Kaposis Sarcoma-Associated Herpesvirus Transactivartor RTA Promotes - - PowerPoint PPT Presentation

1

Kaposi’s Sarcoma-Associated Herpesvirus Transactivartor RTA Promotes Degradation of the Repressors To Regulate Viral Lytic Replication

Zhilong Yang, Zhangcai Yan, and Charles wood

Journal of virology, 82(7): 3590-3603, 2008

Speaker: Hsin-I Tong Advisor: Li-Kwan Chang, Ph.D.

12/23/2008

2

Kaposi’s Sarcoma

‧Tumor, caused by HHV-8/KSHV ‧One of AIDS defining illness ‧assoicated with high mortality

3

‧Human Herpes Virus 8 (HHV-8), γherpesvirus ‧DNA Virus, linear dsDNA, with envelop ‧infect B cell, endothelial cell, monocyte, nerve cells ‧cause of Kaposi’s Sarcoma, primary effusion lymphoma,multicentric castleman’s disease (rare) Kaposi’s Sarcoma-Associated HerpesVirus (KSHV)

4

latent/lytic switch

Immediate-early

Early

Late Transactivator Replication machinerises, kinase Glycoprotein, capsid protein

RTA

The establishment of latency Reactivation from latency lytic replication

5

‧KSHV Immediate early protein (ORF 50) ‧ Necessary and sufficient for virus reactivation ‧ Strong/only transcription activator ‧Ubiquitine E3 ligase function

pEarly gene pEarly gene

Viral genome

RTA

Early proteins Viral reactivation, enter of the lytic replication

regulation of transcription activation

6

RTA

Early proteins

pEarly gene pEarly gene

Viral genome

Viral reactivation, enter of the lytic replication

pEarly gene pEarly gene

Viral genome

Repressor

Virus stays in latency

Repressor

?

RTA

Viral/Cellular factors:

K-RBP, KbZIP, NF-κB , LANA

7

KSHV RTA binding protein (K-RBP)

‧Cellular RTA binding protein ‧ transcriptional repressor

RTA

pEarly gene pORF57

Viral genome

K-RBP Virus stays in latency

8

Objectives

‧How RTA overcome K-RBP repression?

Proposed model

9

Framework

Proposed Model

• RTA expression promotes K-RBP degradation
• K-RBP degradation is mediated by the ubiquitin-

proteasome pathway

• Correlation of RTA-mediated transactivation and

degradation

10

RTA expression promotes K-RBP degradation

control cotransfected Western blot analysis

11

RTA enhances KRBP turnover rate

CHX=Cycloheximide Protein synthesis inhibitor

12

Framework

Proposed Model

• RTA expression promotes K-RBP degradation
• K-RBP degradation is mediated by the ubiquitin-

proteasome pathway

• Correlation of RTA-mediated transactivation and

degradation

13

Protein degradation pathways 1.Lysosome-dependent pathway 2.Proteasome-dependent pathway

?

14

Early proteins

pEarly gene pORF57

Viral genome

Viral reactivation, enter of the lytic replication

RTA reduce K-RBP protein level

K-RBP

?

RTA

pEarly gene pORF57

Viral genome

Fail entering viral reactivation

RTA do not reduce K-RBP protein level

K-RBP

?

RTA

15

Proteasome-dependent inhibitor Lysosome-dependent inhibitor RTA promotes K-RBP degradation via the proteasome-dependent pathway

16

Ubiquitination

Ubiquitin-activating enzyme Ubiquitin-conjugating enzyme Ubiquitin-protein ligase

Proteasome-dependent degradation

17

Immunoprecipitation (IP) protein-protein binding

K-RBP

His Ub

HA

Anti-His

Anti-HA

In Vivo ubiqutination of K-RBP (withou RTA)

Anti-KRBP

Ub

HA

Ub

HA

Ub

HA

18

K-RBP Ubiquitination increased in the presence of RTA

In Vivo In Vitro

RTA(E3) enhance ubiquitination of K-RBP

Human E2

1 2 3 4 1 2 3 4

19

Framework

Proposed Model

• RTA expression promotes K-RBP degradation
• K-RBP degradation is mediated by the ubiquitin-

proteasome pathway

• Correlation of RTA-mediated transactivation and

degradation

20

Framework

• Correlation of RTA-mediated transactivation and

degradation

pEarly gene pORF57

Viral genome

Fail entering viral reactivation

K-RBP

RTA K-RBP repress RTA-mediated transactivation

U b Ub U b U b

Enter lytic replication

21

pORF57 Luciferase

RTA

luciferase luciferase luciferase

Detection

Luciferase assay

22

Luciferase activity represent RTA transactivation activity

23 pEarly gene pORF57

Viral genome

RTA

RTA Transactivity set as 100% For the White bar

Luciferase gene

24

Luciferase activity represent RTA transactivation activity

Set as 100% in each concentration

25 pEarly gene pORF57

Viral genome

K-RBP

RTA

U b Ub U b U b

RTA transactivity partically blocked by K-RBP

RTA Transactivity Luciferase gene

26

Luciferase activity represent RTA transactivation activity

74% 17% % inhibited by K-RBP Set as 100% in each concentration

27

RTA wild type vs. RTA mutant (lower degradation ability)

Lower degradation ability associates with lower transactivation ability

28

Summary

29

Thank you Thank you

Special Thanks

• Dr. Li-Kwan Chang