HSV fusion and entry: A pathway governed by four viral ... · governed by four viral glycoproteins Roselyn J. Eisenberg Gary H. Cohen June, 2012 . 1. 2.A 3. 2.B Nucleus Cytosol HSV

HSV fusion and entry: A pathway governed by four viral glycoproteins

Roselyn J. Eisenberg

Gary H. Cohen

June, 2012

1. 2.A

3.

2.B

Nucleus

Cytosol

HSV Virion

Virus entry involves fusion with plasma membrane or endosomal membrane

2

*Pathway is cell type dependent * But same 4 entry proteins are used - gD, gB, gH/gL

OR

*Structures for gD, gH/gL and gB have been solved

*Structures of HVEM/gD and nectin/gD are also known

gH gL (Blue) gB

gD

Nectin-1 HVEM

or

What do we know about each of the 4 HSV proteins? gD: receptor binding protein of HSV: receptors are HVEM and nectin-1

gH /gL: heterodimer that functions as one unit (protein with 2 pp chains)

gB: a Class III viral fusion protein

gB functions as a fusion protein only when gH/gL is also present

gB and gH/gL are found in all herpesviruses: core fusion machinery

Viral membrane

Cartoon of each

gD

Cell membrane

Nectin-1

gH/gL

1

gB

Our Hypothesis: HSV induced fusion occurs in a series of steps

Viral membrane

gD

Cell membrane

Nectin-1

gH/gL

1

gB

Receptor Binding

activates gD

1

gD binds gH/gL

and activates it

2

FUSION

4

3 gB binds gH/gL

gB inserts fusion loops into cell membrane

One of our Cell-cell fusion assays: fusion in cys (all glycoproteins are in the same membrane)

Original data and method: Turner et al., JVI 1998 gD, gB, gH/gL: fusion

Method: Transfect cells with plasmids as shown in graph, incubate 24h and stain with Giemsa, look for syncytia (fusion)

*First – examine step 1

gD, gH/gL (no gB) – no fusion

All 4 entry Glycoproteins are necessary and sufficient for cell-cell fusion


Viral membrane

gD

Cell membrane

Nectin-1

gH/gL

1

gB

Receptor Binding

activates gD

1

gD binds gH/gL

and activates it

2

FUSION

4

3 gB binds gH/gL


Activation of gD by receptor: conformational changes

Carfi, et al Mol. Cell 2001; Krummenacher et al, Embo, 2005; Lazear et al, JVI 2008

Nectin-1 binding

C C C C C C 23 185 1

NH2 234 306

HVEM binding

C-terminus of ectodomain

Viral envelope

*Receptor binding sites covered by ectodomain C-terminus;

* This triggers next steps leading to fusion

rotate 90°

* Receptor binding requires C-terminus to movel; lots of data for this

Receptor binds

*Although this mutant can trigger next step without receptor, it always works better when receptor is present. Partially activated form of gD?

*How it works may alter our ideas of how gD is activated

9

gD WT (+) Syncytia

gD V231W (+) Syncytia

with receptor (nectin-1)

gD WT

(-) Syncytia

gD V231W

(+) Syncytia

with no receptor

Some gD Mutants function in cell-cell fusion in the absence of receptor (bypass step 1): one example


Viral membrane

gD

Cell membrane

Nectin-1

gH/gL

1

gB

Receptor Binding

activates gD

1

gD binds gH/gL

and activates it

2

FUSION

4

3 A. gB binds gH/gL

B. gB inserts fusion loops into cell membrane

How did we determine which protein is activated by gD (step 2)? Varied fusion assay: fusion in trans

Fusion in trans: First a control: keep all gp’s in cis, receptor in trans

Doina Atanasiu, et al , JVI, 2010

n=80 n=80 n=80

Result: fusion (many syncytia form) Similar to virus/cell BUT: Can fusion occur when gB and gH/gL are in trans? (Not like virus/cell)

stain for nuclei

stain for gH/gL

Transfect 2 sets of cells: 1. B78 cells (no receptor

with gB, gH, gL, gD 2. B78-C10 cells: express nectin 3. At 8h, co-culture for 24h 4. Fix, stain with anti-gH/gL and propidium iodide (for nuclei) Again: Fusion is seen as development of syncytia.

gB, gH/gL, gD

B78-C10 cells

+

B78 cells (no receptor)

nectin

Does fusion occur when gB and gH/gL are in trans ?

