Hepatitis C Virus and Hepatocellular Carcinogenesis May 19, 2009 - - PowerPoint PPT Presentation

1

Hepatitis C Virus and Hepatocellular Carcinogenesis

May 19, 2009

Stanley M. Lemon, MD Center for Hepatitis Research, Institute for Human Infections and Immunity, and the Department of Microbiology and Immunology, University of Texas Medical Branch Galveston TX University of Texas Medical Branch, Galveston, TX

2

The Spectrum of Hepatitis Viruses p p

3

A “Richter Scale” of Global Mortality Rates

Weiss and McMichael, Nat Medicine 10:S70-76, 2004

4

Hepatitis C Virus

4A

5’ E1 NS2

p 7

E2 NS3 NS4B NS5A NS5B 3’ C

4 A

Structural Proteins ER Lumen E1 E2 p7 Serine protease RNA-dependent RNA polymerase NS2 NS3 4A NS4B NS5A NS5B p7 Core NS3 NS5A NS5B

Signal peptidase Signal peptidase

N t t l P t i RNA helicase

Signal peptidase Signal peptide peptidase Signal peptidase Signal peptide peptidase

Cytoplasm

NS2/3 cysteine proteinase NS3/4A serine protease Signal peptide peptidase NS2/3 cysteine proteinase NS2/3 cysteine proteinase NS3/4A serine protease Signal peptide peptidase

Nonstructural Proteins

NS2/3 cysteine proteinase NS3/4A serine protease g p p p p NS2/3 cysteine proteinase NS2/3 cysteine proteinase NS3/4A serine protease g p p p p

5

The Hepatitis C Virus Life Cycle

NS2 E1 E2 4A NS4B p7

Polyprotein Processing and Replicase Assembly

Core NS3 4A NS5A NS5B

5’ 5’

Attachment and Entry RNA

5’ 3’ 5’ 5 +

• Synthesis

Assembly Release Assembly Translation

6

Natural History of Hepatitis C

Necroinflammation Steatosis Progressive Fibrosis Cirrhosis Hepatocellular Carcinoma

ALT Anti-HCV Anti-HCV Viremia

1 2 3 4 5 10 10 15 15 20 20 25 25 30 30

Months Years

7

Hepatitis C Pathogenesis

Virus-Cell Interactions Perturbed intracellular milieu Innate antiviral defenses System Level Interactions Innate immune responses p NK, NKT cells, cyto/chemokines Adaptive immune responses Inflammation Steatosis HSC activation, fibrosis (ROS) Carcinogenesis (DNA damage) Host Consequences Hepatic failure Cryoglobulinemia

8

Hepatitis C Pathogenesis

Virus-Cell Interactions Perturbed intracellular milieu Innate antiviral defenses System Level Interactions Innate immune responses p NK, NKT cells, cyto/chemokines Adaptive immune responses Inflammation Steatosis HSC activation, fibrosis (ROS) Carcinogenesis (DNA damage) Host Consequences Hepatic failure Liver cancer Cryoglobulinemia

9

Emergence of HCV-Related Liver Cancer g

Japan 1971-1990

15 17

000/yr

B C 1971-80

11 13

per 100,

B+C B 1981-90

9 11

eaths p

C B+C

7

1968 1968-

• 77

77 1978 1978-

• 79

79 1980 1980-

• 81

81 1982 1982-

• 83

83 1984 1984-

• 85

85 1986 1986-

• 87

87

Year De

9

10

11

How does HCV cause liver cancer? How does HCV cause liver cancer? Any hypothesis must consider: Contribution of chronic inflammation Increased risk with EtOH consumption HCV transgenic mice develop HCC HCV transgenic mice develop HCC Association with cirrhosis Genetically heterogeneous nature of HCC

12

How does HCV cause liver cancer? How does HCV cause liver cancer? Any hypothesis must consider: Contribution of chronic inflammation Increased risk with EtOH consumption HCV transgenic mice develop HCC HCV transgenic mice develop HCC Association with cirrhosis Genetically heterogeneous nature of HCC Other potential causes of HCC: Aflatoxin Hepatitis B virus Helicobacter sp. Hereditary hemachromatosis (30%) Hereditary hemachromatosis (30%) α1-antitrypsin deficiency

13

Central Role of c-Myc Responsive Genes in Malignant Conversion of Dysplastic Nodules to Early HCC y p y

