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

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.

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Mutational analysis of hepatitis C virus NS3-associated helicase

Microbiology ◽

10.1099/0022-1317-81-7-1649 ◽

2000 ◽

Vol 81 (7) ◽

pp. 1649-1658 ◽

Cited By ~ 15

Author(s):

Chantal Paolini ◽

Armin Lahm ◽

Raffaele De Francesco ◽

Paola Gallinari

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Atpase Activity ◽

Mutational Analysis ◽

Nonstructural Protein ◽

Binding Capacity ◽

Molecular Model ◽

Helicase Domain ◽

Amino Acid Residues

Nonstructural protein 3 (NS3) of hepatitis C virus contains a bipartite structure consisting of an N-terminal serine protease and a C-terminal DEXH box helicase. To investigate the roles of individual amino acid residues in the overall mechanism of unwinding, a mutational–functional analysis was performed based on a molecular model of the NS3 helicase domain bound to ssDNA, which has largely been confirmed by a recently published crystal structure of the NS3 helicase–ssDNA complex. Three full-length mutated NS3 proteins containing Tyr(392)Ala, Val(432)Gly and Trp(501)Ala single substitutions, respectively, together with a Tyr(392)Ala/Trp(501)Ala double-substituted protein were expressed in Escherichia coli and purified to homogeneity. All individually mutated forms showed a reduction in duplex unwinding activity, single-stranded polynucleotide binding capacity and polynucleotide-stimulated ATPase activity compared to wild-type, though to different extents. Simultaneous replacement of both Tyr392 and Trp501 with Ala completely abolished all these enzymatic functions. On the other hand, the introduced amino acid substitutions had no influence on NS3 intrinsic ATPase activity and proteolytic efficiency. The results obtained with Trp(501)Ala and Val(432)Gly single-substituted enzymes are in agreement with a recently proposed model for NS3 unwinding activity. The mutant phenotype of the Tyr(392)Ala and Tyr(392)Ala/Trp(501)Ala enzymes, however, represents a completely novel finding.

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The Arginine-1493 Residue in QRRGRTGR1493G Motif IV of the Hepatitis C Virus NS3 Helicase Domain Is Essential for NS3 Protein Methylation by the Protein Arginine Methyltransferase 1

Journal of Virology ◽

10.1128/jvi.75.17.8031-8044.2001 ◽

2001 ◽

Vol 75 (17) ◽

pp. 8031-8044 ◽

Cited By ~ 38

Author(s):

Jaerang Rho ◽

Seeyoung Choi ◽

Young Rim Seong ◽

Joonho Choi ◽

Dong-Soo Im

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Binding ◽

Rna Helicase ◽

Full Length ◽

Arginine Residue ◽

Protein Methylation ◽

Helicase Domain ◽

Rna Helicases ◽

Ns3 Protein

ABSTRACT The NS3 protein of hepatitis C virus (HCV) contains protease and RNA helicase activities, both of which are likely to be essential for HCV propagation. An arginine residue present in the arginine-glycine (RG)-rich region of many RNA-binding proteins is posttranslationally methylated by protein arginine methyltransferases (PRMTs). Amino acid sequence analysis revealed that the NS3 protein contains seven RG motifs, including two potential RG motifs in the 1486-QRRGRTGRG-1494 motif IV of the RNA helicase domain, in which arginines are potentially methylated by PRMTs. Indeed, we found that the full-length NS3 protein is arginine methylated in vivo. The full-length NS3 protein and the NS3 RNA helicase domain were methylated by a crude human cell extract. The purified PRMT1 methylated the full-length NS3 and the RNA helicase domain, but not the NS3 protease domain. The NS3 helicase bound specifically and comigrated with PRMT1 in vitro. Mutational analyses indicate that the Arg1493 in the QRR1488GRTGR1493G region of the NS3 RNA helicase is essential for NS3 protein methylation and that Arg1488 is likely methylated. NS3 protein methylation by the PRMT1 was decreased in the presence of homoribopolymers, suggesting that the arginine-rich motif IV is involved in RNA binding. The results suggest that an arginine residue(s) in QRXGRXGR motif IV conserved in the virus-encoded RNA helicases can be posttranslationally methylated by the PRMT1.

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Structure-Based Mutagenesis Study of Hepatitis C Virus NS3 Helicase

Journal of Virology ◽

10.1128/jvi.73.10.8798-8807.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8798-8807 ◽

Cited By ~ 66

Author(s):

