The mechanism of HCV The mechanism of HCV extrahepatic infectionextrahepatic infection

Sept. 21, 2012 at WuhanSept. 21, 2012 at Wuhan

Michael M.C. LaiMichael M.C. LaiInstitute of Molecular BiologyInstitute of Molecular Biology

Academia SinicaAcademia Sinica

Clinical manifestations of HCVClinical manifestations of HCV

Liver: Asymptomatic or acute hepatitis chronic hepatitis liver cirrhosis hepatocellular carcinoma

B cells: Mixed cryoglobulinemia (B-cell oligoclonal proliferation) non-Hodgkin’s B-cell lymphoma

Other extrahepatic manifestations:

Sjogren’s SyndromePseudomembranous glomerulonephritisPurpura, Diabetes mellitus, thyroid diseaseLichen planus

Evidence that PBMC infection by HCV is Evidence that PBMC infection by HCV is

causally linked to B cell lymphomacausally linked to B cell lymphoma• High frequency of HCV infection (20-40%) among B-

cell lymphoma patients in some geographical regions

• HCV-transgenic mice develop B-cell lymphoma (Kasama et al. 2011)

• Antiviral therapy (interferon plus ribavirin) of HCV(+) lymphoma patients can induce tumor remission (Hermine et al., 2002: Levine et al., 2003)

• A lymphotropic HCV (SB virus) was isolated from a B-cell lymphoma (Sung et al., 2003)

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A lymphotropic HCV (HCV-SB) isolated from a B-cell A lymphotropic HCV (HCV-SB) isolated from a B-cell lymphomalymphoma

• Isolated from the spleen of an HCV-positive patient with B-cell lymphoma and mixed cryoglobulinemia

• HCV-SB virus particles can infect B cells and T cells but not hepatocytes.

• HCV-SB has typical characteristics of hepatotropic HCV strains

Candidate HCV receptorsCandidate HCV receptors

• CD81 o ubiquitous, initial virus binding

• SR-B1 (scavenger receptor B1)o Hepatocyte, dendritic cells

• LDLR o ubiquitous

• L-SIGN, DC-SIGNo LSEC, Kupffer cells

• Glycosaminoglycans (heparan sulfate)

• Claudin-1, Occludin (entry molecules) o tight junction molecules (liver, epithelial cells)

Hecker, E. & Ott, M. 2011. Ubique ties between hepatitis C virus replication and intracellular lipids. Cell. 22(6).243

Molecular mechanism of viral lympho- vs

hepatotropism

Harvest

Detection (qRT-PCR)

T7 in vitro transcripts

Electroporation into Huh7.5 and Raji

Cloned cDNA

SB virus RNA, but not JFH-1 Virus, Replicates in Raji Cells

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

1.0E+09

4 8 12 16 20 24 28 42 56 80 96

Day

HCV RNA (copies/ug RNA)

JFH-1

JFH-1 GND

1b

SB

SB GND

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

1.0E+09

4 8 12 16 20 24 28 42 56 80 96

Day

HCV RNA (copies/ug RNA)

JFH-1

JFH-1 GND

1b

SB

SB GND

Raji

JFH-1 Virus, but not SB Virus RNA, Replicates in Huh7.5 CellsJFH-1 Virus, but not SB Virus RNA, Replicates in Huh7.5 Cells

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

1.0E+09

4 8 12 16 20 24 28 42 56 80 96

Day

HC

V R

NA

(co

pie

s/u

g R

NA

) JFH-1 JFH-1-GND 1b

SB SB GND

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

1.0E+09

4 8 12 16 20 24 28 42 56 80 96

Day

HCV RNA (copies/ug RNA)

JFH-1

JFH-1-GND

1b

SB

SB GND Huh7.5

•SB virus is lymphotropic. JFH1 virus is hepatotropic.

0 20000 40000

SB

SB GND

JFH-1

JFH-1 GND

SB (JFH1 5UTR-NS2)

JFH1 (SB 5UTR-NS2)

SB (JFH1 5UTR-E2)

JFH1 (SB 5UTR-E2)

SB (JFH1 5UTR-Core)

JFH1 (SB 5UTR-Core)

SB (JFH1 5'UTR)

JFH1 (SB 5'UTR)

SB (JFH1 E1+E2)

JFH1 (SB E1+E2)

SB (JFH1 E2)

JFH1 (SB E2)

HCV RNA (copies/mg of RNA)

Chimera Virus containing E1-E2 of SB Strain Produces High Levels of HCV RNA in Raji Cells

150000

HCV Pseudoparticle System (HCVpp)A retrovirus reporter virus with HCV envelope proteins

gag-pol (retrovirus)

GFP or Luciferase

HCV E1/E2

Study virus entry, receptors, host range, etc.

Infection of target cells

HCVpp entryGFP or luciferase

expression

Raji or Huh7.5

Infectivity of HCV Pseudo-particles is Determined by E1-E2 of SB Strain in Raji Cells

Normalized for p24 HIV core antigen FACS for GFP reporter with HCVpp at 72 h p.i

Huh7.5

Raji

1/10

0

1/10

0

Anti-CD81 and IFN Treatment Inhibit HCV Anti-CD81 and IFN Treatment Inhibit HCV

ReplicationReplication

Consequences of HCV infection of B cellsConsequences of HCV infection of B cells

• Inhibition of DNA damage repair: chromosomal translocation

• Uncoupling of mitotic checkpoint: chromosomal polyploidy

• Induction of error-prone DNA polymerases: hypermutation of immunoglobulin and protooncogenes

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Mutation frequencies of p53 and VH

in HCV-infected human B cell lines

Locus

p53

VH

HCVinfection

-++

-++

iNOSinhibitor1400W

--+

--+

% clonesmutated

0 (0/18)15 (3/20) 0 (0/20)

15 (3/20)55 (11/20)35 (7/20)

Mutationfrequency

X10-4

07.25

0

4.120.39.6

Higher mutation frequency in oncogenes in HCV-Higher mutation frequency in oncogenes in HCV-

associated B cell lymphoma and HCCassociated B cell lymphoma and HCC

B celllymphomas

Hepatocellularcarcinomas(HCC)

30 3.9

35 5.9

3.90

2.5

HCV-associatedNon-HCV

HCV tumor nontumor

HBV tumor nontumor

Nonviral tissues

Mutation frequency(mutation/bp) x10-4

p53 -catenin

33 0

30 11

7.4 7.4

4.4

HCV-SB also infects T cells and suppresses interferon HCV-SB also infects T cells and suppresses interferon

signaling signaling

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Extrahepatic infection, particularly Extrahepatic infection, particularly

PBMC infection, plays an important PBMC infection, plays an important

role in HCV pathogenesisrole in HCV pathogenesis

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AcknowledgmentAcknowledgment• Keigo Machida

o University of Southern California

• Chao Kuen Lai• King-Song Jeng

o Academia Sinica, taiwan

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Thanks for your Thanks for your attention. attention.

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