Cirrhotic Low Grade High Grade Early Cirrhotic Nodule Dysplastic Nodule High Grade Dysplastic Nodule Hepatocellular Carcinoma

• Accumulating genetic and epigenetic modifications
• Increasing c-Myc copy number with 8q duplications

Kaposi-Novak et al. Cancer Res, 69:2775-2782, 2009

14

c-Myc and E2F1 Cooperatively Promote Hepatocellular c Myc and E2F1 Cooperatively Promote Hepatocellular Carcinogenesis in Double Transgenic Mice

Proliferation Apoptosis

CNA PC

• S. Ladu et al. Gastroenterology 135:1322-1332, 2008

15

Cooperative Actions of c-Myc and E2F1 p y

• n
• liferatio
• sis

Pro Apopto

• S. Ladu et al. Gastroenterology 135:1322-1332, 2008

Hepatocellular Proliferation and Survival

16

HCV-infected Cells within a Regenerative Nodule

Elastin 40X 10X

Imaged by 2PE Microscopy

HCV Core DAPI

• Y. Liang et al., Submitted, 2009

17

T i FL N/35 Mi Transgenic FL-N/35 Mice

Tg-FLN/35 NS3 Non-Tg Lerat et al., Gastroenterology 122: 352-365, 2002

18

Centrilobular Hepatic Steatosis

FL-N/35 Lineage, 11-12 months old Normal

19

H ti T i FL N/35 HCV T i M Hepatic Tumor in an FL-N/35 HCV Transgenic Mouse

(FL-N/35-171, 13 mo Male)

Lerat et al., Gastroenterology 122: 352-365, 2002

20

Hepatic Adenoma and Hepatocellular Carcinomas in HCV Transgenic FL-N/35 Mice in HCV Transgenic FL N/35 Mice

Lerat et al., Gastroenterology 122: 352-365, 2002

BrDU Staining for DNA Synthesis

FL N/35 Lineage FL-N/35 Lineage

Lerat et al., Gastroenterology 122: 352-365, 2002

22

Host Factors in HCV Replication Host Factors in HCV Replication and Pathogenesis

STAT1, gC1qR hnRNP K p53 DDX3 TRIF p53 Bin1 Src kinases Grb2 LT-βR TNFR-1 CD81 PKR IPS1/MAVS p53 2’,5’OAS PKR pRb DDX5

4A

5’ E1 NS2

p 7

E2 NS3 NS4B NS5A NS5B 3’ C

4 A

CD81 SR-B1 miR-122 eIF3 Hsp90 FBL2 VAP-A,B VAP-A,B CypA,B PTB La

7 A

SR B1 (Claudin-1) (Occludin) L-SIGN DC-SIGN eIF3 PTB La PCBP-1,2 VAP A,B TBC1D20 CypA,B PRK2 ESR2 La PCBP-1,2 hnRNP C HuR

23

Host Factors in HCV Replication Host Factors in HCV Replication and Pathogenesis

STAT1, gC1qR hnRNP K p53 DDX3 TRIF p53 Bin1 Src kinases Grb2 Tumor Suppressors LT-βR TNFR-1 CD81 PKR IPS1/MAVS p53 2’,5’OAS PKR pRb DDX5

4A

5’ E1 NS2

p 7

E2 NS3 NS4B NS5A NS5B 3’ C

4 A

pp CD81 SR-B1 miR-122 eIF3 Hsp90 FBL2 VAP-A,B VAP-A,B CypA,B PTB La

7 A

SR B1 (Claudin-1) (Occludin) L-SIGN DC-SIGN eIF3 PTB La PCBP-1,2 VAP A,B TBC1D20 CypA,B PRK2 ESR2 La PCBP-1,2 hnRNP C HuR

24

Host Factors in HCV Replication Host Factors in HCV Replication and Pathogenesis

STAT1, gC1qR hnRNP K p53 DDX3 TRIF p53 Bin1 Src kinases Grb2 LT-βR TNFR-1 CD81 PKR IPS1/MAVS p53 2’,5’OAS PKR pRb DDX5

