The Amino Terminal Deletion Mutants of Hepatitis C Virus Nonstructural Protein NS5A Function as Transcriptional Activators in Yeast

1997 ◽  
Vol 236 (2) ◽  
pp. 360-364 ◽  
Author(s):  
Akihide Tanimoto ◽  
Yoshihiro Ide ◽  
Nobuyuki Arima ◽  
Yasuyuki Sasaguri ◽  
R. Padmanabhan
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nonstructural Protein ◽  
Terminal Deletion ◽  
Deletion Mutants ◽  
Transcriptional Activators ◽  
Amino Terminal

scholarly journals Efficient Rescue of Hepatitis C Virus RNA Replication by trans-Complementation with Nonstructural Protein 5A

2005 ◽  
Vol 79 (2) ◽  
pp. 896-909 ◽  
Author(s):  
Nicole Appel ◽  
Ulrike Herian ◽  
Ralf Bartenschlager
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Functional Organization ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Hcv Rna ◽  
Replication Complex ◽  
Diarrhea Virus ◽  
Amino Terminal ◽  
Essential Components

ABSTRACT Studies of Hepatitis C virus (HCV) RNA replication have become possible with the development of subgenomic replicons. This system allows the functional analysis of the essential components of the viral replication complex, which so far are poorly defined. In the present study we wanted to investigate whether lethal mutations in HCV nonstructural genes can be rescued by trans-complementation. Therefore, a series of replicon RNAs carrying mutations in NS3, NS4B, NS5A, and NS5B that abolish replication were transfected into Huh-7 hepatoma cells harboring autonomously replicating helper RNAs. Similar to data described for the Bovine viral diarrhea virus (C. W. Grassmann, O. Isken, N. Tautz, and S. E. Behrens, J. Virol. 75:7791-7802, 2001), we found that only NS5A mutants could be efficiently rescued. There was no evidence for RNA recombination between helper and mutant RNAs, and we did not observe reversions in the transfected mutants. Furthermore, we established a transient complementation assay based on the cotransfection of helper and mutant RNAs. Using this assay, we extended our results and demonstrated that (i) inactivating NS5A mutations affecting the amino-terminal amphipathic helix cannot be complemented in trans; (ii) replication of the helper RNA is not necessary to allow efficient trans-complementation; and (iii) the minimal sequence required for trans-complementation of lethal NS5A mutations is NS3 to -5A, whereas NS5A expressed alone does not restore RNA replication. In summary, our results provide the first insight into the functional organization of the HCV replication complex.

scholarly journals Adaptive Mutations Producing Efficient Replication of Genotype 1a Hepatitis C Virus RNA in Normal Huh7 Cells

2004 ◽  
Vol 78 (15) ◽  
pp. 7904-7915 ◽  
Author(s):  
MinKyung Yi ◽  
Stanley M. Lemon
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Host Cell ◽  
Nonstructural Protein ◽  
Northern Analysis ◽  
Adaptive Mutations ◽  
Amino Terminal ◽  
Genotype 1A ◽  
Genotype 1B ◽  
Huh7 Cells

ABSTRACT Despite recent successes in generating subgenomic RNA replicons derived from genotype 1b strains of hepatitis C virus (HCV) that replicate efficiently in cultured cells, it has proven difficult to generate efficiently replicating RNAs from any other genotype of HCV. This includes genotype 1a, even though it is closely related to genotype 1b. We show here that an important restriction to replication of the genotype 1a H77c strain RNA in normal Huh7 cells resides within the amino-terminal 75 residues of the NS3 protease. We identified adaptive mutations located within this NS3 domain and within NS4A, in close proximity to the essential protease cofactor sequence, that act cooperative to substantially enhance the replication of this genotype 1a RNA in Huh7 cells. These and additional adaptive mutations, identified through a series of iterative transfections and the selection of G418-resistant cell clones, form two groups associating with distinct nonstructural protein domains: the NS3/4A protease and NS5A. A combination of mutations from both groups led to robust replication of otherwise unmodified H77c genomic RNA that was readily detectable by northern analysis within 4 days of transfection into Huh7 cells. We speculate that these adaptive mutations favorably influence assembly of the replicase complex with host cell-specific proteins, or alternatively promote interactions of NS3/4A and/or NS5A with cellular proteins involved in host cell antiviral defenses.

