scholarly journals ISGylation of Hepatitis C Virus NS5A Protein Promotes Viral RNA Replication via Recruitment of Cyclophilin A

2020 ◽  
Vol 94 (20) ◽  
Author(s):  
Takayuki Abe ◽  
Nanae Minami ◽  
Rheza Gandi Bawono ◽  
Chieko Matsui ◽  
Lin Deng ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Host Defense ◽  
E3 Ligase ◽  
Cyclophilin A ◽  
Rna Replication ◽  
Host Factor ◽  
Hcv Genotypes ◽  
Ns5a Protein

ABSTRACT Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like protein that is covalently conjugated to many substrate proteins in order to modulate their functions; this conjugation is called ISGylation. Several groups reported that the ISGylation of hepatitis C virus (HCV) NS5A protein affects HCV replication. However, the ISG15 conjugation sites on NS5A are not well determined, and it is unclear whether the role of NS5A ISGylation in HCV replication is proviral or antiviral. Here, we investigated the role of NS5A ISGylation in HCV replication by using HCV RNA replicons that encode a mutation at each lysine (Lys) residue of the NS5A protein. Immunoblot analyses revealed that 5 Lys residues (K44, K68, K166, K215, and K308) of the 14 Lys residues within NS5A (genotype 1b, Con1) have the potential to accept ISGylation. We tested the NS5A ISGylation among different HCV genotypes and observed that the NS5A proteins of all of the HCV genotypes accept ISGylation at multiple Lys residues. Using an HCV luciferase reporter replicon assay revealed that residue K308 of NS5A is important for HCV (1b, Con1) RNA replication. We observed that K308, one of the Lys residues for NS5A ISGylation, is located within the binding region of cyclophilin A (CypA), which is the critical host factor for HCV replication. We obtained evidence derived from all of the HCV genotypes suggesting that NS5A ISGylation enhances the interaction between NS5A and CypA. Taken together, these results suggest that NS5A ISGylation functions as a proviral factor and promotes HCV replication via the recruitment of CypA. IMPORTANCE Host cells have evolved host defense machinery (such as innate immunity) to eliminate viral infections. Viruses have evolved several counteracting strategies for achieving an immune escape from host defense machinery, including type I interferons (IFNs) and inflammatory cytokines. ISG15 is an IFN-inducible ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation. Here, we demonstrate that HCV NS5A protein accepts ISG15 conjugation at specific Lys residues and that the HERC5 E3 ligase specifically promotes NS5A ISGylation. We obtained evidence suggesting that NS5A ISGylation facilitates the recruitment of CypA, which is the critical host factor for HCV replication, thereby promoting HCV replication. These findings indicate that E3 ligase HERC5 is a potential therapeutic target for HCV infection. We propose that HCV hijacks an intracellular ISG15 function to escape the host defense machinery in order to establish a persistent infection.

scholarly journals Dehydrojuncusol, a Natural Phenanthrene Compound Extracted fromJuncus maritimus, Is a New Inhibitor of Hepatitis C Virus RNA Replication

2019 ◽  
Vol 93 (10) ◽  
Author(s):  
Marie-Emmanuelle Sahuc ◽  
Ramla Sahli ◽  
Céline Rivière ◽  
Véronique Pène ◽  
Muriel Lavie ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Activity ◽  
Treatment Failure ◽  
Natural Compound ◽  
Rna Replication ◽  
Direct Acting Antivirals ◽  
Ns5a Protein ◽  
Juncus Maritimus ◽  
Direct Acting

ABSTRACTRecent emergence of direct-acting antivirals (DAAs) targeting hepatitis C virus (HCV) proteins has considerably enhanced the success of antiviral therapy. However, the appearance of DAA-resistant-associated variants is a cause of treatment failure, and the high cost of DAAs renders the therapy not accessible in countries with inadequate medical infrastructures. Therefore, the search for new inhibitors with a lower cost of production should be pursued. In this context, the crude extract ofJuncus maritimusLam. was shown to exhibit high antiviral activity against HCV in cell culture. Bio-guided fractionation allowed the isolation and identification of the active compound, dehydrojuncusol. A time-of-addition assay showed that dehydrojuncusol significantly inhibited HCV infection when added after virus inoculation of HCV genotype 2a (50% effective concentration [EC50] = 1.35 µM). This antiviral activity was confirmed with an HCV subgenomic replicon, and no effect on HCV pseudoparticle entry was observed. Antiviral activity of dehydrojuncusol was also demonstrated in primary human hepatocytes. Noin vitrotoxicity was observed at active concentrations. Dehydrojuncusol is also efficient on HCV genotype 3a and can be used in combination with sofosbuvir. Interestingly, dehydrojuncusol was able to inhibit RNA replication of two frequent daclatasvir-resistant mutants (L31M or Y93H in NS5A). Finally, mutants resistant to dehydrojuncusol were obtained and showed that the HCV NS5A protein is the target of the molecule. In conclusion, dehydrojuncusol, a natural compound extracted fromJ. maritimus, inhibits infection of different HCV genotypes by targeting the NS5A protein and is active against resistant HCV variants frequently found in patients with treatment failure.IMPORTANCETens of millions of people are infected with hepatitis C virus (HCV) worldwide. Recently marketed direct-acting antivirals (DAAs) targeting HCV proteins have enhanced the efficacy of treatment. However, due to its high cost, this new therapy is not accessible to the vast majority of infected patients. Furthermore, treatment failures have also been reported due to the appearance of viral resistance. Here, we report on the identification of a new HCV inhibitor, dehydrojuncusol, that targets HCV NS5A and is able to inhibit RNA replication of replicons harboring resistance mutations to anti-NS5A DAAs used in current therapy. Dehydrojuncusol is a natural compound isolated fromJuncus maritimus, a halophilic plant species that is very common in coastlines worldwide. This molecule might serve as a lead for the development of a new therapy that is more accessible to hepatitis C patients in the future.