4A

5’ E1 NS2

p 7

E2 NS3 NS4B NS5A NS5B 3’ C

4 A

CD81 SR-B1 miR-122 eIF3 Hsp90 FBL2 VAP-A,B VAP-A,B CypA,B PTB La

7 A

SR B1 (Claudin-1) (Occludin) L-SIGN DC-SIGN eIF3 PTB La PCBP-1,2 VAP A,B TBC1D20 CypA,B PRK2 ESR2 La PCBP-1,2 hnRNP C HuR

25

NS5B Regulation of pRb g p

Host genome instability NS5B

Rb

UbUbUb…

instability NS5B

E6AP

Hepatocellular proliferation p p

Modifed from Classon and Harlow, Nature Rev Cancer 2: 910, 2002

26

pRb Abundance is Post-Transcriptionally Down-Regulated in HCV RNA Replicon Cells Down-Regulated in HCV RNA Replicon Cells

4A

N Neo ΔC 5’ E1 NS2

p

E2 NS3 NS4B NS5A NS5B 3’ EMCV IRES C

4A

Neo Neo 5’ E1 NS2

7

E2 NS3 NS4B NS5A NS5B 3’ C

Northern Immunoblot Pulse-chase

Munakata et al., PNAS 102:18159-64, 2005

27

pRb Abundance is Reduced by Genotype 2a JFH1 Virus Infection of Huh-7.5 Cells

120 96 72 48 24 0 Hrs

• = infection

= harvest

NS5B pRb 0 48 72 96 120 Hrs

60 80 100

nt Relative bundance

60 80 100

nt Relative bundance

GAPDH

1 2 3 4 5 20 40 48 72 96 120 Hours Post-inoculation

Percen pRb Ab

20 40 48 72 96 120 Hours Post-inoculation

Percen pRb Ab

Hours Post inoculation Hours Post inoculation

Munakata et al., PLoS Pathogens, 3(9): e139, 2007

28

NS5B Forms a Complex With pRb

ured) plicon) 2-3c (Cu 2-3 (Rep Rb Ig HC pRb Ig HC IB:α-pRb IP:α-pRb )

• n)

g NS5B IB: α-NS5B g IB: α-NS5B IB: α-NS5B IP:α-pRb

• 3c (Cured
• 3 (Replico

IB: α-Core Core IP:α-pRb IB: α-Core Core Core IB: α-Core IB: α-Core 2- 2-

Munakata et al., PNAS 102:18159-64, 2005

1 2 3 4 1 2 3 4

29

Cytoplasmic Colocalization of pRb and NS5A (replicase) in Huh 7 5 cells 48 hrs Post infection with JFH1 Virus in Huh-7.5 cells 48 hrs Post-infection with JFH1 Virus

Rb DAPI NS5A NS5A+Rb+DAPI

Munakata et al., PLoS Pathogens, 3(9): e139, 2007

30

HCV Regulation of pRb in Human Liver

pRb

Normal liver Chronic Hepatitis C Chronic Hepatitis C

• Y. Liang

31

Epoxomicin, a Potent Inhibitor of the Proteasome, Restores pRb and PO4-pRb Abundance

pRb Phospho- pRb nM EPX EPX 250 nM + + Ubi-pRb 2-3 GAPDH NS5B pRb 50 500 pRb NS5B Phospho- pRb 2-3C GAPDH NS5B pRb GAPDH

1 2 3 4

GAPDH

1 2 3 Munakata et al., PLoS Pathogens, 3(9): e139, 2007

32

HCV Infection Induces Ubiquitination of pRb HCV Infection Induces Ubiquitination of pRb in Cultured Hepatoma Cells

kD 250

EPX 250nM JFH1 HCV + + + +

kD 250

EPX 250nM JFH1 HCV + + + +

75 105 160 kD 250

IP: pRb IB: Ubi Ubi-pRb

75 105 160 kD 250

IP: pRb IB: Ubi Ubi-pRb

50

Ig HC

50

Ig HC IP: pRb IB: pRb Ig HC pRb IP: pRb IB: pRb Ig HC pRb

1 2 3 4 1 2 3 4

Munakata et al., PLoS Pathogens, 3(9): e139, 2007

33

siRNA Knock-down of E6AP Restores pRb Abundance in Replicon Cells

2-3 (Replicon) 2-3c (Cured)

