title: Live Cell Analysis and Mathematical Modeling Identify Determinants of Attenuation of Dengue Virus 2'-O-Methylation Mutant.
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.
authors.affiliation: Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Bianca
authors.initials: B
authors.lastname: Schmid
authors.affiliation: Division of Theoretical Systems Biology, German Cancer Research Center (DKFZ), Heidelberg, Germany.|BioQuant Center, University of Heidelberg, Heidelberg, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Rinas
authors.affiliation: Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Alessia
authors.initials: A
authors.lastname: Ruggieri
authors.affiliation: Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Eliana Gisela
authors.initials: EG
authors.lastname: Acosta
authors.affiliation: Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Marie
authors.initials: M
authors.lastname: Bartenschlager
authors.affiliation: Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Antje
authors.initials: A
authors.lastname: Reuter
authors.affiliation: Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Fischl
authors.affiliation: BioQuant Center, University of Heidelberg, Heidelberg, Germany.|Department of Bioinformatics and Functional Genomics, Biomedical Computer Vision Group, Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Harder
authors.affiliation: BioQuant Center, University of Heidelberg, Heidelberg, Germany.|Department of Bioinformatics and Functional Genomics, Biomedical Computer Vision Group, Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Jan-Philip
authors.initials: JP
authors.lastname: Bergeest
authors.affiliation: Division of Theoretical Systems Biology, German Cancer Research Center (DKFZ), Heidelberg, Germany.|BioQuant Center, University of Heidelberg, Heidelberg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Flossdorf
authors.affiliation: BioQuant Center, University of Heidelberg, Heidelberg, Germany.|Department of Bioinformatics and Functional Genomics, Biomedical Computer Vision Group, Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Karl
authors.initials: K
authors.lastname: Rohr
authors.affiliation: Division of Theoretical Systems Biology, German Cancer Research Center (DKFZ), Heidelberg, Germany.|BioQuant Center, University of Heidelberg, Heidelberg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Höfer
authors.affiliation: Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Bartenschlager
mesh_terms: D002460:Cell Line; D002470:Cell Survival; D003715:Dengue / Q000276:immunology*; D053059:Dengue Vaccines / Q000235:genetics / Q000276:immunology*; D003716:Dengue Virus / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D007372:Interferons / Q000276:immunology*; D008745:Methylation; D008962:Models, Theoretical; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D053059:Dengue Vaccines; D012367:RNA, Viral; D007372:Interferons