Chao Lin ◽

Joseph L. Kim

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Nucleic Acid ◽

Atpase Activity ◽

Rna Binding ◽

Crystallographic Structure ◽

Binary Complex ◽

Helicase Domain ◽

Wild Type ◽

Ns3 Protein

ABSTRACT The NS3 protein of hepatitis C virus (HCV) is a bifunctional protein containing a serine protease in the N-terminal one-third, which is stimulated upon binding of the NS4A cofactor, and an RNA helicase in the C-terminal two-thirds. In this study, a C-terminal hexahistidine-tagged helicase domain of the HCV NS3 protein was expressed in Escherichia coli and purified to homogeneity by conventional chromatography. The purified HCV helicase domain has a basal ATPase activity, a polynucleotide-stimulated ATPase activity, and a nucleic acid unwinding activity and binds efficiently to single-stranded polynucleotide. Detailed characterization of the purified HCV helicase domain with regard to all four activities is presented. Recently, we published an X-ray crystallographic structure of a binary complex of the HCV helicase with a (dU)8oligonucleotide, in which several conserved residues of the HCV helicase were shown to be involved in interactions between the HCV helicase and oligonucleotide. Here, site-directed mutagenesis was used to elucidate the roles of these residues in helicase function. Four individual mutations, Thr to Ala at position 269, Thr to Ala at position 411, Trp to Leu at position 501, and Trp to Ala at position 501, produced a severe reduction of RNA binding and completely abolished unwinding activity and stimulation of ATPase activity by poly(U), although the basal ATPase activity (activity in the absence of polynucleotide) of these mutants remained intact. Alanine substitution at Ser-231 or Ser-370 resulted in enzymes that were indistinguishable from wild-type HCV helicase with regard to all four activities. A mutant bearing Phe at Trp-501 showed wild-type levels of basal ATPase, unwinding activity, and single-stranded RNA binding activity. Interestingly, ATPase activity of this mutant became less responsive to stimulation by poly(U) but not to stimulation by other polynucleotides, such as poly(C). Given the conservation of some of these residues in other DNA and RNA helicases, their role in the mechanism of unwinding of double-stranded nucleic acid is discussed.

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Structural insights into RNA recognition by the Chikungunya virus nsP2 helicase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1900656116 ◽

2019 ◽

Vol 116 (19) ◽

pp. 9558-9567 ◽

Cited By ~ 11

Author(s):

Yee-Song Law ◽

Age Utt ◽

Yaw Bia Tan ◽

Jie Zheng ◽

Sainan Wang ◽

...

Keyword(s):

Atpase Activity ◽

Chikungunya Virus ◽

Rna Binding ◽

Nonstructural Protein ◽

Rna Helicase ◽

Viral Rna ◽

Helicase Domain ◽

Stacking Interactions ◽

Adaptive Mutations ◽

Rna Backbone

Chikungunya virus (CHIKV) is transmitted to humans through mosquitoes and causes Chikungunya fever. Nonstructural protein 2 (nsP2) exhibits the protease and RNA helicase activities that are required for viral RNA replication and transcription. Unlike for the C-terminal protease, the structure of the N-terminal RNA helicase (nsP2h) has not been determined. Here, we report the crystal structure of the nsP2h bound to the conserved 3′-end 14 nucleotides of the CHIKV genome and the nonhydrolyzable transition-state nucleotide analog ADP-AlF4. Overall, the structural analysis revealed that nsP2h adopts a uniquely folded N-terminal domain followed by a superfamily 1 RNA helicase fold. The conserved helicase motifs establish polar contacts with the RNA backbone. There are three hydrophobic residues (Y161, F164, and F287) which form stacking interactions with RNA bases and thereby bend the RNA backbone. An F287A substitution that disrupted these stacking interactions increased the basal ATPase activity but decreased the RNA binding affinity. Furthermore, the F287A substitution reduced viral infectivity by attenuating subgenomic RNA synthesis. Replication of the mutant virus was restored by pseudoreversion (A287V) or adaptive mutations in the RecA2 helicase domain (T358S or V410I). Y161A and/or F164A substitutions, which were designed to disrupt the interactions with the RNA molecule, did not affect the ATPase activity but completely abolished the replication and transcription of viral RNA and the infectivity of CHIKV. Our study sheds light on the roles of the RNA helicase region in viral replication and provides insights that might be applicable to alphaviruses and other RNA viruses in general.

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Characterization of RNA Binding Activity and RNA Helicase Activity of the Hepatitis C Virus NS3 Protein

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1996.1225 ◽

1996 ◽

Vol 225 (2) ◽

pp. 654-659 ◽

Cited By ~ 105

Author(s):

Yousang Gwack ◽

Dong Wook Kim ◽

Jang H. Han ◽

Joonho Choe

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Binding ◽

Rna Helicase ◽

Binding Activity ◽

Helicase Activity ◽

Ns3 Protein

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The NS4A Protein of Hepatitis C Virus Promotes RNA-Coupled ATP Hydrolysis by the NS3 Helicase

Journal of Virology ◽

10.1128/jvi.01849-08 ◽

2009 ◽

Vol 83 (7) ◽

pp. 3268-3275 ◽

Cited By ~ 46

Author(s):

