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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

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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 l.jpg
Canine parvovirus (CPV) & feline panleukopenia virus (FPV) functions, cell biology, & evolution.

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.

~5120 nts

3’

5’

VP1

NS1

VP2

NS2


Slide3 l.jpg

Emergence and evolution of CPV. functions, cell biology, & evolution.

FPV

MEV

RPV

CPV type-2a

CPV type-2b

1990

1984

1970s

1

1979

1

5

17

1

1978-

1980

CPV type-2

  • Questions:

  • What viral changes allowed the new canine host range?

  • How do changes alter virus-host interaction(s) (receptors)?

  • Other selections – antigenic; sialic acid binding;?

  • Epidemiology, selection and spread of mutants.

  • Evolutionary questions – population size, errors, bottlenecks, etc.


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1978 functions, cell biology, & evolution.

~1970

1979

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.

9 changes

3 changes


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3’-end DNA functions, cell biology, & evolution.

VP2

VP1

Methods Used:

1) Protein expression – baculovirus

expression and cell infection.

2) X-ray crystallography and cryoEM

(Rossmann lab).

3) Biochemistry – binding assays.

4) Cell biology and processes of cell entry.

5) Evolutionary biology – processes of cell selection.


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32 functions, cell biology, & evolution.Å

20Å

30Å

Residues controlling canine host range:

Located on threefold spike of capsid:

323

299

93

323


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VP2 93 and 323 structures: functions, cell biology, & evolution.

Asp323Asn:

Lys93Asn:

323


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VP2 “300 region” structures: functions, cell biology, & evolution.

controlling canine and feline host range

E

Block canine

host range

D

G299

A300

G

A/G568


Cell receptor transferrin receptor l.jpg

YTRF functions, cell biology, & evolution.

*

Cell receptor = Transferrin Receptor

11nm

Apical

Helical

Homodimeric type II membrane protein.

Binds diferric transferrin.

Highly expressed on dividing cells.

Clathrin-mediated uptake.

Protease-like

FRTY

Clathrin-AP2

*


Slide10 l.jpg

Transferrin binding functions, cell biology, & evolution.

Virus binding

Virus

TfR

Tf

Capsid binding to TfRs:

Feline TfR: binds FPV & CPV.

Canine TfR: only CPV

Canine TfR

Feline TfR

FPV

CPV-2

CPV-

G299E


Slide11 l.jpg

Canine TfR and cell infection by FPV: functions, cell biology, & evolution.

Single changes in the canine TfR allow FPV infection.

% infected cells

0

10

20

30

40

50

FPV

Feline TfR

CPV-2

CPV-2a

Canine TfR

Canine TfR

N383K

Canine TfR

Ins205


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Virus binding to TfR: functions, cell biology, & evolution.

Determined by 1 face of the apical domain.

11Å

13Å

11Å


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A6 functions, cell biology, & evolution.

A8

A9

A10

A11

A6

A8

A9

A10

A11

Baculovirus

Expression and purification of the TfR for in vitro binding studies.

Viral stock

High 5

Supernatant

Insect cells

Purification of dimeric TfR ectodomain

Nickel

column

Purification of His-tag proteins

Gel filtration

Gel filtration


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In vitro binding to the feline TfR functions, cell biology, & evolution.

- VP2 93 and 323 have no effect on feline TfR binding.

F

P

V

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


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Low binding to canine TfR. functions, cell biology, & evolution.

Canine TfR-N383K distinguishes K93N/D323N mutants.


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Feline TfR:CPV-2 complex: functions, cell biology, & evolution.

Preliminary results:

~1-3 TfR per capsid.

Attachment near top of 3-fold spike.

See Susan Hafenstein’s

Poster #20


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Global selection of CPV VP2 mutations functions, cell biology, & evolution.

100

3045-T

3088-C

3685-G

80

3699-T

(Global)

3045-A

60

3088-T

4062-G

3685-C

(USA)

3699-G

(Global)

3675-G

(Germany)

40

3675-G

4062/4064-GT

(USA)

(Italy – Asia))

20

4062-G

(Germany)

4062/4064-GT

(USA)

0

1976

1978

1980

1982

1984

1986

1988

1990

1992

1994

1996

1998

2000

2002

2004

Year of Virus Isolation


Slide18 l.jpg

Antibody binding and neutralization: functions, cell biology, & evolution.

Two dominant antigenic sites on virus.

Overlap the TfR binding sites.

Host range mutations alter antigenicity.

Site A

Site B


Testing 8 monoclonal fabs high or low neutralizing activity l.jpg
Testing 8 monoclonal Fabs: functions, cell biology, & evolution.High or low neutralizing activity.


Competition by fabs for soluble feline tfr all compete highly neutralizing more efficient l.jpg
Competition by Fabs for soluble feline TfR: functions, cell biology, & evolution.All compete, highly neutralizing more efficient.


Cryoem of antibody binding sites l.jpg
CryoEM of antibody binding sites. functions, cell biology, & evolution.

CPV + 14

FPV + B

  • 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


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J-Y Sgro functions, cell biology, & evolution.

Viruses of vertebrates compared to insect viruses

Raised regions – targets of host antibodies?

AAV2

CPV

Artemis and Actaeon devoured by Artemis's dogs.

Densovirus


Slide23 l.jpg

3’-end DNA functions, cell biology, & evolution.

VP2

VP1

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.

4) TfR binding required for cell infection.

5) Complex interplay between antibody and TfR binding.