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Live Cell Analysis and Mathematical Modeling Identify Determinants of Attenuation of Dengue Virus 2'-O-Methylation Mutant.

Schmid B, Rinas M, Ruggieri A, Acosta EG, Bartenschlager M, Reuter A, Fischl W, Harder N, Bergeest JP, Flossdorf M, Rohr K, Höfer T, Bartenschlager R - PLoS Pathog. (2015)

Bottom Line: Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN.By contrast, a vaccine candidate DENV mutant, which lacks 2'-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells.In conclusion, these results show that attenuation of the 2'-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany.

ABSTRACT
Dengue virus (DENV) is the most common mosquito-transmitted virus infecting ~390 million people worldwide. In spite of this high medical relevance, neither a vaccine nor antiviral therapy is currently available. DENV elicits a strong interferon (IFN) response in infected cells, but at the same time actively counteracts IFN production and signaling. Although the kinetics of activation of this innate antiviral defense and the timing of viral counteraction critically determine the magnitude of infection and thus disease, quantitative and kinetic analyses are lacking and it remains poorly understood how DENV spreads in IFN-competent cell systems. To dissect the dynamics of replication versus antiviral defense at the single cell level, we generated a fully viable reporter DENV and host cells with authentic reporters for IFN-stimulated antiviral genes. We find that IFN controls DENV infection in a kinetically determined manner that at the single cell level is highly heterogeneous and stochastic. Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN. By contrast, a vaccine candidate DENV mutant, which lacks 2'-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. Through mathematical modeling of time-resolved data and validation experiments we show that the primary determinant for attenuation is the accelerated kinetics of IFN production. This rapid induction triggered by mutant DENV precedes establishment of IFN-resistance in infected cells, thus causing a massive reduction of virus production rate. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. In conclusion, these results show that attenuation of the 2'-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.

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IFN pre-treatment protects against DENV infection.(A) IFN-competent A549 cells were treated with 100 IU/ml IFN-α prior to (-4 h, -2 h) or after (2 h, 4 h, 6 h, 8 h) or at the time point of infection (0 h) with the DENV2 strain 16681 at a MOI of 10 TCID50/cell. Cells were fixed 24 h post infection and analyzed by immunofluorescence using a NS5-specific antiserum. Mock-treated and DENV-infected cells without IFN treatment served as reference. (B) Quantification of infection efficiency of cells shown in panel (A). For each time point, 500–1,000 cells detected in 3 view fields were analyzed for DENV infection. Mean values and SDs are shown for each time point (** p-value <0.005). (C) IFN-competent A549 cells were infected with the DENV2 strain 16681 at a MOI of 10 TCID50/cell. Cells were harvested at given time points after infection (h p.i.) and lysates were analyzed by Western blot using STAT2-, E- (DENV envelope protein) and β-actin-specific antisera. A representative immunoblot from 3 independent experiments is shown. Numbers in the left refer to the positions of molecular weight standards in kiloDalton (kDa).
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ppat.1005345.g001: IFN pre-treatment protects against DENV infection.(A) IFN-competent A549 cells were treated with 100 IU/ml IFN-α prior to (-4 h, -2 h) or after (2 h, 4 h, 6 h, 8 h) or at the time point of infection (0 h) with the DENV2 strain 16681 at a MOI of 10 TCID50/cell. Cells were fixed 24 h post infection and analyzed by immunofluorescence using a NS5-specific antiserum. Mock-treated and DENV-infected cells without IFN treatment served as reference. (B) Quantification of infection efficiency of cells shown in panel (A). For each time point, 500–1,000 cells detected in 3 view fields were analyzed for DENV infection. Mean values and SDs are shown for each time point (** p-value <0.005). (C) IFN-competent A549 cells were infected with the DENV2 strain 16681 at a MOI of 10 TCID50/cell. Cells were harvested at given time points after infection (h p.i.) and lysates were analyzed by Western blot using STAT2-, E- (DENV envelope protein) and β-actin-specific antisera. A representative immunoblot from 3 independent experiments is shown. Numbers in the left refer to the positions of molecular weight standards in kiloDalton (kDa).

Mentions: We reasoned that the efficiency of virus spread and ultimately the outcome of infection should depend on the dynamics of virus replication versus activation of the IFN response. To study the dynamics of this interplay we utilized A549 cells that are permissive for DENV and capable of producing type 1 and 3 IFNs. In the first set of experiments, we determined the time-dependency of DENV-2 (strain 16681) IFN sensitivity by treating cells with 100 IU/ml IFN-α prior to (-4 and -2 h) or after DENV infection (2, 4, 6 and 8 h) with MOI 10 TCID50/cell. Cells were fixed 24 h post infection and the fraction of NS5-expressing cells was determined by immunofluorescence (Fig 1A and 1B). In the absence of IFN treatment, ~60% of the cells were infected. However, pre-treatment of cells with IFN-α 4 h prior to infection reduced the number of DENV-positive cells to ~15%. This number steadily increased when IFN treatment was delayed until 6 h post infection and, after this time point, inhibition of DENV replication was no longer detectable. A similar time-dependent IFN sensitivity was observed with the same concentration of IFN-α after DENV infection with MOI 0.1 TCID50/cell (S1A Fig), a condition used later on in live cell imaging. Measurement of the titers of virus released from cells revealed a ~2 log decrease when cells were treated with IFN-α prior to or until 2 h after infection (S1B Fig).


