scholarly journals Mutational Analysis of Hepatitis C Virus Nonstructural Protein 5A: Potential Role of Differential Phosphorylation in RNA Replication and Identification of a Genetically Flexible Domain

2005 ◽  
Vol 79 (5) ◽  
pp. 3187-3194 ◽  
Author(s):  
Nicole Appel ◽  
Thomas Pietschmann ◽  
Ralf Bartenschlager
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mutational Analysis ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Adaptive Mutations ◽  
Phosphorylation Pattern ◽  
Molecular Masses ◽  
Nonstructural Protein 5A

ABSTRACT Nonstructural protein 5A of the hepatitis C virus (HCV) is a highly phosphorylated molecule implicated in multiple interactions with the host cell and most likely involved in RNA replication. Two phosphorylated variants of NS5A have been described, designated according to their apparent molecular masses (in kilodaltons) as p56 and p58, which correspond to the basal and hyperphosphorylated forms, respectively. With the aim of identifying a possible role of NS5A phosphorylation for RNA replication, we performed an extensive mutation analysis of three serine clusters that are involved in phosphorylation and hyperphosphorylation of NS5A. In most cases, alanine substitutions for serine residues in the central cluster 1 that enhanced RNA replication to the highest levels led to a reduction of NS5A hyperphosphorylation. Likewise, several highly adaptive mutations in NS4B, which is also part of the replication complex, resulted in a reduction of NS5A hyperphosphorylation too, arguing that alterations of the NS5A phosphorylation pattern play an important role for RNA replication. On the other hand, a deletion encompassing all highly conserved serine residues in the C-terminal region of NS5A that are involved in basal phosphorylation did not significantly affect RNA replication but reduced formation of p56. This region was found to tolerate even large insertions with only a moderate effect on replication. Based on these results, we propose a model of the role of NS5A phosphorylation in the viral life cycle.

scholarly journals Insertion of Green Fluorescent Protein into Nonstructural Protein 5A Allows Direct Visualization of Functional Hepatitis C Virus Replication Complexes

2004 ◽  
Vol 78 (14) ◽  
pp. 7400-7409 ◽  
Author(s):  
Darius Moradpour ◽  
Matthew J. Evans ◽  
Rainer Gosert ◽  
Zhenghong Yuan ◽  
Hubert E. Blum ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Green Fluorescent Protein ◽  
Hepatitis C ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Direct Visualization ◽  
Green Fluorescent ◽  
Nonstructural Protein 5A

ABSTRACT Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.

scholarly journals Nonstructural Protein 5A of Hepatitis C Virus Inhibits the Function of Karyopherin β3

2000 ◽  
Vol 74 (11) ◽  
pp. 5233-5241 ◽  
Author(s):  
Kyung Min Chung ◽  
Juhang Lee ◽  
Jung-Eun Kim ◽  
Ok-Kyu Song ◽  
Sungchan Cho ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Interaction ◽  
Double Mutant ◽  
Nonstructural Protein ◽  
Protein Protein Interaction ◽  
Human Liver Cell ◽  
Mutant Cells ◽  
Nonstructural Protein 5A

ABSTRACT It has been suggested that nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) plays a role in the incapacitation of interferon by inactivation of RNA-dependent protein kinase PKR. In order to further investigate the role of NS5A, we tried to identify cellular proteins interacting with NS5A by using the yeast two-hybrid system. The karyopherin β3 gene was isolated from a human liver cell library as a protein interacting with NS5A. The protein-protein interaction between NS5A and karyopherin β3 was confirmed by in vitro binding assay and an in vivo coimmunoprecipitation method. The effect of NS5A on the karyopherin β3 activity was investigated using a yeast cell line containing mutations in both PSE1 andKAP123, genes that are homologous to the human karyopherin β3 gene. Human karyopherin β3 complemented the loss of thePSE1 and KAP123 functions, supporting growth of the double mutant cells. However, expression of NS5A hampered the growth of the double mutant cells supplemented with human karyopherin β3. On the other hand, expression of NS5A by itself had no effect on the growth of the double mutant expressing wild-type yeastPSE1. This indicates that NS5A may inhibit karyopherin β3 function via protein-protein interaction. The role of NS5A in HCV replication is discussed.

scholarly journals Essential Role of Domain III of Nonstructural Protein 5A for Hepatitis C Virus Infectious Particle Assembly

2008 ◽  
Vol 4 (3) ◽  
pp. e1000035 ◽  
Author(s):  
Nicole Appel ◽  
Margarita Zayas ◽  
Sven Miller ◽  
Jacomine Krijnse-Locker ◽  
Torsten Schaller ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Essential Role ◽  
Nonstructural Protein ◽  
Particle Assembly ◽  
Infectious Particle ◽  
Domain Iii ◽  
Nonstructural Protein 5A

scholarly journals Glycine-zipper motifs in hepatitis C virus nonstructural protein 4B are required for the establishment of viral replication organelles

2017 ◽  
pp. JVI.01890-17 ◽  
Author(s):  
David Paul ◽  
Vanesa Madan ◽  
Omar Ramirez ◽  
Maja Bencun ◽  
Ina Karen Stoeck ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Molecular Mechanisms ◽  
Nonstructural Protein ◽  
Membrane Vesicles ◽  
Rna Replication ◽  
Viral Rna ◽  
Transmembrane Helices ◽  
Double Membrane

Hepatitis C virus (HCV) RNA replication occurs in tight association with remodeled host cell membranes, presenting as cytoplasmic accumulations of single, double and multi membrane vesicles in infected cells. Formation of these so-called replication organelles is mediated by a complex interplay of host cell factors and viral replicase proteins. Of these, nonstructural protein 4B (NS4B), an integral transmembrane protein, appears to play a key role, but little is known about the molecular mechanisms how this protein contributes to organelle biogenesis. Using forward and reverse genetics we identified glycine-zipper motifs within transmembrane helices 2 and 3 of NS4B that are critically involved in viral RNA replication. Foerster resonance energy transfer analysis revealed the importance of the glycine-zippers in NS4B homo and heterotypic self-interactions. Additionally, ultrastructural analysis using electron microscopy unraveled a prominent role of glycine-zipper residues for the subcellular distribution and the morphology of HCV-induced double membrane vesicles. Notably, loss-of-function NS4B glycine-zipper mutants prominently induced single membrane vesicles with secondary invaginations that might represent an arrested intermediate state in double membrane vesicle formation. These findings highlight a so far unknown role of glycine residues within the membrane integral core domain for NS4B self-interaction and functional as well as structural integrity of HCV replication organelles.IMPORTANCERemodeling of the cellular endomembrane system leading to the establishment of replication organelles is a hallmark of positive-strand RNA viruses. In the case of hepatitis C virus (HCV), expression of the nonstructural proteins induces the accumulation of double membrane vesicles that likely arise from a concerted action of viral and co-opted cellular factors. However, the underlying molecular mechanisms are incompletely understood. Here, we identify glycine-zipper motifs within HCV nonstructural protein 4B (NS4B) transmembrane segments 2 and 3 that are crucial for the protein's self-interaction. Moreover, glycine residues within NS4B transmembrane helices critically contribute to the biogenesis of functional replication organelles and thus, efficient viral RNA replication. These results reveal how glycine-zipper motifs in NS4B contribute to structural and functional integrity of the HCV replication organelles and thus, viral RNA replication.

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 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
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