scholarly journals Molecular and Structural Basis for the Roles of Hepatitis C Virus Polymerase NS5B Amino Acids 15, 223, and 321 in Viral Replication and Drug Resistance

2014 ◽  
Vol 58 (11) ◽  
pp. 6861-6869 ◽  
Author(s):  
Angela M. Lam ◽  
Thomas E. Edwards ◽  
Ralph T. Mosley ◽  
Eisuke Murakami ◽  
Shalini Bansal ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Replication ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Point Mutations ◽  
Resistance Mutation ◽  
Structural Basis ◽  
Nucleoside Triphosphate ◽  
Multiple Point

ABSTRACTResistance to the 2′-F-2′-C-methylguanosine monophosphate nucleotide hepatitis C virus (HCV) inhibitors PSI-352938 and PSI-353661 was associated with a combination of amino acid changes (changes of S to G at position 15 [S15G], C223H, and V321I) within the genotype 2a nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase. To understand the role of these residues in viral replication, we examined the effects of single and multiple point mutations on replication fitness and inhibition by a series of nucleotide analog inhibitors. An acidic residue at position 15 reduced replicon fitness, consistent with its proximity to the RNA template. A change of the residue at position 223 to an acidic or large residue reduced replicon fitness, consistent with its proposed proximity to the incoming nucleoside triphosphate (NTP). A change of the residue at position 321 to a charged residue was not tolerated, consistent with its position within a hydrophobic cavity. This triple resistance mutation was specific to both genotype 2a virus and 2′-F-2′-C-methylguanosine inhibitors. A crystal structure of the NS5B S15G/C223H/V321I mutant of the JFH-1 isolate exhibited rearrangement to a conformation potentially consistent with short primer-template RNA binding, which could suggest a mechanism of resistance accomplished through a change in the NS5B conformation, which was better tolerated by genotype 2a virus than by viruses of other genotypes.

scholarly journals Roles of the AX4GKS and Arginine-Rich Motifs of Hepatitis C Virus RNA Helicase in ATP- and Viral RNA-Binding Activity

2000 ◽  
Vol 74 (20) ◽  
pp. 9732-9737 ◽  
Author(s):  
Shin C. Chang ◽  
Ju-Chien Cheng ◽  
Yi-Hen Kou ◽  
Chuan-Hong Kao ◽  
Chiung-Hui Chiu ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Binding ◽  
Atp Hydrolysis ◽  
Nonstructural Protein ◽  
Binding Activity ◽  
Viral Rna ◽  
Atp Binding ◽  
Rna Unwinding ◽  
Arginine Rich Motif

ABSTRACT The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) possesses protease, nucleoside triphosphatase, and helicase activities. Although the enzymatic activities have been extensively studied, the ATP- and RNA-binding domains of the NS3 helicase are not well-characterized. In this study, NS3 proteins with point mutations in the conserved helicase motifs were expressed inEscherichia coli, purified, and analyzed for their effects on ATP binding, RNA binding, ATP hydrolysis, and RNA unwinding. UV cross-linking experiments indicate that the lysine residue in the AX4GKS motif is directly involved in ATP binding, whereas the NS3(GR1490DT) mutant in which the arginine-rich motif (1486-QRRGRTGR-1493) was changed to QRRDTTGR bound ATP as well as the wild type. The binding activity of HCV NS3 helicase to the viral RNA was drastically reduced with the mutation at Arg1488 (R1488A) and was also affected by the K1236E substitution in the AX4GKS motif and the R1490A and GR1490DT mutations in the arginine-rich motif. Previously, Arg1490 was suggested, based on the crystal structure of an NS3-deoxyuridine octamer complex, to directly interact with the γ-phosphate group of ATP. Nevertheless, our functional analysis demonstrated the critical roles of Arg1490 in binding to the viral RNA, ATP hydrolysis, and RNA unwinding, but not in ATP binding.

scholarly journals HMGB1 Promotes Hepatitis C Virus Replication by Interaction with Stem-Loop 4 in the Viral 5′ Untranslated Region

