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

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.