interacts with TRAF2. This interaction was mapped to the middle one-third (amino acids 148-301) of NS5A and the TRAF domain of TRAF2. Our findings suggest a possible molecular mechanism that could explain the ability of NS5A to negatively regulate TNF-alpha-induced NF-kappaB activation.
Namiki Izumi - One of the best experts on this subject based on the ideXlab platform.
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hepatitis c virus Nonstructural Protein 5A inhibits tumor necrosis factor α mediated apoptosis in huh7 cells
The Journal of Infectious Diseases, 2003Co-Authors: Yuka Miyasaka, Nobuyuki Enomoto, Masayuki Kurosaki, Naoya Sakamoto, Nobuhiko Kanazawa, Takahiro Kohashi, Eri Ueda, Shinya Maekawa, Hideki Watanabe, Namiki IzumiAbstract:To analyze the influence of hepatitis C virus Nonstructural Protein 5A (NS5A) on apoptosis, we established Huh7 cells that stably express NS5A, and induced apoptosis using tumor necrosis factor (TNF)-alpha. The viability of control Huh7 cells was reduced to 40%, compared with untreated cells, after TNF-alpha treatment, whereas that of Huh7-NS5A cells was reduced only to 80%. DNA fragmentation also decreased to <50% in Huh7-NS5A compared with control cells. Nuclear factor-kappaB activation was the same in both cell types, whereas caspase-8, -9, and -3 activity was decreased in Huh7-NS5A cells, compared with control cells, which indicates that the inhibition of caspase-8 activation is responsible for the antiapoptotic effect of the NS5A Protein. The coexpression of NS5A did not inhibit apoptosis induced by caspase-8 or Fas-associating death domain Protein expression. These findings suggest that the NS5A Protein inhibits the apoptotic effect of TNF-alpha upstream of caspase-8 in the apoptosis cascade.
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differential effect of interferon on hepatitis c virus 1b quasispecies in the Nonstructural Protein 5A gene
The Journal of Infectious Diseases, 1999Co-Authors: Ikuo Sakuma, Nobuyuki Enomoto, Masayuki Kurosaki, Namiki Izumi, Fumiaki Marumo, Chifumi SatoAbstract:A close correlation was reported between amino acid mutations in the Nonstructural Protein 5A (interferon [IFN] sensitivity-determining region [ISDR]) of hepatitis C virus (HCV)-1b and the response to IFN therapy. The dynamic change of ISDR quasispecies during IFN treatment was investigated in 22 patients. In 18 nonresponders, the number of ISDR mutations in major quasispecies decreased after therapy (P =.039). In each nonresponder, the percentage of wild-type (no mutations in the ISDR) quasispecies increased after treatment (P =.008), whereas the percentages of intermediate- (1-3 mutations) and mutant-type (≥4 mutations) quasispecies decreased (P =.037 and P =.043, respectively). No mutant-type quasispecies were detected after therapy. Four complete responders had only quasispecies with ≥3 mutations before therapy. Thus, HCVs have fewer mutations in the ISDR after IFN therapy than those before therapy. These IFN-resistant HCVs were already present before therapy as minor quasispecies and were selected by IFN in nonresponders.
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mutations in Nonstructural Protein 5A gene and response to interferon in hepatitis c virus genotype 2 infection
Hepatology, 1999Co-Authors: Takeshi Murakami, Nobuyuki Enomoto, Masayuki Kurosaki, Namiki Izumi, Fumiaki Marumo, M Chifumi D SatoAbstract:An association has been reported between mutations in the amino acid residues 2209-2248 of the Nonstructural Protein 5A (NS5A) gene (interferon-sensitivity determining region [ISDR]) and interferon efficacy in hepatitis C virus (HCV)-1b infection. This relationship was analyzed in chronic HCV-2 infection. Forty patients with HCV-2a and 35 with HCV-2b were treated with interferon alfa for 6 months with a total dose of 468 to 860 million units. Pretreatment NS5A sequences were determined by direct sequencing. A higher complete and sustained response rate was observed in HCV-2a than in HCV-2b (70% vs. 34%; P =.003). Serum HCV-RNA levels were lower in complete responders than nonresponders in HCV-2a (P =.049) and HCV-2b (P =. 02). The number of amino acid mutations was greater in complete responders than nonresponders in NS5A2193-2228 (the region corresponding to the ISDR of HCV-1b) alone (P =.049), or NS5A2163-2228 consisting of NS5A2193-2228 plus its upstream region (P =.02) in HCV-2a, but not in HCV-2b. A significant inverse correlation was observed between serum HCV-RNA levels and the number of amino acid mutations in NS5A2193-2228 (P =.003) or NS5A2163-2228 (P =.005) in HCV-2a. With multivariate analysis, the number of substitutions in NS5A was an independent predictor for complete response in HCV-2a (odds ratio: 6.4; P =.03). Interferon efficacy is associated with amino acid variations in the NS5A Protein in HCV-2a infection.
