Spatiotemporal analysis of Herpesvirus genome presentation and transcriptional progression

Abstract

The initiation of herpesvirus transcription requires the selective recruitment and activity of the host transcriptional apparatus on virus immediate-early (IE) promoters. Despite general understanding of the broad principles of these early processes that have stemmed from population studies, we have comparatively limited knowledge of transcriptional events at the level of single cell and single genomes. I have developed methods combining click chemistry to visualise individual viral genomes with single molecule RNA transcript analysis and immunofluorescence to gain high resolution in-sight into the earliest processes of RNA polymerase recruitment, transcriptional bursting and RNA transport during the initiation of herpes simplex (HSV) infection. Analysing ICP0 and ICP4, as key representatives of the HSV IE class, my results allow quantitative identification of genomes with transcriptionally active IE RNA bursting and the discrimination of active genomes from inactive ones. From simultaneous analysis of the ICP0 intron and exon, I show the highly restricted localisation of intron containing transcripts on genomes within the large mature transcript bursts. The results indicate the highly rapid and likely co transcriptional splicing of the ICP0 pre-mRNA before general dispersal from the genome. I also show that while genomes show bursting activity for both ICP4 and ICP0, individual genomes can also be highly active for only one or other of these IE genes, indicating a spatial and temporal uncoupling of IE transcription at the single genome level. Combining genome, RNA and protein localisation, and discriminating transcriptionally active genomes from inactive ones, my results also indicate that there is no significant correlation between PML-genome association and transcriptional bursting. Using pulse labelling with clickable nucleotide precursors to label nascent infected cell DNA synthesis I have examined the organisation and spatial relationship between later DNA synthesis and virus transcription during lytic HSV infection to reveal new qualitative features of the spatial relationship between viral transcription and DNA synthesis. In infected cells that are in S-phase my results allow the spatial discrimination between cellular and viral DNA synthesis. Moreover, ICP4 shows pronounced selectivity by being recruited only to viral and not cellular sites of DNA replication, in contrast ICP8 is recruited to both types. I also show that different individual replication compartments within a single cell can be functionally distinct in terms of association with transcriptional activity. These and other results yield insight into the processes of HSV gene expression. They demonstrate the utility of single cell and single molecule based imaging approaches for investigation of DNA virus genomes and their relationship with the host cell during infection. Finally, they prompt a re-evaluation of current models in aspects both of host suppression of viral transcription and the temporal progression of DNA replication.