scholarly journals Hepatitis C Virus Nonstructural Protein 5A: Biochemical Characterization of a Novel Structural Class of RNA-Binding Proteins

2010 ◽  
Vol 84 (24) ◽  
pp. 12480-12491 ◽  
Author(s):  
Jungwook Hwang ◽  
Luyun Huang ◽  
Daniel G. Cordek ◽  
Robert Vaughan ◽  
Shelley L. Reynolds ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Binding Activity ◽  
Cross Linking ◽  
Amino Terminal ◽  
Rna Complex ◽  
Domain I ◽  
Nonstructural Protein 5A

ABSTRACT Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) exhibits a preference for G/U-rich RNA in vitro. Biological analysis of the NS5A RNA-binding activity and its target sites in the genome will be facilitated by a description of the NS5A-RNA complex. We demonstrate that the C-4 carbonyl of the uracil base and, by inference, the C-6 carbonyl of the guanine base interact with NS5A. U-rich RNA of 5 to 6 nucleotides (nt) is sufficient for high-affinity binding to NS5A. The minimal RNA-binding domain of NS5A consists of residues 2005 to 2221 (referred to as domain I-plus). This region of the protein includes the amino-terminal domain I as well as the subsequent linker that separates domains I and II. This linker region is the site of adaptive mutations. U-rich RNA-binding activity is not observed for an NS5A derivative containing only residues 2194 to 2419 (domains II and III). Mass spectrometric analysis of an NS5A-poly(rU) complex identified domains I and II as sites for interaction with RNA. Dimerization of NS5A was demonstrated by glutaraldehyde cross-linking. This dimerization is likely mediated by domain I-plus, as dimers of this protein are trapped by cross-linking. Dimers of the domain II-III protein are not observed. The monomer-dimer equilibrium of NS5A shifts in favor of dimer when U-rich RNA is present but not when A-rich RNA is present, consistent with an NS5A dimer being the RNA-binding-competent form of the protein. These data provide a molecular perspective of the NS5A-RNA complex and suggest possible mechanisms for regulation of HCV and cellular gene expression.

scholarly journals An Amino-terminal Amphipathic α-Helix Mediates Membrane Association of the Hepatitis C Virus Nonstructural Protein 5A

2001 ◽  
Vol 277 (10) ◽  
pp. 8130-8139 ◽  
Author(s):  
Volker Brass ◽  
Elke Bieck ◽  
Roland Montserret ◽  
Benno Wölk ◽  
Jan Albert Hellings ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nonstructural Protein ◽  
Membrane Association ◽  
Amino Terminal ◽  
Α Helix ◽  
Nonstructural Protein 5A

scholarly journals Role for TBC1D20 and Rab1 in Hepatitis C Virus Replication via Interaction with Lipid Droplet-Bound Nonstructural Protein 5A

2012 ◽  
Vol 86 (12) ◽  
pp. 6491-6502 ◽  
Author(s):  
I. Nevo-Yassaf ◽  
Y. Yaffe ◽  
M. Asher ◽  
O. Ravid ◽  
S. Eizenberg ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Replication ◽  
Lipid Droplet ◽  
Nonstructural Protein ◽  
Nonstructural Protein 5A

Mechanism of Zinc Ejection by Disulfiram in Nonstructural Protein 5A

2021 ◽  
Author(s):  
Ashfaq Ur Rehman ◽  
Guodong Zheng ◽  
Bozitao Zhong ◽  
Duan Ni ◽  
Jia-Yi Li ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nonstructural Protein ◽  
Structural Protein ◽  
Positive Strand ◽  
Positive Strand Rna ◽  
Nonstructural Protein 5A

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...

scholarly journals Hepatitis C Virus Deletion Mutants Are Found in Individuals Chronically Infected with Genotype 1 Hepatitis C Virus in Association with Age, High Viral Load and Liver Inflammatory Activity

PLoS ONE ◽  
2015 ◽  
Vol 10 (9) ◽  
pp. e0138546 ◽  
Author(s):  
Cristina Cheroni ◽  
Lorena Donnici ◽  
Alessio Aghemo ◽  
Francesca Balistreri ◽  
Annalisa Bianco ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Viral Load ◽  
Hepatitis C ◽  
High Viral Load ◽  
Inflammatory Activity ◽  
Deletion Mutants ◽  
Genotype 1

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.

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