Human Cytomegalovirus Nuclear Egress: Molecular Mechanisms and Drug Targeting

Harvard Medical School, Boston MA

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Abstract

Project Summary 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, HCMV proteins that are involved in the transit of nucleocapsids from the nucleus to the cytoplasm (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 serum-starved (non-dividing) and serum-fed (dividing) cells;in particular, whether the crucial role of UL97 in nuclear egress is phosphorylation of lamin A/C. A principal approach will be to construct and analyze a recombinant HCMV expressing a dominant negative mutant of lamin A/C in place of UL97. Specific aim 2 is to investigate the function(s) of the NEC. HCMV UL50 or UL53 null mutants, mutants that are defective in UL50-UL53 interactions, and mutants lacking non-conserved segments will be constructed and their block(s) in the viral replication cycle determined with the aid of techniques including confocal immunofluorescence and electron microscopy. Why the NEC is not sufficient to disrupt nuclear lamina in infected cells in the absence of UL97 will be studied. Interactions of the NEC with proteins in HCMV-infected cells will be investigated using epitope-tagged virus, and candidate interacting proteins will be investigated 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 methods including RNA interference. Specific aim 3 is to determine the structure of the NEC. The structures of truncated versions of UL50 and UL53 that retain all sequences that are conserved among herpesviruses will be determined by nuclear magnetic resonance, as will the location of the UL53 binding site on UL50. Efforts to improve crystals of a complex of these two proteins will continue, with the goal of obtaining a high resolution crystal structure. Specific aim 4 is to establish an amplified luminescent proximity homogeneous assay to discover small molecules that inhibit subunit interactions of the NEC. This assay will be used to screen libraries of small molecules and natural products. "Hits" will be assayed for specificity, and for anti-HCMV activity and cytotoxicity, and then studied for their mechanisms of action.