Herpesvirus protein kinases: Substrate recognition and pathway targeting.

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DESCRIPTION (provided by applicant): Herpes viruses are significant human pathogens causing a wide range of clinical diseases ranging from relatively benign reactivated infections such as cold sores to more deadly cancers. The very young, the elderly and immunocompromised populations, such as transplant patients and those with HIV-1 infections, are susceptible to more serious disease outcomes. Epstein Barr Virus (EBV) and Kaposi's sarcoma associated herpesvirus (KSHV) are associated with AIDS related cancers and human cytomegalovirus (HCMV) with severe retinitis and pneumonitis in HIV-1 positive individuals. In transplant patients, herpes simplex virus (HSV) and varicella zoster virus can reactivate as disseminated life-threatening infections, HCMV causes an increased incidence of graft rejection and EBV is associated with post-transplant lymphoproliferative disease. There are a limited number of drugs available to treat herpesvirus infections. A problem arising from this limited repertoire is the development of drug resistant virus strains. New therapeutic approaches for treating herpesvirus infections are therefore needed. The human herpesviruses each encode a serine/threonine protein kinase. The dependence on these protein kinases for efficient virus replication and spread, their conservation across the herpesvirus family and their enzymatic nature make these proteins attractive targets for anti-viral drug development. We also propose that identifying key cellular proteins or pathways activated by the virus protein kinases would permit the development of combinatorial anti-cell protein plus anti-protein kinase therapeutic strategies that would limit the development of drug resistant virus variants. We present two approaches designed to obtain basic information that would facilitate the development of anti-herpesvirus treatments targeted at the viral protein kinases. Viral protein kinase substrate recognition overlaps with that of cellular cdc2/CDK1 kinase but is extended beyond cdc2 sites. The degree to which the extended site recognition is unique to, or common to, the different herpesvirus protein kinases is not known. A bioinformatic approach to protein kinase site identification will be undertaken using an algorithm developed by the Co-Investigator and a database of 644 EBV, HCMV, KSHV and HSV-1 substrates identified in my laboratory using human proteomic arrays. The predicted motifs will be validated in in vitro phosphorylation assays and in transfected and infected cell extracts. We recently identified TIP60 as a cell substrate of the protein kinases that is critical for herpesvirus replication. Using the EBV system as the model, we will identify cell genes whose expression is regulated by BGLF4/TIP60 mediated chromatin remodeling by interrogating genome wide CpG methylation of Akata B cell DNA after doxycycline induction of BGLF4 and control kinase dead BGLF4. Data from the screen will be mined using bioinformatic analyses. Selected targets will be validated and the effect of their knockdown on virus replication determined. These experiments will uncover additional cell proteins or networks that could be targets for anti-viral therapeutic strategies.