Research Interest
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My studies have been
focused on cellular intrinsic defense against infecting viruses.
The cell uses widely different cellular proteins, in its
defensive arsenal, such as promyelocytic leukemia (PML) bodies
components, nuclear suppressors, apoptotic pathway molecules,
and pre-mRNA splicing inhibitors (PTB family proteins). Viruses
have also evolved molecular anti-defense mechanisms. For
example, cytomegalovirus (CMV) gene products IE2 and Kaposi’s
sarcoma herpes virus (KSHV) ORF45 gene products suppresses IFN
production, ul36-37 has anti-apoptotic effects, IE1 disperses
PML bodies and represses HDAC activity. These studies will
advance our understanding of the mechanisms of CMV and KSHV
latency and reactivation and may lead to the development of new
therapies to prevent CMV- and KSHV-caused disease.
A. INTERACTION OF CELLULAR SPLICING FACTORS WITH VIRUSES.
There is a fundamental gap in understanding how the
cytomegalovirus (CMV) major-immediate early (MIE) gene is
regulated at the splicing level, and how the virus usurps
cellular gene-splicing machinery for viral gene splicing.
Continued existence of this gap represents an important problem
because, until it is filled, the interaction of CMV with host
cells at the early stage remains incomprehensible. The MIE gene
is essential to the replication of CMV because MIE gene products
(such as IE1 and IE2) are essential for CMV early and late gene
expression, and HCMV with a deleted MIE gene can only replicate
in MIE gene-complemented cells. In addition, understanding the
mechanisms CMV uses for MIE gene splicing regulation might aid
the development of novel strategies against CMV infection. The
long-term goal is to understand how MIE gene splicing can be
manipulated by the interaction of CMV with host cells. The
objectives in this proposal, which represent our next steps in
pursuit of that goal, are to determine how the MIE gene products
are regulated at the splicing level and to determine, as well,
the biological importance of MIE gene splicing with regard to
CMV replication in vitro and in vivo.
B. KSHV PROTEINS POSTTRANSLATIONAL MODIFICATION AND
REACTIVATION. I recently received an award (RSG) from the
American Cancer Society that will support me in a Kaposi’s
sarcoma-associated herpesvirus (KSHV) project for four years. I
have always been interested in the roles of K8 and RTA
(replication and transcription activator) proteins on viral
reactivation, especially in the epigenetic modifications of the
two proteins. Various new technologies will be employed for the
project including KSHV DNA BAC techniques, epigenetic analysis
and construction of KSHV latency.
KSHV (also known as Human herpesvirus 8) has been determined to
be the most frequent cause of malignancies in AIDS patients. It
is associated primarily with Kaposi’s sarcoma and primary
effusion lymphoma (PEL), as well as with multicentric
Castleman’s disease (MCD). The switch from the latent to the
lytic stage is important both in maintenance of malignancy and
viral infection. Therefore, strategies for the treatment of KSHV-related
malignancies need to both prevent cellular proliferation and
block viral production. Only a few genes can be expressed during
latency, and these gene products tether KSHV DNA episomes with
chromosomes in order to keep KSHV in its latent state. Several
chemicals, including 12-O-Tetradecanoyl-phorbol-13-acetate (TPA),
sodium butyrate (NaB), and 5-azacytidine (5-AC), can reactivate
KSHV from latency in cell cultures. RTA (also called ORF50) gene
expression is the switch point from latency to the lytic cycle
because RTA is essential and sufficient for the reactivation of
KSHV, but the pathological mechanism of the reactivation of KSHV
is poorly understood. Prior studies on the reactivation of KSHV
using chemical inducers implied that epigenetic modification,
especially chromatin remodeling by acetylation, is critical for
transactivators to access lytic gene promoters.
The studies in this proposal emphasize that the reactivation of
KSHV is critical to its pathogenesis and especially so to
tumorigenesis. By studying the two essential genes, RTA and K8,
we expect to develop a clearer understanding of the importance
of two different factors on KSHV reactivation: the epigenetic
modulation (acetylation and deacetylation) of RTA and the
interaction of HDAC with K8. We also expect to provide
information through this approach for anti-KSHV-caused tumor
therapy. These studies will contribute to our understanding of
the mechanism of how KSHV can be reactivated
C. CYTOMEGALOVIRUS-host interaction in Human
cytomegalovirus (HCMV) infection causes life-threatening
diseases in immunocompromised hosts, thus representing a large
worldwide population. HCMV can infect all types of organs and
tissues and therefore causes a broad range of diseases,
including CNS abnormalities, pneumonia, hepatitis and
gastrointestinal problems. How to explain the protean
manifestations of HCMV infection has been a challenge for
decades. Cell culture is a powerful tool to analyze whether a
given cell type is able to support virus entry, gene expression,
virus production and release, and for identification of viral
genes with apparent cell type specific activity. Unfortunately
the information obtained from cell culture experiments cannot be
simply transferred to the host situation or patient.
Furthermore, the in vitro milieu can alter cell physiology,
which might transform cellular properties with respect to virus
infection. Therefore, the virus-cell interactions in CMV
infection should be studied in the physiological environment of
the intact host organism, namely an animal model. Human CMV (HCMV)
only infects humans, and to date the infection of mice with
murine CMV (MCMV) is the most informative small animal model for
HCMV infection. Therefore, more detailed studies of the
pathogenesis for different cell and tissue types during MCMV
infection in mouse models are crucial.
Our long-term goal is to understand the interactions of
virus-host in specific organs and tissues and to determine how
CMV infection can cause adaptively variant damage to different
tissues. Our objective is to set up a mouse model for
tissue-specific infection of cytomegalovirus so that we can
investigate viral pathogenesis in both a spatial and temporal
manner.
Conditional or inducible Cre-transgenic mice have been available
commercially for several years. These mice provide a convenient
resource for studying viral infections in tissue- or
cell-specific manner. In our laboratory, we have routinely used
the BAC system to make mutations of large DNA viruses. We will
combine the BAC mutagenesis techniques and Cre/lox system to
localize CMV infection in the tissues of our interest. By
studying tissue-specific infection of CMV, we will instigate a
new field in viral pathology.
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