The effect of viruses on the host can be observed at different levels of complexity and resolution depending on the technique used. The single-cell observation combined with various labeling techniques has provided great insight into host-virus interactions; however, the observations are made on fixed (dead) infected cells. We propose to develop an in vivo virus genome labeling system that will allow observation of virus genomes in live cells. Visualization of a viral genome in vivo requires tagging its DNA sequence. The construction of a """"""""green"""""""" genome is possible by labeling a DNA tag in the viral genome as it enters the nucleus or before packaging with green fluorescent protein (GFP). Cells inducibly producing the GFP-fusion protein to bind to the DNA tag will be generated to assemble a system where upon virus entry into the nucleus or during packaging the viral genome is rendered """"""""green"""""""" through very tight binding of the DNA tag and the reporter protein. These genomes can be visualized by confocal microscopy and documented in a time-resolved fashion by time-lapse microscopy. We propose to produce DNA-tagged recombinant herpes simplex virus and mouse cytomegalovirus. To complete the system, we will develop cell lines that inducibly produce GFP-labeled DNA-binding protein, where HSV-1 and MCMV can replicate and which are useful as quiescent (""""""""latent"""""""") virus containing cultured cell model systems. Such a new system would recognize single viral genomes directly in the live cells and obviate in situ hybridization. The """"""""green"""""""" viral genomes will open up new lines of inquiry into the dynamics of virus entry into the nucleus, the sequence of degradation by endonucleases and/or retention in a nuclease-resistant episome, replication and segregation by multiple observation of single cells or populations during quiescence, affinity immune separation of quiescent viral genomes and identification of viral genomes by immunoelectron microscopy.
| Ayyanathan, Kasirajan; Lechner, Mark S; Bell, Peter et al. (2003) Regulated recruitment of HP1 to a euchromatic gene induces mitotically heritable, epigenetic gene silencing: a mammalian cell culture model of gene variegation. Genes Dev 17:1855-69 |
