Georg KochsInstitute for VIROLOGY

UNIVERSITY OF FREIBURG

Influenza A virus &

the antiviral action of

Mx proteins

GEIG-Paris-03.04.14-30 min

Susceptibilité genetique de l‘hote a l‘infection grippale !

Why some individuals get severe influenza, mild influenza,or do not develope a disease upon virus infection ?

=> Host-Virus-interaction determines the mild or fatal outcome of an infection.

viral genetics has also an effect.

pH1N1 H5N1

What means: Host genetic susceptibility to influenza A virus infection ?

- Large differences in the susceptibility to pathogenic influenza virus infection,

indicating a clear influence of host genetics on susceptibility.

- Host heritable resistance is polygenic - not linked to one specific gene - !

- A strong, non-restricted replication of the virus leads toelevated inflammatory response (cytokine storm) and severe lung pathology.

strain1

strain1

influenza A virus

strain2 strain2

influenza A virus

Analysis of different inbred mouse strains:

Human genetic susceptibility to influenza A virus infection !

- How to identify candidates that determine host susceptibility ?

Host genetic susceptibility to severe influenza in humans remains unanswered.

(Peter Horby et al., (2012) Plos One 7, e33180)

- Need of coordinated efforts to define and assemble cohorts of severe influenza cases -

Alick Isaacs & Jean Lindenmann

1957 – Discovery of Interferon

NIMR-Mill Hill-London

virus-inhibiting factor = Interferon (IFN)

- anti-viral state against influenza A virus

primary chicken fibroblasts

M

P

B

2

P

B

1

P

A N

PN

A

H

A N

S

inactivated

Influenza A virus

primary chicken fibroblasts

M

P

B

2

P

B

1

P

A N

PN

A

H

A N

S

replication-competent

Influenza A virus

1957 – Discovery of Interferon

virus

IFN-a/

RLRs TLRs

IFN-a/

IFN-a/

RLR – Rig-I-like receptors

TLR – Toll-like receptors

Influenza A virus induces Interferons

ISGISRE

IFNAR

JAK-1 / TYK-2

STAT-1 -2

IRF-9

ISGF-3

virus

IFN-a/

RLRs TLRs

IFN-a/

IFN-a/

IRF – IFN-regulatory factor

JAK – Janus Kinase

STAT – Signal transducer and activator of transcription

ISGF3 – IFN-stimulated gene factor

ISRE – IFN-stimulated response element

ISG - IFN-stimulated genes

IFN induces an antiviral state

ISGISRE

IFNAR

JAK-1 / TYK-2

STAT-1 -2

IRF-9

ISGF-3

?

? ?

ISG

virus

IFN-a/

RLRs TLRs

IFN-a/

IFN-a/

Ralph Steinman Jules Hoffmann Bruce Beutler

Steinman&Cohn (1973)

JEM. 137, 1142-1162.

First description of

Dendritic cells as

important components

of the host defence.

Lemaitre, B. et al. (1996)

Cell 86, 973-983.

First description of

Toll- receptors in Drosophila

as components of

the immune system

Poltorak, A. et al. (1998)

Science 282, 2085-2088.

First description of

Toll-like receptors in mice

as a component of LPS sensing.

Identification of other components

of PAMP recognition and

innate Immunity, via RIG-I.

2011 – Nobel prize in medicine

Is the IFN system of any relevance for infections in vivo ?

Antiviral effect of IFN in vivo !

0 2 4 6 8 12

days p.i.

10

IFN

mock

0

25

50

75

100

Surv

ival

[%

]

mice were treated i.n. with IFN-a 10h,before intranasal infection with 100 LD50 of hvPR8.(Grimm et al., (2007) PNAS 104, 6806-6811)

Vir

us

tite

r [l

og 1

0p

fu/m

l]

3

1

5

7

IFN mock

detection limit

8

6

4

2

IFN induces an antiviral state against Flu,

Search for effector molecules !

IFN induces an antiviral state

Search for effector molecules !

Pan-viral specificity of ISGs.

Expression of 350 human ISGs for inhibition of 14 gfp-viruses.Schoggins et al. (Rice-Rockefeller), (2014) Nature 505, 691-696.