P siRNA DD4 siRNA M2 siRNA trol siRNA #2 trol siRNA #1 P siRNA DD4 siRNA M2 siRNA trol siRNA #2 trol siRNA #1

pRb

E6A NED MDM Cont Cont E6A NED MDM Cont Cont 100 kDa

E6AP NEDD4

150 100

Actin MDM2

75 37

1 2 3 4 5 6 7 8 9 10

37 Munakata et al., PLoS Pathogens, 3(9): e139, 2007

34

Over expression of Dominant negative E6AP C840A Over-expression of Dominant-negative E6AP-C840A Partially Restores pRb Abundance in 2-3 Cells

2-3c (Cured) 2-3 (Rep) Vector

+ +

3

nce 2-3

IB: pRb

Vector E6AP E6AP-C840A

+ + + + + +

2

b Abunda

IB: E6AP IB: pRb IB A ti

1

lative pRb

1 2 3 4 5 6

IB: Actin

Rel

Munakata et al., PLoS Pathogens, 3(9): e139, 2007

35

E6AP-pRb Complex p p in Cells Ectopically Expressing wt NS5B

N tor /4A B A B wt 8N/D319N tor /4A B A B wt 8N/D319N E6AP Vect NS3/ NS4B NS5A NS5B D318 IB: E6AP Vect NS3/ NS4B NS5A NS5B D318 IB: pRb E6AP IP: pRb pRb E6AP IP: pRb Input

1 2 3 4 5 6

pRb E6AP Input

1 2 3 4 5 6

pRb E6AP

1 2 3 4 5 6 1 2 3 4 5 6

Munakata et al., PLoS Pathogens, 3(9): e139, 2007

36

2b AY232746 MD2b9-1 2b AY232732 MD2b2-1 2b AB030907 JPUT 2b AY232730 MD2b1-1 2b AY232742 MD2b7-1 2b D10988 J8 2b AY232736 MD2b4-1 2b AY232748 MD2b10-1 2b AY232740 MD2b6-1 2b AY232744 MD2b8-1 2b AF238486 MD2b-1 2b AY232734 MD2b3-1 2b AY232738 MD2b5-1 2c D50409 BEBE1 2k AB031663 VAT96 2a AB047639 JFH1 2a AB047643 JCH4 2a AB047641 JCH2 2 D00944 J6

JFH1

2b

2a D00944 J6 2a AB047642 JCH3 2a AF238483 MD2a-4 2a AF169002 NDM228 2a AB047640 JCH1 2a AF238485 MD2a-7 2a AF238481 MD2a-1 2a AF238482 MD2a-2 2a AB047645 JCH6 2a AB047644 JCH5 2a AF169005 NDM59 2a AF169003 G2aK1 2a AF238484 MD2a-5 2a AF169004 G2aK3 6a Y12083 HK2 6b D84262 Th580 11a D63822 JK046 7a D84263 VN235 8a D84264 VN405 9a D84265 VN004 5a Y13184 H1480

2a

5a Y13184 H1480 5a AF064490 SA13 10a D63821 JK049 3b D49374 Tr 3a D28917 K3a650 3a X76918 3a AF046866 CB 3a D17763 NZL1 4a Y11604 ED43 1c D14853 G9 1c AY051292 India 1a AJ278830 British 1a AF511950 1a AF511949 1a AF511948 1a D10749 J1 1a AF009606 H77Rice INF 1a AF271632 HCV1Lanford INF 1b AF356827 S1 1bN AF483269 TR1 1b AJ000009 A 1bN AF176573 274933RU <<<<<<<<< One 2736N outlier

1a

One 316N Outlier

Phylogenetic analysis of 82 full-length HCV genomes ( )

1bN AF176573 274933RU 1b AJ132997 AD78P1 1b U45476 HD1 1b restrict LB Feinstone 1b D50485 IFNS2 1b AF207760 1b AF207761 1b AJ238799 Con1 1b AF313916 Ireland1977 1b D89815 NIHJ1 1b D30613 PP 1b AB049090 T140 1b AF207773 1b AF207764 1b AB049095 T169 1b U89019 1b AF165063 MD10-1 1b X61596 JK1 1b AB049096 T191 1b AB049094 T161 1b AF207771

<<<<<<<<< One 2736N outlier

Con-1

1b

One 316N Outlier

(nucleotide sequences)

