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Differences in type I interferon signaling antagonism by dengue viruses in human and non-human primate cell lines.

Medina FA, Torres-Malavé G, Chase AJ, Santiago GA, Medina JF, Santiago LM, Muñoz-Jordán JL - PLoS Negl Trop Dis (2015)

Bottom Line: The ability of DENVs to inhibit IFN-α/β signaling is conserved.Although some variation in the inhibition was observed, the moderate differences may be difficult to correlate with clinical outcomes.DENVs were unable to inhibit pSTAT1 in NHP cell lines, but their ability to inhibit pSTAT1 in primary Rhesus macaque dendritic cells suggests that this may be a cell specific phenomena or due to the transformed nature of the cell lines.

View Article: PubMed Central - PubMed

Affiliation: Centers for Disease Control and Prevention, Division of Vector-Borne Diseases, Dengue Branch, San Juan, Puerto Rico, United States of America.

ABSTRACT

Background/objectives: In vitro studies have shown that dengue virus (DENV) can thwart the actions of interferon (IFN)-α/β and prevent the development of an antiviral state in infected cells. Clinical studies looking at gene expression in patients with severe dengue show a reduced expression of interferon stimulated genes compared to patients with dengue fever. Interestingly, there are conflicting reports as to the ability of DENV or other flaviviruses to inhibit IFN-α/β signaling.

Methodology/principal findings: In order to determine the relative inhibition of IFN-α/β signaling by DENVs, a method combining flow cytometry and a four-parameter logistic regression model was established. A representative isolate from DENV-1, -3 and -4 and seventeen representative isolates encompassing all DENV-2 genotypes were evaluated. All of the DENVs evaluated in this study were capable of inhibiting IFN-α/β signaling. Most of the strains were able to inhibit IFN-α/β to a degree similar to DENV strain 16681; however, DENV-2 sylvatic strains demonstrated an increased inhibition of phosphorylated signal transducer and activator of transcription (pSTAT1). Surprisingly, we were unable to observe inhibition of pSTAT1 by DENV-2 sylvatic strains or the Asian strain 16681 in non-human primate (NHP) cell lines. Analysis in primary Rhesus macaque dendritic cells suggests that DENVs are capable of inhibiting IFN signaling in these cells. However, contrary to human dendritic cells, production of IFN-α was detected in the supernatant of DENV-infected Rhesus macaque dendritic cells.

Conclusions: The ability of DENVs to inhibit IFN-α/β signaling is conserved. Although some variation in the inhibition was observed, the moderate differences may be difficult to correlate with clinical outcomes. DENVs were unable to inhibit pSTAT1 in NHP cell lines, but their ability to inhibit pSTAT1 in primary Rhesus macaque dendritic cells suggests that this may be a cell specific phenomena or due to the transformed nature of the cell lines.

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Inhibition of IFN-β signaling in DENV-2 16681 in human and non-human primate cell lines.DENV-2 16681 was used to infect human (A549 & Huh7) and NHP (LLCMK2 & Vero) cell lines at a MOI of 5 for 24 hours. Cells were then stimulated for 30 min. with IFN-β (500 U/ml) and processed for (A) flow cytometry of pSTAT1 or (B) Western blots of pSTAT1, STAT1, STAT2, NS4B and GAPDH. Results shown are representative of two independent experiments.
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pntd.0003468.g006: Inhibition of IFN-β signaling in DENV-2 16681 in human and non-human primate cell lines.DENV-2 16681 was used to infect human (A549 & Huh7) and NHP (LLCMK2 & Vero) cell lines at a MOI of 5 for 24 hours. Cells were then stimulated for 30 min. with IFN-β (500 U/ml) and processed for (A) flow cytometry of pSTAT1 or (B) Western blots of pSTAT1, STAT1, STAT2, NS4B and GAPDH. Results shown are representative of two independent experiments.

Mentions: The unexpected finding that sylvatic DENVs were unable to inhibit STAT1 phosphorylation in NHP cell lines led us to question whether this observation was unique to these viruses. For this reason, we compared inhibition of IFN signaling by DENV-2 strain 16681 in both human (A549, Huh7) and NHP (LLCMK2, Vero) cell lines. Flow cytometry analysis of STAT1 phosphorylation in the DENV+ population revealed that there is a slight reduction in NHP cells compared to uninfected cells stimulated with IFN-β (Fig. 6A). As expected, DENV 16681 inhibited phosphorylation of STAT1 and caused STAT2 degradation in human cell lines. However, in NHP cell lines, STAT1 was phosphorylated in infected cells after IFN stimulation, but degradation of STAT2 was observed (Fig. 6A & B). This demonstrates a significantly reduced ability of DENVs to inhibit STAT1 phosphorylation in NHP cell lines.