IFIT

M3

IFITM3 is a potent inhibitor of influenza virus replication !

What is IFITM3 ?

(-) vRNA

(+) cRNA (+) mRNA

virale Proteins

IFITM3

Interferon-induced transmembrane protein-3.

- trans-membrane protein of the endosomal compartment.

- alters membrane fluidity.

- inhibits entry of influenza-, Ebola-, West Nile- and other viruses.

Is there a role of IFITM3 in vivo ?

Antiviral action of IFITM3 in vivo ?

wt

Ifitm3-/-

- IFITM3 is a potent inhibitor of influenza virus replication in vivo.

wt

IFITM3-/-

mice were intranasal infected with A sublethal dose of pH1N1/09.(Everitt et al., Kellam&Brass-Lab, UCL&MIT (2012) Nature 484, 519-523)

? Is there a link to severe influenza in humans ?

Role of IFITM3 in human influenza ?

- Hospitalized patients with severe Flu symptoms in 2009, n=53 in England and Scotland.

Everitt et al., Kellam&Brass-Lab, UCL&MIT-Harvard (2012) Nature 484, 519-523.

Zhang et al., (2013) Nature Communication 4, 1418-1423.

T-allel/93%C-allel/0.3%

- Analysis for genotypic differences in IFITM3 gene: 10-fold higher frequency of C-allele.

What is the effect

of C/C for Flu ?

Role of IFITM3 in human influenza ?

- Hospitalized patients with severe Flu symptoms in 2009, n=53 in England and Scotland.

Everitt et al., Kellam&Brass-Lab, UCL&MIT-Harvard (2012) Nature 484, 519-523.

Zhang et al., (2012) Nature Communication 4, 1418-1423.

T-allel/93%C-allel/0.3%

- Analysis for genotypic differences in IFITM3 gene: 10-fold higher frequency of C-allele.

Cells expressing IFITM3 cDNAs, infected with pH1N1 for 12 h, stained for HA expression.

Role of IFITM3 in human influenza ?

- Hospitalized patients with severe Flu symptoms in 2009, n=53 in England and Scotland.

Everitt et al., Kellam&Brass-Lab, UCL&MIT-Harvard (2012) Nature 484, 519-523.

Zhang et al., (2012) Nature Communication 4, 1418-1423.

T-allel/93%C-allel/0.3%

- Analysis for genotypic differences in IFITM3 gene: 10-fold higher frequency of C-allele.

Cells expressing IFITM3 cDNAs, infected with pH1N1 for 12 h, stained for HA expression.- IFITM3 is antivirally active against Flu in cell culture and in vivo.

- Changes in IFITM3 expression correlate with 6-fold greater risk for severe influenza.

0 2 4 6 8 12

days p.i.

10

IFN

mock

0

25

50

75

100

Surv

ival

[%

]

C57BL/6 mice were treated i.n. with IFN-a,before intranasal infection with 100 LD50 of hvPR8.(Grimm et al., (2007) PNAS 104, 6806-6811)

Antiviral effect of IFN, other candidates ?

Mx1+/+

Antiviral action of Mx Proteins !

C57BL/6 and B6.A2G-Mx1 mice were treated i.n. with IFN-a,before intranasal infection with 100 LD50 of hvPR8.(Grimm et al., (2007) PNAS 104, 6806-6811)

0 2 4 6 8 12days p.i.

10

Mx1+/+

IFN

mock

0

25

50

75

100

Surv

ival

[%] Mx1-/-

0

25

50

75

100

0 2 4 6 8days p.i.

IFN

mock

Vir

us

tite

r [l

og 1

0p

fu/m

l]

3

1

5

7

Mx1-/-

IFN mock IFN mock

detection limit

8

6

4

2

Mx1+/+

Antiviral action of Mx Proteins !

pH1N1-1918 H5N1 – A/Vietnam/1203/04

(Tumpey et al., (CDC, Atlanta) (2007) JVI 81, 10818-10821)Mx1 is a dominant antiviral restriction factor for influenza in the mouse.

