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 NS5A Domain 1 and Polyprotein Cleavage Kinetics Are Critical for Induction of Double-Membrane Vesicles Associated with Hepatitis C Virus Replication

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Inés Romero-Brey ◽  
Carola Berger ◽  
Stephanie Kallis ◽  
Androniki Kolovou ◽  
David Paul ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Viruses ◽  
Nonstructural Protein ◽  
Membrane Vesicles ◽  
Rna Replication ◽  
Concerted Action ◽  
Double Membrane ◽  
Positive Strand ◽  
Positive Strand Rna

ABSTRACTInduction of membrane rearrangements in the cytoplasm of infected cells is a hallmark of positive-strand RNA viruses. These altered membranes serve as scaffolds for the assembly of viral replication factories (RFs). We have recently shown that hepatitis C virus (HCV) infection induces endoplasmic reticulum-derived double-membrane vesicles (DMVs) representing the major constituent of the RF within the infected cell. RF formation requires the concerted action of nonstructural action of nonstructural protein (NS)3, -4A, protein (NS)3 -4A, -4B, -5A, and -5B. Although the sole expression of NS5A is sufficient to induce DMV formation, its efficiency is very low. In this study, we dissected the determinants within NS5A responsible for DMV formation and found that RNA-binding domain 1 (D1) and the amino-terminal membrane anchor are indispensable for this process. In contrast, deletion of NS5A D2 or D3 did not affect DMV formation but disrupted RNA replication and virus assembly, respectively. To identifycis- andtrans-acting factors of DMV formation, we established atranscleavage assay. We found that induction of DMVs requires full-length NS3, whereas a helicase-lacking mutant was unable to trigger DMV formation in spite of efficient polyprotein cleavage. Importantly, a mutation accelerating cleavage kinetics at the NS4B-5A site diminished DMV formation, while the insertion of an internal ribosome entry site mimicking constitutive cleavage at this boundary completely abolished this process. These results identify key determinants governing the biogenesis of the HCV RF with possible implications for our understanding of how RFs are formed in other positive-strand RNA viruses.IMPORTANCELike all positive-strand RNA viruses, hepatitis C virus (HCV) extensively reorganizes intracellular membranes to allow efficient RNA replication. Double-membrane vesicles (DMVs) that putatively represent sites of HCV RNA amplification are induced by the concerted action of viral and cellular factors. However, the contribution of individual proteins to this process remains poorly understood. Here we identify determinants in the HCV replicase that are required for DMV biogenesis. Major contributors to this process are domain 1 of nonstructural protein 5A and the helicase domain of nonstructural protein 3. In addition, efficient DMV induction depends onciscleavage of the viral polyprotein, as well as tightly regulated cleavage kinetics. These results identify key determinants governing the biogenesis of the HCV replication factory with possible implications for our understanding of how this central compartment is formed in other positive-strand RNA viruses.

scholarly journals Template Requirements for De Novo RNA Synthesis by Hepatitis C Virus Nonstructural Protein 5B Polymerase on the Viral X RNA

2002 ◽  
Vol 76 (14) ◽  
pp. 6944-6956 ◽  
Author(s):  
Meehyein Kim ◽  
Hajeong Kim ◽  
Seong-Pil Cho ◽  
Mi-Kyung Min
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Synthesis ◽  
De Novo ◽  
Nonstructural Protein ◽  
Initiation Site ◽  
Viral Rna ◽  
Positive Strand ◽  
Esherichia Coli ◽  
Positive Strand Rna

