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PILOT
PROJECT #5 |
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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. |
