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Abstract

Events controlling herpesvirus nuclear genome uncoating, nuclear transport, and the onset of transcription remain poorly understood. We have now developed procedures to examine these processes within individual cells and at the single molecule level for both the genome and the transcripts produced from it. We have combined two novel techniques of, firstly, bioorthogonal chemistry to visualise genomes which incorporate an alkyne-nucleoside analogue (ethynyl deoxycytidine, EdC) and secondly, single molecule RNA in-situ hybridisation (smFISH) which allows detection of individual mRNA transcripts. Using these techniques simultaneously, we can now qualitatively and quantitatively analyse individual transcript abundances and their intracellular localisation, in relation to the genome itself at single molecule resolution during the progression of infection. Moreover, we are able to examine these parameters when a single genome infects a cell. We have examined the transcripts of the immediate-early mRNA for ICP0, and features revealed from this work include; transcriptional ‘bursting’ with clustered transcripts around individual genomes; mean mRNA transcript number, variance, and intracellular localisation produced from a single genome; the progressive abundant ICP0 transcription occurring selectively from replicated genomes; an increasing bottleneck in cytoplasmic transport of transcripts emanating from replicated genomes; and increased transcription bursts from virtually every uncoated genome when protein synthesis is suppressed. Further, by multiplexing probes, we can simultaneously analyse distinct transcription outputs from different genes of the same or classes, and genomes, in the same individual cell. Our results reveal completely new perspectives on the very early events of genome presentation and transcription from those genomes.

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/content/journal/acmi/10.1099/acmi.ac2019.po0468
2019-04-08
2020-01-27
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http://instance.metastore.ingenta.com/content/journal/acmi/10.1099/acmi.ac2019.po0468
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