Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

Hepatitis C virus (HCV) is a notorious member of the enveloped, positive-strand RNA flavivirus family. Non-structural protein 5A (NS5A) plays a key role in HCV replication and assembly. NS5A is...

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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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Hepatitis C virus-encoded nonstructural protein NS4A has versatile functions in viral protein processing.

Journal of Virology ◽

10.1128/jvi.69.3.1575-1581.1995 ◽

1995 ◽

Vol 69 (3) ◽

pp. 1575-1581 ◽

Cited By ~ 153

Author(s):

Y Tanji ◽

M Hijikata ◽

S Satoh ◽

T Kaneko ◽

K Shimotohno

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Viral Protein ◽

Nonstructural Protein ◽

Protein Processing

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Screening of genes differentially expressed in HepG2 cells transfected with gene 3 transactivated by hepatitis C virus nonstructural protein 5A (NS5ATP3) using cDNA microarray

World Chinese Journal of Digestology ◽

10.11569/wcjd.v12.i2.306 ◽

2004 ◽

Vol 12 (2) ◽

pp. 306

Author(s):

Yan Liu ◽

Qian Yang ◽

Jun Cheng ◽

Jian-Jun Wang ◽

Dong Ji ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cdna Microarray ◽

Hepg2 Cells ◽

Nonstructural Protein ◽

Differentially Expressed ◽

Nonstructural Protein 5A

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Regulation of Neuronal Proapoptotic Potassium Currents by the Hepatitis C Virus Nonstructural Protein 5A

Journal of Neuroscience ◽

10.1523/jneurosci.0937-12.2012 ◽

2012 ◽

Vol 32 (26) ◽

pp. 8865-8870 ◽

Cited By ~ 13

Author(s):

C. A. Norris ◽

K. He ◽

M. G. Springer ◽

K. A. Hartnett ◽

J. P. Horn ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Nonstructural Protein ◽

Potassium Currents ◽

Nonstructural Protein 5A

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Serine 229 Balances the Hepatitis C Virus Nonstructural Protein NS5A between Hypo- and Hyperphosphorylated States

Journal of Virology ◽

10.1128/jvi.01028-19 ◽

2019 ◽

Vol 93 (23) ◽

Cited By ~ 1

Author(s):

Chia-Ni Tsai ◽

Ting-Chun Pan ◽

Cho-Han Chiang ◽

Chun-Chiao Yu ◽

Shih-Han Su ◽

...

Keyword(s):

Life Cycle ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Nonstructural Protein ◽

Feedback Regulation ◽

Wild Type ◽

Serine Residue ◽

Large Structures ◽

Phosphorylation Level ◽

In The Wild

ABSTRACT The nonstructural protein NS5A of hepatitis C virus (HCV) is a phosphorylated protein that is indispensable for viral replication and assembly. We previously showed that NS5A undergoes sequential serine S232/S235/S238 phosphorylation resulting in NS5A transition from a hypo- to a hyperphosphorylated state. Here, we studied functions of S229 with a newly generated antibody specific to S229 phosphorylation. In contrast to S232, S235, or S238 phosphorylation detected only in the hyperphosphorylated NS5A, S229 phosphorylation was found in both hypo- and hyperphosphorylated NS5A, suggesting that S229 phosphorylation initiates NS5A sequential phosphorylation. Immunoblotting showed an inverse relationship between S229 phosphorylation and S235 phosphorylation. When S235 was phosphorylated as in the wild-type NS5A, the S229 phosphorylation level was low; when S235 could not be phosphorylated as in the S235A mutant NS5A, the S229 phosphorylation level was high. These results suggest an intrinsic feedback regulation between S229 phosphorylation and S235 phosphorylation. It has been known that NS5A distributes in large static and small dynamic intracellular structures and that both structures are required for the HCV life cycle. We found that S229A or S229D mutation was lethal to the virus and that both increased NS5A in large intracellular structures. Similarly, the lethal S235A mutation also increased NS5A in large structures. Likewise, the replication-compromised S235D mutation also increased NS5A in large structures, albeit to a lesser extent. Our data suggest that S229 probably cycles through phosphorylation and dephosphorylation to maintain a delicate balance of NS5A between hypo- and hyperphosphorylated states and the intracellular distribution necessary for the HCV life cycle. IMPORTANCE This study joins our previous efforts to elucidate how NS5A transits between hypo- and hyperphosphorylated states via phosphorylation on a series of highly conserved serine residues. Of the serine residues, serine 229 is the most interesting since phosphorylation-mimicking and phosphorylation-ablating mutations at this serine residue are both lethal. With a new high-quality antibody specific to serine 229 phosphorylation, we concluded that serine 229 must remain wild type so that it can dynamically cycle through phosphorylation and dephosphorylation that govern NS5A between hypo- and hyperphosphorylated states. Both are required for the HCV life cycle. When phosphorylated, serine 229 signals phosphorylation on serine 232 and 235 in a sequential manner, leading NS5A to the hyperphosphorylated state. As serine 235 phosphorylation is reached, serine 229 is dephosphorylated, stopping signal for hyperphosphorylation. This balances NS5A between two phosphorylation states and in intracellular structures that warrant a productive HCV life cycle.

