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1.
FIG. 7.

FIG. 7. From: Differential Effects of Mutations in NS4B on West Nile Virus Replication and Inhibition of Interferon Signaling .

Mutant viruses exhibit growth kinetics similar to wild type. Vero cells were infected at an MOI of 0.5 with WNII, WNII-ER, WNII-KUN4B, or WNII-KUN4B-EK for 12, 24, 48, and 72 h. Virus titers in medium from infected cells were determined by plaque assay on BHK-21 cells.

Jared D. Evans, et al. J Virol. 2007 November;81(21):11809-11816.
2.
FIG. 1.

FIG. 1. From: Differential Effects of Mutations in NS4B on West Nile Virus Replication and Inhibition of Interferon Signaling .

Schematic of C20DX-BLaM. WNV subgenomic replicon containing the beta-lactamase (BLaM) transgene. The coding regions for NS4A (gray box) and NS4B are magnified. The transmembrane domains of NS4B are represented by dashed boxes. The double-alanine site-directed substitutions are highlighted. UTR, untranslated region; IRES, internal ribosome entry cite; EMCV, encephalomyocarditis virus.

Jared D. Evans, et al. J Virol. 2007 November;81(21):11809-11816.
3.
FIG. 2.

FIG. 2. From: Differential Effects of Mutations in NS4B on West Nile Virus Replication and Inhibition of Interferon Signaling .

C20DX-BLaM replicons are stable in HeLa cells. HeLa cells were electroporated with wild type or mutant RNA. The transfected cells were selected with G418. Colonies were pooled and passed as cell lines. Parental HeLa (A), C20DX-BLaM (B), or NS4B-E22K24 (C) cells were analyzed for beta-lactamase activity. (D) Northern blot analysis of cell lines for the amount of WNV RNA present. Actin served as the loading control. WT, wild type; E22K24, NS4B-E22K24.

Jared D. Evans, et al. J Virol. 2007 November;81(21):11809-11816.
4.
FIG. 3.

FIG. 3. From: Differential Effects of Mutations in NS4B on West Nile Virus Replication and Inhibition of Interferon Signaling .

The NS4B-E22K24 mutation relieves WNV repression of IFN antiviral response. Parental HeLa, C20DX-BLaM, or NS4B-E22K24 (C20DX-EK) cells were incubated for 24 h with the indicated concentrations of IFN-α and then infected with VSV at an MOI of 1. Twenty-four hours postinfection, cells were stained with crystal violet to determine CPE. The cells in the control column were not infected or treated with IFN. WT, wild type.

Jared D. Evans, et al. J Virol. 2007 November;81(21):11809-11816.
5.
FIG. 6.

FIG. 6. From: Differential Effects of Mutations in NS4B on West Nile Virus Replication and Inhibition of Interferon Signaling .

Individually expressed NS4B proteins reflect activity in replicons. HeLa cells were transfected with each of indicated plasmids. At 24 h posttransfection, cells were treated with 100 U of IFN-α. The firefly luciferase activities were normalized to Renilla luciferase to determine ISRE-specific gene induction. Firefly luciferase activities were determined as mean values from three independent experiments performed in triplicate (P values of 0.005 to 0.01). WT, wild type; NS4B-EK, NS4B-E22K24.

Jared D. Evans, et al. J Virol. 2007 November;81(21):11809-11816.
6.
FIG. 5.

FIG. 5. From: Differential Effects of Mutations in NS4B on West Nile Virus Replication and Inhibition of Interferon Signaling .

IFN-stimulated gene expression in replicon cells. Parental HeLa, C20DX-BLaM (C20DX-WT), and NS4B-E22K24 (C20DX-E22K24) cells were left untreated or treated with 100 IU of IFN-α for 6 h. Total RNA was isolated by TRIzol reagent extraction. Equivalent amounts of total RNA were reverse transcribed with gene-specific primers to create cDNA pools. Real-time PCR with ISG-specific TaqMan primer-probe sets was performed. Results shown are the average of two independent experiments.

Jared D. Evans, et al. J Virol. 2007 November;81(21):11809-11816.
7.
FIG. 9.

FIG. 9. From: Differential Effects of Mutations in NS4B on West Nile Virus Replication and Inhibition of Interferon Signaling .

Mutant viruses exhibit growth kinetics comparable to wild type. Wild-type (A and C) or IFN-α/β receptor knockout (B and D) B6 MEFs were infected at an MOI of 0.2 (A and B) or 0.002 (C and D) with WNII, WNII-ER, WNII-KUN4B, or WNII-KUN4B-EK for 12, 24, 48, and 72 h. Virus titers in medium from infected cells were determined by plaque assay on BHK-21 cells. Means are shown with standard error bars.

Jared D. Evans, et al. J Virol. 2007 November;81(21):11809-11816.
8.
FIG. 4.

FIG. 4. From: Differential Effects of Mutations in NS4B on West Nile Virus Replication and Inhibition of Interferon Signaling .

The NS4B-E22K24 mutation cannot inhibit IFN-induced STAT phosphorylation in replicon cells. (A) Parental HeLa, C20DX-BLaM (wild type [WT]) and NS4B-E22K24 (E22K24) cells were left untreated or treated with 1,000 IU of IFN-α for 20 min. (B) A second pool of replicon-bearing HeLa cells was generated with the C20DXrep/neo replicon harboring the NS4B-E22K24 mutation without the beta-lactamase transgene. Cells were treated with IFN-α. Equal amounts of cell lysates were separated by SDS-PAGE and transferred to Immobilon-P membranes. The expression of STAT1, phosphorylated STAT (P-STAT), and NS4B proteins was detected by Western blotting with the corresponding specific antibodies. Actin served as the lane loading control. One representative experiment of three is shown.

Jared D. Evans, et al. J Virol. 2007 November;81(21):11809-11816.
9.
FIG. 8.

FIG. 8. From: Differential Effects of Mutations in NS4B on West Nile Virus Replication and Inhibition of Interferon Signaling .

Mutant viruses inhibit STAT1 phosphorylation. HeLa cells were infected at an MOI of 1 with WNII or WNII-ER for 24 h. The infected (lanes 3 and 4) or uninfected (Uninf; lane 2) cells were treated with 1,000 U of IFN-α (+IFN) for 20 min. As a control, cells were not infected (Uninf; lane 1) and left untreated (−IFN). Equal amounts (15 μg) of cell lysates were separated by SDS-PAGE and transferred to Immobilon-P membranes. The expression of phosphorylated STAT1 (P-STAT1), STAT1, and NS4B proteins was detected by Western blotting with the corresponding specific antibodies. Actin served as the lane loading control. One representative experiment of three is shown.

Jared D. Evans, et al. J Virol. 2007 November;81(21):11809-11816.

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