2015 ◽  
Vol 90 (5) ◽  
pp. 2332-2344 ◽  
Author(s):  
Rong Yu ◽  
Darong Yang ◽  
Shaohua Lei ◽  
Xiaohong Wang ◽  
Xianghe Meng ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Replication ◽  
Untranslated Region ◽  
Rna Binding ◽  
High Mobility ◽  
Hcv Rna ◽  
Functional Domain ◽  
Hmgb1 Protein ◽  
Stem Loop

ABSTRACTHigh-mobility group box 1 (HMGB1) protein is a highly conserved nuclear protein involved in multiple human diseases, including infectious diseases, immune disorders, metabolic disorders, and cancer. HMGB1 is comprised of two tandem HMG boxes (the A box and the B box) containing DNA-binding domains and an acidic C-terminal peptide. It has been reported that HMGB1 enhances viral replication by binding to viral proteins. However, its role in hepatitis C virus (HCV) replication is unknown. Here, we show that HMGB1 promoted HCV replication but had no effect on HCV translation. RNA immunoprecipitation experiments indicated that the positive strand, not the negative strand, of HCV RNA interacted with HMGB1. HCV infection triggered HMGB1 protein translocation from the nucleus to the cytoplasm, in which it interacted with the HCV genome. Moreover, the A box of HMGB1 is the pivotal domain to interact with stem-loop 4 (SL4) of the HCV 5′ untranslated region. Deletion of the HMGB1 A box abrogated the enhancement of HCV replication by HMGB1. Our data suggested that HMGB1 serves as a proviral factor of HCV to facilitate viral replication in hepatocytes by interaction with the HCV genome.IMPORTANCEHepatitis C virus (HCV) is a major global health threat, affecting more than 170 million people infection worldwide. These patients are at high risk of developing severe liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Currently, no vaccine is available. Many host factors may be implicated in the pathogenesis of HCV-related diseases. In this study, we found a novel HCV RNA-binding protein, HMGB1, that promotes HCV RNA replication. Moreover, SL4 in the 5′ untranslated region of the HCV genome is the key region for HMGB1 binding, and the A box of HMGB1 protein is the functional domain to interact with HCV RNA and enhance viral replication. HMGB1 appears to play an important role in HCV-related diseases, and further investigation is warranted to elucidate the specific actions of HMGB1 in HCV pathogenesis.

scholarly journals Phosphorylated tyrosine 93 of hepatitis C virus nonstructural protein 5A is essential for interaction with host c-Src and efficient viral replication

2019 ◽  
Vol 294 (18) ◽  
pp. 7388-7402 ◽  
Author(s):  
Stefan Klinker ◽  
Sabine Stindt ◽  
Lothar Gremer ◽  
Johannes G. Bode ◽  
Christoph G. W. Gertzen ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Replication ◽  
Nonstructural Protein ◽  
Efficient Viral Replication ◽  
Nonstructural Protein 5A

scholarly journals Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase

2001 ◽  
Vol 75 (17) ◽  
pp. 8289-8297 ◽  
Author(s):  
Chun-Ling Tai ◽  
Wen-Ching Pan ◽  
Shwu-Huey Liaw ◽  
Ueng-Cheng Yang ◽  
Lih-Hwa Hwang ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Atpase Activity ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Nonstructural Protein ◽  
Rna Helicase ◽  
Helicase Domain ◽  
Helicase Activity ◽  
Conserved Sequence

