Oncogenic herpesvirus hijacks components of the DNArepair machinery to promote its own replication

Xiaofan Li, Ph.D.Bhaduri-McIntosh laboratory

The DNA Damage Response cascade and ATM

Yosef Shiloh et al., Nature Reviews, 2013

Phosphorylation of Krüppel -associated Box (KRAB)-associated protein (KAP)-1by ATM is a key event during DDR

Yael Ziv et al., Nature Cell Biology, 2006

Krüppel -associated Box (KRAB)-associated protein (KAP)-1

376 493 628 801 835

651RBCC

BromoHP1 box

KAP1/TRIM28 (tripartite motif-containing protein 28) structure

KAP1 recruits HDACs and HMT to modify the epigenetic status around its binding locus

KAP1

DAPI

PS824

NuRD

CHD HDAC1/2

SETDB1

PS824

H3-K9 trimethylation H3-acetylation

KAP1 facilitates repair of DNA breaks in heterochromatin by allowing access to repair factors

Epstein-Barr virus (EBV)

Member of human gammaherpesvirus family

Enveloped, encapsidated large dsDNA genome

Mainly lymphoid, epithelial tropism

Associated with numerous human malignanciesBurkitt lymphoma Nasopharyngeal cell carcinoma Hodgkin’s diseasePost-transplant Lymphoproliferative diseases (PTLD)NK/T-Cell lymphomasGastric carcinoma

A. B.

C. D.

In situ hybridization for Epstein-Barr virus–encoded small RNAs (EBERs) inA. Burkitt lymphoma, B. Nasopharyngeal cell carcinoma, C. Hodgkin’s disease; D. PTLD

Lytic (re)activationLatent infection

Latency to lytic switch of EBV

Limited viral genes expressed

Viral genome duplicates in S-phase

No progeny production

HDACi, TGF-β, hypoxia, etc

ImmediateEarly

Early Late

Cellular components that regulate latency-to-lytic switch

-STAT3

-PCBP2

-KRAB-ZFPs: large family of transcriptional repressors

thought to function via the KAP1 corepressor

Derek Daigle, et al., JVI, 2010; Erik Hill, et al., JVI, 2013;

Siva Koganti, et al., JVI, 2015;

Mechanisms underlying EBV latency to lytic switch

Manipulation of KAP1 levels modulates EBV lytic activation

CA

E

0.5

1.0

1.5

0

0.5

1.0

1.5

0

0.5

1.0

1.5

0

BZ

LF

1m

RN

A

(rela

tive

am

ou

nt)

BM

RF

1m

RN

A

(rela

tive

am

ou

nt)

BF

RF

3m

RN

A

(rela

tive

am

ou

nt)

ZEBRA

KAP1

β-actin

0h 24h 48h 72h

Empty vector FLAG-KAP1

0h 24h 48h 72h

50kD

98kD

36kD1.00 0.85 0.66 0.36

D F

0h 13h 26h 39h

scrambled

ZEBRA

KAP1

β-actin

0h 13h 26h 39h

si-KAP1

50kD

98kD

36kD1.00 3.771.90 1.35

0.5

1.0

1.5

2.0

0

BZ

LF

1m

RN

A

(rela

tive

am

ou

nt)

BM

RF

1m

RN

A

(rela

tive

am

ou

nt)

BF

RF

3m

RN

A

(rela

tive

am

ou

nt)

0

1.5

*

3.0

4.5

6.0

0

1

2

3

4

0.5

1.0

1.5

0

EB

V D

NA

co

pie

s

(rela

tive

am

ou

nt)

0.5

1.0

1.5

2.0

0

EB

V D

NA

co

pie

s

(rela

tive

am

ou

nt)