Mx1-/- Mx1-/-

Mx1+/+ Mx1+/+

FLUAV

[adapted from: Haller O. et al: Microbes and Infection, 2007]

Mx family members

orthomyxoviruses

Influenza-, paramyxo- bunya-, rhabdo-, picorna- hepadna-, African swine fever viruses

anti- HIV-1 activity

hsMxA (MX1)

hsMxB (MX2)

hsMxA active in vivo?

human MxA transgenic mouse

Peter StaeheliVirology-Freiburg

MEF+IFN 18h

Antiviral activity of human MxA ?

Mx-/-

tgMxA

mice intranasal infected with lethal dose of flu (titers 5 d.p.i).(Deeg&Staeheli (2014) in preparation)

Human MxA protects against lethal influenza A virus infection

in the transgenic mouse model.

• Mx = Myxovirus resistance (1962 by Jean Lindenmann)

• induced by IFN-a/ and l (used as a marker for IFN activity in vivo)

unstructured

loop 4

Oliver DaumkeMDC, Berlin

What are Mx proteins ?

• large dynamin-like GTPase (75 kDa)

• antivirally active against influenza viruses and other RNA viruses

Gao S., von der Malsburg A. et al.; Immunity 2011

Dr. Harmit MalikCancer Center, Seattle

Dr. Corinna PatzinaVirology, Freiburg

Evolution guided analysis of primate MxA

Comparative analysis for positive selection

as marker of host-target interfaces

Structure of MxA is highly conserved.

L4 is a hotspot for positive selection.

(Mitchell&Patzina et al., (2012) CHM 12, 598-604)

L4

MxA proteins from primates

hsMxAagmMxA

558 571

561 566

MxA proteins from primates

Chimeric proteins:

hsMxA agmL4

agmMxA hsL4

hsMxA

hsMxA

delL4

hsMxA

agm

L4

agm

MxA

agm

MxA

hsL

4

0

20

40

60

80

100

% i

nfe

cte

d,

Mx-p

osit

ive c

ell

s

African green monkey

old world human

hsMxAagmMxA

558 571

561 566

MxA proteins from primates

Chimeric proteins:

hsMxA agmL4

agmMxA hsL4

hsMxA

hsMxA

delL4

hsMxA

agm

L4

agm

MxA

agm

MxA

hsL

4

0

20

40

60

80

100

% i

nfe

cte

d,

Mx-p

osit

ive c

ell

s

F561V

hsMxAagmMxA

558 571

561 566

MxA proteins from primates

Chimeric proteins:

hsMxA agmL4

agmMxA hsL4

hsMxA

hsMxA

delL4

hsMxA

agm

L4

agm

MxA

agm

MxA

hsL

4

0

20

40

60

80

100

% i

nfe

cte

d,

Mx-p

osit

ive c

ell

s

- positive selection in Loop L4 of primate MxAs.

- L4 and position 561 are essential for antiviral specificity against influenza A virus.

- L4 represents the interface of MxA to contact the viral target.

What is the viral target ?

F561V

Viral target of Mx Proteins ?

MxA

avian influenza virusesA/Thailand/1/04 (H5N1)

Human flu isolatesA/Hamburg/4/09 (pH1N1)

A/BM/01/18

- Flu strains differ in their Mx sensitivity (Dittmann, 2008).

- The viral nucleoprotein (NP) determines Mx sensitivity (Zimmermann, 2011).

Viral target of Mx Proteins ?

? Why avian influenza viruses are so sensitive to MxA ?

NP

Analysis of viral NP for Mx sensitivity

NP

AvianH5N1

32 aadifferences

2009pH1N1

191814 aa

differences

Martin Schwemmle,Virology, Freiburg

NP

- Specific positions in NP determine Mx sensitivity

and might serve as interaction partners of MxA (Mänz, 2013)

NP

pH1N1 1918

Model of MxA action

Loop 4+

from Gao et al., (2011) Immunity 35,1-12

Summary:

- Analysis of candidates influencing susceptibility is difficult, polygenic effect.

- Two canidates, IFITM3 and MxA, block virus replication in vivo.