Cell 1: gB, gD (no receptor) Cell 2: gH/gL + nectin-1 After 8h, versene treat first cells and overlay on second set Incubate 40h. Stain with anti-gH/gL (red) and propidium iodide (grey) (nuclei)

*Fusion occurs when gB & gH/gL are in trans This allows us to see effect of gD on each protein separately

n=2

gB

C10

gH/gL nectin

+

B78

no gD control

n=0 n=95

Syncytium

n = number of syncytia per coverslip

gB

C10

gH/gL nectin

gD

+

B78

+ gD

Which gp does gD contact after it binds to receptor: gB or gH/gL?

Briefly expose one set of cells to soluble gD, then

co-culture with second set

CONCLUSION

*gD contacts gH/gL

*gD activates gH/gL

*Activated gH/gL activates

gB to carry out fusion

C10

HL nectin

+ gD306

Incubate o/n

Wash our gD;

trypsinize

Co-culture with cells

transfected with

gB

Doina Atanasiu, JVI, 2010

C10

B nectin

+ gD306

Incubate o/n

Wash out gD;

trypsinize

Co-culture for 24h

with cels

transfected with

gH/gL

n=110 n=110

n=0 Fusion occurs

No Fusion

gL

* gH and gL form a boot

shaped heterodimer

* Heterodiimer has no

resemblance to any

known fusion protein

Why can gH/gL and gB carry out fusion in trans ?

Heldwein et al, solved both structures in collaboration with our lab

H1

H2

H3

Hypothesis: gB is the fusion protein and gH/gL up-regulates gB’s fusogenic activity

*gB is a trimer *Resembles VSV G

*Base contains fusion loops * Looks like a fusion protein

Step 3: interaction of gH/gL with gB Used bimolecular complementation assay

Viral membrane

gD

Cell membrane

Nectin-1

gH/gL

1

gB

Receptor Binding

activates gD

1

gD* binds gH/gL

and activates it

2

3

FUSION

4

A. gB binds actvated gH/gL*


Atanasiu, et al PNAS 2007

* Put half EYFP tags on gB and gH

* Looked for BiFC and fusion by confocal microscopy

* EYFP complementation = interaction; fusion= syncytia

173 EYFP (Venus)

nEYFP cEYFP

N

gB

YFPc

YFPn

outside gL

N

N gH

inside

pro

t A

pro

t B

comp

lex

YFPc YFPn

Interaction between gB and gH/gL

Bimolecular Fluorescence Complementation (BiFC)

gH/gL gB

- Co-transfect receptor bearing cells with gL + YFP tagged of B+H (no gD)

- At 20h post-transfection add soluble gD(306) protein

•Important: Soluble gD triggers gB and gH/gL to interact

•Soluble gD triggers fusion and interaction between gB and gH/gL

•Not shown here: fusion requires this interaction

BcHnL (no gD)

YFP

anti-gB

40x

gB

cYFP

gL gH

nYFP

BcHnL+gD(306)

100x

Atanasiu, et al, 2007, 2010

Assay: Put tags on gB and gH Fusion is triggered with soluble gD; do B and H interact?

Still Step 3: insertion of gB fusion loops into target membrane

Viral membrane

gD

Cell membrane

Nectin-1

gH/gL

1

gB

Receptor Binding

activates gD

1

gD binds gH/gL

and activates it

2

3

FUSION

4

A. gB binds gH/gL


Step 3: Insertion of gB fusion loops

Heldwein, Harrison, 2006.

gB trimer

III

IV

V

II

I

gB protomer

Long central helix

* Five structural domains thaat look

similar to those of VSV G which is a

fusion protein on its own

* Suggests that gB is a fusion protein

* But gB des not work on its own

* Requires gH/gL to function

* Like VSV G – 2 loops at base of each

protomor that are very hydrophobic

and may be fusion loops

* Are they really fusion loops?