B

**

*

**

*

*

scrambled

si-KAP1

VectorFLAG-KAP1

Xiaofan Li, et al., PLOS Pathogens, 2017

A

p-S824 KAP1

KAP1

β-actin

ZEBRA

EA-D

98kD

98kD

36kD

50kD

50kD

NaB (mM) 0 0.375 0.75 1.5 3

p-S824 KAP1

KAP1

β-actin

ZEBRA

EA-D

98kD

98kD

36kD

50kD

50kD

Dox (μg/ml) 0 0.15 0.6 2.5 10

p-S824 KAP1 ZEBRA DAPI

p-S824 KAP1 EA-D DAPI

B

D E

F

100

101

102

103

104

100

101

102

103

104

006

FL1-H

FL

4-H

0.778 40.2

6.8252.2

100

101

102

103

104

100

101

102

103

104

005

FL1-H

FL

4-H

1.04 0.846

0.26597.9

100

101

102

103

104

100

101

102

103

104

010

FL1-H

FL

4-H

1.04 39.6

5.0454.3

100

101

102

103

104

100

101

102

103

104

009

FL1-H

FL

4-H

0.907 1.07

1.296.8

1.04 0.846

97.9 0.265

0.778 40.2

52.2 6.82

0.907 1.07

96.8 1.2

1.04 39.6

54.3 5.04

ZEBRA

p-S

824 K

AP

1

EAD

p-S

824 K

AP

1

Dox- Dox+

EAD

100

101

102

103

104

100

101

102

103

104

012

FL1-H

FL

4-H

1.07 42.8

6.6249.5

100

101

102

103

104

100

101

102

103

104

007

FL1-H

FL

4-H

1.82 0.619

0.14397.4

100

101

102

103

104

100

101

102

103

104

008

FL1-H

FL

4-H

1.15 43.7

7.5147.6

100

101

102

103

104

100

101

102

103

104

011

FL1-H

FL

4-H

0.633 0.586

0.24198.5

1.82 0.619

97.4 0.143

1.15 43.7

47.6 7.51

0.633 0.586

98.5 0.241

1.07 42.8

49.5 6.62

ZEBRA

p-S

824 K

AP

1

NaB- NaB+

p-S

824 K

AP

1

KAP1 is phosphorylated at Serine 824 upon virallytic activation in EBV+ BL cells

C

p-S824 KAP1 ZEBRA DAPI

p-S824 KAP1 EA-D DAPI

Xiaofan Li, et al., PLOS Pathogens, 2017

36kD

50kD

98kD

98kD

ZEBRA

β-actin

KAP1

FLAG

0h 48h

1.00 0.76 0.72 1.25

NaB

EVKAP1

wt

KAP1

S824A

KAP1

S824D

A

0.5

1.0

1.5

0

BM

RF

1m

RN

A

(rela

tiv

e a

mo

un

t)

p<0.01

p<0.01

BF

RF

3m

RN

A

(rela

tiv

e a

mo

un

t)

0.5

1.0

1.5

0

p<0.01

p<0.01

0.5

1.0

1.5

2.0

0

BZ

LF

1m

RN

A

(rela

tiv

e a

mo

un

t)

p<0.01

p<0.01

B

0.5

1.0

1.5

2.0

0

EB

V D

NA

co

pie

s

(rela

tiv

e a

mo

un

t)

p<0.01

p<0.01

C

Figure 4

EVKAP1

wt

KAP1

S824A

KAP1

S824D

Phosphorylated KAP1 at S824 is impaired in its ability to restrain EBV lytic activation

C

H

Alanine

Phospho-dead phosphomimetic phosphorylated

Xiaofan Li, et al., PLOS Pathogens, 2017;

FLAG-KAP1-wt

Empty vector

FLAG-KAP1-S824A

FLAG-KAP1-S824D

How is KAP1 phosphorylated at S824?

