The mechanism of HCV extrahepatic infection Sept. 21, 2012 at Wuhan Michael M.C. Lai Institute of...
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Transcript of The mechanism of HCV extrahepatic infection Sept. 21, 2012 at Wuhan Michael M.C. Lai Institute of...
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)
3
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
18
Extrahepatic infection, particularly Extrahepatic infection, particularly
PBMC infection, plays an important PBMC infection, plays an important
role in HCV pathogenesisrole in HCV pathogenesis
19
AcknowledgmentAcknowledgment• Keigo Machida
o University of Southern California
• Chao Kuen Lai• King-Song Jeng
o Academia Sinica, taiwan
20
Thanks for your Thanks for your attention. attention.
21