PRINCIPLE INVESTIGATOR
Dr. Roy Duncan
B.Sc. University of Guelph, M.Sc. Queens University, Ph.D. University of Guelph
After completing a Ph.D. degree on birnavirus translation, I completed
postdoctoral training in Dr. Patrick Lee’s laboratory at the University
of Calgary working on structure-function relationships in the mammalian
reovirus cell attachment protein. After joining the department of Microbiology
and Immunology in 1991, my laboratory explored avian reoviruses, their
interactions with the host cell, and virus pathogenicity. Since 1996, much
of the lab has focused on the study of the fusion-associated small transmembrane
proteins (FAST) encoded by the fusogenic reoviruses. We have also continued
our interest in the control of translation initiation and viral pathogenesis.
RESEARCH ASSOCIATE
Dr. Roberto de Antueno
B. Sc., M. Sc., Ph.D., La Plata National University
I am using model membrane vesicles called liposomes in combination with the
purified FAST protein, RRV p14, to generate proteoliposomes. p14-proteoliposomes,
by virtue of the presence of a fusogenic protein in the membrane, deliver their
contents to target cells with greater efficiency than liposomes alone. I am
currently optimizing this delivery system using three approaches: monitoring
the quality of the p14 protein produced to improve proteoliposome production,
altering protein:lipid ratios and lipid formulations within the proteoliposome
to improve liposome-cell fusion, and optimizing the encapsulation of pharmacologically-relevant
cargo.
POSTDOCTORAL FELLOW
Dr. Jennifer Corcoran
B.Sc. Dalhousie University, Ph.D. Dalhousie University
My Ph.D. thesis characterized the p14 fusion protein encoded by reptilian reovirus
and identified many of the important structural components that are essential
for its membrane fusion activity. I am currently focused on two aspects of
the mechanism of p14-mediated fusion: 1) the dependence of the fusion mechanism
on cholesterol and its correlation with p14 subcellular localization and 2)
the oligomer status of p14.
GRADUATE STUDENTS
Chris Barry
B.Sc. University of British Columbia
My project is focused on the homologous p10 proteins of ARV and NBV. I am curious
to understand why these two FAST proteins, with 33% amino acid sequence identity,
display such markedly different fusion kinetics. I hope to utilize plasma membrane
vesicles to elucidate which relevant motifs contribute to this difference,
and have also developed the T7-vaccinia virus fusion assay to analyze mutant
p10 proteins. I am also interested in the possibility that the p10 homologues,
like RRV p14, also localize to cholesterol-rich membrane microdomains. I plan
to characterize p10-containing lipid rafts by immune purification.
Eileen Clancy
B.Sc. University of Guelph
The various FAST proteins have little sequence similarity, yet contain shared
domains including a transmembrane domain with an adjacent polybasic region,
a hydrophobic patch, and modification by acylation. However, the nature and
position of these domains varies among the FAST proteins suggesting that these
proteins may have evolved as modular fusion machines comprised of more than
one domain capable of carrying out a given function. To investigate this hypothesis,
I am utilizing PCR to create chimeric FAST proteins with different domains
of interest exchanged. My initial studies have indicated that the transmembrane
domain may be interchangeable between RRV p14 and BRV p15, but neither can
be exchanged with ARV p10. This suggests that the FAST protein transmembrane
domain may play a direct role in the fusion process.
Jayme Salsman
B.Sc. Dalhousie University
I began my thesis research by asking why certain reoviruses encode membrane
fusion proteins. Our data suggests that FAST-mediated cell-cell fusion plays
a dual role in the virus replication cycle. While early in infection multinucleated
syncytium formation facilitates cell-cell spread and the establishment of a
localized infection, at late infection times when syncytia become very large,
membrane integrity is lost, facilitating systemic dissemination of the virus.
I am currently investigating the influence of membrane lipids, cellular adhesion
molecules, and the actin cytoskeleton on FAST protein-mediated fusion. The
FAST proteins may traffic to cholesterol-enriched membrane microdomains as
a mechanism for increasing the local concentration of protein as well as placing
the FAST protein in association with key cellular proteins, such as adhesion
molecules. I am currently attempting to identify which cellular junctions may
serve as sites for fusion initiation, and am investigating the role of actin
remodeling in FAST-induced syncytium formation.
