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Results: 7

1.
FIG. 1.

FIG. 1. From: Identification of Hepatitis C Virus NS5A Inhibitors .

Chemical structures of compounds used in this study. (A) Structure of screen hit BMS-858. (B) Structure of library compound BMS-824. (C) Structure of BMS-665.

Julie A. Lemm, et al. J Virol. 2010 January;84(1):482-491.
2.
FIG. 2.

FIG. 2. From: Identification of Hepatitis C Virus NS5A Inhibitors .

Resistance to BMS-858 in cells transfected with RNA from wild-type or 858R cells. Naive Huh-7 cells were electroporated with total cellular RNA containing 2 ng HCV RNA isolated from wild-type (A) or 858R (B) cell lines and incubated with or without compound. HCV RNA levels were determined at various times posttransfection (pt).

Julie A. Lemm, et al. J Virol. 2010 January;84(1):482-491.
3.
FIG. 6.

FIG. 6. From: Identification of Hepatitis C Virus NS5A Inhibitors .

Inhibitor effect on genotype 1a/1b NS5A hybrids. DNAs encoding various HCV replicons were expressed in a vaccinia virus transient expression system treated with either DMSO (−) or 0.1 μM BMS-824 (+). Cell lysates were separated by SDS-PAGE on 8% gels, and NS5A proteins were identified by Western immunoblotting using an anti-NS5A antibody. Cmpd, compound.

Julie A. Lemm, et al. J Virol. 2010 January;84(1):482-491.
4.
FIG. 3.

FIG. 3. From: Identification of Hepatitis C Virus NS5A Inhibitors .

Transient replication of wild-type and Y93H HCV genomes. Huh-7 cells were transfected with wild-type (WT) or Y93H replicon RNA and incubated in the presence or absence of 2 μM PI or 1 μM BMS-824. Luciferase activities (relative light units [RLU]) were determined with lysates of cells harvested 72 h after transfection. The 4-h value was used to correct for differences in transfection efficiencies. This graph represents data from one of two experiments giving similar results.

Julie A. Lemm, et al. J Virol. 2010 January;84(1):482-491.
5.
FIG. 5.

FIG. 5. From: Identification of Hepatitis C Virus NS5A Inhibitors .

Effect of compound on p58 production. DNAs encoding the wild-type (wt)-, Y93H-, or D168V-containing 1-377 1b HCV replicons were expressed in a vaccinia virus transient expression system treated with either DMSO (no cmpd) or a titration of BMS-824 (0.05 to 0.002 μM) or NS3 PI (0.1 to 0.01 μM). NS5A (Y93H) and NS3 (D168V) mutants were treated with 0.05 μM BMS-824 and 0.1 μM PI, respectively. Cell lysates were separated by SDS-PAGE on 8% gels, and NS5A proteins were identified by Western immunoblotting using an anti-NS5A antibody. NS5A-specific bands, both p56 and p58, were quantified by phosphorimaging.

Julie A. Lemm, et al. J Virol. 2010 January;84(1):482-491.
6.
FIG. 4.

FIG. 4. From: Identification of Hepatitis C Virus NS5A Inhibitors .

Schematic depicting the nonstructural region of HCV replicon RNAs and alignment of the N-terminal 100 amino acids of genotype 1a and 1b NS5A. (A) Genotype 1a and 1b sequences are shown, with the genotype 1a sequence being represented by a hatched fill pattern, and the positions of the nonstructural proteins are indicated. The nomenclature used for each construct is shown on the left, and the EC50 of BMS-824 or BMS-665 for each construct is indicated at the right. nd, not determined. (B) The genotype 1a sequence is derived from the H77 strain, and genotype 1b is Con1. Two dots indicate identical amino acid residues between genotypes 1a and 1b.

Julie A. Lemm, et al. J Virol. 2010 January;84(1):482-491.
7.
FIG. 7.

FIG. 7. From: Identification of Hepatitis C Virus NS5A Inhibitors .

Blocking NS5A hyperphosphorylation. (A) Cells transiently expressing the HCV replicon were [33P]orthophosphate labeled in the presence or absence of BMS-824, and extracts were immunoprecipitated with antibody to NS5A. Precipitated samples were then incubated in phosphatase buffer with or without CIP or BMS-824. A lysate from mock-transfected cells (no DNA) that had been infected only with vaccinia virus was included as a control. Samples treated with BMS-824 during labeling are indicated at the top of the gel. (B) Cells transiently expressing the HCV replicon were treated with or without BMS-824 and harvested at various times posttransfection. Western immunoblotting was performed on cell extracts by using an NS5A-specific antibody. The times of harvest are indicated above the gel. (C) Cells transiently expressing the HCV replicon were labeled with [35S]methionine for 30 min and chased in the presence (+) or absence (−) of either BMS-824 or PI (PI not shown). Cells were harvested at 4 h postlabeling and immunoprecipitated with NS5A-specific antibody.

Julie A. Lemm, et al. J Virol. 2010 January;84(1):482-491.

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