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NS5B

About: NS5B is a research topic. Over the lifetime, 1314 publications have been published within this topic receiving 59534 citations.


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Journal ArticleDOI
TL;DR: Limited proteolysis, homology modeling, and mutagenesis data were used to aid the design of polymerase constructs that might crystallize more readily, and two proteins were successfully crystallized.
Abstract: Bovine viral diarrhea virus (BVDV) nonstructural protein 5B is an RNA-dependent RNA polymerase, essential for viral replication. Initial attempts to crystallize a soluble form of the 695-residue BVDV polymerase did not produce any crystals. Limited proteolysis, homology modeling, and mutagenesis data were used to aid the design of polymerase constructs that might crystallize more readily. Limited proteolysis of the polymerase with trypsin identified a domain boundary within the protein. Homology modeling of the polymerase, based on the structure of hepatitis C virus polymerase, indicated that the two polymerases share a 23% identical “core,” although overall sequence identity is low. Eighty-four expression clones of the BVDV polymerase were designed by fine-sampling of chain termini at the boundaries of domain and of active truncated forms of the polymerase. The resulting constructs were expressed in Escherichia coli and purified using high-throughput methods. Soluble truncated proteins were subjected to crystallization trials in a 96-well format, and two of these proteins were successfully crystallized.

16 citations

Journal ArticleDOI
TL;DR: An important role for mitochondria is suggested in the modulation HCV NS4A-induced inhibition of HCV core-mediated cell growth.
Abstract: Hepatitis C virus (HCV) is a significant health problem facing the world. More than 170 million people are infected with HCV worldwide. HCV encodes a large polyprotein precursor that is processed into at least 10 distinct products including structural (core, E1 and E2) and non-structural (NS2, NS3, NS4A, NS4B, NS5A and NS5B). Besides its importance in virus replication, NS4A functions as a cofactor for NS3 and contributes to viral pathogenesis by influencing cellular functions. Here, we investigated the effect of NS4A protein on the growth rate induced by core protein in liver cells. Using our established tetracycline inducible system, we demonstrated the ability of NS4A protein to inhibit core protein-induced cell growth in Hepatoma cell line, HepG2. Induction of both core and NS4A proteins in HepG2-core/NS4A transfectants inhibited core-induced growth advantage in HepG2-core transfectants and blocked NS4A protein-induced cell growth inhibition in HepG2-NS4A transfectants. Using both immune fluorescence staining and Western blot analysis, we confirmed the localization of NS4A protein to the mitochondria in HepG2-NS4A transfectants expressing NS4A protein. Data obtained from flow cytometry analysis, using JC-1 demonstrated the loss of mitochondrial membrane potential (DeltaPsim) by the expression of NS4A protein in HepG2-NS4A transfectants, but not by the expression of core protein in HepG2-core transfectants. Whereas, the induction of the expression of both core and NS4A proteins in HepG2-core/NS4A transfectants blocked NS4A-induced loss of DeltaPsim in HepG2 cells. Taken together, our data suggest an important role for mitochondria in the modulation HCV NS4A-induced inhibition of HCV core-mediated cell growth.

16 citations

Patent
19 May 2008
TL;DR: The 7a/JFH1 recombinants provided a robust in vitro tool for research in HCV genotype 7, including vaccine studies and functional analyses as discussed by the authors, which showed growth kinetics, infectivity and RNA titers comparable to a previously developed 3a/jFH1 reference virus.
Abstract: Genotype 7a has been identified recently, thus not much is known about the biology of this new, major HCV genotype. The present inventors developed hepatitis C virus 7a/2a intergenotypic recombinants in which the JFH1 structural genes (Core, E1 and E2), p7 and the complete NS2 were replaced by the corresponding genes of the genotype 7a strain QC69 and characterized them in Huh7.5 cells. Sequence analysis of 7a/JFH1 recombinants recovered after viral passage in Huh7.5 cells following 4 independent transfection experiments revealed adaptive mutations in Core, E2, NS2, NS5A and NS5B. In reverse genetic studies the importance of these mutations for improved growth kinetics was shown. Adapted 7a/JFH1 viruses showed growth kinetics, infectivity and RNA titers comparable to a previously developed 3a/JFH1 reference virus. Conclusion: The developed 7a/JFH1 viruses provide a robust in vitro tool for research in HCV genotype 7, including vaccine studies and functional analyses.

16 citations

Journal ArticleDOI
TL;DR: The findings reveal that NS5B/Core and NS3-4A exhibit conflicting effects (activation and inhibition) on the IFN system in PH5CH8 cells, and suggest that such effects may promote the distraction of the host defense system to lead to persistent infection.

16 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
202310
202258
202128
202033
201943
201842