(-) vRNA

(+) cRNA (+) mRNA

virale Proteins

IFITM3MxA

- MxA provides a barrier for zoonotic transmission of avian influenza viruses,

might depict new strategies for antiviral treatment.

- IFITM3 polymorphism might determine susceptibility to severe influenza.

helps to identify patients at higher risk and adequate treatment.

Martin SchwemmleOtto Haller Peter Stäheli

Oliver DaumkeMDC, Berlin

Harmit MalikFred Hutchinson Cancer Res. Center,

Seattle

Fine

Analysis of viral NP for Mx sensitivity

NP

200919571918

H3N2

Swine

16D

100I

283P

313Y

16D

100V

283P

313Y

Avian

pH1N1

100I

53D

313V

100I/V

Human

16D

100V

283P

313Y

H2N2H1N1

Evasion of human influenza A viruses

from MxA restriction

Bold letters indicate amino acids shown to significantly increase Mx resistance,

whereas amino acids highlighted in grey are minor contributors.

adaptive mutations that newly emerged with the appearance of the 2009 pandemic pH1N1

are depicted in red. 53D got partially lost after re-introduction into the swine host.

Temporal appearance of Mx resistance enhancing mutations

in influenza A viruses (Mänz et al., 2013)

HA + Receptor

Polymerase + cell. factors

Adaptation

Avian influenza Human adapted influenza

MxA

MxA

Hypothesis: Mx Proteins prevent Influenza A

viruses from crossing the species barrier

Influenza A virus & Mx proteins

A/HH/01/09 (pH1N1)A/Thailand/1/04 (H5N1)-Kan1

Verhelst, Hulpiau, Salens “Mx Proteins: Antiviral Gatekeepers That Restrain the Uninvited”, MMBR, 2013

INTRODUCTION: The Mx-Patch

0

20

40

60

80

100

120

0

20

40

60

80

100

120

NP

(wt)

NP

(V1

00

R/P

23

8L)

NP

(V1

00

R/Y

31

3F)

NP

(P2

83

L/Y3

13

F)

NP

(V1

00

R/P

28

3L/

Y31

3F)

******

*** ***

NSNS *

NP

(wt)

NP

(V1

00

R)

NP

(P2

83

L)

NP

(Y3

13

F)

****** ***

α-Mx1

α-Η1Ν1-NP

α-Actin

Mx-sensitivity of hvPR8-NP(V100R); (P283L); (Y313F) constructs in the minireplicon

Creation of NP constructs

PR8-NP(V100R/P283L) PR8-NP(V100R/Y313F)

PR8-NP(P283L/Y313F) PR8-NP(V100R/P283L/Y313F)

Rescue of PR8(mut) viruses

Rescue failed

Rescue of NP(mut) viruses

NP(w

t)

NP(V

100R

/P28

3L)

NP(V

100R

/Y31

3F)

NP(P

283L

/Y31

3F)

6

7

8

9

NP(w

t)

NP(V

100R

/P28

3L)

NP(V

100R

/Y31

3F)

NP(P

283L

/Y31

3F)

6

7

8

9

NP(w

t)

NP(V

100R

/P28

3L)

NP(V

100R

/Y31

3F)

NP(P

283L

/Y31

3F)

5

6

7

8

9

Virus C57BL/6 BL6-Mx1

hvPR8-NP(wt) <10 31,6

NP(V100R/P283L) <10 100000

NP(V100R/Y313F) <10 562

NP(P283L/Y313F) <10 450000

LD 50

BL6-Mx1200 PFU; 48h

BL6-Mx1100 PFU; 48h

C57BL/6200 PFU; 48h

Fitness of hvPR8-NP(V100R); (P283L); (Y313F) viruses in vivoLu

ng

tite

rlo

g 10

(PFU

/ml)

Finding the right candidate for the passaging experiment

1.A

1.B

1.C

1.D

Start: Infection with hvPR/8-NP(P283L/Y313F)

t=0: Infection with 100 PFU intra nasal

+48h: Preparation of lungs; homogenisation; virustitration

PASSAGING EXPERIMENT

0 1 2 3 4 5 6 7 8 9 105

6

7

8

B-Line

A-Line

Y52N

N309K

Passage Number

Lun

g ti

ter

log 1

0(P

FU/m

l)

Inp

ut

1. P

ass.

