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      Spatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy

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          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

          We investigated the spatiotemporal dynamics of HSV genome transport during the initiation of infection using viruses containing bioorthogonal traceable precursors incorporated into their genomes (HSV EdC). In vitro assays revealed a structural alteration in the capsid induced upon HSV EdC binding to solid supports that allowed coupling to external capture agents and demonstrated that the vast majority of individual virions contained bioorthogonally-tagged genomes. Using HSV EdC in vivo we reveal novel aspects of the kinetics, localisation, mechanistic entry requirements and morphological transitions of infecting genomes. Uncoating and nuclear import was observed within 30 min, with genomes in a defined compaction state (ca. 3-fold volume increase from capsids). Free cytosolic uncoated genomes were infrequent (7–10% of the total uncoated genomes), likely a consequence of subpopulations of cells receiving high particle numbers. Uncoated nuclear genomes underwent temporal transitions in condensation state and while ICP4 efficiently associated with condensed foci of initial infecting genomes, this relationship switched away from residual longer lived condensed foci to increasingly decondensed genomes as infection progressed. Inhibition of transcription had no effect on nuclear entry but in the absence of transcription, genomes persisted as tightly condensed foci. Ongoing transcription, in the absence of protein synthesis, revealed a distinct spatial clustering of genomes, which we have termed genome congregation, not seen with non-transcribing genomes. Genomes expanded to more decondensed forms in the absence of DNA replication indicating additional transitional steps. During full progression of infection, genomes decondensed further, with a diffuse low intensity signal dissipated within replication compartments, but frequently with tight foci remaining peripherally, representing unreplicated genomes or condensed parental strands of replicated DNA. Uncoating and nuclear entry was independent of proteasome function and resistant to inhibitors of nuclear export. Together with additional data our results reveal new insight into the spatiotemporal dynamics of HSV genome uncoating, transport and organisation.

          Author summary

          Virtually all DNA virus classes as well as many RNA viruses must deposit their genomes within the nucleus for transcription, genome replication and subsequent capsid assembly. While infecting capsids have been studied by various methods and biochemical approaches have been used to investigate the bulk genome population characteristics, quantitative spatiotemporal information of the infecting genome itself at the single particle level has been lacking. This is required for any complete understanding of many critical aspects of virus infection and virus pathogenesis. Using novel techniques in bioorthogonal chemistry to produce normal non-recombinant viruses with readily traceable genomes, we provide the first direct quantitative spatiotemporal analysis of HSV genome transport and presentation to the cellular environment. Using these techniques which discriminate encapsidated from uncoated genomes and input from replicated DNA, our work provides a comprehensive analysis, using direct measures for genome detection not dependant on surrogate outputs. The results reveal completely novel aspects of early genome localisation and organisation not previously appreciated or amenable to study. Furthermore the work also provides a roadmap for similar studies in other systems and for future analysis of many aspects in different fields of the biology of infecting virus genomes early during cell infection.

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          Most cited references 79

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          A chemical method for fast and sensitive detection of DNA synthesis in vivo.

          We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)-catalyzed [3 + 2] cycloaddition reaction ("click" chemistry). Detection of the EdU label is highly sensitive and can be accomplished in minutes. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration, allowing the staining of whole-mount preparations of large tissue and organ explants. In contrast to BrdU, the method does not require sample fixation or DNA denaturation and permits good structural preservation. We demonstrate the use of the method in cultured cells and in the intestine and brain of whole animals.
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            Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination.

            Structured illumination microscopy is a method that can increase the spatial resolution of wide-field fluorescence microscopy beyond its classical limit by using spatially structured illumination light. Here we describe how this method can be applied in three dimensions to double the axial as well as the lateral resolution, with true optical sectioning. A grating is used to generate three mutually coherent light beams, which interfere in the specimen to form an illumination pattern that varies both laterally and axially. The spatially structured excitation intensity causes normally unreachable high-resolution information to become encoded into the observed images through spatial frequency mixing. This new information is computationally extracted and used to generate a three-dimensional reconstruction with twice as high resolution, in all three dimensions, as is possible in a conventional wide-field microscope. The method has been demonstrated on both test objects and biological specimens, and has produced the first light microscopy images of the synaptonemal complex in which the lateral elements are clearly resolved.
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              Icy: an open bioimage informatics platform for extended reproducible research.

              Current research in biology uses evermore complex computational and imaging tools. Here we describe Icy, a collaborative bioimage informatics platform that combines a community website for contributing and sharing tools and material, and software with a high-end visual programming framework for seamless development of sophisticated imaging workflows. Icy extends the reproducible research principles, by encouraging and facilitating the reusability, modularity, standardization and management of algorithms and protocols. Icy is free, open-source and available at http://icy.bioimageanalysis.org/.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: InvestigationRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: Formal analysisRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: SoftwareRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                9 November 2017
                November 2017
                : 13
                : 11
                PPATHOGENS-D-17-01897
                10.1371/journal.ppat.1006721
                5697887
                29121649
                © 2017 Sekine 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.

                Counts
                Figures: 12, Tables: 0, Pages: 36
                Product
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100004440, Wellcome Trust;
                Award ID: LCII-P54163
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100000268, Biotechnology and Biological Sciences Research Council;
                Award ID: BB/L015129/1
                Award Recipient :
                This work was funded in part by grants from the Biotechnology and Biological Sciences Research Council, GB which support the Facility for Light Microscopy Imperial College London to DG ( http://www.bbsrc.ac.uk/) and by a PhD studentship from the Wellcome Trust, UK ( https://wellcome.ac.uk/) to ES. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Microbiology
                Virology
                Viral Replication
                Viral Packaging
                Biology and Life Sciences
                Genetics
                Genomics
                Biology and Life Sciences
                Genetics
                Genomics
                Microbial Genomics
                Viral Genomics
                Biology and Life Sciences
                Microbiology
                Microbial Genomics
                Viral Genomics
                Biology and Life Sciences
                Microbiology
                Virology
                Viral Genomics
                Biology and Life Sciences
                Cell Biology
                Signal Transduction
                Cell Signaling
                Genomic Signal Processing
                Biology and life sciences
                Genetics
                DNA
                DNA replication
                Biology and life sciences
                Biochemistry
                Nucleic acids
                DNA
                DNA replication
                Biology and Life Sciences
                Computational Biology
                Comparative Genomics
                Biology and Life Sciences
                Genetics
                Genomics
                Comparative Genomics
                Biology and Life Sciences
                Microbiology
                Virology
                Viral Structure
                Virions
                Research and Analysis Methods
                Immunologic Techniques
                Immunoassays
                Immunofluorescence
                Custom metadata
                vor-update-to-uncorrected-proof
                2017-11-21
                All relevant data are within the paper and its Supporting Information files.

                Infectious disease & Microbiology

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