The long-term objective of this research is to identify, characterize, and exploit drug targets of human herpesviruses. This research is especially health-related, as new drugs are needed for treatment of herpesvirus infections, particularly those of human cytomegalovirus (HCMV). In this application, two HCMV proteins UL50 and UL53 are investigated for involvement in the unusual process by which nucleocapsids transit from the nucleus to the cytoplasm (nuclear egress). These proteins interact to form a nuclear egress complex (NEC) that can serve as a new drug target. The roles of these proteins in two important steps of nuclear egress - movement of capsids towards the nuclear rim, and budding through the inner nuclear membrane - are poorly understood and are a major focus of this application.
Specific aim 1 is to investigate how HCMV capsids move from the nuclear interior to the nuclear rim. The roles of nuclear actin and myosin Va in this process will be investigated using live cell imaging and single particle tracking analyses of infected cells that either express dominant negative mutants of these proteins or have been treated with actin inhibitors. Similar analyses will be performed on UL53 mutant viruses. Whether UL53 can link capsids to myosin Va will be tested biochemically using purified capsids and proteins.
Specific aim 2 is to investigate how the NEC orchestrates budding through the inner nuclear membrane. A possible role for the AAA ATPase, VCP, and its co-factors in this process will be examined initially by assessing associations of these host proteins with the NEC using immunofluorescence, immuno-electron microscopy, and co-immunoprecipitation. These studies will be followed by measuring the effects of siRNAs that block expression of the host proteins and VCP inhibitors on nuclear egress in infected cells, and vesiculation in cells expressing the NEC or its subunits, but no other viral proteins. Whether VCP and its co-factors can promote budding in vitro will be studied using giant unilamellar vesicles in collaboration with the Heldwein laboratory.
Specific aim 3 is to determine structures of the NEC and its subunits. The structures of a complex of truncated versions of UL50 and UL53 that retain all sequences that are conserved among herpesviruses, and of a similar version of UL53 will be determined by X- ray crystallography. In collaboration with the Wagner laboratory, nuclear magnetic resonance (NMR) will be used to solve similar versions of UL50, and the mouse CMV homologs of UL53 and the complex. A longer term goal is to solve the structure of a near-full length complex using X-ray crystallography.
Specific aim 4 is to use a high throughput assay and, with the Wagner laboratory, NMR-based fragment screening for small molecules that inhibit subunit interactions of the NEC. Hits will be then assayed for specificity, for anti-HCMV activity and cytotoxicity, and for their mechanism of inhibition, and will be developed into leads in collaboration with medicinal chemists.
HCMV causes severe disease in people with impaired immunity, and is associated with a number of chronic 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 about virus biology and proteins that could aid in understanding drug targets and mechanisms, but aims directly to discover new anti-HCMV drugs.
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