Transcript ppt - Erice Crystallography 2006 IT Support

Integrating studies of viral, receptor, antibody
structures, functions, cell biology, & evolution.
Example: Parvovirus capsids, receptors, and
antibodies – controlling host range, cell
infection and/or neutralization.
Colin Parrish
Cornell
• Laura Palermo
• Christian Nelson
• Wendy Weichert
• Gail Sullivan
• Karsten Hueffer
Collaborators
• Michael Rossmann
• Susan Hafenstein
• Mavis Agbandje-McKenna
• Alan Simpson
• Eddie Holmes
• Laura Shackelton
• Pamela Bjorkman
Canine parvovirus (CPV) & feline
panleukopenia virus (FPV)
Non-enveloped, T =1
icosahedral capsid, 26 nm
diameter.
Single-stranded DNA
genome
2 genes – 4 overlapping
proteins.
Capsid - 60 copies of VP2
and VP1 (90% VP2; 10%
VP1).
VP2 forms capsids; VP1
required for cell infection.
3’
~5120 nts
VP1
NS1
NS2
VP2
5’
Emergence and evolution of CPV.
FPV
MEV
RPV
CPV type-2a CPV type-2b
1984 1990
1970s
5
17
1
1979 1
1
19781980
CPV type-2
Questions:
1) What viral changes allowed the new canine host range?
2) How do changes alter virus-host interaction(s) (receptors)?
3) Other selections – antigenic; sialic acid binding;?
4) Epidemiology, selection and spread of mutants.
5) Evolutionary questions – population size, errors,
bottlenecks, etc.
VP2 gene evolution:
CPV in a separate clade,
divided into 2 subclades.
Substitution rates:
FPV to CPV: ~7 x 10-3
(nt/site/yr)
CPV clade: 2 x 10-4
(nt/site/yr)
CPV ancestor ~1970.
1978
~1970
9 changes
1979
3 changes
Methods Used:
1) Protein expression – baculovirus
expression and cell infection.
2) X-ray crystallography and cryoEM
(Rossmann lab).
3’-end DNA
VP2
3) Biochemistry – binding assays.
4) Cell biology and processes of
cell entry.
5) Evolutionary biology – processes
of cell selection.
VP1
Residues controlling canine host range:
Located on threefold spike of capsid:
323
299
93
32Å
323
20Å
30Å
VP2 93 and 323
structures:
Lys93Asn:
Asp323Asn:
323
VP2 “300 region” structures:
controlling canine and feline host range
G299
A300
E Block canine
D host range
G
A/G568
Cell receptor = Transferrin Receptor
Apical
Helical
Protease-like
*
FRTY
Clathrin-AP2
YTRF
Homodimeric type II
membrane protein.
Binds diferric
transferrin.
Highly expressed on
dividing cells.
Clathrin-mediated
uptake.
11nm
*
Capsid binding to TfRs:
Feline TfR: binds FPV &
CPV.
Canine TfR: only CPV
FPV
Transferrin binding
Virus
TfR
Tf
CPV-2
CPVG299E
Virus binding
Feline TfR
Canine TfR
Canine TfR and cell infection by FPV:
Single changes in the canine TfR allow FPV infection.
0
Feline TfR
Canine TfR
Canine TfR
N383K
Canine TfR
Ins205
% infected cells
10
20
30
40
FPV
CPV-2
CPV-2a
50
Virus binding to TfR:
Determined by 1 face of the apical domain.
11Å
11Å
13Å
Expression and purification of the TfR for in
vitro binding studies.
Baculovirus
Viral
stock
High 5
Insect cells
Purification of
dimeric TfR
Purification ectodomain
of His-tag
Gel filtration
proteins
Nickel
column
A6 A8
Gel filtration
800 2bacthesS300 020604:1_UV
800 2bacthesS300 020604:1_Fractions
800 2bacthesS300 020604:1_Inject
800 2bacthesS300 020604:1_Logbook
mAU
25.0
20.0
15.0
10.0
5.0
0.0
A1
60
A2
A3
A4
A5
80
Supernatant
A6
A7
A8
A9
100
A10
A11
A12
B12
120
A6 A8 A9A10A11
B11
B10
B9
B8
140
B7
B6
B5
B4
160
B3
B2
ml
A9 A10 A11
In vitro binding to the feline TfR
- VP2 93 and 323 have no effect on feline TfR binding.
FPV
C P V -2
C P V -2 b
F P V-K 9 3 N /D 3 2 3 N
C P V -N 9 3 K /N 3 2 3 D
Low binding to canine TfR.
Canine TfR-N383K distinguishes K93N/D323N mutants.
Feline TfR:CPV-2
complex:
Preliminary results:
~1-3 TfR per capsid.
Attachment near top of 3-fold
spike.
See Susan Hafenstein’s
Poster #20
Global selection of CPV VP2 mutations
100
3045-T
3088-C
3685-G
3699-T
(Global)
80
60
3045-A
3088-T
3685-C
3699-G
(Global)
4062-G
(USA)
3675-G
(Germany)
40
3675-G
(USA)
4062/4064-GT
(Italy – Asia))
20
0
1976
4062-G
(Germany)
1978 1980
1982
1984 1986
1988 1990
Year of Virus Isolation
1992
1994 1996
4062/4064-GT
(USA)
1998
2000
2002
2004
Antibody binding and neutralization:
Two dominant antigenic sites on virus.
Overlap the TfR binding sites.
Host range mutations alter antigenicity.
Year of
emergenc
e
Ag.
site
Residue
~1970
A
93 K-N
B
300 A-G
87 M-L
305 D-Y
FPV-CPV
~1979
CPV2 –
CPV2a
~1984
A
426 N-D
Site B
CPV-2a CPV-2b
~2000
CPV2bCPVx
Site A
A
426 D-E
Testing 8 monoclonal Fabs:
High or low neutralizing activity.
Competition by Fabs for soluble feline TfR:
All compete, highly neutralizing more efficient.
CPV + 14
FPV + B
CryoEM of antibody
binding sites.
• A site - around 3-fold
axis.
• B site - near two-fold
axis.
• MAb 8 and MAb F
differ in
neutralization.
FPV + 8
FPV + F
Susan Hafenstein & Christian
Nelson
J-Y Sgro
CPV
Viruses of
vertebrates
compared
to insect
viruses
AAV2
Raised
regions –
targets of
host
antibodies?
Artemis and
Actaeon
devoured by
Artemis's
dogs.
Densovirus
Conclusions:
1) The TfR-capsid interactions are
optimized for each combination; affinity
can differ widely.
2) Canine TfR binding controls both
canine and feline host ranges.
3) Glycosylation of canine TfR important.
3’-end DNA
VP2
4) TfR binding required for cell infection.
5) Complex interplay between antibody
and TfR binding.
VP1