Poliovirus Genome Organization

Genomic RNA

VPg IRES 3'-UT

743 7370 7440

AAA

PolyproteinP1 P2 P3

1B1A

3AB 3C 3D2C2B2A1D1AB 1C

VP4 VP2 VP3 VP1

Capsid Proteins Replication Proteins

Polymerase

Protease

Virus entry: The final stages of the morphogenic pathwayor "How does a simple nonenveloped virus enter a cell"

160SNative

135SPVR altered

80SEmpty

Infection160S Particle

+ PVRRNA release in cytoplasm

Attachment (Docking)

?

Inf+ PVR–

“EnvironmentallyStable”

“EnvironmentallyUnstable”

135S Particle

Entry Active

Particle

160S Particle

+ PVR

Heat

Ea=~120-140 kcals

“Energy”~100 kcals

(Soluble PVR)

“Docking”

HighAffinityBinding

(Kinetically Trapped)

160S 135S

Structural Differences

Fricks et al 1990 JVBubeck et al 2005 JV

RNA Release

VP4 Mutants

4028T.G

4028T.V

4028T.S

15-20 min

80SParticle

“Environmentally

Stable”

“Environmentally

Unstable”

Membrane insertion

135S Particle

160S

Particle

Entry

Active

Particle

“Docking”

“Energy”

+ PVR X?

Mutant VP4 localize to membranes

Western

VP4

min pi 0 30T.GWT

0 30

VP3VP2VP1

0 30WT

0 30T.S

300T.V

VP3VP2VP1

VP4

Poliovirus 160S particles form ion channels in lipid bilayers

Current Distribution @ 40mV

Current (pA)

Voltage Dependence

Voltage (mV)

Channel Parameters1. Conductances2. Mean open time and

probability3. Voltage dependence

(gating, +vs–)4. Current ion

VP4 entry mutants form ion channels with different properties

T.G

WT

T.S

T.V

--

0.2

0.02

ND

Cond.(pS)

--

47

28±10

15

Mempoten.

--

+

+/-

+/-

--

15-18 m

15-18 m

25-30 m

PoT1/2-135S

4028T.S

200ms 2 p

A

Control

4028T.G

200ms 1p

A

4028T.V

2s 1 pA

+40mV

+40mV

RNA Release

VP4

mutants

“Environmentally

Stable”

“Environmentally

Unstable”

15-20 min

Ion Channel/RNA delivery

80SParticle

?Membrane insertion

135S Particle

160S

Particle

Entry

Active

Particle

“Docking”

“Energy”

+ PVR

X

Functional reconstitution of purified PVR/gpi onto cells

M PVR M PVR

Silver Western

Mock Tf

PVR/gpi

Phase GFP+

Functional Reconstitution of PVR in lipid bilayers

WT21°C

21°C

4028T.G 21°C

21°C

31°C

+Virus

PVR-GPI

1s5

pA

+Virus

PVR-GPI

+Virus

500ms

2 p

A

21°C

31°C

21°C

Temperature Dependent Conformational Change of PVR - Virus Channel in Lipid Bilayers

~120-140

~90-100

~60

+PVR (sol)

Heat

+Membrane

Ea (kcals/mole)160S - 135S

asolecitin/hPVR

Lipid/PVR

asolecitin/PVRgpi

PE:PS/PVRgpi

Single Channel Conductance (pS)

65±15 ~460

360±56

~7

8

6

Temperature (°C)

45±7

1450±350264±5

3121Voltage -Gated

No

No

No

Q10

160S

PVR

Channel model of poliovirus entry

160S

PVR

135S

delivery of viral RNA genome into

cell cytoplasm

Bubeck et al (2005) Nat Struct. Mol.Biol. 12:615-618

QuickTime™ and aDV/DVCPRO - NTSC decompressor

are needed to see this picture.

Future Directions

1. Define the architecture and structure of the channel

2. Corelate channel behavior in bilayers with mutant entry phenotypes

3. Direct in vivo measurements during infection process

+PVR ?

Functional Transitions of Poliovirus

Storageand Protection

(160S)

Delivery(135S)

Empty (80S)

Mutant Analyses

Pranav Danthi

Xiaojing Ning

Qi-Han Li

Nicola Moscufo

Receptor

Yan Huang

Hong Wang

Anatoli Naumov

Electrophysiology

Magdalena Tosteson (Harvard)

Khaled Machaca (UAMS)

Structure

Jim Hogle

Dave Filman

Doryen Bubeck

Mihnea Bostina

his collaborators:

A. Stevens (NIH)

R. McIntosh (UC Boulder)

V 10 20 300 45Medium

VP4

-VP4

MembraneV 10 20 300 45min pi

VP3

VP2VP1

Autorad

QuickTime™ and aGraphics decompressorare needed to see this picture.

◆◆◆◆◆

◆

●●●●●

●

▲▲▲▲▲▲

media

membrane

cytoplasm

100

80

60

40

0

20

0 20 30 40 50 6010

Time Post-Infection (min.)

% T

ota

l C

PM

VP4 is inserted into microsomal membranes