AA 52A-Line

AA 309B-Line

1. P

ass.In

pu

t6

. Pass.

7. P

ass.8

. Pass.

9. P

ass.1

0. P

ass.

2. P

ass.

3. P

ass.

4. P

ass.

5. P

ass.

10

. Pas

s.

Development of lung titers during passaging and outcome of segment 5 sequencing

PASSAGING EXPERIMENT

Two escape mutants were found

The new mutations are inside the Mx-patch next to the original knock-down mutations

P283L

Y313F

Y52N

0

20

40

60

80

100

120

0

20

40

60

80

100

120

0

20

40

60

80

100

120R

el. L

uc

-acti

vit

y (

%)

α-Mx1

α-NP

α-ActinN

P(w

t)

NP

(Y5

2N

)

NP

(P2

83

L/Y3

13

F)

NP

(Y5

2N

/P2

83

L/Y3

13

F)

***NS NS

NP

(wt)

NP

(N3

09

K)

NP

(P2

83

L/Y3

13

F)

NP

(P2

83

L/N

30

9K

/Y3

13

F)

***NS ***

NP

(wt)

NP

(Y5

2N

/N3

09

K)

NP

(P2

83

L/Y3

13

F)

NP

(Y5

2N

/P2

83

L/N

30

9K

/Y3

13

F)

***NS NS

Mx-sensitivity of hvPR8-NP(Y52N) & NP(N309K) constructs in the minireplicon

The Y52N mutation decreases Mx-sensitivity, N309K mutation has no effect

0 12 24 36 48 602

3

4

5

6

7

8

hvPR8-NP(wt)

hvPR8-NP(283L/313F)hvPR8-NP(52N)hvPR8-NP(52N/283L/313F)

hours post infection

Vir

us

tite

rlo

g 10

(PFU

/ml)

Fitness of hvPR8-NP(Y52N) & NP(N309K) viruses in vitro

Calu-3 cells; MOI 0,001

Rescue and test of the interesting 52N mutant

Lun

g ti

ters

log 1

0(P

FU/m

l)

C57BL/6

200 PFU; 48hC57BL/6

100 PFU; 48h

Lung titers in Mx-/- and Mx+/+ mice

Fitness of hvPR8-NP(Y52N); (P283L); (Y313F) viruses in vivo

Rescue and test of the interesting 52N mutant

BL6.A2G-Mx1

200 PFU; 48h

Survival of hvPR8-NP(Y52N/P283L/Y313F) virus in Mx+/+ mice

LETHALITY

n=4

NP(wt) is aggressive, NP(283L/313F) is attenuated in Mx+/+ mice

Survival of hvPR8-NP(Y52N/P283L/Y313F) virus in Mx+/+ mice

LETHALITY

n=4

NP(52N) is comparable to NP(wt)

Survival of hvPR8-NP(Y52N/P283L/Y313F) virus in Mx+/+ mice

LETHALITY

NP(52N/283L/313F) is nearly as aggressive as NP(wt)

***

NS ***

***

NS

*

Relative polymerase activityHow do the mutations affect polymerase speed?

NP(283L/313F) is nearly on the same level as NP(52N/283L/313F)

qPCR:Do the mutations affect Mx-induction??

All mutations induce Mx1 to a comparable level

Mx induction of hvPR8-NP(mut) ISG15 induction of SC35M(ΔNS1)

Project:Doomsday

Outlook: H7N9

MASQG TKRS YEQ METGG ERQ NA TEIRASVGR MVSG IGR FYIQ MC TELKLSD NEGRL IQ NS ITIER MVLSA FD ERR NR YL EEHPSAGKDPKK TGGP IYRRRD

GK WVR EL IL YDK EEIRR IWRQA NNG EDA TAGL THL MIWHS NL NDA TYQR TRALVR TG MDPR MCSL MQGS TLPRRSGAAGAAVKG IG TMV MEL IR MIKRG INDR NFWRG ENGRR TR I

