Molecular biology of
eukaryotic DNA viruses, regulation of gene expression, programmed cell
death (apoptosis), and retrotransposons
Research
in our laboratory involves the molecular biology and replication of
eukaryotic DNA viruses. We are particularly interested in the
baculoviruses, a group of large DNA viruses that are pathogenic to
invertebrates. These unique viruses are known for their prolific
multiplication in insect (moth) cells and are the most popular
eukaryotic vectors for high level expression of foreign gene products
(proteins). Moreover, these DNA viruses exhibit a highly controlled but
complex program of transcription during replication which makes them
useful and attractive models for studies on eukaryotic gene regulation.
Our goal is to investigate the molecular mechanisms that govern
baculovirus replication to better understand fundamental problems in
regulation of eukaryotic gene expression and the interaction of viruses
with their host cell.
We study the early and late
replication stages of the prototype baculovirus, Autographa californica
nuclear polyhedrosis virus (AcMNPV) in cultured cells. AcMNPV uses a
complex regulatory scheme to ensure the proper timing and level of
expression of essential genes. Early genes encode transcriptional
regulatory factors, whereas late genes encode virus structural
proteins. We are examining the mechanisms that control the proper turn
on and turn off of viral genes during infection by focusing on the
molecular interactions between cis-acting DNA regulatory sequences and
virus/host transcription factors. We have characterized the promoters
and enhancers required for proper transcription of early AcMNPV genes
and continue to investigate the trans-acting factors involved.
AcMNPV
also causes programmed cell death. PCD or apoptosis is a built-in,
signal-induced process by which a cell self-destructs. Whereas
regulated apoptosis is critical for normal development, aberrant
apoptosis is associated with tumorigenesis, neurodegenerative diseases,
immunodeficiency, and viral pathogenesis. The baculoviruses encode
novel PCD suppressors (including p35 and iap) that function to block
apoptosis in diverse organisms, including humans. Thus, baculovirus
infection provides a powerful and convenient system for molecular
analysis of the induction and suppression of apoptosis. Such studies
are yielding important insight into the regulation of PCD and should
facilitate the long term design of novel therapeutic strategies for
treatment of apoptosis-linked diseases in humans.
Techniques
used in the laboratory include gene cloning and sequencing,
site-directed mutagenesis, in vitro transcription and RNA mapping,
construction of recombinant baculoviruses, protein purification, and
immunoblot analysis, and others pertinent to molecular virology such as
cell culture and virus propagation.
Diagram showing apoptosis pathway and steps inhibited by baculovirus
apoptotic suppressors IAP, P49, and P35