“MIE Gene Splicing is a New Target for HCMV Caused Diseases”

Biographical Sketch

Human cytomegalovirus (HCMV) is ubiquitous and infects a majority of the population (50%-90% of the general population) and remains the most frequent infectious pathogen in AIDS patients and other immunocompromised populations. HCMV infection in AIDS patients and transplantation patients is a serious problem worldwide and it is a highly prevalent cause of birth defects and life-threatening infections in fetuses and neonates. To date, the most effective drugs used to treat HCMV infections are DNA replication inhibitors; however, these drugs are not appropriate for use in pregnant women and neonates due to their capacity to interfere with development. Therefore, new drugs need to be developed by new strategies. We recently discovered a novel target for HCMV therapy, which takes advantage of the observation that HCMV replicates more efficiently in cells that do not express the polypyrimidine tract-binding protein (PTB). This protein strongly inhibits major immediate-early (MIE) gene splicing and expression by competing with U2 small nuclear RNP auxiliary factor (U2aF) for the polypyrimidine-tract (Py) binding site. The Py is different and specific for each gene in the virus and in mammalian cells. We hypothesize that a small RNA complementary to the polypyrimidine tract (scRNA-Py) in the HCMV MIE gene can interfere specifically with pre-mRNA splicing of the HCMV MIE gene; and that this interference takes place in nucleus. In contrast, siRNA interferes with MIE gene expression only in the cytoplasm, so we further hypothesize that a combination of an scRNA Py that interferes specifically with the binding of U2AF to the Py of the MIE gene-with an siRNA that interferes with MIE gene exon 5, will synergistically block HCMV production. Our long-term goal is to protect against HCMV infection by designing specific, safe and effective drugs. To test our hypothesis, we plan to pursue the following aims: Aim 1. Determine the role of alternative splicing in regulating the relative amounts of the two major immediate early proteins. IE1 and IE2. We will use mutational analysis to identify the specific exon enhancer and exon silencing sequences. Aim 2. Identify RNA sequences of the Py of the HCMV MIE gene by RNA gel shift assay, and determine the extent to which scRNA-Py can block the binding of U2AF65 to the Py in vitro. Aim 3. Determine the extent to which scRNA-Py can block HCMV production and gene expression and whether a combination of an scRNA-Py that interferes specifically with the binding of U2AF to the Py of the MIE gene and an siRNA that interferes with the MIE gene exon 5, will synergistically block HCMV production.