The Many Faces of Phosphatidylserine (PS) In Virus Entry

David CoilMiller Lab

Why study Virus Entry?

+ =+ =

Basic Biology Disease Gene Therapy

Lipid bilayer

Basic anatomy of a enveloped virion

Virus capsid

Envelope protein

Viral genome

Characterized Virus Receptors

Single Transmembrane Proteins

Multiple Membrane-SpanningProteins

GPI-anchored Proteins

Carbohydrates

Receptor

Mechanism of enveloped virus fusion

Envelopeprotein

1. Membranefusion

Fusion Peptide

Vesicular Stomatitis Virus (VSV)

From Fields Virology p. 1122From F.A. Murphy, UC Davis

Cellular Receptor for VSV

-Infectable cell extracts still inhibited binding after treatment with:

-trypsin-pronase-Neuraminidase-heating to 100ºC

-However the inhibitory factor was soluble in chloroform-methanol and sensitive to PLPC. Conclusion: Phospholipid

-Attempted to inhibit VSV binding with various purified lipids and only phosphatidylserine (PS) totally inhibited binding

Phosphatidylserine (PS)

-Ubiquitous membrane lipid

-Primarily found on inner leaflet of the plasma membrane

-Exposure of PS is a hallmark of apoptosis

Annexin-V

(binds to PS)

Annexin-V Staining

Frog

Minnow

Zebrafish

Mosquito

Unstained Cells

Cell incubated with Annexin-V

PS level (relative fluorescence units)

VS

V-(i

u/m

l)

50 100 150 250200 3503000

107

108

109

106

105

104

<103

1010

400

Chicken

HumanDog

Quail

Hamster

Minnow

Mosquito

Zebrafish

Frog

PS level (relative fluorescence units)

VS

V-G

FP

tit

er

50 100 150 250200 3503000

107

108

109

106

105

104

<103

1010

400

Chicken

HumanDog

Quail

Hamster

Minnow

Mosquito

Zebrafish

Frog

PS levels on cells versus VSV Infection

VS

V-G

- GF

P b

ind

ing

per

un

it c

ell

surf

ace

area

20 40 60 10080 12000

2

4

8

6

Minnow

Hamster

Mosquito

PS levels per unit cell surface area

VS

V-G

- GF

P b

ind

ing

per

un

it c

ell

surf

ace

area

20 40 60 10080 12000

2

4

8

6

Minnow

Hamster

Mosquito

Quail

Chicken

Zebrafish

Dog

Human

Frog

1.0

0.8

0.6

0.4

0.2

0.00 1 2 3 4

1.2

Quail

Chicken

Zebrafish

Dog

Human

Frog

1.0

0.8

0.6

0.4

0.2

0.00 1 2 3 4

1.2

PS levels on cells versus VSV Binding

Cel

l co

unts

Annexin-V binding

Preincubation with annexin-V

Preincubation without annexin-V

Unlabeledcells

Saturation of cell-surface PS with annexin-V

Zebrafish Cells

Effect of annexin-V saturationon VSV infection

0

200

400

600

800

1000

1200

# of

In

fect

ed C

ells

(ze

bra

fish

)

VSV alone With annexin-V

Annexin Interference with Binding

VSV Binding

Cellcounts

Unlabeled cells

Preincubation with annexin-V

Preincubation without annexin-V

PS as a “Fusion Receptor” for VSV?

-Some viruses such as HIV, SIV and FELV-T require two component receptors

-Characterization of a small region of VSV-G that interacts with target membranes at low pH, (Durrer et al 1995)

-Increased PS in target membrane enhances VSV fusion in vitro (Carneiro et al 2002)

-A peptide within this region has been shown to have PS binding capability in vitro (Coll 1997)

Summary of VSV Results

-VSV infection does not correlate with PS levels

-VSV binding does not correlate with PS levels

-Saturating concentrations of annexin-V do not inhibit VSV infection

-Saturating concentrations of annexin-V do not inhibit VSV binding

-Potential role for PS as a secondary receptor for VSV

Generation of PS liposomes

-comes as free lipid in chloroform/methanol

-dry under nitrogen

-resuspend in PBS

-sonicate to generate uniform vesicles (bilayer liposomes formed with preference to micells)

Blue = unstained negative controlGreen = Annexin-V stained cellsRed = Annexin-V stained cells (with PS)

PS liposome addition to cells increases cell surface PS levels

(8-fold change)

Mouse Cells

VSV-GNone

VSV-G+ PS

20

25

10

15

30

5

0

GF

P

Vector Env:Liposomes:

VSV-GNone

VSV-G+ PS

20

25

10

15

30

5

0

-pos

itive

cel

ls p

er 1

0 3

cells

Vector Env:Liposomes:

Effect of PS on Virus Infection

RD114None

RD114+ PS

RD114None

RD114+ PS

Enhancement of enveloped virus infection following treatment of cells with PS

Virus Fold increase Maximum

Cell type envelope in infection n titer

3-6 4 1.8 x 104

RD114 8-11 4 2.4 x 104

GALV 3 2 6.6 x 104

A-MLV 3-5 2 1.3 x 105

RD114 2-5 5 3.0 x 106

JSRV 4-8 3 3.4 x 104

JSRV 2-7 9 6.0 x 103

RD114 10-20 10 4.5 x 105

ZF4

HTX

Rat-2/Hyal2

NIH 3T3/RDR

NIH 3T3/Pit1 GALV 3-8 2 1.8 x 104

VSV-G

PS treatment does not allow infection whena functional receptor is not present

Virus Envelope Cell Type Titer Titer with PS(iu/ml) (iu/ml)

