Human Immunodeficiency Virus (HIV) infection is characterized by recurrent oral disease caused by HSV-1. The defining hallmark of HSV-1 is reactivation from latency. Following stress (heat, trauma, ultra violet light, etc.) viral transcription is induced resulting in HSV-1 reactivation. The mechanism regulating the switch between latency and reactivation is poorly understood. The broad objectives of this proposal are to determine the viral gene sequences required for HSV-1 reactivation in vitro and in vivo during normal and compromised immune function. The central hypothesis to be tested is that stress induced HSV-1 genes are critical for reactivation from latency.
Aim one will test the hypothesis that specific HSV-1 genes are induced by stress. Transient transfection of neurally differentiated (ND)-PC12 cells and the firefly lucifease (LUC) assay will be used to determine whether HSV-1 (i.e., alpha genes: alpha 0, alpha 4, alpha 22, alpha 27, and alpha 47, beta genes: ul9 ul23, ul39 and the gamma gene ul48 and LAT) are transcriptionally induced by heat stress and forskolin treatment in the absence of other viral gene products.
Aim 2 will identify cis elements that mediate stress responsiveness of HSV-1 promoters using the LUC assay, electrophoretic mobility shift assay (EMSA) and DNA footprinting. Sequentially deleted constructs of HSV-1 promoters will be analyzed for loss of stress responsiveness in the (ND)-PCI2 cells using the LUC assay. Regions identified by standard, competition and super shift EMSA and foot printing will be assessed for stress responsiveness by mutational analysis in the LUC assay.
Aim 3 will test the hypothesis that stress response elements in HSV-1 genes are required for reactivation in vitro and in vivo. The cis regulatory elements of HSV-1 genes governing the stress response will be mutated and introduced into the viral genome using recombination techniques. Mutant viruses will be tested in parallel with the rescued virus and parental virus in the PC12 cell culture model for quiescent HSV infection that was developed during their previous NIH funding. Mutant viruses that maintain wild type lytic growth properties and display at least 50 percent reduction in reactivation phenotype will be tested in the immunocompetent and immunosuppressed mouse and rabbit eye models of reactivation to determine whether altered reactivation in vitro correlates with that seen in vivo. The coordinate sequential pattern of gene expression (immediate early, early, late) of the parental, mutant and rescued viruses during the establishment and reactivation phases of quiescence will be analyzed by RT-PCR. These studies will advance the understanding of the molecular biological principals that govern the process of HSV-1 reactivation in immunocompetent and immunosuppressed individuals and could lead to the development of novel antiviral agents that block reactivation in patients with Acquired Immunodeficiency Syndrome (AIDS).
