Amy Odegard
Research Summary
The goal of my research is to identify and characterize the protein receptor(s) that facilitate attachment and
uptake of Flock House virus (FHV) into host cells. The long-term goal is to characterize interactions between
FHV and the receptor to develop a model of virus cell entry that can be applied to several medically important
viruses and may potentially lead to the development of new and effective antiviral therapies.
Introduction
FHV is a small insect virus (~30 nm in
diameter) that was originally isolated from grass
grubs in New Zealand and infects Drosophila
melanogaster cells in tissue culture. Each virus
particle consists of two single-stranded RNA
genome segments surrounded by a protective
protein shell, or capsid, made up of 180 copies
of a single coat protein (Figure 1). The simplicity
of the viral capsid and genome make FHV an
excellent model system for understanding how
viruses enter and infect cells.
Figure 1. FHV structure. (A) Schematic drawing of a FHV
particle. The virus protein capsid, which is represented as a
blue hexagon, surrounds the two RNA genome segments. (B)
Space-filling model of the FHV capsid (1). 180 copies of the
coat protein make up the FHV capsid. The coat protein is
colored blue, red, or green to indicate different icosahedral
symmetry positions. (C) Ribbon diagram of a single coat
protein subunit (1). An exposed loop on the FHV capsid
surface is indicated. This loop is predicted to be important for
binding the cellular receptor.
To initiate infection, animal viruses must bind
to one or more specific receptors on the surface
of the host cell. The expression of the receptor
on specific cells or tissues is a major
determinant of viral tropism and disease. In
addition to mediating attachment of the virus to
the cell surface, many virus receptors serve
essential functions during virus entry, such as
trafficking the virus to the proper intracellular compartment or triggering the virus particle to undergo entryrelated structural changes. Although virus receptors have been identified for a number of medically important
viruses, the processes of virus binding and entry are complex and many questions remain. A complete
understanding of these events is essential for defining the molecular details of virus-receptor interactions,
understanding how these interactions lead to entry-related structural rearrangements, and for the rational
design of anti-viral drugs and novel therapeutic treatments.
Current Research
Reserach goal 1: Identify candidate FHV receptor proteins. To identify membrane proteins that bind FHV, I
plan to use the three biochemistry-based experimental approaches summarized below.
1. Virus Overlay Protein Blot Assay (VOPBA). This assay is a well-established method for identifying virus
receptor proteins (9). Briefly, membrane extracts from Drosophila cells will be separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane, and probed
with FHV particles. Gel bands that bind FHV will be excised from a corresponding gel and subjected to mass
spectrometry and proteomic analysis to identify putative receptor proteins in the sample (5).
2. Affinity chromatography. An FHV affinity column will be constructed by coupling the virus to Sepharose
beads (10). Membrane extracts from Drosophila cells will be passed over this column, and the column will be
washed to eliminate unbound cellular proteins. FHV-associated proteins will be eluted and visualized by SDSPAGE and coomassie staining. To identify the FHV-binding proteins, gel bands will be excised and subjected
to mass spectrometry analysis as described above.
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3. Chemical cross-linking. FHV-binding
proteins will be identified by chemically
cross-linking the virus to proteins on the
surface of cells (8). To accomplish this, FHV
will be incubated with Drosophila cells at
4°C, a temperature that will allow the virus to
bind to the cell surface, but not enter inside
the cell. Following attachment, the virus will
be covalently linked to associated proteins
using commercially available cross-linking
agents. Virus-receptor complexes will be
purified by sucrose sedimentation (a
procedure routinely used to purify FHV
particles from infect cells), separated by
SDS-PAGE, excised from the gel, and
analyzed by mass spectrometry as
described above.
Goal 2: Confirm putative FHV receptor.
Once a candidate protein is identified, multiple approaches will be used to confirm that this protein functions as
a FHV receptor. Three main approaches are summarized below.
1. Express the protein in a non-permissive cell line to establish FHV binding. Candidate receptor proteins will
be expressed a cell line that does not naturally bind FHV (I have already identified several insect and
mammalian cell lines that do not bind FHV). It will then be tested whether the transiently expressed putative
receptor protein can establish virus attachment to a non-permissive cell line.
2. Demonstrate binding of FHV to a soluble form of the protein. The candidate receptor protein will be
expressed and purified in either a bacterial or insect cell protein expression system. The purified receptor will
be added to FHV to test whether the candidate protein can bind to virus in solution.
3. Block FHV binding to cells using an antibody or a reagent specific to the protein. Drosophila cells will be
incubated with antibodies specific for the candidate receptor and it will then be tested whether the antibodies
can block FHV binding to cells. It will also be examined whether a soluble form of the candidate receptor
(described above) can competitively inhibit FHV binding to cells.