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mutations in the Nonstructural Protein 5A gene and response to interferon in patients with chronic hepatitis c virus 1b infection
The New England Journal of Medicine, 1996Co-Authors: Nobuyuki Enomoto, Masayuki Kurosaki, Namiki Izumi, Ikuo Sakuma, Fumiaki Marumo, Yasuhiro Asahina, Takeshi Murakami, Chikara Yamamoto, Yuki Ogura, Chifumi SatoAbstract:Background A region associated with sensitivity to interferon has been identified in the Nonstructural Protein 5A (NS5A) of hepatitis C virus (HCV) genotype 1b. The region spans amino acid residues 2209 to 2248 (NS5A2209–2248) of HCV-J, a strain of HCV-1b whose complete genomic sequence has been identified. We examined whether the NS5A2209–2248 sequence present before therapy could be used as a predictor of the response to interferon therapy in patients with chronic HCV-1b infection. Methods We retrospectively analyzed 84 patients with chronic HCV-1b infection who had received interferon alfa (total dose, 516 million to 880 million units) for six months. Pretreatment serum samples were analyzed. The amino acid sequence of NS5A2209–2248 was determined by direct sequencing of the HCV genome amplified by the polymerase chain reaction (PCR) and was compared with the established sequence for HCV-J. Results A complete response, as evidenced by the absence of HCV RNA in serum on nested reverse-transcription PCR ...
Kenji Ikeda - One of the best experts on this subject based on the ideXlab platform.
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dual therapy with the Nonstructural Protein 5A inhibitor daclatasvir and the Nonstructural Protein 3 protease inhibitor asunaprevir in hepatitis c virus genotype 1b infected null responders
Hepatology, 2012Co-Authors: Kazuaki Chayama, Shoichi Takahashi, Fiona Mcphee, Joji Toyota, Yoshiyasu Karino, Kenji Ikeda, Eric Hughes, Hideaki Watanabe, Hiroki Ishikawa, Hiromitsu KumadaAbstract:Patients with chronic hepatitis C virus (HCV) infection and previous null response to pegylated interferon (Peg-IFN) and ribavirin (RBV) have limited therapeutic options. HCV genotype 1 is the most common worldwide and the most difficult to treat; genotype 1b is the most common subtype of genotype 1 outside North America. The enhanced antiviral activity achieved by combining two direct-acting antiviral (DAA) agents may improve clinical outcomes. This open-label, phase IIa study included 10 patients with chronic HCV genotype 1b infection and previous null response (<2 log10 reduction in HCV RNA after 12 weeks) to Peg-IFN and RBV. Patients received dual DAA treatment for 24 weeks with the Nonstructural Protein 5A replication complex inhibitor, daclatasvir (60 mg once-daily), and the Nonstructural Protein 3 protease inhibitor, asunaprevir (initially 600 mg twice-daily, then subsequently reduced to 200 mg twice-daily). The primary efficacy endpoint was the proportion of patients with sustained virologic response (SVR) at 12 weeks post-treatment (SVR12). Nine patients completed 24 weeks of treatment; 1 patient discontinued treatment after 2 weeks. In the 9 patients who completed the full course of treatment, HCV RNA was undetectable at week 8 and remained undetectable through the end of treatment; all 9 patients achieved SVR12 and SVR24. HCV RNA also remained undetectable post-treatment in the patient who discontinued after 2 weeks. There was no viral breakthrough. Diarrhea and headache, generally mild, were the most common adverse events; transaminase elevations were reported in 3 patients, but did not result in discontinuation. Conclusions: Dual therapy with daclatasvir and asunaprevir, without Peg-IFN and RBV, can achieve high SVR rates in difficult-to-treat patients with HCV genotype 1b infection and previous null response to Peg-IFN and RBV. (HEPATOLOGY 2011)
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association of amino acid substitution pattern in Nonstructural Protein 5A of hepatitis c virus genotype2a low viral load and response to interferon monotherapy
Journal of Medical Virology, 2003Co-Authors: Norio Akuta, Fumitaka Suzuki, Akihito Tsubota, Yoshiyuki Suzuki, Tetsuya Hosaka, Takashi Someya, Masahiro Kobayashi, Satoshi Saitoh, Yasuji Arase, Kenji IkedaAbstract:Patients with low titer (<0.5 mEq/ml) of hepatitis C virus (HCV) genotype 2a achieve high and sustained response (SR) rates to interferon (IFN) monotherapy, but we also encounter patients who are resistant to therapy. We explored the relationship between response to IFN and virological differences in such patients. We evaluated 159 consecutive naive patients with low titer of HCV genotype 2a who received IFN monotherapy. A case-control study matched for age, sex, and viral load was conducted to examine the substitution patterns in amino acid positions (amino acids) 2163–2254 of Nonstructural (NS) 5A between nonresponders to ideal IFN dose (≥500 million units) (nonresponders; NR) and responder to less than ideal dose. Overall, 82.4% achieved SR. The substitution numbers in amino acids 2193–2254 were higher in SR than NR patients (P < 0.05). High proportions of patients with substitution at amino acid 2205 (mainly threonine [T] instead of alanine [A]), dual amino acids 2169 and 2205 (mainly A-T instead of T-A), and those without substitution at amino acids 2227 were NR (P < 0.05). Four of 7 NR patients achieved SR after receiving a second course of IFN. Their amino acids patterns at positions probably associated with sensitivity to IFN did not change at the start of initial and second therapies except for one patient, and they had lower viral load and were treated with higher IFN dose in the second course compared with the initial course. Our results suggest that substitution patterns in NS5A in patients with low titer of HCV genotype 2a may affect their response to IFN, but the response to therapy may be affected by mechanisms other than substitutions in this region. J. Med. Virol. 69:376–383, 2003. © 2003 Wiley-Liss, Inc.