Internal fusion loops

We showed these were the fusion loops by mutagenizing each amino acid (grey = hydrophobic; white = hydrophilic)

Hannah, Cairns et al, JVI 2010

Conclusions 1. Most mutations to hydrophobic residues

impaired fusion

2. Mutation of charged or hydrophilic residues closer to WT 3. Results support concept of fusion loops CAVEAT: Fusion measured using firefly luciferase assay of cell lysates at one time point. Does not measure rates Wanted a way to look at fusion rate to get closer to what happens in real time

- Use two plasmids each encoding fragment of Renilla luciferase

coupled to half GFP

- Key: there are membrane permeable substrates for Renilla luciferase

- Therefore – can measure rate of fusion in intact live cells

- Co-transfect two sets of cells along with gD, gB, gH/gL and receptor

- If fusion occurs, split luciferase reforms; stabilized by reformation of GFP

- Add substrate 20h post transfection; take readings of luminescent product

Split Luciferase Assay of Fusion:

A way to measure rate of fusion in live cells in a 96 well plate

DSP1-7 nRL GFP1-7

DSP8-11 GFP8-11 cRL

Plasmid 1

Plasmid 2

Kondo et al: J.Biol.Chem. Vol. 285: 19681-88, 2010

Split luciferase assay

Add EnduRen live cell substrate At T=0 *At 1h intervals, measure RU in plate 1 for 8-10 h

Co-culture in plate 1 for 24h post-transfection

Luciferase activity

GFP

FUSION OF THE TWO CELLS

DSP1-7 nRL GFP1-7

DSP8-11 GFP8-11 cRL

B78-C10

DSP8-11

R

Transfect

with

Nectin+ cells (R)

Plate 2 B78

DSP1-7

B D HL

no receptor

Plate 1

Transfect with

0

20

40

60

80

100

120

0h 1h 2h 3h 4h 5h 6h 7h

fusi

on

(%

7h

)

time

IFA

SLA

*For luciferase: measured in luminometer at hourly intervals: one set of wells

*For GFP, counted syncytia (fusion events) on separate cover slips hourly

*The rates look very similar so can do kinetics using either luciferase or GFP

Comparison of split luciferase (SLA) with green syncytia (IFA)

0

20

40

60

80

100

120

0h 1h 2h 3h 4h 5h 6h 7h 8h

Lum

ine

sce

ne

c (%

wt)

Wt r=15.5 (100%)

E260A r=13.8 (89%)

H263A r=1.7 (11%)

Y265R r=8.9 (57%)

F175K r=1.9 (12%)

Y179K r=0.2 (1.3%)

0

20

40

60

80

100

120

wt E260A Y265R H263A F175K Y179K

rate

of

fusi

oo

n (

% w

t)

Rates

Curves are linear from 2-8 h

Rates of fusion of fusion loop mutants

Are the phenotypes of the fusion loop mutants due to differences in the rate of fusion?

Hannah, Cairns et al, JVI 2010

Saw, Atanasiu et al, Unpublished, 2012

We are applying this technique to other mutants in gB as well

as gD and gH/gL

Results and Conclusion: *Rate of fusion matches the total amount of fusion * Fusion phenotypes are mostly due to impaired rate of fusion

0

20

40

60

80

100

120

wt E260A Y265R H263A F175K Y179Kra

te o

f fu

sio

on

(%

wt)

Rates

Endpoint fusion assay

Some gB mutants have a faster rate than

WT – results in more fusion – but reason

still not known

607

657

679

0

50

100

150

200

250

0 1 2 3 4 5 6 7 8

Wt

gB* 657


Viral membrane

gD

Cell membrane

Nectin-1

gH/gL

1

gB

Receptor Binding

activates gD

1

gD binds gH/gL

and activates it

2

gB causes fusion

This is doubly

regulated

4

3 A. gB binds gH/gL


* These mutants partially uncouple key interactions at each step.

* When all three are combined, gB is no longer regulated.

Regulation of fusion can be altered by mutations in gD, gH/gL or gB

Each of these mutants can cause cell-cell fusion in absence of receptor

D48 at N

term. of

gH

0

5000

10000

15000

20000

25000

30000

35000

BDHL BDΔ48L2 BD*H2L2 B*DH2L2 B*DΔ48L2 B*D*H2L2 B*D*Δ48L2

tota

l fu

sion

eve

nts

gH* mutant

gD* mutant

gB* mutant

All 3 mutants

WT gp’s

H657R

V231W

Normal situation: Fusion by gB is highly regulated

Viral membrane

gD

Cell membrane

Nectin-1

gH/gL

1

gB

Receptor Binding

activates gD

1

gD binds gH/gL

and activates it

2

gB binds gH/gL 3

FUSION

4


Nucleus

Cytosol

From the point of view of combatting HSV: Virus entry is the first chance to block infection

Block

1.

