2014 russell gordon delta sh new pdf

CONCLUSION • RSV infection leads to an IL-1β

response, from a range of cells.

• The SH protein of RSV inhibits

inflammasome function.

• Deletion of the SH gene modulates the

outcome of secondary RSV infection.

• RSV ΔSH is a vaccine candidate strain

which is protective to RSV infection

• We propose that this is via its pore

structure altering the K+ flux.

INHIBITION OF THE INFLAMMASOME BY THE SMALL

HYDROPHOBIC PROTEIN OF RESPIRATORY SYNCYTIAL VIRUS

Ryan F. Russell1, Jacqueline U. McDonald1, Ziyun Zhong1, Alex Bukreyev2, Peter L. Collins2, John S. Tregoning1 1Mucosal Infection and Immunity group, St Mary’s Campus, Imperial College London, UK.

2 Laboratory of Infectious Diseases, NIAID/NIH, MD, USA.

INTRODUCTION • Respiratory syncytial virus (RSV) infects most children in the first year of life and is a major single

cause of hospitalization in infants and young children.

• The small hydrophobic (SH) protein of RSV has recently been described as a viral pore protein

(viroporin) which forms a proton-selective channel (figure 1).

• The function of SH is unknown, but SH knockout viruses are partially attenuated in vivo and are

potential vaccine candidates.

• Here, we present evidence that SH is involved in evasion of the host immune response, inhibiting the

inflammasome.

Figure 1. Computational analysis by Gan et al

(2013) suggests that the SH protein forms a

pentamer containing a pore forming

transmembrane region, whose diameter allows only

for the passage of small molecules and ions.

Further studies reveal potential ion channel activity.

The SH proteins of other related viruses show

inflammasome modulating activity.

FIGURE 1. The SH protein of RSV encodes a viral pore

protein (viroporin) that can modulate the inflammasome.

4 7100

101

102

103

104

105

106

107

108

109

Days after Challenge

RS

V L

gen

e co

pies

perg

Lun

g R

NA

Control RSV SH RSV A20

5

10

15

20

Ant

i-RS

V Ig

Gg

/ml

***

******


25

50

75

100

125

Ant

i-RS

V Ig

Gg/

ml

***

******

0 1 2 3 4 5 6 7

85

90

95

100

105

Control

RSV SH

RSV A2


% O

rigin

al W

eigh

t

*** ***###

###

*###

*

4 70

5106

1107

2107


Lung

cel

l num

ber

*

**

4 70

2

4

6

8


CD

3+C

D8%

****

4 70

50

100

150

200

250


Lung

IL-1

pg/

ml

***

***


10

20

30

40

50

RS

V (

M2)

Sp

ecifi

c C

D8

(% to

tal C

D8)

***

*

4 70

2

4

6

8


CD

3+C

D4+

(%)

