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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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Characterization of the DENV-faR reporter virus.(A) Schematic of the DENV reporter virus genome. The first 103 nucleotides of the capsid coding sequence were duplicated, because they contain the circularization sequence 1 (CS1; blue bar) that is essential for DENV RNA replication. Thus, the far red reporter is fused N-terminally with ~34 amino acid residues of the capsid protein that contains a nuclear localization sequence and C-terminally with the sequence encoding for the 2A cleavage factor (PCS) of the Thosea asigna virus, thus generating the authentic N-terminus of the capsid protein. (B) A549 cells were infected with the DENV-faR reporter virus or the parental wildtype (wt) lacking a reporter gene at a MOI of 1 TCID50/cell. At time points specified in the bottom, culture supernatants were harvested and titers of infectious virus were determined by limiting dilution assay. (C) Single-step growth curve of DENV-faR in A549 cells. Cells were infected at a MOI of 10 TCID50/cell and harvested at multiple time points. Viral RNA was quantified by RT-qPCR and infectivity released from cells was monitored by limiting dilution assay. Graphs display a representative result out of 3 independent experiments. Values correspond to the mean of triplicate measurements and error bars. (D) Kinetics of activation of IFIT1 transcription and IFN-λ secretion. A549 cells were infected with DENV-faR at a MOI of 10 TCID50/cell and harvested at time points specified in the bottom of the graph. IFIT1 transcription was determined by RT-qPCR, whereas IFN-λ secretion was quantified by ELISA. IFIT1 mRNA values were normalized to GAPDH mRNA levels.
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ppat.1005345.g004: Characterization of the DENV-faR reporter virus.(A) Schematic of the DENV reporter virus genome. The first 103 nucleotides of the capsid coding sequence were duplicated, because they contain the circularization sequence 1 (CS1; blue bar) that is essential for DENV RNA replication. Thus, the far red reporter is fused N-terminally with ~34 amino acid residues of the capsid protein that contains a nuclear localization sequence and C-terminally with the sequence encoding for the 2A cleavage factor (PCS) of the Thosea asigna virus, thus generating the authentic N-terminus of the capsid protein. (B) A549 cells were infected with the DENV-faR reporter virus or the parental wildtype (wt) lacking a reporter gene at a MOI of 1 TCID50/cell. At time points specified in the bottom, culture supernatants were harvested and titers of infectious virus were determined by limiting dilution assay. (C) Single-step growth curve of DENV-faR in A549 cells. Cells were infected at a MOI of 10 TCID50/cell and harvested at multiple time points. Viral RNA was quantified by RT-qPCR and infectivity released from cells was monitored by limiting dilution assay. Graphs display a representative result out of 3 independent experiments. Values correspond to the mean of triplicate measurements and error bars. (D) Kinetics of activation of IFIT1 transcription and IFN-λ secretion. A549 cells were infected with DENV-faR at a MOI of 10 TCID50/cell and harvested at time points specified in the bottom of the graph. IFIT1 transcription was determined by RT-qPCR, whereas IFN-λ secretion was quantified by ELISA. IFIT1 mRNA values were normalized to GAPDH mRNA levels.

Mentions: To study the interplay between the IFN response and the kinetics of DENV replication and spread, we constructed a genetically modified DENV reporter virus genome encoding the far red fluorescent protein TurboFP635 (“faR”) (Fig 4A). This reporter matures rapidly, is pH- and photo-stable and has high signal intensity [50]. The reporter virus, designated DENV-faR, was derived from the DENV-2 isolate 16681 by using the monocistronic construct design we have described earlier [51]. Replication fitness and virus production of this reporter genome was only slightly lower as compared to the parental DENV wildtype (DENV-wt) (Fig 4B). Importantly, reporter virus spread was readily detected in A549 cells. This is best demonstrated by the robust increase of reporter protein-positive cells upon infection with this virus as compared to infection with virus-like particles that support only a single round of infection (S5A and S5B 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)