scholarly journals A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

2015 ◽  
Vol 290 (31) ◽  
pp. 19104-19120 ◽  
Author(s):  
Marie Dujardin ◽  
Vanesa Madan ◽  
Roland Montserret ◽  
Puneet Ahuja ◽  
Isabelle Huvent ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Replication ◽  
Hepatitis C Virus Rna ◽  
Intrinsically Disordered ◽  
Ns5a Protein ◽  
Virus Rna ◽  
Intrinsically Disordered Domain

scholarly journals Lipid Droplet-Binding Protein TIP47 Regulates Hepatitis C Virus RNA Replication through Interaction with the Viral NS5A Protein

2013 ◽  
Vol 9 (4) ◽  
pp. e1003302 ◽  
Author(s):  
Dorothee A. Vogt ◽  
Grégory Camus ◽  
Eva Herker ◽  
Brian R. Webster ◽  
Chia-Lin Tsou ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Droplet ◽  
Binding Protein ◽  
Rna Replication ◽  
Hepatitis C Virus Rna ◽  
Ns5a Protein ◽  
Virus Rna

scholarly journals Identification of Residues Required for RNA Replication in Domains II and III of the Hepatitis C Virus NS5A Protein

2007 ◽  
Vol 82 (3) ◽  
pp. 1073-1083 ◽  
Author(s):  
Timothy L. Tellinghuisen ◽  
Katie L. Foss ◽  
Jason C. Treadaway ◽  
Charles M. Rice
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Rna Replication ◽  
Hcv Rna ◽  
Alanine Scanning Mutagenesis ◽  
Amino Terminal ◽  
Ns5a Protein ◽  
Amino Acid Region ◽  
Domain Iii

ABSTRACT The NS5A protein of hepatitis C virus (HCV) plays an important but undefined role in viral RNA replication. NS5A has been proposed to be a three-domain protein, and the crystal structure of the well-conserved amino-terminal domain I has been determined. The remaining two domains of NS5A, designated domains II and III, and their corresponding interdomain regions are poorly understood. We have conducted a detailed mutagenesis analysis of NS5A domains II and III using the genotype 1b HCV replicon system. The majority of the mutants containing 15 small (8- to 15-amino-acid) deletions analyzed were capable of efficient RNA replication. Only five deletion mutations yielded lethal phenotypes, and these were colinear, spanning a 56-amino-acid region within domain II. This region was further analyzed by combining triple and single alanine scanning mutagenesis to identify individual residues required for RNA replication. Based upon this analysis, 23 amino acids were identified that were found to be essential. In addition, two residues were identified that yielded a small colony phenotype while possessing only a moderate defect in RNA replication. These results indicate that the entire domain III region and large portions of domain II of the NS5A protein are not required for the function of NS5A in HCV RNA replication.

scholarly journals Critical Role of Cyclophilin A and Its Prolyl-Peptidyl Isomerase Activity in the Structure and Function of the Hepatitis C Virus Replication Complex

2009 ◽  
Vol 83 (13) ◽  
pp. 6554-6565 ◽  
Author(s):  
Zhe Liu ◽  
Feng Yang ◽  
Jason M. Robotham ◽  
Hengli Tang
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Critical Role ◽  
Cyclophilin A ◽  
Hcv Rna ◽  
Nonstructural Proteins ◽  
Ppiase Activity ◽  
Replication Complex ◽  
And Function

ABSTRACT Replication of hepatitis C virus (HCV) RNA occurs on intracellular membranes, and the replication complex (RC) contains viral RNA, nonstructural proteins, and cellular cofactors. We previously demonstrated that cyclophilin A (CyPA) is an essential cofactor for HCV infection and the intracellular target of cyclosporine's anti-HCV effect. Here we investigate the mechanism by which CyPA facilitates HCV replication. Cyclosporine treatment specifically blocked the incorporation of NS5B into the RC without affecting either the total protein level or the membrane association of the protein. Other nonstructural proteins or viral RNAs in the RC were not affected. NS5B from the cyclosporine-resistant replicon was resistant to this disruption of RC incorporation. We also isolated membrane fractions from both naïve and HCV-positive cells and found that CyPA is recruited into membrane fractions in HCV-replicating cells via an interaction with RC-associated NS5B, which is sensitive to cyclosporine treatment. Finally, we introduced point mutations in the prolyl-peptidyl isomerase (PPIase) motif of CyPA and demonstrated a critical role of this motif in HCV replication in cDNA rescue experiments. We propose a model in which the incorporation of the HCV polymerase into the RC depends on its interaction with a cellular chaperone protein and in which cyclosporine inhibits HCV replication by blocking this critical interaction and the PPIase activity of CyPA. Our results provide a mechanism of action for the cyclosporine-mediated inhibition of HCV and identify a critical role of CyPA's PPIase activity in the proper assembly and function of the HCV RC.

scholarly journals Mutational Analysis of Hepatitis C Virus Nonstructural Protein 5A: Potential Role of Differential Phosphorylation in RNA Replication and Identification of a Genetically Flexible Domain

2005 ◽  
Vol 79 (5) ◽  
pp. 3187-3194 ◽  
Author(s):  
Nicole Appel ◽  
Thomas Pietschmann ◽  
Ralf Bartenschlager
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mutational Analysis ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Adaptive Mutations ◽  
Phosphorylation Pattern ◽  
Molecular Masses ◽  
Nonstructural Protein 5A

ABSTRACT 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.

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