*

**

A B C D

E F G H I

0 1 2 3 4 5 6 7

90

95

100

105

110

Days after infection

% O

rigin

al W

eig

ht

RSV SHRSV A2

***

0 1 2 3 4 5 6 7

104

105

106

107

108

109


RS

V L

ge

ne c

opie

s

pe

rg L

ung R

NA

0 1 2 3 4 5 6 7

0

100

200

300

400

500


CX

CL1

pg/m

l **

0 1 2 3 4 5 6 7

0

100

200

300

400

500


IL-6

pg

/ml

**

0 1 2 3 4 5 6 7

0

200

400

600

800

1000


IL-1

pg

/ml

0 1 2 3 4 5 6 7

106

107


Lung c

ell

num

ber

0 1 2 3 4 5 6 7

104

105

106


Lung C

D4 T

cells

*

0 1 2 3 4 5 6 7

104

105

106


Lung C

D8 T

cells

*

0 1 2 3 4 5 6 7

104

105

106


Lung N

K c

ells *

A B

D E F

C

G H I

0 20 40 60 80

0

200

400

600

800

1000

**

*

*

*

Hours after infection

IL-1

pg/m

l

0 20 40 60 80

0

500

1000

1500

**

*

*


IL-1

pg/m

l

0 20 40 60 80

0

5

10

15

20


IL-1

pg/m

l

RSV A2 RSV SH Control

0

50

100

150

200

IL-1

pg/m

l

*

0 20 40 60 80

0

5.0105

1.0106

1.5106

2.0106


RS

V p

fu/m

l

RSV A2

RSV SH

A B C D E

P L + C o n P L + 1 A 8 C L + C o n C L + 1 A 8

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

Lu

ng

IL

-1

pg

/ml * *

* * *

* * *


0

1 0 0

2 0 0

3 0 0

4 0 0

Lu

ng

IL

-6 p

g/m

l

* *

* *


0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

Lu

ng

KC

pg

/ml

* *

* *


1 0 4

1 0 5

1 0 6

1 0 7

1 0 8

RS

V L

ge

ne

co

pie

s

pe

r

g L

un

g R

NA


0

5 0 0 0 0

1 0 0 0 0 0

1 5 0 0 0 0

Ly

6G

+ c

ell

co

un

t

* * *

* * *

0 1 2 3 4 5 6 7

0

1 0 0 0 0

2 0 0 0 0

3 0 0 0 0

4 0 0 0 0

5 0 0 0 0

D a y s a fte r in fe c tio n

IL-1

Po

sit

ive

Ly

6G

+

* * * * *

* * *R S V A 2

C o n tro l

0 1 2 3 4 5 6 7

0

5 0 0

1 0 0 0

1 5 0 0

D a y s a fte r in fe c tio n

IL-1

+C

D1

1c

+ M

HC

I lo

F4

/80

+

Ce

ll c

ou

nt

* * *

* * *


0

2 0 0 0

4 0 0 0

6 0 0 0

CD

11

c+

MH

CII

lo

F4

80

+

ce

ll c

ou

nt

* *

* *

*

A B

C D

E F

G H

FIGURE 2. RSV ΔSH is protective against RSV infection.

FIGURE 3. RSV ∆SH induces a greater IL-1B response than wild type in vitro.

FIGURE 4. RSV ∆SH is attenuated in vivo but induces a greater IL-1B

response than wild type.

FIGURE 5. IL-1B is produced by neutrophils and

macrophages in vivo.

Figure 2. Mice were infected with either RSV A2, ΔSH or control treated, 4 weeks later all mice were

challenged with RSV A2. Anti-RSV IgG before RSV challenge (A). Weight change (B), lung viral load

(C), lung IL-1B (D) and lung cell number (E) after infection. Lung CD4+ (F) and CD8+ (G) T cells on

day 4 and day 7. Lung RSV specific CD8 T cells on day 7 (H). Anti-RSV IgG day 7 (I).

Figure 4. Mice were infected intranasally with RSV A2 (wild type) or RSV lacking the SH gene (RSV

∆SH). Weight loss (A), lung viral load (B) and lung cell number (C) were measured after infection.

Lung CD4 T (D), CD8 T (E) and DX5+ NK (G) cells were measured by flow cytometry. IL-6 (G),

CXCL1/KC (H) and IL-1B (I) were measured in lung supernatants.

Figure 3. HEp-2 cells were infected with *** MOI RSV A2 or RSV ∆SH, viral load was assessed by

plaque assay (A). Supernatants were collected and analysed for IL-1B level by ELISA following infection

of HEp-2 cells (B), PHBE cells (C), THP-1 cells (D), and neutrophils (F). Points represent mean +/- SEM

of n=3 repeats of HEp2, PHBE and THP1 cells and 3 individual PBMC and neutrophil donors.

Figure 5. Mice were infected with RSV A2 intranasally. Expression of IL-1B

by Ly6G+ (neutrophils: A) and CD11c/MHCII lo/F480+ (alveolar

macrophages: B) was measured by flow cytometry at various time points

after infection. Mice were treated with anti-Ly6G depleting antibody (1A8) or

control antibody (Con) intraperitoneally, and clodronate liposomes (CL) or

empty liposomes (PL) intranasally prior to RSV A2 infection. Neutrophil (C)

and alveolar macrophage (D) numbers were analysed by flow cytometry,

RSV L gene (E) by RT-PCR and lung IL-1B (F), KC (G) and IL-6 (H) were

measured on day 1 after infection.

SH K+

Casp1

NLRP3

Pro IL-1β

Meet the

authors!

2014 russell gordon delta sh new pdf

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