Live Cell Analysis and Mathematical Modeling Identify Determinants of Attenuation of Dengue Virus 2'-O-Methylation Mutant.

Schmid B, Rinas M, Ruggieri A, Acosta EG, Bartenschlager M, Reuter A, Fischl W, Harder N, Bergeest JP, Flossdorf M, Rohr K, Höfer T, Bartenschlager R - PLoS Pathog. (2015)

IFN pre-treatment protects against DENV infection.(A) IFN-competent A549 cells were treated with 100 IU/ml IFN-α prior to (-4 h, -2 h) or after (2 h, 4 h, 6 h, 8 h) or at the time point of infection (0 h) with the DENV2 strain 16681 at a MOI of 10 TCID50/cell. Cells were fixed 24 h post infection and analyzed by immunofluorescence using a NS5-specific antiserum. Mock-treated and DENV-infected cells without IFN treatment served as reference. (B) Quantification of infection efficiency of cells shown in panel (A). For each time point, 500–1,000 cells detected in 3 view fields were analyzed for DENV infection. Mean values and SDs are shown for each time point (** p-value <0.005). (C) IFN-competent A549 cells were infected with the DENV2 strain 16681 at a MOI of 10 TCID50/cell. Cells were harvested at given time points after infection (h p.i.) and lysates were analyzed by Western blot using STAT2-, E- (DENV envelope protein) and β-actin-specific antisera. A representative immunoblot from 3 independent experiments is shown. Numbers in the left refer to the positions of molecular weight standards in kiloDalton (kDa).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4697809&req=5

ppat.1005345.g001: IFN pre-treatment protects against DENV infection.(A) IFN-competent A549 cells were treated with 100 IU/ml IFN-α prior to (-4 h, -2 h) or after (2 h, 4 h, 6 h, 8 h) or at the time point of infection (0 h) with the DENV2 strain 16681 at a MOI of 10 TCID50/cell. Cells were fixed 24 h post infection and analyzed by immunofluorescence using a NS5-specific antiserum. Mock-treated and DENV-infected cells without IFN treatment served as reference. (B) Quantification of infection efficiency of cells shown in panel (A). For each time point, 500–1,000 cells detected in 3 view fields were analyzed for DENV infection. Mean values and SDs are shown for each time point (** p-value <0.005). (C) IFN-competent A549 cells were infected with the DENV2 strain 16681 at a MOI of 10 TCID50/cell. Cells were harvested at given time points after infection (h p.i.) and lysates were analyzed by Western blot using STAT2-, E- (DENV envelope protein) and β-actin-specific antisera. A representative immunoblot from 3 independent experiments is shown. Numbers in the left refer to the positions of molecular weight standards in kiloDalton (kDa).
Mentions: We reasoned that the efficiency of virus spread and ultimately the outcome of infection should depend on the dynamics of virus replication versus activation of the IFN response. To study the dynamics of this interplay we utilized A549 cells that are permissive for DENV and capable of producing type 1 and 3 IFNs. In the first set of experiments, we determined the time-dependency of DENV-2 (strain 16681) IFN sensitivity by treating cells with 100 IU/ml IFN-α prior to (-4 and -2 h) or after DENV infection (2, 4, 6 and 8 h) with MOI 10 TCID50/cell. Cells were fixed 24 h post infection and the fraction of NS5-expressing cells was determined by immunofluorescence (Fig 1A and 1B). In the absence of IFN treatment, ~60% of the cells were infected. However, pre-treatment of cells with IFN-α 4 h prior to infection reduced the number of DENV-positive cells to ~15%. This number steadily increased when IFN treatment was delayed until 6 h post infection and, after this time point, inhibition of DENV replication was no longer detectable. A similar time-dependent IFN sensitivity was observed with the same concentration of IFN-α after DENV infection with MOI 0.1 TCID50/cell (S1A Fig), a condition used later on in live cell imaging. Measurement of the titers of virus released from cells revealed a ~2 log decrease when cells were treated with IFN-α prior to or until 2 h after infection (S1B Fig).

Bottom Line: Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN.By contrast, a vaccine candidate DENV mutant, which lacks 2'-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells.In conclusion, these results show that attenuation of the 2'-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany.

ABSTRACT
Dengue virus (DENV) is the most common mosquito-transmitted virus infecting ~390 million people worldwide. In spite of this high medical relevance, neither a vaccine nor antiviral therapy is currently available. DENV elicits a strong interferon (IFN) response in infected cells, but at the same time actively counteracts IFN production and signaling. Although the kinetics of activation of this innate antiviral defense and the timing of viral counteraction critically determine the magnitude of infection and thus disease, quantitative and kinetic analyses are lacking and it remains poorly understood how DENV spreads in IFN-competent cell systems. To dissect the dynamics of replication versus antiviral defense at the single cell level, we generated a fully viable reporter DENV and host cells with authentic reporters for IFN-stimulated antiviral genes. We find that IFN controls DENV infection in a kinetically determined manner that at the single cell level is highly heterogeneous and stochastic. Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN. By contrast, a vaccine candidate DENV mutant, which lacks 2'-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. Through mathematical modeling of time-resolved data and validation experiments we show that the primary determinant for attenuation is the accelerated kinetics of IFN production. This rapid induction triggered by mutant DENV precedes establishment of IFN-resistance in infected cells, thus causing a massive reduction of virus production rate. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. In conclusion, these results show that attenuation of the 2'-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.

Show MeSH
Related in: MedlinePlus