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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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Lack of 2’-O-methylation of the DENV RNA genome causes attenuation of virus spread by IFN action on infected cells.(A) Schematic of the extended mathematical model. The inhibition of virus replication by early IFN signals is described explicitly. For further details see legend to Fig 6A. (B) Extent of predicted viral spread simulated by the model as a function of the delay from infection to IFN secretion and the virus production rate by infected cells that have not been inhibited by early IFN signals. The color code gives the relative difference from the spread of wildtype DENV in the full model at 96 h p.i. (C, D) Sensitivity analysis of the model parameters for infection with (C) wildtype DENV and (D) the E217A mutant. The response coefficients Ri of model parameter i were calculated with the full model. Shown are virus-related (blue bars) and IFN-specific (green bars) parameter effects on infected cells.
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ppat.1005345.g009: Lack of 2’-O-methylation of the DENV RNA genome causes attenuation of virus spread by IFN action on infected cells.(A) Schematic of the extended mathematical model. The inhibition of virus replication by early IFN signals is described explicitly. For further details see legend to Fig 6A. (B) Extent of predicted viral spread simulated by the model as a function of the delay from infection to IFN secretion and the virus production rate by infected cells that have not been inhibited by early IFN signals. The color code gives the relative difference from the spread of wildtype DENV in the full model at 96 h p.i. (C, D) Sensitivity analysis of the model parameters for infection with (C) wildtype DENV and (D) the E217A mutant. The response coefficients Ri of model parameter i were calculated with the full model. Shown are virus-related (blue bars) and IFN-specific (green bars) parameter effects on infected cells.

Mentions: Although the model identified the parameters most critical for the attenuation of the E217A mutant, their relative importance was not clear. Specifically, the IFN production delay and the virus production rate might not be independent parameters (as assumed for simplicity in the previous analysis), because virus replication could be affected by IFN in a time-dependent manner through the early induction of ISGs before STAT2 degradation takes effect (see Fig 1). To account for this possibility, we extended the mathematical model (Fig 9A): The inhibition of virus production by IFN was described explicitly and could take place through ISG induction in an initial phase after infection (Fig 9A, solid green inhibition link), assumed to be 8 h. Compared to Fig 1B, this somewhat extended time window accounts for the fact that the initial infection dose in the time-course experiments (Figs 6B and 8B) is much lower, likely delaying the average onset of viral replication. If an infected cell does not perceive IFN within this time period, it becomes a virus producer, albeit with a somewhat reduced production rate (~50% of wildtype DENV), to account for the possibility that basal IFIT1 expression inhibits translation of DENV mutant RNA. With these modifications (S3 Table), the model fitted the kinetics of both wildtype and mutant DENV replication and IFN response as before (Fig 8C), but could now account for ISG induction and the resulting inhibition of viral replication within an early window of opportunity after infection.


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)

Lack of 2’-O-methylation of the DENV RNA genome causes attenuation of virus spread by IFN action on infected cells.(A) Schematic of the extended mathematical model. The inhibition of virus replication by early IFN signals is described explicitly. For further details see legend to Fig 6A. (B) Extent of predicted viral spread simulated by the model as a function of the delay from infection to IFN secretion and the virus production rate by infected cells that have not been inhibited by early IFN signals. The color code gives the relative difference from the spread of wildtype DENV in the full model at 96 h p.i. (C, D) Sensitivity analysis of the model parameters for infection with (C) wildtype DENV and (D) the E217A mutant. The response coefficients Ri of model parameter i were calculated with the full model. Shown are virus-related (blue bars) and IFN-specific (green bars) parameter effects on infected cells.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005345.g009: Lack of 2’-O-methylation of the DENV RNA genome causes attenuation of virus spread by IFN action on infected cells.(A) Schematic of the extended mathematical model. The inhibition of virus replication by early IFN signals is described explicitly. For further details see legend to Fig 6A. (B) Extent of predicted viral spread simulated by the model as a function of the delay from infection to IFN secretion and the virus production rate by infected cells that have not been inhibited by early IFN signals. The color code gives the relative difference from the spread of wildtype DENV in the full model at 96 h p.i. (C, D) Sensitivity analysis of the model parameters for infection with (C) wildtype DENV and (D) the E217A mutant. The response coefficients Ri of model parameter i were calculated with the full model. Shown are virus-related (blue bars) and IFN-specific (green bars) parameter effects on infected cells.
Mentions: Although the model identified the parameters most critical for the attenuation of the E217A mutant, their relative importance was not clear. Specifically, the IFN production delay and the virus production rate might not be independent parameters (as assumed for simplicity in the previous analysis), because virus replication could be affected by IFN in a time-dependent manner through the early induction of ISGs before STAT2 degradation takes effect (see Fig 1). To account for this possibility, we extended the mathematical model (Fig 9A): The inhibition of virus production by IFN was described explicitly and could take place through ISG induction in an initial phase after infection (Fig 9A, solid green inhibition link), assumed to be 8 h. Compared to Fig 1B, this somewhat extended time window accounts for the fact that the initial infection dose in the time-course experiments (Figs 6B and 8B) is much lower, likely delaying the average onset of viral replication. If an infected cell does not perceive IFN within this time period, it becomes a virus producer, albeit with a somewhat reduced production rate (~50% of wildtype DENV), to account for the possibility that basal IFIT1 expression inhibits translation of DENV mutant RNA. With these modifications (S3 Table), the model fitted the kinetics of both wildtype and mutant DENV replication and IFN response as before (Fig 8C), but could now account for ISG induction and the resulting inhibition of viral replication within an early window of opportunity after infection.

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