D C T M L V

HCV-N N316 Con1 C316

D C T M L V N G D D L C G D D L 316

1b AF207771 1b AF207757 1b AF165045 MD1-1 1b AF165053 MD5-1 1b AF165055 MD6-1 1b AB016785 UTR342 1b AF165047 MD2-1 1b AF207768 1b AB049092 T145 1b AB049087 T050 1b D50484 IFNS3 1b AB049089 T109 1b M84754 Taiwan 1b AB049101 T221 1b D50483 IFNS1 1b AF207766 1b AF08024 MD34 1bN U16362 1bN U01214 L2 1bN AF207769 1bN AF207772 1bN AY460204 HCV S 1bN AY460204 HCV-S 1bN AB049093 T150 1bN AF207758 1bN D13558 J483 1bN D11168 JT 1bN D90208 J 1bN D89872 TMORF 1bN D14484 J33 1bN AB049099 T212 1bN D63857 HCVNprime 1bN L02836 HeBei 1bN M58335 BK 1bN AF207774 1bN AF139594 NLemon INF 1bN AB049098 T209 1bN D10934 C2 1bN AF207754 1bN AB049091 T142 1bN AB049088 T094 1bN AF207753 1bN AB049097 T197

Bootstrap support = 74%

HCV-N

316N Clade

0.1

1bN AF207756 1bN AF207763 1bN AF207767 1bN AF165059 MD8-1 1bN AB049100 T217 1bN D85516 JS 1bN AB080299 1bN AF165051 MD4-1 1bN AF207770 1bN AF207752 1bN AF207759 1bN AF165057 MD7-1 1bN AF165049 MD3-1 1bN AF207765 1bN AF207762 1bN AF165061 MD9-1 1bN AY045702 HCR6

• S. Ray, Johns Hopkins Univ.

37

The NS5B LxCxE-homology Domain is Required The NS5B LxCxE-homology Domain is Required for NS5B Binding to pRb

LxCxE LxCxE

Vector

+

• +
• GST

GST-Rb (301-928) 10% Vector

+

• +
• GST

GST-Rb (301-928) Vector

+

• +
• GST

Vector

+

• +
• GST

GST-Rb (301-928) 10%

Flag-NS5B Mutants NS5B-Flag Pull-Down of GST-pRb(301-928)

D C T M L V N G D D L D C T M L V N G N N L D C T M L V C G D D L D C T M L V A G D D L D C T M A V A G D D L D C T M L V N G D D L D C T M L V N G N N L D C T M L V C G D D L D C T M L V A G D D L D C T M A V A G D D L

wt Vector 316C (Con1 wt) L314A/N316A D318N/D319N N316A

• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• GST

GST-Rb 0% Input wt Vector L314A/N316A D318N/D319N N316A

• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• wt

Vector L314A/N316A D318N/D319N N316A

• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• 316N (HCV-N wt)

Vector L314A/N316A D318N/D319N N316A

• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• GST

GST-Rb 0% Input

D C T M A V A G D D L D C T M A V A G D D L

L314A/N316A

• +
• +

G G GST-Rb (301-928) Ig HC IP: α-Flag IB: α-GST L314A/N316A

• +
• +

L314A/N316A

• +
• +

L314A/N316A

• +
• +

G G GST-Rb (301-928) Ig HC IP: α-Flag IB: α-GST

1 3 4 5 6 7 8 9 10 11 12 13 14 2

GST-Rb (301-928) IP: α-Flag IB: α-Rb

1 3 4 5 6 7 8 9 10 11 12 13 14 2

GST-Rb (301-928) IP: α-Flag IB: α-Rb

Munakata et al., PNAS 102:18159-64, 2005

38

The NS5B LxCxE-homology Domain is Required The NS5B LxCxE-homology Domain is Required for NS5B Binding to pRb

LxCxE LxCxE

Vector

+

• +
• GST

GST-Rb (301-928) 10% Vector

+

• +
• GST

GST-Rb (301-928) Vector

+

• +
• GST

Vector

+

• +
• GST

GST-Rb (301-928) 10%

Flag-NS5B Mutants NS5B-Flag Pull-Down of GST-pRb(301-928)

D C T M L V N G D D L D C T M L V N G N N L D C T M L V C G D D L D C T M L V A G D D L D C T M A V A G D D L D C T M L V N G D D L D C T M L V N G N N L D C T M L V C G D D L D C T M L V A G D D L D C T M A V A G D D L

wt Vector L314A/N316A D318N/D319N N316A

• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• GST

GST-Rb 0% Input wt Vector 316C (Con1 wt) L314A/N316A D318N/D319N N316A

• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• wt

Vector L314A/N316A D318N/D319N N316A

• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• 316N (HCV-N wt)

