There is a fundamental gap in our understanding of the contributions of the essential viral protein, pUL34 to human cytomegalovirus (HCMV) replication. This gap remains an important problem, because until it is filled, challenges in understanding the effects of HCMV infection on the human host, and the design of new therapeutic agents remain. The long term goal is to develop specific, new antiviral compounds that target the essential UL34 proteins. The objective of this application is to define functions o UL34 proteins and their contributions to viral replication, a necessary precursor to developing targeted antiviral compounds. The central hypothesis is that UL34 protein (pUL34) interactions with the pUL34-binding sites in the HCMV genome and with the cellular protein filamin A (FlnA) comprise the essential functions of the UL34 gene. This hypothesis has been formulated based on work produced in the applicant's laboratory. The rationale for the proposed research is that defining the functions of the UL34 proteins is essential for understanding HCMV replication. UL34 encodes sequence-specific DNA binding proteins that are expressed throughout the viral replication cycle. Of the 14 pUL34-binding sites within the HCMV genome (AD169), 3 are located near the origin for lytic replication, 6 are within protein-coding regions, and the remainder are located in the regulatory regions of non-essential viral genes. Guided by strong preliminary data, the hypothesis will be tested by pursuing three specific aims: 1) Identify the effects of pUL34- DNA interactions on viral DNA replication;2) Define the effects of UL34-DNA interactions on the expression of the essential genes UL32, UL37 and UL54;and 3) Determine the contribution of filamin A to pUL34 function and viral replication. For the first specific aim, sites within the origin for lytic replication that have been shown by the applicant to bind pUL34, will be mutated and the effects on viral DNA replication will be determined. The plasmids and the HCMV-BAC that are already on hand will be used.
For specific aim 2, a pUL34-binding site contained within an essential viral gene has been shown to decrease gene expression in preliminary studies performed by the applicant. The molecular mechanism that results in decreased gene expression will be identified using RNA analyses.
For specific aim 3, the applicant has identified filamin A as a binding partner for pUL34. The intracellular colocalization patterns of pUL34 and filamin A will be analyzed;the effect of siRNA-mediated knockdown of filamin A expression on viral replication will be determined. The research proposed here is innovative, because it will define the additional roles of the unique UL34 proteins in viral replication and gene expression, including the novel interaction of pUL34 with filamin A. The proposed research is significant because it is will advance and expand our understanding of the complex patterns of HCMV gene regulation that result in viral replication and associated diseases. Ultimately, such knowledge has the potential to contribute to the general understanding of gene regulation, and to the development of new therapeutic agents.
The proposed research is relevant to public health because understanding of human cytomegalovirus (HCMV) replication and the role of the essential UL34 gene is crucial for the design and development of new prevention and treatment modalities. Human cytomegalovirus infection causes a significant health and economic impact on organ transplant recipients, people with HIV infection and newborn infants, causing permanent defects in ~8,000 children yearly in the United States. Thus, the proposed research is relevant to the mission of NIH related to developing fundamental knowledge that has implications for immune-mediated diseases, human immunodeficiency virus infection and vaccine development.