This application's long-term objectives are to understand the central steps by which human cytomegalovirus (HCMV) nucleocapsids transit from the nucleus to the cytoplasm (nuclear egress), and to exploit that understanding for drug discovery. This project is especially health-related, as new drugs are needed for treatment of HCMV. In this application, HCMV proteins that are involved in nuclear egress are investigated. One of these proteins, the UL97 protein kinase, is already an established drug target. Two other proteins, UL50 and UL53, interact to form a nuclear egress complex (NEC) that can serve as a new drug target.
Specific aim 1 is to investigate the roles of UL97 that are important for production of infectious virus in both non-dividing and dividing cells;in particular, whether the only role of UL97 in nuclear egress is phosphorylation of lamin A/C to disrupt the nuclear lamina. A principal approach will be to analyze a recombinant HCMV expressing a dominant negative mutant of lamin A/C in place of UL97 to determine if that virus can replicate and disrupt nuclear lamina as well as wild type virus in dividing cells or as well as a virus expressing human papillomavirus E7 in non-dividing cells.
Specific aim 2 is to investigate the function(s) of the NEC. HCMV mutants that fail to express UL50 or UL53 or that have more specific alterations such as defects in UL50-UL53 interactions will be constructed and their block(s) in nuclear egress will be determined with the aid of techniques including electron microscopy. Why the HCMV NEC is not sufficient to disrupt nuclear lamina in the absence of UL97 will be investigated. Candidate proteins that interact with the NEC in HCMV-infected cells will be investigated to explore the hypothesis that such proteins are recruited to effect budding of nucleocapsids through the inner nuclear membrane. These proteins will be tested for co-localization with the NEC in cells, whether they interact directly with the NEC, and, if so, to map determinants of the interaction. The importance of these proteins for HCMV replication will be investigated using techniques including RNA interference. Should these studies fail to uncover a role for interacting proteins, in vitro studies to test whether the NEC can promote membrane curvature and vesiculation will be undertaken.
Specific aim 3 is to determine the structure of the NEC. The structures of truncated versions of NEC subunits that retain sequences that are conserved among herpesviruses are being determined by NMR, as are the locations of the subunit interfaces, which can lead to an NMR structure of the complex. Efforts to improve crystals of the complex will continue, with the goal of obtaining a high resolution crystal structure.
Specific aim 4 is to establish a high throughput assay for small molecules that inhibit subunit interactions of the NEC using an amplified luminescent proximity homogeneous assay or other assay. This assay will be used to screen libraries of small molecules and natural products. """"""""Hits"""""""" will be then assayed for biochemical activity and specificity, and for anti-HCMV activity and cytotoxicity, with the long range goal of developing them into antiviral drugs.
HCMV causes severe disease in people with impaired immunity, and is associated with a number of diseases in the immunocompetent population. There is considerable need for new drugs to combat HCMV, as current drugs have major limitations. The research proposed should not only provide information that could aid in understanding drug targets and mechanisms, but aims directly to discover new anti-HCMV drugs.
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