This project is concerned with how neuronal cells manage HSV infection at the intracellular level. The intracellular details of HSV/neuronal cell interactions are difficult to study in vivo. Information regarding the mechanisms and kinetics of HSV genome physical organization following neuronal cell infection in vivo and even in vitro is extremely limited. We have, therefore, developed a tissue culture system of quiescent HSV infection using nerve growth factor (NGF) differentiated cells. This system will be used to determine the impact of HSV upon NGF differentiated cells and to track the fate and structure of viral DNA following infection. Briefly, NGF differentiated PC 12 cells have been shown to support long-term """"""""quiescent"""""""" infections of HSV-1. NGF differentiated PC12 cells are not killed by virus infection and, surprisingly, persist longer than uninfected controls. There is little viral transcription and progeny is not detected in the culture medium, despite the presence of an inducible infectious genome. Strangely, the viral genome in quiescently infected PC12 cells persists as a linear form for several weeks before ultimately assuming an endless, presumably circular, state. These cells will, therefore, be used to study (a) if and how HSV can cause populations of PC12 cells to have a survival advantage over uninfected populations; (b) how linear viral genomes can be maintained intact, for weeks in neuronal like cells; (c) the mechanism(s) involved in their assumption of an endless, possibly modified, quiescent viral genomic state. Observations made in this in vitro system will be related to in vivo mouse models of latency by comparing the physical properties of viral DNA derived from tissue derived from infected mice with that from quiescently infected PC12 cells. This work will thus allow for the testing of hypotheses made about HSV latency seen in the vitro system, in mouse models of latency. Some of the information being uncovered in the in vitro, quiescent infection system has the exciting potential to influence our understanding of how HSV genomes are organized and """"""""silenced"""""""".
