Interaction of viruses with Receptors and

Antibodies

Elizabeth A. Hewat

Institut de Biologie Structurale, Grenoble, France

Erice 10-06-06

Interaction of small intact non-enveloped viruses with receptors and antibodies

1990 First virus/Fab reconstruction by cryo-EM

Followed by virus/antibody structures for CPMV, HRVs, Sinbis virus, Ross River virus, RHVD, HSV, Adenovirus, Rotavirus DLP, CPV…….

Rotavirus/FabPrasad et al. 1990

Nature 343, 476

Schematic diagram of an Antibody

Surface representation of a Fab as a function of

resolution

Combining Cryo-EM and X-ray data gives pseudo-atomic

models

Rotavirus/Fab complexThouvenin et al. 2001 J Mol Biol.,

307, 161

How do Antibodies neutralize a virus?

What is the role of

- aggregation (mono-valent vs bivalent attachment )

- inhibition of receptor binding - hiding receptor site

- inhibition of uncoating and delivery of the genome into the cytoplasm e.g. induced changes in the capsid or inhibition of changes?

Cryo-EM image of FMDV / FabSD6

Picornavirus Structure

VP1 with pocket factor

N-terminal VP1

Canyon

VP1

VP4

HRV2

Picornaviruses HRV2, HRV14 and FMDV in complex with

neutralizing antibodies

Antibody neutralization of picornnaviruses

• Bivalent binding neither necessary nor sufficient for strong neutralization

• No evidence of modification of the capsid

• Inhibition of the release of RNA • Strong neutralization = Strong binding to receptor site

• 1 to 6 efficient antibodies can neutralize a virus! Explain?

Antibodies as markersFabs which bind to empty

HRV2 capsids but not native capsids

Fab 2G2 bound to HRV2 after RNA release It binds to the site predicted as the most highly modified but….

How do picornavirus deliver their genome into the cytosol

for replication?

How and where do receptors bind?How is uncoating initiated?How and where is the RNA released?Does the receptor remain attached?How does the RNA cross the

membrane?How do viruses adapt to different receptors?

Questions addressed by cryo-EM

Schematic representation of some of the known picornavirus

receptors

Evans & Almond 1998 Trends in Microbiology, 6, 198

Receptor sites of Picornaviruses

Examples of receptors bound in

the canyon

ECHO1 & 2 integrin

Xing et al. 2004, J Biol. Chem. 279,

11632

Rossmann et al. 2002, TRENDS Microbiol., 10, 324

Ig-like Integrin

Major and minor group human rhinoviruses receptors bind on non-

over lapping sites

HRV2/V123

HRV2/

HRV2/VLDL-R

HRV16/ICAM-1Olsenet al.1993 PNAS

Kolatkar et al., (2000) EMBO J.,

18, 6249

Hewat et al. 2000, EMBO J. 19, 6317

RNA is released from HRVs by a cooperative movement of all capsid

proteins

X-ray cryo-EMNative HRV2 Empty HRV2

5 23

5 2

3

*4% capsid expansion*Iris type movement of VP1s about the 5-fold*N-term VP1 & VP4 absent

Hewat et al. 2000, Mol. Cell. 10, 317

Schematic diagrams of HRV2 uncoating

Model systems of the virus-receptor-membrane

interactions

Doyen et al. 2005 Nature Struct. & Mol Biol, 12, 615

*lipid bilayer / full-length PV-R / Poliovirus

*liposome / His-tagged PV-R / Poliovirus

How do viruses adapt to new receptors?

• In vitro eg. Some HRV’s and FMDV adapt to use heparin sulphate

• In vivo eg HRV (ICAM-1 and LDL-R)• In vivo species shift e.g. Canine Parvovirus

Questions for the near Future

• Where do the other receptors bind? (VCAM-1, integrins, heparin sulphate …)

• Is the pocket factor expelled and when ?• How many receptors are necessary for uncoating?

• Visualise the egress of VP4, the N-terminal VP1 and the RNA (acridine orange) and "pore" formation? (exploit a model system of bi-layer / receptor / virus)

CollaborationsInstitut de Biologie

Structurale,

Grenoble, France

Emmanuelle Neumann

Eric Thouvenin

Guy Schoehn

James F. Conway

Departamento de Biologia

Molecular de Barcelona , Spain

Nuria VerdaguerWendy OchoaIgnacio Fita

Institute of Biochemistry,

University of Vienna, Austria

Dieter Blaas

Rosita Moser

Bernhard Ronacher

Thomas C. Marlovits

Luc Snyers

Division of Structural Biology, University of

Oxford, England

Dave Stuart and colleagues