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Pharmacokinetic Interactions between the Hepatitis C Virus Inhibitors Elbasvir and Grazoprevir and HIV Protease Inhibitors Ritonavir, Atazanavir, Lopinavir, and Darunavir in Healthy Volunteers

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02142-18 ◽

2019 ◽

Vol 63 (4) ◽

Cited By ~ 3

Author(s):

Hwa-Ping Feng ◽

Luzelena Caro ◽

Christine Fandozzi ◽

Xiaoyan Chu ◽

Zifang Guo ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Protease Inhibitors ◽

Nonstructural Protein ◽

Phase 1 ◽

Geometric Mean ◽

Hiv Treatment ◽

Hiv Protease ◽

Hiv Protease Inhibitors ◽

Pharmacokinetic Interactions

ABSTRACT The combination of the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor elbasvir and the NS3/4A protease inhibitor grazoprevir is a potent, once-daily therapy indicated for the treatment of chronic HCV infection in individuals coinfected with human immunodeficiency virus (HIV). We explored the pharmacokinetic interactions of elbasvir and grazoprevir with ritonavir and ritonavir-boosted HIV protease inhibitors in three phase 1 trials. Drug-drug interaction trials with healthy participants were conducted to evaluate the effect of ritonavir on the pharmacokinetics of grazoprevir (n = 10) and the potential two-way pharmacokinetic interactions of elbasvir (n = 30) or grazoprevir (n = 39) when coadministered with ritonavir-boosted atazanavir, lopinavir, or darunavir. Coadministration of ritonavir with grazoprevir increased grazoprevir exposure; the geometric mean ratio (GMR) for grazoprevir plus ritonavir versus grazoprevir alone area under the concentration-time curve from 0 to 24 h (AUC0–24) was 1.91 (90% confidence interval [CI]; 1.31 to 2.79). Grazoprevir exposure was markedly increased with coadministration of atazanavir-ritonavir, lopinavir-ritonavir, and darunavir-ritonavir, with GMRs for grazoprevir AUC0–24 of 10.58 (90% CI, 7.78 to 14.39), 12.86 (90% CI, 10.25 to 16.13), and 7.50 (90% CI, 5.92 to 9.51), respectively. Elbasvir exposure was increased with coadministration of atazanavir-ritonavir, lopinavir-ritonavir, and darunavir-ritonavir, with GMRs for elbasvir AUC0–24 of 4.76 (90% CI, 4.07 to 5.56), 3.71 (90% CI, 3.05 to 4.53), and 1.66 (90% CI, 1.35 to 2.05), respectively. Grazoprevir and elbasvir had little effect on atazanavir, lopinavir, and darunavir pharmacokinetics. Coadministration of elbasvir-grazoprevir with atazanavir-ritonavir, lopinavir-ritonavir, or darunavir-ritonavir is contraindicated, owing to an increase in grazoprevir exposure. Therefore, HIV treatment regimens without HIV protease inhibitors should be considered for HCV/HIV-coinfected individuals who are being treated with elbasvir-grazoprevir.

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Characterization of Soluble Hepatitis C Virus RNA-Dependent RNA Polymerase Expressed in Escherichia coli

Journal of Virology ◽

10.1128/jvi.73.2.1649-1654.1999 ◽

1999 ◽

Vol 73 (2) ◽

pp. 1649-1654 ◽

Cited By ~ 172

Author(s):

Eric Ferrari ◽

Jacquelyn Wright-Minogue ◽

Jane W. S. Fang ◽

Bahige M. Baroudy ◽

Johnson Y. N. Lau ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Nonstructural Protein ◽

Full Length ◽

Dependent Manner ◽

Rdrp Activity ◽

Rna Dependent Rna Polymerase ◽

Hydrophobic Domain ◽

Hcv Ns5b

ABSTRACT Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BΔCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.

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