Vector L314A/N316A D318N/D319N N316A

• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• GST

GST-Rb 0% Input

D C T M A V A G D D L D C T M A V A G D D L

L314A/N316A

• +
• +

G G GST-Rb (301-928) Ig HC IP: α-Flag IB: α-GST L314A/N316A

• +
• +

L314A/N316A

• +
• +

L314A/N316A

• +
• +

G G GST-Rb (301-928) Ig HC IP: α-Flag IB: α-GST

1 3 4 5 6 7 8 9 10 11 12 13 14 2

GST-Rb (301-928) IP: α-Flag IB: α-Rb

1 3 4 5 6 7 8 9 10 11 12 13 14 2

GST-Rb (301-928) IP: α-Flag IB: α-Rb

Munakata et al., PNAS 102:18159-64, 2005

39

The NS5B LxCxE-homology Domain is Required The NS5B LxCxE-homology Domain is Required for NS5B Binding to pRb

LxCxE LxCxE

Vector

+

• +
• GST

GST-Rb (301-928) 10% Vector

+

• +
• GST

GST-Rb (301-928) Vector

+

• +
• GST

Vector

+

• +
• GST

GST-Rb (301-928) 10%

Flag-NS5B Mutants NS5B-Flag Pull-Down of GST-pRb(301-928)

D C T M L V N G D D L D C T M L V N G N N L D C T M L V C G D D L D C T M L V A G D D L D C T M A V A G D D L D C T M L V N G D D L D C T M L V N G N N L D C T M L V C G D D L D C T M L V A G D D L D C T M A V A G D D L

wt Vector 316C (Con1 wt) L314A/N316A D318N/D319N N316A

• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• GST

GST-Rb 0% Input wt Vector L314A/N316A D318N/D319N N316A

• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• wt

Vector L314A/N316A D318N/D319N N316A

• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• 316N (HCV-N wt)

Vector L314A/N316A D318N/D319N N316A

• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• +
• GST

GST-Rb 0% Input

D C T M A V A G D D L D C T M A V A G D D L

L314A/N316A

• +
• +

G G GST-Rb (301-928) Ig HC IP: α-Flag IB: α-GST L314A/N316A

• +
• +

L314A/N316A

• +
• +

L314A/N316A

• +
• +

G G GST-Rb (301-928) Ig HC IP: α-Flag IB: α-GST

1 3 4 5 6 7 8 9 10 11 12 13 14 2

GST-Rb (301-928) IP: α-Flag IB: α-Rb

1 3 4 5 6 7 8 9 10 11 12 13 14 2

GST-Rb (301-928) IP: α-Flag IB: α-Rb

Munakata et al., PNAS 102:18159-64, 2005

40

Surface Plasmon Resonance Measurements Demonstrate NS5B Interacts Physically with pRb Demonstrate NS5B Interacts Physically with pRb in an LxCxE-motif Dependent Manner

GST-NS5BD318ND319N GST-NS5B GST-NS5BD318ND319N GST-NS5B GST-NS5BD318ND319N GST-NS5B

GST-NS5b Rb CM-5

1670 936 1240 624 nM nM 1670 936 1240 624 nM nM 1670 936 1240 624 nM nM 1670 936 1240 624 nM nM 1670 936 1240 624 nM nM 832 520 416 312 312 208 104 52 26

RU

832 520 416 312 312 208 104 52 26

RU

832 520 416 312 312 208 104 52 26

RU

832 520 416 312 312 208 104 52 26 832 520 416 312 312 208 104 52 26

RU

104 52 26

Ti

CM5 Chip: 95nM Rb

104 52 26

Ti

104 52 26

Ti

104 52 26 104 52 26

Ti

CM5 Chip: 95nM Rb

Time Time Time Time

• Y. Liang

41

HJ3/5 Chimeric HCVs With NS5B LxCxD D i M t ti A Vi bl B t H di d Domain Mutations Are Viable But Handicapped

p 4

H77c JFH1

4A

5’ E1 NS2

p 7

E2 NS3 NS4B NS5A NS5B 3’ C

4 A

316

• Leu●Val●Cys●Gly●Asp●Asp●Leu●
• Leu●Val●Cys●Gly●Asp●Asp●Leu●

L x C x E D.R. McGivern et al., J. Virol. In press, 2009

42

Polymerase Activities of wt vs C316A NS5B Polymerase Activities of wt vs. C316A NS5B