HSV Virion

3.

2.B

2.A

Acknowledgments

• Gary H. Cohen

• Our lab:

- Doina Atanasiu - Chuck Whitbeck – Huan Lou – Wan Ting Saw – Tina Cairns – John Gallagher – Samhita Rao – Chwan Hong Foo – Zhenyu Huang

• Collaborators – Katya Heldwein – Kay Grunewald – Claude Krummenacher – Anna Wald

NIH for support

Second: Can we block fusion in trans with neutralizing Mabs to gB?

Cell 1: Bc, gD Cell 2: gH/gL (HnL) After 8h, trypsininze one set (or versene treat) and overlay on second set +/- antibody to gB Incubate 40h. Stain with anti-gH/gL (red) and propidium iodide (grey) (nuclei)

n=80 n=2

NON- neut. Ab to gB Neut. Ab to gB

Syncytia No Syncytia

No ab

n=95

Syncytia

Result: A neutralizing MAb to gB (C226, FR2) blocks fusion in trans Conclusion: Ectodomains of gH/gL and gB interact when they are in trans Evidence that gH/gL is not a co-fusogen but may act as an activator of gB

Hypothesis:

* If the gB-gH/gL interaction is essential, neutralizing MAbs to gB

and gH/gL should block it – expect no BiMC (green), no fusion

* Non-neutralizing Mabs should not block – expect BiMC and fusion

Method Transfect C10 cells with Bc + Hn +L. Add Mab to gH or to gB Wash, add soluble gD306 Fix, examine for BiMC and fusion

Atanasiu, et al JVI, 2010

?

?

We mapped functional regions (FR) of gB to its structure based on epitopes for neutralizing MAbs

Stampfer, Heldwein et at, JVI 2010; Cairns et al, JVI 201o

fusion loops

SS10 and several others

to the crown (FR IV)

block gB binding to cells

and block fusion

C226 blocks fusion

Maps to FR2

(domain II)

SS55 and SS144 block fusion *FR1 contains the fusion loops

MUTATIONS CONFIRMED THAT THEY ARE FUSION LOOPS

FR1

FR2

FR3

Fusion loops

1. Which Mabs gB block fusion?

2. Which Mabs to gB block the interaction, i.e. BiFC?

n=2

a-gH/gL

EYFP

No gD controls

Add gD

Atanasiu, et al JVI, 2010

+neut. Ab to gB, then gD

n=30 n=2 n=2

SS 55

n=2

C226 (FR2)

*Neut. Mabs to FR1 & FR2 block both *Neut. Mabs to FR3 do not block the

interaction but block fusion Therefore the interaction precedes fusion and is necessary for fusion Same results with Mabs to gH/gL

SS10 (FR3)

fusion loops

SS144 SS55(FR1)

+ Neut MAb to gB, then gD

SS55

DL21

+NON- neut. Ab to gB, then gD

A22

Conclusion Non neutralizing Mabs do not block the interaction nor fusion

HL nectin D

+ sol gB

C10

n=0

+ sol gD

C10

HL nectin B

n=600

+ sol gH/gL

nectin B

C10

D

n=30

nectin B

C10

+ sol gH/gL + sol gD

n= 200

FINALLY: Can fusion be triggered by soluble forms of gB or gH/gL ?

Our Conclusions and current hypothesis:

*Fusion can be triggered by soluble gD, gH/gL or combination of gD+gH/gL *Therefore, only gB has to remain in a membrane *gB is the fusion protein; gD and gH/gL are regulatory proteins


Viral membrane

gD

Cell membrane

Nectin-1

gH/gL

1

gB

Receptor Binding

activates gD

1

gD binds gH/gL

and activates it

2

FUSION

4

3 gB binds gH/gL


Is there a way to bypass step 1? Can we bypass receptor to activate gD?

Documents

HSV fusion and entry: A pathway governed by four viral ... · governed by four viral glycoproteins Roselyn J. Eisenberg Gary H. Cohen June, 2012 . 1. 2.A 3. 2.B Nucleus Cytosol HSV