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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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Quantitative model of DENV spread and IFN response.(A) Schematic of the mathematical model: Virus replicates in infected cells after time delay τR and produces virus with time delay τV and rate vV, which can infect susceptible cells (blue arrow). Infected cells produce IFN after time delay τF with rate vF, which protects susceptible cells (solid green arrow) and can induce antiviral genes in infected cells (dashed green arrow). For further details of the mathematical model see S1 Supplementary Methods. (B) Spread of DENV-faR virus and activation of the IFN response. A549-IFIT1deGFP cells were infected with DENV-faR at a MOI of 0.1 TCID50/cell. At time points specified in the top of each panel, cells were fixed and processed for measurement by flow cytometry. (C) Best fit of the mathematical model to two independent experiments; the initial conditions for the number of susceptible cells are specific to each experiment whereas the model parameters are the same for both experiments. The numbers of susceptible, DENV-faR positive (i.e. infected) and IFIT1deGFP positive (i.e. protected) cells have been taken from the flow-cytometry data (the comparatively small number of double-positive cells was counted as infected); IFN-λ was measured in experiment 2 by ELISA. Non-infected cells served as control.
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ppat.1005345.g006: Quantitative model of DENV spread and IFN response.(A) Schematic of the mathematical model: Virus replicates in infected cells after time delay τR and produces virus with time delay τV and rate vV, which can infect susceptible cells (blue arrow). Infected cells produce IFN after time delay τF with rate vF, which protects susceptible cells (solid green arrow) and can induce antiviral genes in infected cells (dashed green arrow). For further details of the mathematical model see S1 Supplementary Methods. (B) Spread of DENV-faR virus and activation of the IFN response. A549-IFIT1deGFP cells were infected with DENV-faR at a MOI of 0.1 TCID50/cell. At time points specified in the top of each panel, cells were fixed and processed for measurement by flow cytometry. (C) Best fit of the mathematical model to two independent experiments; the initial conditions for the number of susceptible cells are specific to each experiment whereas the model parameters are the same for both experiments. The numbers of susceptible, DENV-faR positive (i.e. infected) and IFIT1deGFP positive (i.e. protected) cells have been taken from the flow-cytometry data (the comparatively small number of double-positive cells was counted as infected); IFN-λ was measured in experiment 2 by ELISA. Non-infected cells served as control.

Mentions: To understand which parameters control the kinetics of viral spread and the IFN response, we developed a data-based mathematical model. This model describes the rates of naïve cells becoming infected by extracellular DENV or protected by the action of IFNs released from infected cells (Fig 6A). The model takes into account that in infected cells, DENV replicates after a time delay τR (see Fig 4C). After a time delay τV, which considers the additional time necessary for viral protein expression and particle assembly, infectious virions are released. Moreover, infected cells secrete IFN after time delay τF that corresponds to the time necessary for viral recognition, downstream signal transduction as well as IFN-gene transcription, mRNA translation and IFN secretion (Fig 4D). Released IFNs protect susceptible cells (i.e. cells that are sensitive to infection) from infection (solid green arrow in Fig 6A). Importantly, IFNs inhibit virus replication in infected cells if acting early, i.e. prior to DENV-induced shut-off of JAK-STAT signaling (dashed green arrow in Fig 6A; cf. Fig 1A and 1B).


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)

Quantitative model of DENV spread and IFN response.(A) Schematic of the mathematical model: Virus replicates in infected cells after time delay τR and produces virus with time delay τV and rate vV, which can infect susceptible cells (blue arrow). Infected cells produce IFN after time delay τF with rate vF, which protects susceptible cells (solid green arrow) and can induce antiviral genes in infected cells (dashed green arrow). For further details of the mathematical model see S1 Supplementary Methods. (B) Spread of DENV-faR virus and activation of the IFN response. A549-IFIT1deGFP cells were infected with DENV-faR at a MOI of 0.1 TCID50/cell. At time points specified in the top of each panel, cells were fixed and processed for measurement by flow cytometry. (C) Best fit of the mathematical model to two independent experiments; the initial conditions for the number of susceptible cells are specific to each experiment whereas the model parameters are the same for both experiments. The numbers of susceptible, DENV-faR positive (i.e. infected) and IFIT1deGFP positive (i.e. protected) cells have been taken from the flow-cytometry data (the comparatively small number of double-positive cells was counted as infected); IFN-λ was measured in experiment 2 by ELISA. Non-infected cells served as control.
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ppat.1005345.g006: Quantitative model of DENV spread and IFN response.(A) Schematic of the mathematical model: Virus replicates in infected cells after time delay τR and produces virus with time delay τV and rate vV, which can infect susceptible cells (blue arrow). Infected cells produce IFN after time delay τF with rate vF, which protects susceptible cells (solid green arrow) and can induce antiviral genes in infected cells (dashed green arrow). For further details of the mathematical model see S1 Supplementary Methods. (B) Spread of DENV-faR virus and activation of the IFN response. A549-IFIT1deGFP cells were infected with DENV-faR at a MOI of 0.1 TCID50/cell. At time points specified in the top of each panel, cells were fixed and processed for measurement by flow cytometry. (C) Best fit of the mathematical model to two independent experiments; the initial conditions for the number of susceptible cells are specific to each experiment whereas the model parameters are the same for both experiments. The numbers of susceptible, DENV-faR positive (i.e. infected) and IFIT1deGFP positive (i.e. protected) cells have been taken from the flow-cytometry data (the comparatively small number of double-positive cells was counted as infected); IFN-λ was measured in experiment 2 by ELISA. Non-infected cells served as control.
Mentions: To understand which parameters control the kinetics of viral spread and the IFN response, we developed a data-based mathematical model. This model describes the rates of naïve cells becoming infected by extracellular DENV or protected by the action of IFNs released from infected cells (Fig 6A). The model takes into account that in infected cells, DENV replicates after a time delay τR (see Fig 4C). After a time delay τV, which considers the additional time necessary for viral protein expression and particle assembly, infectious virions are released. Moreover, infected cells secrete IFN after time delay τF that corresponds to the time necessary for viral recognition, downstream signal transduction as well as IFN-gene transcription, mRNA translation and IFN secretion (Fig 4D). Released IFNs protect susceptible cells (i.e. cells that are sensitive to infection) from infection (solid green arrow in Fig 6A). Importantly, IFNs inhibit virus replication in infected cells if acting early, i.e. prior to DENV-induced shut-off of JAK-STAT signaling (dashed green arrow in Fig 6A; cf. Fig 1A and 1B).

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