ABSTRACT The carboxyl terminus of the hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses ATP-dependent RNA helicase activity. Based on the conserved sequence motifs and the crystal structures of the helicase domain, 17 mutants of the HCV NS3 helicase were generated. The ATP hydrolysis, RNA binding, and RNA unwinding activities of the mutant proteins were examined in vitro to determine the functional role of the mutated residues. The data revealed that Lys-210 in the Walker A motif and Asp-290, Glu-291, and His-293 in the Walker B motif were crucial to ATPase activity and that Thr-322 and Thr-324 in motif III and Arg-461 in motif VI significantly influenced ATPase activity. When the pairing between His-293 and Gln-460, referred to as gatekeepers, was replaced with the Asp-293/His-460 pair, which makes the NS3 helicase more like the DEAD helicase subgroup, ATPase activity was not restored. It thus indicated that the whole microenvironment surrounding the gatekeepers, rather than the residues per se, was important to the enzymatic activities. Arg-461 and Trp-501 are important residues for RNA binding, while Val-432 may only play a coadjutant role. The data demonstrated that RNA helicase activity was possibly abolished by the loss of ATPase activity or by reduced RNA binding activity. Nevertheless, a low threshold level of ATPase activity was found sufficient for helicase activity. Results in this study provide a valuable reference for efforts under way to develop anti-HCV therapeutic drugs targeting NS3.

scholarly journals A Single-Amino-Acid Mutation in Hepatitis C Virus NS5A Disrupting FKBP8 Interaction Impairs Viral Replication

2008 ◽  
Vol 82 (7) ◽  
pp. 3480-3489 ◽  
Author(s):  
Toru Okamoto ◽  
Hiroko Omori ◽  
Yuuki Kaname ◽  
Takayuki Abe ◽  
Yorihiro Nishimura ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Fluorescence Microscopy ◽  
Viral Replication ◽  
Nonstructural Protein ◽  
Specific Interaction ◽  
Single Amino Acid ◽  
Single Amino Acid Residue

ABSTRACT Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) regulates viral replication through its interaction with host and other viral proteins. We have previously shown that FK506-binding protein 8 (FKBP8) binds to NS5A and recruits Hsp90 to form a complex that participates in the replication of HCV. In this study, we examined the biochemical characteristics of the interaction and the intracellular localization of NS5A and FKBP8. Surface plasmon resonance analysis revealed that the dissociation constant of the interaction between the purified FKBP8 and NS5A expressed in bacteria was 82 nM. Mutational analyses of NS5A revealed that a single amino acid residue of Val or Ile at position 121, which is well conserved among all genotypes of HCV, is critical for the specific interaction with FKBP8. Substitution of the Val121 to Ala drastically impaired the replication of HCV replicon cells, and the drug-resistant replicon cells emerging after drug selection were shown to have reverted to the original arrangement by replacing Ala121 with Val. Examination of individual fields of the replicon cells by both fluorescence microscopy and electron microscopy (the correlative fluorescence microscopy-electron microscopy technique) revealed that FKBP8 is partially colocalized with NS5A in the cytoplasmic structure known as the membranous web. These results suggest that specific interaction of NS5A with FKBP8 in the cytoplasmic compartment plays a crucial role in the replication of HCV.

scholarly journals The FUSE Binding Protein Is a Cellular Factor Required for Efficient Replication of Hepatitis C Virus

2008 ◽  
Vol 82 (12) ◽  
pp. 5761-5773 ◽  
Author(s):  
Zhengbin Zhang ◽  
Dylan Harris ◽  
Virendra N. Pandey
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Replication ◽  
Molecular Mechanisms ◽  
Nonstructural Protein ◽  
Binding Protein ◽  
Cellular Factor ◽  
Nontranslated Region ◽  
Efficient Replication

ABSTRACT Hepatitis C virus (HCV) infection is the leading cause of liver cirrhosis and hepatocellular carcinoma and one of the primary indications for liver transplantation. The molecular mechanisms underlying the actions of host factors in HCV replication remain poorly defined. FUSE (far upstream element of the c-myc proto-oncogene) binding protein (FBP) is a cellular factor that we have identified as a binder of HCV 3′ nontranslated region (3′NTR). Mapping of the binding site showed that FBP specifically interacts with the poly(U) tract within the poly(U/UC) region of the 3′NTR. Silencing of FBP expression by small interfering RNA in cells carrying HCV subgenomic replicons severely reduced viral replication, while overexpression of FBP significantly enhanced viral replication. We confirmed these observations by an in vitro HCV replication assay in the cell-free replicative lysate, which suggested that there is a direct correlation between the cellular FBP level and HCV replication. FBP immunoprecipitation coprecipitated HCV nonstructural protein 5A (NS5A), indicating that FBP interacts with HCV NS5A, which is known to function as a link between HCV translation and replication. Although FBP is mainly localized in the nucleus, we found that in MH14 cells a significant level of this protein is colocalized with NS5A in the cytosol, a site of HCV replication. While the mechanism of FBP involvement in HCV replication is yet to be delineated, our findings suggest that it may be an important regulatory component that is essential for efficient replication of HCV.