Differences in type I interferon signaling antagonism by dengue viruses in human and non-human primate cell lines.

Medina FA, Torres-Malavé G, Chase AJ, Santiago GA, Medina JF, Santiago LM, Muñoz-Jordán JL - PLoS Negl Trop Dis (2015)

Inhibition of IFN-β signaling in DENV-2 16681 in human and non-human primate cell lines.DENV-2 16681 was used to infect human (A549 & Huh7) and NHP (LLCMK2 & Vero) cell lines at a MOI of 5 for 24 hours. Cells were then stimulated for 30 min. with IFN-β (500 U/ml) and processed for (A) flow cytometry of pSTAT1 or (B) Western blots of pSTAT1, STAT1, STAT2, NS4B and GAPDH. Results shown are representative of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359095&req=5

pntd.0003468.g006: Inhibition of IFN-β signaling in DENV-2 16681 in human and non-human primate cell lines.DENV-2 16681 was used to infect human (A549 & Huh7) and NHP (LLCMK2 & Vero) cell lines at a MOI of 5 for 24 hours. Cells were then stimulated for 30 min. with IFN-β (500 U/ml) and processed for (A) flow cytometry of pSTAT1 or (B) Western blots of pSTAT1, STAT1, STAT2, NS4B and GAPDH. Results shown are representative of two independent experiments.
Mentions: The unexpected finding that sylvatic DENVs were unable to inhibit STAT1 phosphorylation in NHP cell lines led us to question whether this observation was unique to these viruses. For this reason, we compared inhibition of IFN signaling by DENV-2 strain 16681 in both human (A549, Huh7) and NHP (LLCMK2, Vero) cell lines. Flow cytometry analysis of STAT1 phosphorylation in the DENV+ population revealed that there is a slight reduction in NHP cells compared to uninfected cells stimulated with IFN-β (Fig. 6A). As expected, DENV 16681 inhibited phosphorylation of STAT1 and caused STAT2 degradation in human cell lines. However, in NHP cell lines, STAT1 was phosphorylated in infected cells after IFN stimulation, but degradation of STAT2 was observed (Fig. 6A & B). This demonstrates a significantly reduced ability of DENVs to inhibit STAT1 phosphorylation in NHP cell lines.

Bottom Line: The ability of DENVs to inhibit IFN-α/β signaling is conserved.Although some variation in the inhibition was observed, the moderate differences may be difficult to correlate with clinical outcomes.DENVs were unable to inhibit pSTAT1 in NHP cell lines, but their ability to inhibit pSTAT1 in primary Rhesus macaque dendritic cells suggests that this may be a cell specific phenomena or due to the transformed nature of the cell lines.

View Article: PubMed Central - PubMed

Affiliation: Centers for Disease Control and Prevention, Division of Vector-Borne Diseases, Dengue Branch, San Juan, Puerto Rico, United States of America.

ABSTRACT

Background/objectives: In vitro studies have shown that dengue virus (DENV) can thwart the actions of interferon (IFN)-α/β and prevent the development of an antiviral state in infected cells. Clinical studies looking at gene expression in patients with severe dengue show a reduced expression of interferon stimulated genes compared to patients with dengue fever. Interestingly, there are conflicting reports as to the ability of DENV or other flaviviruses to inhibit IFN-α/β signaling.

Methodology/principal findings: In order to determine the relative inhibition of IFN-α/β signaling by DENVs, a method combining flow cytometry and a four-parameter logistic regression model was established. A representative isolate from DENV-1, -3 and -4 and seventeen representative isolates encompassing all DENV-2 genotypes were evaluated. All of the DENVs evaluated in this study were capable of inhibiting IFN-α/β signaling. Most of the strains were able to inhibit IFN-α/β to a degree similar to DENV strain 16681; however, DENV-2 sylvatic strains demonstrated an increased inhibition of phosphorylated signal transducer and activator of transcription (pSTAT1). Surprisingly, we were unable to observe inhibition of pSTAT1 by DENV-2 sylvatic strains or the Asian strain 16681 in non-human primate (NHP) cell lines. Analysis in primary Rhesus macaque dendritic cells suggests that DENVs are capable of inhibiting IFN signaling in these cells. However, contrary to human dendritic cells, production of IFN-α was detected in the supernatant of DENV-infected Rhesus macaque dendritic cells.

Conclusions: The ability of DENVs to inhibit IFN-α/β signaling is conserved. Although some variation in the inhibition was observed, the moderate differences may be difficult to correlate with clinical outcomes. DENVs were unable to inhibit pSTAT1 in NHP cell lines, but their ability to inhibit pSTAT1 in primary Rhesus macaque dendritic cells suggests that this may be a cell specific phenomena or due to the transformed nature of the cell lines.

Show MeSH
Related in: MedlinePlus