ABSTRACT The hepatitis C virus (HCV)-encoded NS5B protein is an RNA-dependent RNA polymerase which plays a substantial role in viral replication. We expressed and purified the recombinant NS5B of an HCV genotype 3a from Esherichia coli, and we investigated its ability to bind to the viral RNA and its enzymatic activity. The results presented here demonstrate that NS5B interacts strongly with the coding region of positive-strand RNA, although not in a sequence-specific manner. It was also determined that more than two molecules of polymerase bound sequentially to this region with the direction 3′ to 5′. Also, we attempted to determine the initiation site(s) of de novo synthesis by NS5B on X RNA, which contains the last 98 nucleotides of HCV positive-strand RNA. The initiation site(s) on X RNA was localized in the pyrimidine-rich region of stem I. However, when more than five of the nucleotides of stem I in X RNA were deleted from the 3′ end, RNA synthesis initiated at another site of the specific ribonucleotide. Our study also showed that the efficiency of RNA synthesis, which was directed by X RNA, was maximized by the GC base pair at the penultimate position from the 3′ end of the stem. These results will provide some clues to understanding the mechanism of HCV genomic RNA replication in terms of viral RNA-NS5B interaction and the initiation of de novo RNA synthesis.

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

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

2012 ◽  
Vol 32 (26) ◽  
pp. 8865-8870 ◽  
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

scholarly journals Cleavage of Hepatitis C Virus Nonstructural Protein 5A by a Caspase-like Protease(s) in Mammalian Cells

Virology ◽  
2000 ◽  
Vol 270 (2) ◽  
pp. 476-487 ◽  
Author(s):  
Shinya Satoh ◽  
Masami Hirota ◽  
Tohru Noguchi ◽  
Makoto Hijikata ◽  
Hiroshi Handa ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mammalian Cells ◽  
Nonstructural Protein ◽  
Nonstructural Protein 5A

scholarly journals Nonstructural Protein 5A Is Incorporated into Hepatitis C Virus Low-Density Particle through Interaction with Core Protein and Microtubules during Intracellular Transport

PLoS ONE ◽  
2014 ◽  
Vol 9 (6) ◽  
pp. e99022 ◽  
Author(s):  
Chao-Kuen Lai ◽  
Vikas Saxena ◽  
Chung-Hsin Tseng ◽  
King-Song Jeng ◽  
Michinori Kohara ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Intracellular Transport ◽  
Core Protein ◽  
Nonstructural Protein ◽  
Low Density ◽  
Nonstructural Protein 5A

scholarly journals Identification of Determinants Involved in Initiation of Hepatitis C Virus RNA Synthesis by Using Intergenotypic Replicase Chimeras

2007 ◽  
Vol 81 (10) ◽  
pp. 5270-5283 ◽  
Author(s):  
Marco Binder ◽  
Doris Quinkert ◽  
Olga Bochkarova ◽  
Rahel Klein ◽  
Nikolina Kezmic ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Synthesis ◽  
Nonstructural Protein ◽  
Negative Strand ◽  
Virus Rna ◽  
Nontranslated Region ◽  
Genotype 2 ◽  
Positive Strand Rna

ABSTRACT The 5′ nontranslated region (NTR) and the X tail in the 3′ NTR are the least variable parts of the hepatitis C virus (HCV) genome and play an important role in the initiation of RNA synthesis. By using subgenomic replicons of the HCV isolates Con1 (genotype 1) and JFH1 (genotype 2), we characterized the genotype specificities of the replication signals contained in the NTRs. The replacement of the JFH1 5′ NTR and X tail with the corresponding Con1 sequence resulted in a significant decrease in replication efficiency. Exchange of the X tail specifically reduced negative-strand synthesis, whereas substitution of the 5′ NTR impaired the generation of progeny positive strands. In search for the proteins involved in the recognition of genotype-specific initiation signals, we analyzed recombinant nonstructural protein 5B (NS5B) RNA polymerases of both isolates and found some genotype-specific template preference for the 3′ end of positive-strand RNA in vitro. To further address genotype specificity, we constructed a series of intergenotypic replicon chimeras. When combining NS3 to NS5A of Con1 with NS5B of JFH1, we observed more-efficient replication with the genotype 2a X tail, indicating that NS5B recognizes genotype-specific signals in this region. In contrast, a combination of the NS3 helicase with NS5A and NS5B was required to confer genotype specificity to the 5′ NTR. These results present the first genetic evidence for an interaction between helicase, NS5A, and NS5B required for the initiation of RNA synthesis and provide a system for the specific analysis of HCV positive- and negative-strand syntheses.

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