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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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Spread of a DENV mutant with defective 2’-O-methylation of the viral RNA genome.(A) Genomic structure of the DENV-faR 2’-O-methylation mutant containing the E217A substitution in the NS5 protein (for further details see legend to Fig 4A). (B) Spread of the DENV-faR mutant in A549 reporter cells and activation of the IFN response. A549-IFIT1deGFP cells were infected with the DENV-faR E217A mutant at a MOI of 0.1 TCID50/cell. At the time points specified in the top of each panel, cells were fixed and processed for measurement by flow cytometry. (C) Model dynamics versus experimental data from (B) (solid lines and squares, respectively); for comparison, the model dynamics and data for wildtype DENV are reproduced from Fig 6C (dashed lines and circles, respectively). IFN-λ was measured by ELISA. Non-infected cells served as control. (D) Best-fit values and confidence bounds for the model parameters that have been allowed to differ between wildtype DENV and the 2’-O-methylation mutant. The model predicts that the virus production rate and the delay of IFN secretion decrease in case of the mutant (red box), whereas the delay of virus production and IFN secretion rate remain unchanged.
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ppat.1005345.g008: Spread of a DENV mutant with defective 2’-O-methylation of the viral RNA genome.(A) Genomic structure of the DENV-faR 2’-O-methylation mutant containing the E217A substitution in the NS5 protein (for further details see legend to Fig 4A). (B) Spread of the DENV-faR mutant in A549 reporter cells and activation of the IFN response. A549-IFIT1deGFP cells were infected with the DENV-faR E217A mutant at a MOI of 0.1 TCID50/cell. At the time points specified in the top of each panel, cells were fixed and processed for measurement by flow cytometry. (C) Model dynamics versus experimental data from (B) (solid lines and squares, respectively); for comparison, the model dynamics and data for wildtype DENV are reproduced from Fig 6C (dashed lines and circles, respectively). IFN-λ was measured by ELISA. Non-infected cells served as control. (D) Best-fit values and confidence bounds for the model parameters that have been allowed to differ between wildtype DENV and the 2’-O-methylation mutant. The model predicts that the virus production rate and the delay of IFN secretion decrease in case of the mutant (red box), whereas the delay of virus production and IFN secretion rate remain unchanged.

Mentions: Next, we asked whether the model could explain the attenuated spread of a proposed vaccine candidate—a DENV mutant that lacks 2'-O-methyltransferase activity [39,40]. To this end, we replaced the glutamic acid residue 217 in NS5 by an alanine residue in our DENV-faR reporter virus (Fig 8A). This modification impairs 2’-O-methylation of the DENV RNA genome.


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)

Spread of a DENV mutant with defective 2’-O-methylation of the viral RNA genome.(A) Genomic structure of the DENV-faR 2’-O-methylation mutant containing the E217A substitution in the NS5 protein (for further details see legend to Fig 4A). (B) Spread of the DENV-faR mutant in A549 reporter cells and activation of the IFN response. A549-IFIT1deGFP cells were infected with the DENV-faR E217A mutant at a MOI of 0.1 TCID50/cell. At the time points specified in the top of each panel, cells were fixed and processed for measurement by flow cytometry. (C) Model dynamics versus experimental data from (B) (solid lines and squares, respectively); for comparison, the model dynamics and data for wildtype DENV are reproduced from Fig 6C (dashed lines and circles, respectively). IFN-λ was measured by ELISA. Non-infected cells served as control. (D) Best-fit values and confidence bounds for the model parameters that have been allowed to differ between wildtype DENV and the 2’-O-methylation mutant. The model predicts that the virus production rate and the delay of IFN secretion decrease in case of the mutant (red box), whereas the delay of virus production and IFN secretion rate remain unchanged.
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Related In: Results  -  Collection

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ppat.1005345.g008: Spread of a DENV mutant with defective 2’-O-methylation of the viral RNA genome.(A) Genomic structure of the DENV-faR 2’-O-methylation mutant containing the E217A substitution in the NS5 protein (for further details see legend to Fig 4A). (B) Spread of the DENV-faR mutant in A549 reporter cells and activation of the IFN response. A549-IFIT1deGFP cells were infected with the DENV-faR E217A mutant at a MOI of 0.1 TCID50/cell. At the time points specified in the top of each panel, cells were fixed and processed for measurement by flow cytometry. (C) Model dynamics versus experimental data from (B) (solid lines and squares, respectively); for comparison, the model dynamics and data for wildtype DENV are reproduced from Fig 6C (dashed lines and circles, respectively). IFN-λ was measured by ELISA. Non-infected cells served as control. (D) Best-fit values and confidence bounds for the model parameters that have been allowed to differ between wildtype DENV and the 2’-O-methylation mutant. The model predicts that the virus production rate and the delay of IFN secretion decrease in case of the mutant (red box), whereas the delay of virus production and IFN secretion rate remain unchanged.
Mentions: Next, we asked whether the model could explain the attenuated spread of a proposed vaccine candidate—a DENV mutant that lacks 2'-O-methyltransferase activity [39,40]. To this end, we replaced the glutamic acid residue 217 in NS5 by an alanine residue in our DENV-faR reporter virus (Fig 8A). This modification impairs 2’-O-methylation of the DENV RNA genome.

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