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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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slide1

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
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.

~5120 nts

3’

5’

VP1

NS1

VP2

NS2

slide3

Emergence and evolution of CPV.

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.
slide4

1978

~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

slide5

3’-end DNA

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.

slide6

32Å

20Å

30Å

Residues controlling canine host range:

Located on threefold spike of capsid:

323

299

93

323

slide7

VP2 93 and 323 structures:

Asp323Asn:

Lys93Asn:

323

slide8

VP2 “300 region” structures:

controlling canine and feline host range

E

Block canine

host range

D

G299

A300

G

A/G568

cell receptor transferrin receptor

YTRF

*

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

Transferrin binding

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

Canine TfR and cell infection by FPV:

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

slide12

Virus binding to TfR:

Determined by 1 face of the apical domain.

11Å

13Å

11Å

slide13

A6

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

slide14

In vitro binding to the feline TfR

- 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

slide15

Low binding to canine TfR.

Canine TfR-N383K distinguishes K93N/D323N mutants.

slide16

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

slide17

Global selection of CPV VP2 mutations

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

Antibody binding and neutralization:

Two dominant antigenic sites on virus.

Overlap the TfR binding sites.

Host range mutations alter antigenicity.

Site A

Site B

cryoem of antibody binding sites
CryoEM of antibody binding sites.

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

slide22

J-Y Sgro

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

3’-end DNA

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.

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