PS824

KAP1KAP1ATM mTOR

HCMV lytic activationDNA damage response

David White et.al, Cancer Res, 2006;

Yael Ziv et.al, Nature Cell Biology, 2006Benjamin Rauwel et al., Elife, 2015

ATM is responsible for phosphorylation of KAP1 at S824 during EBV lytic activation

A

C

p-S824 KAP1

KAP1

β-actin

ATM

98kD

NaB - + - +

scrambled si-ATM

1.0 0.63

98kD

250kD

50kD

p-S824 KAP1

KAP1

β-actin

ZEBRA

KU-55933 (μM) 0 0.1 0.5 1.0 0 0.1 0.5 1.0

-NaB +NaB

98kD

98kD

36kD

50kD

p-S824 KAP1

KAP1

β-actin

ZEBRA

Torin1 (μM) 0 0.1 0.2 0.5 0 0.1 0.2 0.5

-NaB +NaB

98kD

98kD

36kD

50kD

B

Torin1

KU

p-S824 KAP1 ZEBRA DAPI

-

Xiaofan Li, et al., PLOS Pathogens, 2017

KU-55933: ATM inhibitor

Torin1: mTOR inhibitor

Input R-IgG ATM KAP1 Input R-IgG ATM KAP1

IP IP

ATM

KAP1

IgG hc

250kD

98kD

50kD

-NaB +NaB

DAPI

BA

PLA (ATM/KAP1) ZEBRA DAPI

DAPIPLA (ATM/R-IgG) ZEBRA

PLA (G-IgG/KAP1) ZEBRA

ATM binds to KAP1 during EBV lytic activation

Xiaofan Li, et al., PLOS Pathogens, 2017

KAP1KAP1 S824

p

ZEBRA

ATMViral lytic product

(IE, E, L)

EBV genome EBV genome

Viral lytic product

(IE, E, L)

i. ii.

Working model

Chloroquine

F

ATM activator chloroquine induces phosphorylation of KAP1 at S824 and EBV lytic activation.

C

CQ 0h 4h 8h 16h 24h 48h 72h

ZEBRA

β-actin

p-S824 KAP1

KAP1

36kD

98kD

98kD

50kD

B

ZEBRA

β-actin

KAP1

CQ - + - +

KAP1

wt

KAP1

S824A

1.0 0.48

98kD

50kD

36kD

A

-

CQ

ZEBRA DAPIp-S824 KAP1

CQ

+KU

BZ

LF

1m

RN

A

(rela

tiv

e a

mo

un

t)

0

4

8

12

0

10

20

30

BM

RF

1m

RN

A

(rela

tiv

e a

mo

un

t)

0

2.5

5.0

7.5

BF

RF

3m

RN

A

(rela

tiv

e a

mo

un

t)

p<0.01 p<0.01 p<0.01

D E

2700

2600

2500

400

300

100

0

200

Vir

al lo

ad

(10

6/m

l)

NDG

ZEBRA

β-actin

CQ 0h 8h 24h 72h4h 16h 48h

36kD

50kD

0h 8h 24h 72h4h 16h 48h

Jijoye

Raji

ZEBRA

β-actin

CQ

36kD

50kD

Xiaofan Li, et al., PLOS Pathogens, 2017;

10μM CQ

Mock

NaB

200μM CQ

ZEBRA DAPIgH2AX

-

NaB

NaB

+KU

Etoposide

A

B

NaB EtoposideCQ

gH2AX

DAPI

C

-

gH2AX ZEBRA DAPI

CQ

+KU

CQ

Chloroquine and NaB activate ATM in the absence of observable DNA damage

Xiaofan Li, et al., PLOS Pathogens, 2017

Conclusion

The cancer-causing virus EBV exploits a cellular mechanism (involving ATM and KAP1)

that repairs breaks in heterochromatin to promote its own replication

Acknowledgements

Collaborators:

Jizu Zhi, Ph.D.FDA, USA

Funding:

NIAID, NIHStony Brook Research Foundation

Sumita Bhaduri-McIntosh, M.D., Ph.D.

Siva Koganti, Ph.D.

Eric Burton

Lab members:

Ramon Perez

Sandeepta Burgula, Ph.D

Salvatore Spadaro