scholarly journals Serine 235 Is the Primary NS5A Hyperphosphorylation Site Responsible for Hepatitis C Virus Replication

2017 ◽  
Vol 91 (14) ◽  
Author(s):  
Shih-Chin Hsu ◽  
Chieh-Wen Lo ◽  
Ting-Chun Pan ◽  
Kuan-Ying Lee ◽  
Ming-Jiun Yu
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Replication ◽  
Nonstructural Protein ◽  
Genetic Mutation ◽  
Serine Residue ◽  
Phosphorylation Cascade ◽  
Potential Interactions ◽  
First Time ◽  
Nonstructural Protein 5A

ABSTRACT The nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) is a phosphoprotein with two phosphorylation states: hypo- and hyperphosphorylation. Genetic mutation studies have demonstrated a cluster of serine residues responsible for NS5A hyperphosphorylation and functions in viral replication and assembly; however, the phosphorylation levels and potential interactions among the serine residues are unclear. We used three specific antibodies to measure NS5A phosphorylation at S222, S235, and S238 that were identified in our previous proteomics study. In the HCV (J6/JFH-1)-infected Huh7.5.1 cells, S222 phosphorylation was barely detected, whereas S235 phosphorylation and S238 phosphorylation were always detected in parallel in time and intracellular spaces. S235A mutation eliminated S238 phosphorylation whereas S238A mutation did not affect S235 phosphorylation, indicating that S235 phosphorylation occurs independently of S238 phosphorylation while S238 phosphorylation depends on S235 phosphorylation. In line with this, immunoprecipitation coupled with immunoblotting showed that S235 phosphorylation existed alone without S238 phosphorylation, whereas S238 phosphorylation existed only when S235 was phosphorylated on the same NS5A molecule. S235-phosphorylated NS5A constituted the primary hyperphosphorylated NS5A species. S235A mutation blunted viral replication, whereas S238A mutation did not affect replication. We concluded that S235 is the primary NS5A hyperphosphorylation site required for HCV replication. S238 is likely phosphorylated by casein kinase Iα, which requires a priming phosphorylation at S235. IMPORTANCE It has been known for years that the hepatitis C virus nonstructural protein 5A (NS5A) undergoes transition between two phosphorylation states: hypo- and hyperphosphorylation. It is also known that a cluster of serine residues is responsible for NS5A hyperphosphorylation and functions; however, the primary serine residue responsible for NS5A hyperphosphorylation is not clear. Here, we show for the first time that serine 235-phosphorylated NS5A constitutes the primary hyperphosphorylated NS5A species required for viral replication. We also show that NS5A phosphorylation among the serine residues is interdependent and occurs in a directional manner, i.e., phosphorylation at serine 235 leads to phosphorylation at serine 238. Our data provide the first proof-of-principle evidence that NS5A undergoes a sequential phosphorylation cascade.

scholarly journals All Three Domains of the Hepatitis C Virus Nonstructural NS5A Protein Contribute to RNA Binding

2010 ◽  
Vol 84 (18) ◽  
pp. 9267-9277 ◽  
Author(s):  
Toshana L. Foster ◽  
Tamara Belyaeva ◽  
Nicola J. Stonehouse ◽  
Arwen R. Pearson ◽  
Mark Harris
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Binding Capacity ◽  
Specific Interaction ◽  
Genome Replication ◽  
Mobility Shift ◽  
Positive Strand Rna