Rudolf K. F. Beran ◽

Brett D. Lindenbach ◽

Anna Marie Pyle

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Serine Protease ◽

Protease Activity ◽

Rna Binding ◽

Nonstructural Protein ◽

Helicase Activity ◽

Protease Domain

ABSTRACT Nonstructural protein 3 (NS3) is an essential replicative component of the hepatitis C virus (HCV) and a member of the DExH/D-box family of proteins. The C-terminal region of NS3 (NS3hel) exhibits RNA-stimulated NTPase and helicase activity, while the N-terminal serine protease domain of NS3 enhances RNA binding and unwinding by NS3hel. The nonstructural protein 4A (NS4A) binds to the NS3 protease domain and serves as an obligate cofactor for NS3 serine protease activity. Given its role in stimulating protease activity, we sought to determine whether NS4A also influences the activity of NS3hel. Here we show that NS4A enhances the ability of NS3hel to bind RNA in the presence of ATP, thereby acting as a cofactor for helicase activity. This effect is mediated by amino acids in the C-terminal acidic domain of NS4A. When these residues are mutated, one observes drastic reductions in ATP-coupled RNA binding and duplex unwinding by NS3. These same mutations are lethal in HCV replicons, thereby establishing in vitro and in vivo that NS4A plays an important role in the helicase mechanism of NS3 and its function in replication.

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

Journal of Virology ◽

10.1128/jvi.74.20.9732-9737.2000 ◽

2000 ◽

Vol 74 (20) ◽

pp. 9732-9737 ◽

Cited By ~ 17

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.

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Regulating Intracellular Antiviral Defense and Permissiveness to Hepatitis C Virus RNA Replication through a Cellular RNA Helicase, RIG-I

Journal of Virology ◽

10.1128/jvi.79.5.2689-2699.2005 ◽

2005 ◽

Vol 79 (5) ◽

pp. 2689-2699 ◽

Cited By ~ 641

Author(s):

Rhea Sumpter ◽

Yueh-Ming Loo ◽

Eileen Foy ◽

Kui Li ◽

Mitsutoshi Yoneyama ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Viral Infections ◽

Cultured Cells ◽

Rna Replication ◽

Rna Helicase ◽

Hcv Rna ◽

Helicase Domain ◽

Replication Studies ◽

Virus Rna

ABSTRACT Virus-responsive signaling pathways that induce alpha/beta interferon production and engage intracellular immune defenses influence the outcome of many viral infections. The processes that trigger these defenses and their effect upon host permissiveness for specific viral pathogens are not well understood. We show that structured hepatitis C virus (HCV) genomic RNA activates interferon regulatory factor 3 (IRF3), thereby inducing interferon in cultured cells. This response is absent in cells selected for permissiveness for HCV RNA replication. Studies including genetic complementation revealed that permissiveness is due to mutational inactivation of RIG-I, an interferon-inducible cellular DExD/H box RNA helicase. Its helicase domain binds HCV RNA and transduces the activation signal for IRF3 by its caspase recruiting domain homolog. RIG-I is thus a pathogen receptor that regulates cellular permissiveness to HCV replication and, as an interferon-responsive gene, may play a key role in interferon-based therapies for the treatment of HCV infection.

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A minimal region in the NTPase/helicase domain of the TGBp1 plant virus movement protein is responsible for ATPase activity and cooperative RNA binding

Journal of General Virology ◽

10.1099/vir.0.81971-0 ◽

2006 ◽

Vol 87 (10) ◽

pp. 3087-3095 ◽

Cited By ~ 33

Author(s):

Anna D. Leshchiner ◽

Andrey G. Solovyev ◽

Sergey Yu. Morozov ◽

Natalia O. Kalinina

Keyword(s):

Amino Acid ◽

Atpase Activity ◽

Amino Acid Residue ◽

Plant Virus ◽

Rna Binding ◽

Triple Gene Block ◽

Basic Amino Acid ◽

Helicase Domain ◽

Basic Amino Acid Residue ◽

Conserved Sequence

The TGBp1 protein, encoded in the genomes of a number of plant virus genera as the first gene of the ‘triple gene block’, possesses an NTPase/helicase domain characterized by seven conserved sequence motifs. It has been shown that the TGBp1 NTPase/helicase domain exhibits NTPase, RNA helicase and RNA-binding activities. In this paper, we have analysed a series of deletion and point mutants in the TGBp1 proteins encoded by Potato virus X (PVX, genus Potexvirus) and Poa semilatent virus (PSLV, genus Hordeivirus) to map functional regions responsible for their biochemical activities in vitro. It was found that, in both PVX and PSLV, the N-terminal part of the TGBp1 NTPase/helicase domain comprising conserved motifs I, Ia and II was sufficient for ATP hydrolysis, RNA binding and homologous protein–protein interactions. Point mutations in a single conserved basic amino acid residue upstream of motif I had little effect on the activities of C-terminally truncated mutants of both TGBp1 proteins. However, when introduced into the full-length NTPase/helicase domains, these mutations caused a substantial decrease in the ATPase activity of the protein, suggesting that the conserved basic amino acid residue upstream of motif I was required to maintain a reaction-competent conformation of the TGBp1 ATPase active site.

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