A YER MC NILKGK FQ TAAQRA MMDQVR ESR NPG NA EIEDL IFLARSAL ILRGSVAHKSCLPACV YGLAVASG YD FER EG YSLVG IDP FRLLQ NSQV FSL IRP NENPAHKSQLV WMAC

HSAA FEDLRVSS FIRG TR MVPRGQLS TRGVQ IAS NENMEA MDS NTL ELRSR YWA IR TRSGG NTNQQRASAGQVSVQP TFSVQR NLP FERA TIMAA FTG NTEGR TSD MR TEIIR MME

SARP EDVS FQGRGV FELSD EKA TNP IVPS FD MNNEGS YFFGD NA EEYD N

MASQG TKRS YEQ METGG ERQ NA TEIRASVGR MVSG IGR FYIQ MC TELKLSD NEGRL IQ NS ITIER MVLSA FD ERR NR YL EEHPSAGKDPKK TGGP IYRRRD

GK WVR EL IL YDK EEIRR IWRQA NNG EDA TAGL THL MIWHS NL NDA TYQR TRALVR TG MDPR MCSL MQGS TLPRRSGAAGAAVKG IG TMV MEL IR MIKRG INDR NFWRG ENGRR TR I

A YER MC NILKGK FQ TAAQRA MVDQVR ESR NPG NA EIEDL IFLARSAL ILRGSVAHKSCLPACV YGLAVASG YD FER EG YSLVG IDP FRLLQ NSQV FSL IRP NENPAHKSQLV WMAC

HSAA FEDLRVSS FIRG TR MVPRGQLS TRGVQ IAS NENIEA MES NTL ELRSR YWA IR TRSGG NTNQQRASAGQ IS IQP TFSVQR NLP FERA TIMAA FTG NTEGR TSD MR TEIIR MME

SARP EDVS FQGRGV FELSD EKA TNP IVPS FD MNNEGS YFFGD NA EEYD N

MASQG TKRS YEQ METGG ERQ NA TEIRASVGR MVSG IGR FYIQ MC TELKLSD NEGRL IQ NS ITIER MVLSA FD ERR NR YL EEHPSAGKDPKK TGGP IYRRRD

GK WVR EL IL YDK EEIRR IWRQA NNG EDA TAGL THL MIWHS NL NDA TYQR TRALVR TG MDPR MCSL MQGS TLPRRSGAAGAAVKG IG TMV MEL IR MIKRG INDR NFWRG ENGRR TR I

A YER MC NILKGK FQ TAAQRA MMDQVR ESR NPG NA EIEDL IFLARSAL ILRGSVAHKSCLPACV YGLAVASG YD FER EG YSLVG IDP FRLLQ NSQV FSL IRP NENPAHKSQLV WMAC

HSAA FEDLRVSS FIRG TR MVPRGQLS TRGVQ IAS NENMEA MDS NTL ELRSR YWA IR TRSGG NTNQQRASAGQVSVQP TFSVQR NLP FERA TIMAA FTG NTEGR TSD MR TEIIR MME

SARP EDVS FQGRGV FELSD EKA TNP IVPS FD MNNEGS YFFGD NA EEYD N

SKSRVD NHSPSD INIMASQG TKRS YEQ METGG ERQ NA TEIRASVGR MVSG IGR FYIQ MC TELKLSD YEGRL IQ NS ITIER MVLSA FD ERR NR YL EEHPSAGKDPKK TGGP IYRRRD

GK WVR EL IL YDK EEIRR IWRQA NNG EDA TAGL THL MIWHS NL NDA TYQR TRALVR TG MDPR MCSL MQGS TLPRRSGAAGAAVKGVG TMV MEL IR MIKRG INDR NFWRG ENGRR TR I

A YER MC NILKGK FQ TAAQRA MMDQVR ESR NPG NA EIEDL IFLARSAL ILRGSVAHKSCLPACV YGLAVASG YD FER EG YSLVG IDP FRLLQ NSQV FSL IRP NENPAHKSQLV WMAC

