177
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Live Cell Analysis and Mathematical Modeling Identify Determinants of Attenuation of Dengue Virus 2’-O-Methylation Mutant

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          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.

          Author Summary

          Dengue virus (DENV) infection is a global health problem for which no selective therapy or vaccine exists. The magnitude of infection critically depends on the induction kinetics of the interferon (IFN) response and the kinetics of viral countermeasures. Here we established a novel live cell imaging system to dissect the dynamics of this interplay. We find that IFN controls DENV infection in a kinetically determined manner. At the single cell level, the IFN response is highly heterogeneous and stochastic, likely accounting for viral spread in the presence of IFN. Mathematical modeling and validation experiments show that the kinetics of activation of the IFN response critically determines control of virus replication and spread. A vaccine candidate DENV mutant lacking 2’-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. This attenuation is primarily due to accelerated kinetics of IFN production acting on infected cells in an autocrine manner. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. Thus, attenuation of the 2’-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.

          Related collections

          Most cited references57

          • Record: found
          • Abstract: found
          • Article: not found

          Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins.

          Through the study of transcriptional activation in response to interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma), a previously unrecognized direct signal transduction pathway to the nucleus has been uncovered: IFN-receptor interaction at the cell surface leads to the activation of kinases of the Jak family that then phosphorylate substrate proteins called STATs (signal transducers and activators of transcription). The phosphorylated STAT proteins move to the nucleus, bind specific DNA elements, and direct transcription. Recognition of the molecules involved in the IFN-alpha and IFN-gamma pathway has led to discoveries that a number of STAT family members exist and that other polypeptide ligands also use the Jak-STAT molecules in signal transduction.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Composition and Three-Dimensional Architecture of the Dengue Virus Replication and Assembly Sites

            Summary Positive-strand RNA viruses are known to rearrange cellular membranes to facilitate viral genome replication. The biogenesis and three-dimensional organization of these membranes and the link between replication and virus assembly sites is not fully clear. Using electron microscopy, we find Dengue virus (DENV)-induced vesicles, convoluted membranes, and virus particles to be endoplasmic reticulum (ER)-derived, and we detect double-stranded RNA, a presumed marker of RNA replication, inside virus-induced vesicles. Electron tomography (ET) shows DENV-induced membrane structures to be part of one ER-derived network. Furthermore, ET reveals vesicle pores that could enable release of newly synthesized viral RNA and reveals budding of DENV particles on ER membranes directly apposed to vesicle pores. Thus, DENV modifies ER membrane structure to promote replication and efficient encapsidation of the genome into progeny virus. This architecture of DENV replication and assembly sites could explain the coordination of distinct steps of the flavivirus replication cycle.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              A variant upstream of IFNL3 (IL28B) creating a new interferon gene IFNL4 is associated with impaired clearance of hepatitis C virus.

              Chronic infection with hepatitis C virus (HCV) is a common cause of liver cirrhosis and cancer. We performed RNA sequencing in primary human hepatocytes activated with synthetic double-stranded RNA to mimic HCV infection. Upstream of IFNL3 (IL28B) on chromosome 19q13.13, we discovered a new transiently induced region that harbors a dinucleotide variant ss469415590 (TT or ΔG), which is in high linkage disequilibrium with rs12979860, a genetic marker strongly associated with HCV clearance. ss469415590[ΔG] is a frameshift variant that creates a novel gene, designated IFNL4, encoding the interferon-λ4 protein (IFNL4), which is moderately similar to IFNL3. Compared to rs12979860, ss469415590 is more strongly associated with HCV clearance in individuals of African ancestry, although it provides comparable information in Europeans and Asians. Transient overexpression of IFNL4 in a hepatoma cell line induced STAT1 and STAT2 phosphorylation and the expression of interferon-stimulated genes. Our findings provide new insights into the genetic regulation of HCV clearance and its clinical management.
                Bookmark

                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                31 December 2015
                December 2015
                : 11
                : 12
                : e1005345
                Affiliations
                [1 ]Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany
                [2 ]Division of Theoretical Systems Biology, German Cancer Research Center (DKFZ), Heidelberg, Germany
                [3 ]BioQuant Center, University of Heidelberg, Heidelberg, Germany
                [4 ]Department of Bioinformatics and Functional Genomics, Biomedical Computer Vision Group, Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany
                Purdue University, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: BS MR ARu TH RB. Performed the experiments: BS MR ARu EGA MB ARe WF. Analyzed the data: BS MR ARu EGA ARe NH JPB MF KR TH RB. Contributed reagents/materials/analysis tools: WF KR. Wrote the paper: BS MR ARu TH RB.

                [¤]

                Current address: Haplogen GmbH, Vienna, Austria

                Article
                PPATHOGENS-D-15-00028
                10.1371/journal.ppat.1005345
                4697809
                26720415
                101adccb-7c9f-40b8-b081-a6c1c865f46f
                © 2015 Schmid et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 13 January 2015
                : 26 November 2015
                Page count
                Figures: 10, Tables: 0, Pages: 36
                Funding
                This work was funded by the Deutsche Forschungsgemeinschaft, FOR1202, TP1 (to RB) and the Bundesministerium für Bildung und Forschung (BMBF), project Immunoquant (to TH and RB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

                Infectious disease & Microbiology
                Infectious disease & Microbiology

                Comments

                Comment on this article