MoMLV (ecotropic) 293 <1 <1

HTX <1 <1

JSRV NIH 3T3 <1 <1

208F <1 <1

NRK <1 <1

Rat-2 <1 <1

GALV NIH 3T3 <1 <1

AKR6 (xenotropic MLV) CHO-K1 <1 <1

PS treatment does not allow infection whena functional receptor is not present

Virus Envelope Cell Type Titer Titer with PS(iu/ml) (iu/ml)

MoMLV (ecotropic) 293 <1 <1

HTX <1 <1

JSRV NIH 3T3 <1 <1

208F <1 <1

NRK <1 <1

Rat-2 <1 <1

GALV NIH 3T3 <1 <1

AKR6 (xenotropic MLV) CHO-K1 <1 <1

Can related phospholipids enhance virus infection?

Phosphatidylserine

Phosphatidylcholine

Phosphatidylglycercol

Phosphatidylethanolamine

Liposomes

0

40

80

120

160

None PS

Liposomes

0

40

80

120

160

None PS PC PE PGPC PE PG

JSR

V In

fect

ion

of h

uman

cel

ls (

foci

/wel

l)Enhancement of infection is specific to PS

Summary of PS effects

-Increases enveloped virus infection 2 to 20-fold

-Receptor-dependent

-Specific to PS

-Does not affect receptor levels or virus binding

-Does not enhance non-enveloped virus infection

-Rapid timecourse

Model for PS Fusion Effect(normal cells)

Virus Membrane

Target Cell Membrane

HighEnergy

Virus Membrane

Target Cell Membrane

LowerEnergy

Model for PS Fusion Effect(PS treated cells)

PS is a powerful tool used toenhance virus infection

-generated in large batches

-freezes well

- synergistic effects with Polybrene (PB)

-effects in vivo?

Receptor present Receptor absent

Infection No infection

Receptor blocked byglycosylation

No infection

Glycosylation-Blocked Receptors

Target Cells Virus Type PS treatment Titer (iu/ml)

Mouse RD114 - <1

RD114 +

Hamster MoMLV - <5

MoMLV +

Infection of cells containing glycosylation-blocked receptors

2.3 x 104

2.3 x 103

Hypothesis: PS treatment affects theglycosylation machinery of the cell.

RD114 Receptor(glycosylated)

RD114 Receptor(unglycosylated)

PNGaseF

PS

−

−

+

−

Effect of PS treatment on receptor glycosylation

RD114 Receptor(glycosylated)

RD114 Receptor(unglycosylated)

PNGaseF

PS

−

− +

−+ +

+−

Effect of PS treatment on receptor glycosylation

Conclusion: PS treatment does not affect theglycosylation machinery of the cell.

Glycosylation-Blocked Receptors

Receptor present Receptor absent

Infection No infection

Receptor present Receptor absent

Infection No infection

Receptor blocked byglycosylation

No infection

Receptor blocked byglycosylation

No infection

Receptor blocked byglycosylation

No infection

PS treatment allowsinfection

No infectionNo infectionInfection

+PS

Huh?

Normal mouse cells

No infection

Overexpressed RD114 Receptor

RD114 virus

Mouse cells overexpressingthe human RD114 receptor (RDR)

Infection

0

20

40

60

80

100

120

0 5 10 15 20 25 30

PS Addition Timecourse (24 hours)PS Levels

Time after PS addition (hours)

An

nex

in-V

sta

inin

g

0

20

40

60

80

100

120

0 5 10 15 20 25 30

Normal mouse cells

Time after PS addition (hours)

RD

114

infe

ctio

n

0

20

40

60

80

100

120

0 5 10 15 20 25 30

Mouse cells with hRDR

Time after PS addition (hours)

RD

114

infe

ctio

nThreshold effect?

PS Level

200µM

400µM

0

40

80

120

0 5 10 15 20 25 30

PS Exposure Time (h)

RD114 Infection(mouse cells)

400µM

200µM200

400

600

0

RD

114

Infe

ctio

n(a

vera

ge

foci

/wel

l)A

nn

exin

-V b

ind

ing

(flu

ore

scen

ce u

nit

s)

Standard Glycosylation Block Model

Virus

Cell

No recognition

Requirement for multi-valent contact

Virus

CellAssociationprevented

Reduced requirement for multi-valent contact with PS treatment

Virus

Cell

+PS

Summary

-PS is not the cell-surface receptor for VSV

-PS treatment allows certain viruses to overcome glycosylation-blocked receptors

No infectionNo infectionInfectionNo infectionNo infection

-Treatment of target cells with PS enhances infection by enveloped viruses, most likely through an effect on virus fusion

-Utility of PS as a tool for virus infection

Acknowledgments

Thesis Committee:Dusty MillerMichael EmermanLarry RohrschneiderStan McknightJohn Albers

Michele KarantsavelosMaryEllin Robinson

Human Biology Office

Miller Lab:John AlfanoJosh DankeClarissa DirksChristine HalbertNeal Van HoevenShan-Lu LiuSiu Ling LamVladimir VigdorovichSarah Wooten

Strong LabGeballe Lab

Funding:MCBVOTG Training GrantDusty Miller

Dusty Miller

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