Characterization of the DENV-faR reporter virus.(A) Schematic of the DENV reporter virus genome. The first 103 nucleotides of the capsid coding sequence were duplicated, because they contain the circularization sequence 1 (CS1; blue bar) that is essential for DENV RNA replication. Thus, the far red reporter is fused N-terminally with ~34 amino acid residues of the capsid protein that contains a nuclear localization sequence and C-terminally with the sequence encoding for the 2A cleavage factor (PCS) of the Thosea asigna virus, thus generating the authentic N-terminus of the capsid protein. (B) A549 cells were infected with the DENV-faR reporter virus or the parental wildtype (wt) lacking a reporter gene at a MOI of 1 TCID50/cell. At time points specified in the bottom, culture supernatants were harvested and titers of infectious virus were determined by limiting dilution assay. (C) Single-step growth curve of DENV-faR in A549 cells. Cells were infected at a MOI of 10 TCID50/cell and harvested at multiple time points. Viral RNA was quantified by RT-qPCR and infectivity released from cells was monitored by limiting dilution assay. Graphs display a representative result out of 3 independent experiments. Values correspond to the mean of triplicate measurements and error bars. (D) Kinetics of activation of IFIT1 transcription and IFN-λ secretion. A549 cells were infected with DENV-faR at a MOI of 10 TCID50/cell and harvested at time points specified in the bottom of the graph. IFIT1 transcription was determined by RT-qPCR, whereas IFN-λ secretion was quantified by ELISA. IFIT1 mRNA values were normalized to GAPDH mRNA levels.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4697809&req=5

ppat.1005345.g004: Characterization of the DENV-faR reporter virus.(A) Schematic of the DENV reporter virus genome. The first 103 nucleotides of the capsid coding sequence were duplicated, because they contain the circularization sequence 1 (CS1; blue bar) that is essential for DENV RNA replication. Thus, the far red reporter is fused N-terminally with ~34 amino acid residues of the capsid protein that contains a nuclear localization sequence and C-terminally with the sequence encoding for the 2A cleavage factor (PCS) of the Thosea asigna virus, thus generating the authentic N-terminus of the capsid protein. (B) A549 cells were infected with the DENV-faR reporter virus or the parental wildtype (wt) lacking a reporter gene at a MOI of 1 TCID50/cell. At time points specified in the bottom, culture supernatants were harvested and titers of infectious virus were determined by limiting dilution assay. (C) Single-step growth curve of DENV-faR in A549 cells. Cells were infected at a MOI of 10 TCID50/cell and harvested at multiple time points. Viral RNA was quantified by RT-qPCR and infectivity released from cells was monitored by limiting dilution assay. Graphs display a representative result out of 3 independent experiments. Values correspond to the mean of triplicate measurements and error bars. (D) Kinetics of activation of IFIT1 transcription and IFN-λ secretion. A549 cells were infected with DENV-faR at a MOI of 10 TCID50/cell and harvested at time points specified in the bottom of the graph. IFIT1 transcription was determined by RT-qPCR, whereas IFN-λ secretion was quantified by ELISA. IFIT1 mRNA values were normalized to GAPDH mRNA levels.
Mentions: To study the interplay between the IFN response and the kinetics of DENV replication and spread, we constructed a genetically modified DENV reporter virus genome encoding the far red fluorescent protein TurboFP635 (“faR”) (Fig 4A). This reporter matures rapidly, is pH- and photo-stable and has high signal intensity [50]. The reporter virus, designated DENV-faR, was derived from the DENV-2 isolate 16681 by using the monocistronic construct design we have described earlier [51]. Replication fitness and virus production of this reporter genome was only slightly lower as compared to the parental DENV wildtype (DENV-wt) (Fig 4B). Importantly, reporter virus spread was readily detected in A549 cells. This is best demonstrated by the robust increase of reporter protein-positive cells upon infection with this virus as compared to infection with virus-like particles that support only a single round of infection (S5A and S5B 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