HSAA FEDLRVSS FIRG TRVVPRGQLS TRGVQ IAS NENMEA MDS NTL ELRSR YWA IR TRSGG NTNQQRASAGQ ISVQP TFSVQR NLP FERA TIMAA FTG NTEGR TSD MR TEIIR MME

SARP EDVS FQGRGV FELSD EKA TNP IVPS FD MNNEGS YFFGD NA EEYD N*RK IPL FL

1 10 20 30 40 50 60 70 80 90 100 110

120 130 140 150 160 170 180 190 200 210 220 230

240 250 260 270 280 290 300 310 320 330 340

350 360 370 380 390 400 410 420 430 440 450 460

470 480 490 500 510 521

A/Anhui/1/2013 NP

A/Anhui/1/2013 NP

A/Anhui/1/2013 NP

A/Anhui/1/2013 NP

A/Anhui/1/2013 NP

A/Shanghai/1/2013 NP

A/Shanghai/1/2013 NP

A/Shanghai/1/2013 NP

A/Shanghai/1/2013 NP

A/Shanghai/1/2013 NP

A/Shanghai/2/2013 NP

A/Shanghai/2/2013 NP

A/Shanghai/2/2013 NP

A/Shanghai/2/2013 NP

A/Shanghai/2/2013 NP

H5N1 (KAN-1) NP

H5N1 (KAN-1) NP

H5N1 (KAN-1) NP

H5N1 (KAN-1) NP

H5N1 (KAN-1) NP

Identity

Identity

Identity

Identity

Identity

Analysis of H7N9-NP(wt), H7N9-NP(N52Y) and H7N9-NP(4X)

Together with Dominik Dornfeld (AG Schwemmle):Checking H7N9 for Mx-sensitivity

NP Comparison

52N and 352M are close to Mx-patch positions

Minireplicon by Dominik

H7N9 MinirepliconIntermediate Mx-sensitivity in the minireplicon

H7N9 has an intermediate Mx-sensitiviy between H5N1 and H1N1

Cryo-TEM purified

MxA on lipid membrane

G domain

Stalk

[von der Malsburg A, et al., J. Biol. Chem, 286, 37858-37865 (2011)]

Loop 4

T- bar shape of MxA

Loop L4: binding site for lipid membranes

Homo-oligomer of MxA

L4 L4 L4 L4 L4 L4

[Gao S, von der Malsburg A, et al., Immunity, 35: 514-525 (2011)]

Most recent model of mouse Mx1 action

GTPase

BSE

Stalk

Mx

oligomerization

Mx dimerMx ring

cc c c c cc c

Mx1 binding to vRNPs

cc c c c cc c

Disruption of vRNPs

(GTPase-dependent)

Viral Polymerase

complex

Viral RNA

Viral

nucleoproteinDissociation between polymerase

complex and RNA-bound NP

[Verhelst … Fiers….Saelens, J Virol 86: 13445 - 13455 (2012)]

Verhelst et al., (2012) JVI in press

Model of Mx action against influenza A viruses

Model of Mx antiviral action

Gao S., von der Malsburg A. et al.; Immunity 2011

Loop L4 of MxA

from Gao et al., (2011) Immunity 35,1-

1. Human MxA and MxB are IFN-induced antiviral restriction factors

1. MxA provides a barrier against trans-species transmission of FLUAV

2. Disordered loop L4 is a target interface under positive selection

3. Adaptive mutations in viral NP allow a degree of MxA evasion

4. Host Mx proteins and viral components are „genes in conflict“

in an evolutionary „arms - race“

CONCLUSIONS

Influenza A virus induces Interferons

virus

IFN-a/

RLRs TLRs

IFN-a/

IFN-a/

NS1

ss(-)RNA-Segments

M

PB2PB1PANP

HANA

NS

AG - Kochs

Valentina, Barbara, David,

Mirjam, Basti, Corinna, Jonas

Martin Schwemmle

Benjamin Mänz

Dominik Dornfeld

Otto Haller

Petra Zimmermann

Corinna Patzina

Peter Stäheli

Oliver Daumke

Song GaoMDC, Berlin

Harmit Malik

Patrick MitchellFred Hutchinson Cancer Res.

Center, Seattle