Spatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy.
We investigated the spatiotemporal dynamics of HSV genome transport during the initiation of infection using viruses containing bioorthogonal traceable precursors incorporated into their genomes (HSVEdC). In vitro assays revealed a structural alteration in the capsid induced upon HSVEdC binding to s...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2017-11-01
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| Series: | PLoS Pathogens |
| Online Access: | https://doi.org/10.1371/journal.ppat.1006721 |
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| author | Eiki Sekine Nora Schmidt David Gaboriau Peter O'Hare |
| author_facet | Eiki Sekine Nora Schmidt David Gaboriau Peter O'Hare |
| author_sort | Eiki Sekine |
| collection | DOAJ |
| description | We investigated the spatiotemporal dynamics of HSV genome transport during the initiation of infection using viruses containing bioorthogonal traceable precursors incorporated into their genomes (HSVEdC). In vitro assays revealed a structural alteration in the capsid induced upon HSVEdC 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 HSVEdC 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. |
| format | Article |
| id | doaj-art-9b9523ad00924e428e42bbb58f1c4782 |
| institution | Kabale University |
| issn | 1553-7366 1553-7374 |
| language | English |
| publishDate | 2017-11-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Pathogens |
| spelling | doaj-art-9b9523ad00924e428e42bbb58f1c47822025-08-20T03:28:51ZengPublic Library of Science (PLoS)PLoS Pathogens1553-73661553-73742017-11-011311e100672110.1371/journal.ppat.1006721Spatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy.Eiki SekineNora SchmidtDavid GaboriauPeter O'HareWe investigated the spatiotemporal dynamics of HSV genome transport during the initiation of infection using viruses containing bioorthogonal traceable precursors incorporated into their genomes (HSVEdC). In vitro assays revealed a structural alteration in the capsid induced upon HSVEdC 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 HSVEdC 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.https://doi.org/10.1371/journal.ppat.1006721 |
| spellingShingle | Eiki Sekine Nora Schmidt David Gaboriau Peter O'Hare Spatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy. PLoS Pathogens |
| title | Spatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy. |
| title_full | Spatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy. |
| title_fullStr | Spatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy. |
| title_full_unstemmed | Spatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy. |
| title_short | Spatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy. |
| title_sort | spatiotemporal dynamics of hsv genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super resolution microscopy |
| url | https://doi.org/10.1371/journal.ppat.1006721 |
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