ABSTRACT The hepatitis C virus (HCV) nonstructural protein NS5A is critical for viral genome replication and is thought to interact directly with both the RNA-dependent RNA polymerase, NS5B, and viral RNA. NS5A consists of three domains which have, as yet, undefined roles in viral replication and assembly. In order to define the regions that mediate the interaction with RNA, specifically the HCV 3′ untranslated region (UTR) positive-strand RNA, constructs of different domain combinations were cloned, bacterially expressed, and purified to homogeneity. Each of these purified proteins was probed for its ability to interact with the 3′ UTR RNA using filter binding and gel electrophoretic mobility shift assays, revealing differences in their RNA binding efficiencies and affinities. A specific interaction between domains I and II of NS5A and the 3′ UTR RNA was identified, suggesting that these are the RNA binding domains of NS5A. Domain III showed low in vitro RNA binding capacity. Filter binding and competition analyses identified differences between NS5A and NS5B in their specificities for defined regions of the 3′ UTR. The preference of NS5A, in contrast to NS5B, for the polypyrimidine tract highlights an aspect of 3′ UTR RNA recognition by NS5A which may play a role in the control or enhancement of HCV genome replication.

scholarly journals Sequential S232/S235/S238 Phosphorylation of the Hepatitis C Virus Nonstructural Protein 5A

2018 ◽  
Vol 92 (20) ◽  
Author(s):  
Shih-Chin Hsu ◽  
Chia-Ni Tsai ◽  
Kuan-Ying Lee ◽  
Ting-Chun Pan ◽  
Chieh-Wen Lo ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Direct Evidence ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Low Complexity ◽  
Phosphorylation Cascade ◽  
Binding Groove ◽  
Domain I ◽  
Nonstructural Protein 5A

ABSTRACTThe hepatitis C virus (HCV) protein NS5A is a phosphorylated protein with crucial roles in viral replication and assembly. NS5A was thought to undergo sequential phosphorylation on a series of conserved serine residues; however, the phosphorylation cascade remained obscure. Using three phosphorylation-specific antibodies, we found that phosphorylation at S232, S235, and S238 occurred in parallel in HCV-infected Huh7.5.1 cells, suggestive of intramolecular sequential NS5A phosphorylation from S232 through S235 to S238 by casein kinase Iα (CKIα). In line with this, alanine mutation at S225, S229, or S232 reduced, whereas aspartate mutation at the same sites rescued, NS5A phosphorylation at S232, S235, and S238. In contrast, alanine or aspartate mutation at S235 or S238 had little or no effect on S232 or S235 phosphorylation. Consistent with an intramolecular sequential phosphorylation cascade, S232, S235, and S238 phosphorylation coexisted on one single NS5A molecule. Phosphorylation of NH2-terminal serine residues in one NS5A molecule did not rescue phosphorylation of COOH-terminal serine residues in another NS5A molecule. CKIα inhibition reduced NS5A phosphorylation at S232, S235, and S238. In summary, our results are indicative of a CKIα-mediated intramolecular, sequential phosphorylation cascade from S232 through S235 to S238 of the HCV NS5A protein. S225 and S229 also contribute substantially to the above sequential phosphorylation cascade of NS5A.IMPORTANCEThe nonstructural protein 5A (NS5A) of the hepatitis C virus was thought to undergo sequential intramolecular phosphorylation on a series of serine residues; however, direct evidence was missing. We offer the first direct evidence of a CKIα-mediated intramolecular sequential NS5A phosphorylation cascade from serine 232 through 235 to 238. This sequential phosphorylation cascade occurs in the disordered low-complexity sequence I region, which together with the domain I region forms an RNA-binding groove in an NS5A dimer. Sequential phosphorylation in the disordered region adds charge-charge repulsion to the RNA-binding groove and probably thereby regulates NS5A's RNA-binding ability and functions in viral RNA replication and assembly.

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