Final Activity Report Summary - DYNAMICS OF HCMV GEN (Localisation, dynamics and regulation of human cytomegalovirus genomes during lytic and quiescent infection)
Human cytomegalovirus (HCMV) is a common human pathogen of considerable clinical interest. The relative locations of active and repressed genes within the nucleus are becoming recognised as a significant factor in the control of gene expression. We have developed systems to visualise parental HCMV amplicon genomes, the replication compartments that develop from those genomes and the association of these structures with PML nuclear bodies (ND10) in live infected cells. Plasmids containing viral replication and packaging signals, a gene expressing Enhanced yellow fluorescent protein (EYFP) linked to the Tetracycline repressor DNA binding domain (TetR) and 14 copies of the Tetracycline operator (TetO) sequence were used to produce amplicon genomes, packaged into otherwise normal viral particles.
The frequency of viral genome juxtaposition to ND10 was found to be substantially increased by inclusion of an active HCVM Early gene transcription unit, indicating that the association is neither random nor passive. Furthermore, the ND10 associated genomes preferentially progressed to form viral replication compartments. Thus, active viral transcription contributes to the efficiency of viral genome association with ND10 and this in turn increases the probability that the genome will engage in active DNA replication. These studies in live cells provide novel insight into virus-ND10 interactions and provide compelling visualisation of their functional relevance.
The frequency of viral genome juxtaposition to ND10 was found to be substantially increased by inclusion of an active HCVM Early gene transcription unit, indicating that the association is neither random nor passive. Furthermore, the ND10 associated genomes preferentially progressed to form viral replication compartments. Thus, active viral transcription contributes to the efficiency of viral genome association with ND10 and this in turn increases the probability that the genome will engage in active DNA replication. These studies in live cells provide novel insight into virus-ND10 interactions and provide compelling visualisation of their functional relevance.