Article Figures & Data
Figures
- FIG. 1.
Schematic showing construction of plasmid pDENΔCprME-PAC2A. (A) Dengue virus type 2 infectious clone cDNA (in plasmid pDVWS601 [29]), showing a single open reading frame carrying three structural genes (C, core; prM, premembrane; E, envelope), seven nonstructural (NS) genes, and flanking 5′ and 3′ untranslated regions (UTR). (B) pDENΔCprME. A large in-frame deletion was introduced within the region carrying the structural genes. (C) pDENΔCprME-PAC2A. An antibiotic selection cassette encoding PAC and the foot-and-mouth disease virus protein 2A was cloned in place of the deleted structural genes.
- FIG. 2.
Dengue virus replicon RNA replication is resistant to IFN-α. (A) K562.ΔCprME-PAC2A cells were grown in the presence of various concentrations of IFN-α2a, as indicated, for 24 h. Dengue virus replicon RNA levels were measured by quantitative PCR and normalized to GAPDH mRNA levels. (B) K562.ΔCprME-PAC2A cells were grown in the presence of 100 IU of IFN-α2a/ml for 0, 6, and 24 h. Cell lysates (2 × 105 cells per reaction) were separated by SDS-PAGE, and then the dengue virus NS1 protein was analyzed by immunoblotting. K562 cells were included as a negative control. (C) K562.ΔCprME-PAC2A cells were grown in the absence (black bars) or presence (white bars) of 100 IU of IFN-α2a/ml for 24 and 48 h. The cumulative concentrations of NS1 in the culture supernatants at each time point were measured by ELISA.
- FIG. 3.
Antiviral effect of IFN-α is blocked in dengue virus replicon-containing cells. K562 cells (solid lines) and K562.ΔCprME-PAC2A cells (broken lines) were treated with 0 (•), 10 (□), or 100 (○) IU of IFN-α2a/ml for 24 h. The cells were then infected with EMCV, and after a further 24 h, the supernatants were harvested and serially diluted on confluent A549 cells. After 24 h, the A549 cells were fixed and stained with methyl violet. The amount of staining was quantified by measuring the optical density (OD) of each well at 570 nm. More EMCV replication resulted in increased cell death and lower optical density readings.
- FIG. 4.
Induction of classical ISGs by IFN-α is inhibited in dengue virus replicon-containing cells. K562 cells (black bars) and K562.ΔCprME-PAC2A cells (white bars) were stimulated with 100 IU of IFN-α2a for 6 and 24 h. MxA (A) and PKR (B) gene transcription was measured by real-time PCR and normalized to the housekeeping gene GAPDH.
- FIG. 5.
Dengue virus RNA replication inhibits STAT1 and STAT2 phosphorylation in response to IFN-α and reduces steady-state levels of STAT2. (A) K562, K562.ΔCprME-PAC2A, and cured K562 cells; (B) THP-1 and THP-1.ΔCprME-PAC2A cells. Cells were left untreated or treated with 100 IU of IFN-α or IFN-γ/ml for 30 min and then lysed in SDS loading buffer. Proteins were separated by SDS-PAGE and then analyzed by immunoblotting with specific antibodies for STAT1, phosphorylated STAT1, STAT2, and phosphorylated STAT2, as indicated.
- FIG. 6.
Dengue virus RNA replication does not affect IFNAR protein levels. (A) Cell lysates from K562 and K562.ΔCprME-PAC2A cells were separated by SDS-PAGE and then analyzed by immunoblotting with specific antibodies for IFNAR1 and IFNAR2, as indicated. (B) K562 and K562.ΔCprME-PAC2A cells were stained with specific anti-IFNAR2 antibodies and then analyzed by flow cytometry. Cells stained with the secondary antibody alone (gray-filled plot) were included as a negative staining control.
- FIG. 7.
Dengue virus infection reduces STAT2 levels. K562 cells were infected with dengue virus for 48 h and then dually stained with an anti-dengue virus NS1 mouse monoclonal antibody followed by a Texas Red-labeled secondary antibody to detect dengue virus-infected cells (A) and an anti-STAT2 rabbit polyclonal antibody followed by a fluorescein isothiocyanate-labeled secondary antibody to detect STAT2 (B). Cells were visualized by confocal microscopy. Arrows show infected cells. (C) Cell lysates were separated by SDS-PAGE and then analyzed by immunoblotting with specific antibodies for dengue virus NS1, STAT1, and STAT2, as indicated. Mock-infected cells were included for comparison.
Tables
- TABLE 1.
ISG transcription in response to IFN-α in cells that do and do not contain dengue virus repliconsa
ISG product Fold induction K562 cells K562 ΔCprME-PAC2A cells IFN-α-induced protein 27 55.8 11.5 VCAM-1 30.6 2.6 MxA 17.9 1.8 IFN-α-induced protein (clone IFI-616) 12.9 1.1 Met proto-oncogene product (hepatocyte growth factor) 7.9 1.8 PSMB9 7.9 0.4 IFN-induced protein 17 7.5 1.3 Vipirin (Cig5) 7.2 1.3 Interleukin-15 6.5 1.1 9-27mrna 6.4 1.2 STAT1 6.2 1.1 STAT4 6.2 1.5 IFIT1 6.1 1.3 KIAA0284 5.6 1.5 STAT1 (91 kDa) 4.9 1.8 IFN-induced transmembrane protein 3 4.9 1.4 INDO 4.8 0.6 Interleukin-6 4.8 0.6 IFN-induced transmembrane protein 2 4.5 1.1 MAP2K4 4.5 1.0 IFI35 4.4 1.1 Homo sapiens STAT 4.1 1.6 ↵ a ISGs up-regulated more than fourfold in K562 cells compared to K562.ΔCprME-PAC2A cells are shown.
