DNA viruses are the causative agents of a large number of debilitating diseases. They are also used as vehicles in gene therapy. We are investigating the early host-virus interaction with the long-term goal of identifying common sites of interference and of prolonging the transcriptional life of non-replicative gene therapy vehicles. DNA viruses are deposited at very few precisely circumscribed nuclear domains named NDI0 suggesting the existence of a targeted DNA deposition mechanism within the nucleus. We mapped the targeting DNA sequence for simian virus 40 (SV40) to the core origin of replication. Also DNA viruses begin to transcribe in association with ND10. Thus, an essential functional interaction appears to reside at ND10. On the other hand the major ND10-associated proteins are interferon-upregulate. We will determine, whether deposition of the viruses at ND10 is essential and beneficial for infection or instead, reduces productive viral infection, and whether the mechanism of deposition is shared by the different DNA virus families that deposit their infectious virus at ND10. Elucidation of such a mechanism could provide general means of interference with viral infections. Therefore, basic biological properties need to be defined. We will continue to define the mechanism that targets viral DNA to ND10 by identifying the DNA targeting sequence using a new method where DNA is directly labeled in vivo. 2. We will define the functional consequences of viral deposition at ND10 by determining whether deposition of a reporter gene at ND10 by the viral DNA targeting sequence determines transcriptional potential. 3. We will determine where quiescent herpes viruses (EBV and HSV-l) transcribe and replicate relative to ND10 in a cell culture model system and whether upon reactivation they start their immediate early transcription at ND10. 4. We will determine whether the absence of certain ND10-associated proteins influences productive infection using knock-out cells and focusing on ND10-associated proteins that are thought to be transcriptional repressors or involved in heterochromatin formation. 5. We will determine whether the SUMO-1 iso-peptidase dependent release of ND10-associated proteins induces or suppresses activation of repressed viral genomes. The long-term goal is to define the mechanism by which the infecting virus deposits DNA at specific highly localized sites within the nucleus, what advantage the virus (or the cell) derives from such specific localization, and to determine whether this specific localization could be used as a target for anti-viral therapy, or has properties to improve long-term transcription of viral vectors for gene therapy.
