Herpesvirus transport and genome presentation during the initiation of infection

Abstract

Events controlling herpesvirus nuclear genome uncoating, nuclear transport, and early transcription remain poorly understood. I have developed methods to quantitatively examine these processes within individual cells and at the level of single molecules for both the genome and the mRNA transcripts produced from it. Using bioorthogonal nucleoside precursors for genome labelling in HSV-1 infected cells and quantitative single particle analysis, I demonstrate extremely efficient precursor incorporation resulting in quantitative genome detection on a single particle basis in the population of progeny virus. I report a comprehensive analysis in infected cells of genome dynamics during capsid exit and nuclear import in which I, (i) demonstrate qualitative transitions in genome condensation state linked to transcription and replication, (ii) reveal novel processes in genome congregation dependent upon transcription and (iii) show the temporal switching in regulatory protein recruitment (represented by ICP4) to distinct genome compartments. Furthermore, I have developed the simultaneous visualisation of HSV-1 genomes and their product transcripts. This has enabled me to conduct spatiotemporal analysis of immediate early (IE) gene transcription kinetics in relation to the template genomes. I demonstrate a biphasic nature of ICP0 transcription, heterogeneity of genome transcription bursting, and differences in IE gene transcript behaviour with multiplex analysis, all of which have laid the foundations for further multivariate quantitative analysis. Finally, I have started to decipher the relationship of infectious genomes with host cell antiviral factors, demonstrating the association of PML and γH2AX with genomes, as well as increased viral activity in the absence of antiviral DNA sensors IFI16 and cGAS, and potentially a new mechanism for general suppression of incoming viral genomes. Altogether my results reveal novel aspects of the spatiotemporal dynamics of HSV-1 genome uncoating and transport, together with their relationship with nascent transcript that can be integrated with previous biochemically based analysis and provide a framework for future investigation of the virus genome and its relationship with the host cell.