Deniz Top
B.Sc. University of Toronto
I am currently working on understanding the importance of the ectodomain of
RRV p14 and its relation to the fusion function of the p14 protein as a whole.
In my studies on p14, I utilize a proteoliposome-cell fusion assay. This assay
will be instrumental in identifying how other molecules contribute to FAST
protein-induced membrane fusion, including lipids, receptors, and protein partners.
This assay will also help identify regions of RRV p14 that are essential for
protein function. That the N-terminal ectodomain of p14 is myristoylated is
of particular interest to me, not only because the modification is essential,
but also because of its peculiar topology. To further understand the role of
myristoylation, I am developing a liposome-to-liposome hemifusion assay to
analyze a synthetic peptide of the p14 ectodomain. This assay can also be used
to analyze p14 constructs that harbour point mutations in the ectodomain. In
collaboration with Dr. Richard Epand at MacMaster University, I also hope to
conduct calorimetric studies on the p14 protein and p14 ectodomain peptide
to understand the thermodynamic parameters associated with membrane fusion.
TECHNICIANS
Tara Read
B.Sc. Dalhousie University
After completing my honours research on the degradation of the FAST proteins,
I joined the Duncan lab full-time as a technician. I am responsible for expression
and purification of the FAST proteins, particularly various constructs of RRV
p14, using the baculovirus-insect cell expression system. These purified proteins
are being used to develop the FAST-liposome intracellular delivery system,
and for atomic-level structural determination of the FAST proteins by NMR.
Jingyun Shou
B.Sc. Zhejiang University
Since 1996, I have been responsible for the administrative needs of the lab
and maintaining stocks of tissue culture media, cell lines, viruses, antibodies,
and other reagents essential to the Duncan lab. I was responsible for the cloning
and sequencing of over 30 cDNAs representing the dsRNA genome segments of seven
different orthoreoviruses. I am currently involved in the creation of recombinant
baculoviruses to express different FAST protein constructs, and in the analysis
of aquareovirus translation.
UNDERGRADUATE RESEARCH STUDENTS
Angela Richardson
I have recently joined the lab as a summer student and plan to complete my
honours thesis in the Duncan lab. My project is on the expression, purification,
and characterization of FAST proteins that have been C-terminally modified
to contain motifs designed to target specific cell receptors (e.g. RGD motif
binds integrins). We hope that proteoliposomes created with modified FAST
proteins will demonstrate improved attachment to target cells, thereby facilitating
improved delivery of the encapsulated cargo.
RECENT GRADUATES
OF THE DUNCAN LAB
Jennifer Corcoran
B.Sc. Dalhousie University, Ph.D. 2003
Jennifer’s Ph.D. thesis characterized the p14 fusion protein encoded
by reptilian reovirus. She is currently working as a postdoctoral fellow in
the lab, completing studies on p14 subcellular localization and oligomerization,
before pursuing postdoctoral research in Dr. James Smiley’s laboratory
at the University of Alberta.
Sandra Dawe
B.Sc. University of Guelph, Ph.D. 2004
Sandra completed her thesis on the characterization of the BRV p15 fusion protein,
and is currently a postdoctoral fellow in the laboratory of Dr. Theresa Compton
at the University of Wisconsin-Madison.
Tara Hurst
B.Sc. Bishops University, M.Sc. 2003
Tara completed her M.Sc. thesis on the unusual translation mechanism of a polycistronic
aquareovirus genome segment that encodes the fusion gene. She has since moved
to Dublin, Ireland and is currently pursuing a Ph.D. degree.
David O'Hara
B.Sc. Dalhousie University, M.Sc. 2002
David’s M.Sc. thesis investigated strain-specific differences in avian
reovirus cell entry and pathogenicity. He is now a member of the Tissue Harvesting
transplant team in New York City.
Maya Shmulevitz
B.Sc. University of Alberta, Ph.D 2001
Maya’s thesis research characterized the p10 homologues encoded by avian
reovirus and Nelson Bay reovirus. After a postdoctoral position at the Veterinary
Infectious Disease Organization (VIDO) in Saskatoon, she returned to the department
and is currently a postdoctoral fellow in Dr. Patrick Lee’s laboratory. |