Goal 3: Characterization of virus-receptor interactions. Once a cellular receptor has been identified, it will
be essential to characterize interactions between FHV and the receptor to develop a model of how the receptor
facilitates virus entry into the host cell.
1. Identify FHV surface residues involved in receptor binding. Based on the X-ray crystal structure of the virus
(1), an exposed loop on the FHV capsid surface is predicted to be important for binding to the cellular receptor
(Figure 1C). To examine the role of this loop in receptor binding, residues in and near this loop will be replaced
by site-directed mutagenesis, mutant particles will be generated, and cell binding and infectivity will be tested.
2. Localize regions of the receptor involved in virus binding. The X-ray crystal structure (if available) or
secondary structure predictions of the receptor will be used to identify regions of the protein that may be
important for virus binding. These regions will be targeted by site-directed mutagenesis and the ability of
mutant receptor proteins to bind to the virus will be examined.
3. Structural analysis of virus-receptor complexes. Detailed structural analysis of virus-receptor complexes,
such as x-ray crystallography and cryo-electron microscopy.
Additional Research
In addition to the research proposed above, I plan to carry out alternative projects that do not depend upon
identifying FHV receptors. These research projects are described briefly below.
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1. Selection and analysis of FHV heat-stable mutants. To identify regions of the capsid that are important for
maintaining structure and stability, spontaneous FHV mutants that are resistant to heat treatment will be
selected. Genetic analysis of these heat-resistant, hyper-stable FHV mutants will reveal changes in the coat
protein that confer increased stability. FHV normally replicates at 27°C (the temperature Drosophila cells are
maintained in tissue culture). Heat-resistant viruses will be selected by incubating the virus stock at elevated
temperature (30-70°C) prior to infection; this process will be repeated for several rounds to ensure selection of
mutant viruses. These studies are directly related to my interest in virus entry since increased capsid stability
often correlates with defects in virus entry (2, 6). If the viral capsid cannot disassemble efficiently during entry,
the viral genome will not be released and infectivity will be compromised. Therefore, heat-resistant particles will
also be examined for defects in cell entry and infectivity.
2. Analysis of the role of disulfide bonds in FHV assembly and infectivity. The FHV coat protein encodes four
cysteine residues. Based upon the FHV x-ray crystal structure (1), two of these cysteine residues are
positioned to form an intra-molecular disulfide bond. We predict that this disulfide bond may be required for
proper capsid assembly and virus infectivity. To test this hypothesis, these cysteine residues will be replaced
by site-directed mutagenesis and mutant virus particles will be generated. These mutant particles will then be
examined for any assembly defects and virus infectivity will be tested.
References
1.
Fisher, A. J., and J. E. Johnson. 1993. Ordered duplex RNA controls capsid architecture in an
icosahedral animal virus. Nature 361:176-9.
2.
Hooper, J. W., and B. N. Fields. 1996. Role of the mu 1 protein in reovirus stability and capacity to
cause chromium release from host cells. J Virol 70:459-67.
3.
Karlstrom, R. O., L. P. Wilder, and M. J. Bastiani. 1993. Lachesin: an immunoglobulin superfamily
protein whose expression correlates with neurogenesis in grasshopper embryos. Development
118:509-22.
4.
Odegard, A. L., E. Wu, M. H. Kwan, A. Scheemann, and J. E. Johnson. The Drosophila Plasma
Membrane Protein Lachesin is a Receptor for Flock House Virus. Manuscript in preparation.
5.
Peng, J., and S. P. Gygi. 2001. Proteomics: the move to mixtures. J Mass Spectrom 36:1083-91.
6.
Shiomi, H., T. Urasawa, S. Urasawa, N. Kobayashi, S. Abe, and K. Taniguchi. 2004. Isolation and
characterization of poliovirus mutants resistant to heating at 50 degrees Celsius for 30 min. J Med Virol
74:484-91.
7.
Sieczkarski, S. B., and G. R. Whittaker. 2005. Viral entry. Curr Top Microbiol Immunol 285:1-23.
8.
Trakselis, M. A., S. C. Alley, and F. T. Ishmael. 2005. Identification and mapping of protein-protein
interactions by a combination of cross-linking, cleavage, and proteomics. Bioconjug Chem 16:741-50.
9.
Trauger, S. A., E. Wu, S. J. Bark, G. R. Nemerow, and G. Siuzdak. 2004. The identification of an
adenovirus receptor by using affinity capture and mass spectrometry. ChemBioChem 5:1095-9.
10.
Upanan, S., A. Kuadkitkan, and D. R. Smith. 2008. Identification of dengue virus binding proteins using
affinity chromatography. J Virol Methods 151:325-8.
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