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 (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.

Recombination between co-infecting herpesviruses occurs where replication compartments coalesce

“…Therefore, we developed a quantitative evaluation of the RC overlapping areas in each cell. We found that common pixel based co-localization methods do not provide an accurate estimate of the degree of overlap between HSV-1 RCs, probably due to the relaxed form of the replicating viral DNA (34). Our results following infection with one virus carrying the two tag sequences (OK31) showed limited co-localization at the pixel level, similar to results obtained by two probes corresponding to adjacent loci on HSV-1 genomes (53).…”

“…Second, the smaller RC could interact with two larger RCs that flank both sides of the small RC and eventually unite into a single larger RC. A third mechanism could involve the recently observed condensed viral genomes at the edge of RCs (34). Sekine et al showed that at 3HPI several incoming genomes remain condensed at the periphery of an enlarging RC (34).…”

“…A third mechanism could involve the recently observed condensed viral genomes at the edge of RCs (34). Sekine et al showed that at 3HPI several incoming genomes remain condensed at the periphery of an enlarging RC (34). These condensed genomes might be silent throughout the lytic infection or may serve as templates for recombination at later time points (which will result in our observed third interaction).…”

“…Since our images represent snapshots during the infection process, we cannot distinguish between the possibilities that larger nuclei allow a larger number of incoming viral genomes to initiate replication or that the nuclei in which more genomes initiated replication expand faster. The use of new methods to visualise incoming genomes using click chemistry (34,35,63) should be able to resolve these two alternatives.…”

“…A recent study showed that viral genomes entering the nucleus are observed as condensed foci, and suggested that viral expression and DNA replication allow decondensation of these genomes and formation of RCs (34). Interestingly, some genomes remain highly condensed at the edge of newly developing RCs (34,35). Here, we visualised co-infecting HSV-1 genomes and confirmed that alphaherpesviruses RCs initiate from single genomes.…”