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association of amino acid substitution pattern in Nonstructural Protein 5A of hepatitis c virus genotype2a low viral load and response to interferon monotherapy
Journal of Medical Virology, 2003Co-Authors: Norio Akuta, Fumitaka Suzuki, Akihito Tsubota, Yoshiyuki Suzuki, Tetsuya Hosaka, Takashi Someya, Masahiro Kobayashi, Satoshi Saitoh, Yasuji Arase, Kenji IkedaAbstract:Patients with low titer ( /=500 million units) (nonresponders; NR) and responder to less than ideal dose. Overall, 82.4% achieved SR. The substitution numbers in amino acids 2193-2254 were higher in SR than NR patients (P < 0.05). High proportions of patients with substitution at amino acid 2205 (mainly threonine [T] instead of alanine [A]), dual amino acids 2169 and 2205 (mainly A-T instead of T-A), and those without substitution at amino acids 2227 were NR (P < 0.05). Four of 7 NR patients achieved SR after receiving a second course of IFN. Their amino acids patterns at positions probably associated with sensitivity to IFN did not change at the start of initial and second therapies except for one patient, and they had lower viral load and were treated with higher IFN dose in the second course compared with the initial course. Our results suggest that substitution patterns in NS5A in patients with low titer of HCV genotype 2a may affect their response to IFN, but the response to therapy may be affected by mechanisms other than substitutions in this region.
Ling Tong - One of the best experts on this subject based on the ideXlab platform.
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Discovery of Ruzasvir (MK-8408): A Potent, Pan-Genotype HCV NS5A Inhibitor with Optimized Activity against Common Resistance-Associated Polymorphisms.
Journal of Medicinal Chemistry, 2016Co-Authors: Ling Tong, Oleg Selyutin, Michael P Dwyer, Anilkumar G Nair, Wensheng Yu, Lei Chen, Robert MazzolaAbstract:We describe the research that led to the discovery of compound 40 (ruzasvir, MK-8408), a pan-genotypic HCV Nonstructural Protein 5A (NS5A) inhibitor with a “flat” GT1 mutant profile. This NS5A inhibitor contains a unique tetracyclic indole core while maintaining the imidazole–proline–valine Moc motifs of our previous NS5A inhibitors. Compound 40 is currently in early clinical trials and is under evaluation as part of an all-oral DAA regimen for the treatment of chronic HCV infection.
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structure activity relationships of proline modifications around the tetracyclic indole class of ns5A inhibitors
Bioorganic & Medicinal Chemistry Letters, 2016Co-Authors: Ling Tong, Oleg Selyutin, Qingbei Zeng, Michael P Dwyer, Anilkumar G Nair, Bandarpalle B Shankar, Wensheng Yu, Craig A Coburn, Lei Chen, Deyi YangAbstract:Abstract We describe the impact of proline modifications, in our tetracyclic-indole based series of Nonstructural Protein 5A (NS5A) inhibitors, to their replicon profiles. This work identified NS5A inhibitors with an improved and flattened resistance profiles.
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alternative core development around the tetracyclic indole class of hcv ns5A inhibitors
Bioorganic & Medicinal Chemistry Letters, 2016Co-Authors: Ling Tong, Stuart B Rosenblum, Oleg Selyutin, Michael P Dwyer, Anilkumar G Nair, Peter T Meinke, Wensheng Yu, Craig A Coburn, Lei Chen, Yueheng JiangAbstract:Abstract Herein, we describe our research efforts to develop unique cores in molecules which function as HCV Nonstructural Protein 5A (NS5A) inhibitors. In particular, various fused tetracyclic cores were identified which showed genotype and mutant activities comparable to the indole-based tetracyclic core.