• E. coli-expressed

RdRp P16 post-nuclear membrane fractions RdRp membrane fractions

D.R. McGivern et al., J. Virol. In press, 2009

pRb Expression in Cells Infected with HJ3/5 vs. HJ3/5-C316A Viruses

NS5A

Mock HJ3/5 HJ3/5-NS5B/C316A

HJ3/5 vs. HJ3/5 C316A Viruses

pRb

D.R. McGivern et al., J. Virol. In press, 2009

44

Passage of L314A led to Emergence of L314V Passage of L314A led to Emergence of L314V which Restored Replication Capacity

105 104 104 103

U/ml

102

FF

wt L314A L314V C316A D318N

10

wt L314A L314V C316A D318N

D.R. McGivern et al., J. Virol. In press, 2009

The L314V Revertant Regulates Rb

wt L314V L314A DAPI Rb HCV Rb/HCV

D.R. McGivern et al., J. Virol. In press, 2009

46

D NS B R l i f Rb A Does NS5B Regulation of pRb Attenuate pRb Repression of E2F Transcription Factors?

Rb

NS5B

?

Modified from Classon and Harlow, Nature Rev Cancer 2: 910-917, 2002

47

NS5B Activates E2F-Responsive Promoters p in an LxCxE Domain-Dependent Fashion

Ectopic NS5B Expression Mad2 Transcripts in HCV Replicon Cells Ectopic NS5B Expression in Huh7 Cells

Munakata et al., PNAS 102:18159-64, 2005 Lauren Evers, David McGivern

48

Viral Proteins That Bind pRb and Suppress pRb Function

LxCxE E7 HPV AdV …LxCxE… E1A T

Chromatin Remodeling Complexes

AdV SV-40 Rb EBNA3C EBV NS5B HCV

Modified from Classon and Harlow, Nature Rev Cancer 2: 910-917, 2002

49

Acute pRb Loss De-Regulates Many Genes

MP Markey et al., Oncogene 10.1038/sj.onc.1210450

50

HCV Interactions withTumor Suppressors and HCC

D.R. McGivern & S,M. Lemon, Annu Rev Pathol Mech Dis 4:399-415, 2008

51

Potential Protective Role of NS5B in c-Myc Malignant Conversion of Dysplastic Nodules to Early HCC y p y

NS5B

Rb E2F Cirrhotic Low Grade High Grade Early Apoptosis Cirrhotic Nodule Dysplastic Nodule High Grade Dysplastic Nodule Hepatocellular Carcinoma

• Accumulating genetic and epigenetic modifications
• Increasing c-Myc copy number with 8q duplications

Kaposi-Novak et al. Cancer Res, 69:2775-2782, 2009

52

Acknowledgements

Lemon Lab: University of Texas Medical Branch

David R. McGivern, Yuqiong Liang, Rodrigo Villanueva, Seungtaek Kim, MinKyung Yi F l b b F k S h ll M i Ik d Former lab members: Frank Scholle, Masanori Ikeda Bob Fox Lab UTMB Center for Biomedical Engineering: Gracie Vargas, Tuya Shilagard UTMB O ti l I i C E K t Th Alb ht UTMB Optical Imaging Core: Eugene Knutsen, Thomas Albrecht

Uni ersit

• f Tok o

University of Texas at Austin Texas A&M University

University of Tokyo

Tsubasa Munakata, Akio Nomoto University of Texas at Austin

Jon Huibregtse

Texas A&M University

Cheng Kao, Sreedhar Chinnaswamy

Penn State University

Craig Cameron

IRBM Merck

Raffaele de Francesco

Johns Hopkins University

Stuart Ray

Supported by grants U19-AI40035 and N01-AI25488 from the National Institute of Allergy and Infectious Diseases, NIH D.M. was a Kempner Postdoctoral Scholar

Craig Cameron Raffaele de Francesco y

53

Putative HCV Interactions with p53

54

D.R. McGivern & S,M. Lemon, Annu Rev Pathol Mech Dis 4:399-415, 2008