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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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Heterogeneity of IFN induction and DENV-faR replication in A549 reporter cells at the single cell level.A549 reporter cell lines were infected with DENV-faR and time-lapse live cell imaging was started one hour later. Images were recorded in 30 min intervals for IFIT1 and 1 h intervals for Mx1 until 72 h p.i. Images were analyzed by using the ImageJ software package and the MTrackJ plug-in. (A, B) A549-IFIT1deGFP cells were infected with DENV-faR at a MOI of 0.2 TCID50/cell. (A) Representative still images taken at time points specified in the bottom left. Arrowheads point to faR—IFIT1deGFP double positive cells. (B) Kinetics of onset of detectable expression of the viral faR reporter gene (upper panel) and the ISG-reporter IFIT1deGFP (lower panel), respectively. (C, D) A549-Mx1deGFP cells were infected with DENV-faR at a MOI of 0.1 TCID50/cell. Shown are representative still images (C) and kinetics of detectable onset of expression (D) of the viral faR (upper panel) as well as the Mx1deGFP reporter (lower panel), respectively. In panel B, 91 DENV+ and 119 IFIT1+ cells were used for the analysis, respectively; in panel D 79 DENV+ and 83 Mx1+ cells, respectively. The corresponding movies are shown in the supplementary section.
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ppat.1005345.g005: Heterogeneity of IFN induction and DENV-faR replication in A549 reporter cells at the single cell level.A549 reporter cell lines were infected with DENV-faR and time-lapse live cell imaging was started one hour later. Images were recorded in 30 min intervals for IFIT1 and 1 h intervals for Mx1 until 72 h p.i. Images were analyzed by using the ImageJ software package and the MTrackJ plug-in. (A, B) A549-IFIT1deGFP cells were infected with DENV-faR at a MOI of 0.2 TCID50/cell. (A) Representative still images taken at time points specified in the bottom left. Arrowheads point to faR—IFIT1deGFP double positive cells. (B) Kinetics of onset of detectable expression of the viral faR reporter gene (upper panel) and the ISG-reporter IFIT1deGFP (lower panel), respectively. (C, D) A549-Mx1deGFP cells were infected with DENV-faR at a MOI of 0.1 TCID50/cell. Shown are representative still images (C) and kinetics of detectable onset of expression (D) of the viral faR (upper panel) as well as the Mx1deGFP reporter (lower panel), respectively. In panel B, 91 DENV+ and 119 IFIT1+ cells were used for the analysis, respectively; in panel D 79 DENV+ and 83 Mx1+ cells, respectively. The corresponding movies are shown in the supplementary section.

Mentions: Taking advantage of our reporter cells and virus system, we determined the kinetics of DENV replication/spread and activation of the IFN response by time-lapse microscopy. Experiments were conducted with A549-IFIT1deGFP and A549-Mx1deGFP cells (Fig 5), using low MOI to allow for viral spread through secondary infection events. In both cell lines DENV-faR-expressing cells were first detected 22 h after infection, reflecting the delay from infection to the rise in intracellular viral RNA and expression of the reporter protein to a detectable level (cf. Fig 4C). Given that infected cells start to produce viral particles from 16 h post infection (cf. Fig 4C and S7B Fig) and that infection takes ~20 h to become visible with the faR reporter, the majority of cells becoming DENV-faR-positive between 36 and 72 h p.i. likely reflects viral spread by secondary infection events. Consistent with these kinetics we found that A549-IFIT1deGFP reporter cells infected with the reporter virus at high MOI (10 TCID50/cell) displayed a faster onset of faR expression concomitant with a lower number of IFIT1deGFP-positive cells, likely as a result of the JAK-STAT signaling block (S9 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)

Heterogeneity of IFN induction and DENV-faR replication in A549 reporter cells at the single cell level.A549 reporter cell lines were infected with DENV-faR and time-lapse live cell imaging was started one hour later. Images were recorded in 30 min intervals for IFIT1 and 1 h intervals for Mx1 until 72 h p.i. Images were analyzed by using the ImageJ software package and the MTrackJ plug-in. (A, B) A549-IFIT1deGFP cells were infected with DENV-faR at a MOI of 0.2 TCID50/cell. (A) Representative still images taken at time points specified in the bottom left. Arrowheads point to faR—IFIT1deGFP double positive cells. (B) Kinetics of onset of detectable expression of the viral faR reporter gene (upper panel) and the ISG-reporter IFIT1deGFP (lower panel), respectively. (C, D) A549-Mx1deGFP cells were infected with DENV-faR at a MOI of 0.1 TCID50/cell. Shown are representative still images (C) and kinetics of detectable onset of expression (D) of the viral faR (upper panel) as well as the Mx1deGFP reporter (lower panel), respectively. In panel B, 91 DENV+ and 119 IFIT1+ cells were used for the analysis, respectively; in panel D 79 DENV+ and 83 Mx1+ cells, respectively. The corresponding movies are shown in the supplementary section.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4697809&req=5

ppat.1005345.g005: Heterogeneity of IFN induction and DENV-faR replication in A549 reporter cells at the single cell level.A549 reporter cell lines were infected with DENV-faR and time-lapse live cell imaging was started one hour later. Images were recorded in 30 min intervals for IFIT1 and 1 h intervals for Mx1 until 72 h p.i. Images were analyzed by using the ImageJ software package and the MTrackJ plug-in. (A, B) A549-IFIT1deGFP cells were infected with DENV-faR at a MOI of 0.2 TCID50/cell. (A) Representative still images taken at time points specified in the bottom left. Arrowheads point to faR—IFIT1deGFP double positive cells. (B) Kinetics of onset of detectable expression of the viral faR reporter gene (upper panel) and the ISG-reporter IFIT1deGFP (lower panel), respectively. (C, D) A549-Mx1deGFP cells were infected with DENV-faR at a MOI of 0.1 TCID50/cell. Shown are representative still images (C) and kinetics of detectable onset of expression (D) of the viral faR (upper panel) as well as the Mx1deGFP reporter (lower panel), respectively. In panel B, 91 DENV+ and 119 IFIT1+ cells were used for the analysis, respectively; in panel D 79 DENV+ and 83 Mx1+ cells, respectively. The corresponding movies are shown in the supplementary section.
Mentions: Taking advantage of our reporter cells and virus system, we determined the kinetics of DENV replication/spread and activation of the IFN response by time-lapse microscopy. Experiments were conducted with A549-IFIT1deGFP and A549-Mx1deGFP cells (Fig 5), using low MOI to allow for viral spread through secondary infection events. In both cell lines DENV-faR-expressing cells were first detected 22 h after infection, reflecting the delay from infection to the rise in intracellular viral RNA and expression of the reporter protein to a detectable level (cf. Fig 4C). Given that infected cells start to produce viral particles from 16 h post infection (cf. Fig 4C and S7B Fig) and that infection takes ~20 h to become visible with the faR reporter, the majority of cells becoming DENV-faR-positive between 36 and 72 h p.i. likely reflects viral spread by secondary infection events. Consistent with these kinetics we found that A549-IFIT1deGFP reporter cells infected with the reporter virus at high MOI (10 TCID50/cell) displayed a faster onset of faR expression concomitant with a lower number of IFIT1deGFP-positive cells, likely as a result of the JAK-STAT signaling block (S9 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