Hepatitis Viruses and

1
Hepatitis Viruses and Hepatocellular Carcinoma
(HCC)
2
Hepatitis Viruses
Blood borne pathogens
Hepatitis B virus (HBV)
Hepatitis C virus (HCV)
Hepatitis D virus (delta) - viriod-like and
requires HBV as a helper
Hepatitis G virus
Faecal-oral pathogens
Hepatitis A virus
Hepatitis E virus
3
Etiology of HCC (2001)
Other 22
HBV 53
HCV 25
HCC is the third leading cause of cancer death
HCC often unresponsive to treatment
4

• gt250 million people worldwide are persistently
  infected with HBV

• Vaccination has been effective in decreasing the
  incidence of
• HBV infection where implemented

5
Global prevalence of HCV infection
170 million of the worlds population is
infected
6
Progression to HCC in HBV- HCV- infected
individuals
HBV
10 (adults) 40-80 (newborn)
Minimal liver disease
Chronic infection
70-80
Chronic active hepatitis
HCV
Acute infection
Cirrhosis
gt20-30 yr
HCC (1-6/yr 30-50/10 yr)
7
Normal Liver Architecture

• Most hepatocytes in a normal liver are in a
  quiescent state

8
Hepatitis-induced liver injury
Infection
Inflammation
Fibrosis
Cirrhosis

• HBV and HCV are non-cytopathic

• Hepatocyte damage is immune-mediated

• Liver injury promotes hepatocyte regeneration

9
Pathology of chronic hepatitis and HCC
Prominent necrosis inflammation
Cirrhosis
HCC
10
Hepatitis-associated oncogenesis

• Continual liver damage and cirrhosis are
  predisposing factors

• All HCC cases occur after many years of chronic
  hepatitis

• Viral agent provokes liver injury ---gt Growth
  deregulating
• mutations responsible for tumor formation
  come from the host

• Viral genome directly contributes to the lesion
• - cis-acting sequences that activate or
  suppress host growth
• regulating genes
• - Trans-acting factors alter the program
  of cellular growth control

11
HBV particles

• Non-infectious 20 nm spheres
• and filaments

• Infectious 42-47 nm Dane particle

HBsAg
polymerase
genome
HBcAg
Titers lt104/ml to gt109/ml
12
HBV Genome
13
Hepadnaviral Life Cycle
RNA pol II
14
Defining the mechanism(s) of HBV-mediated HCC is
challenging

• Narrow host range of HBV

• Natural animal models of viral infection
• - Wood chuck hepatitis virus (WHV)
• - Duck hepatitis virus
• - Ground squirrel hepatitis

• Woodchucks infected with WHV develop HCC

• Transgenic mouse models for liver disease and
  HCC progression

15
Immune injury is sufficient to drive hepatic
oncogenesis
Transgenic mice expressing HBsAg in hepatocytes
Adoptive transfer of sensitized CTLs
from HBsAg-immunized syngeneic mice
Liver injury chronic hepatitis HCC
16
Genome integration and WHV- associated HCC

• gt40 of all HCCs contain an integrated
• WHV genome in close proximity to
• cellular N-myc

• Insertional activation
• - N-myc genes are transcriptionally
• activated by an integration event

17
Genome integration and HBV- associated HCC

• gt85 of HBV tumors harbor integrated viral DNA

• Tumors are clonal with respect to their
  integrants
• ----gt Lead to growth changes due to
  deletions of flanking DNA

• No common integration sites in human HCC

• Sites of integration in HBV DNA appear
  restricted
• - Occurs within one or two of the direct repeat
  sequences
• in HBV DNA
• - DNA fragments from X and S ORFs commonly
  integrated

• In advanced tumors most viral gene expression is
  ablated

18
The X protein of HBV (HBx)

• Predominantly cytoplasmic protein (17 kD)

• Essential for viral infectivity

• Poorly immunogenic short half life

• HBx-expressing transgenic mice can develop HCC
• - highly dependent on HBx expression levels and
  mouse lineage

• HBx transforms cells in culture

• HBx transcripts and HBx expression detected in
  malignant cells

• Promiscuous transcriptional activator (HBV and
  cellular)

19
HBx mechanism of transcriptional
transactivation?

• HBx does not bind DNA directly

• HBx may interact directly with transcriptional
  factors in the nucleus?

• Cytoplasmic localization of X is consistent with
  stimulation of
• transcription indirectly by activating
  cellular signaling pathways

20
HBx activates the Ras-Raf-MAPK cascade Influence
on cell proliferation?

• Unlike the magnitude of Ras activation by
• growth factors, HBx stimulation is modest
• but sustained

• Downstream signaling activate transcription
• factors (eg. AP-1, NF-KB and c-myc)

• Activation of the Ras-Raf-MAPK signaling
• pathway is essential for HBx activation
• of AP-1 and NF-KB

• HBx-mediated activation of the
• Ras-Raf-MAPK pathway has been linked
• to accelerated entry of cells into S phase

21
Influence cellular DNA repair?
Functional inactivation - p53?
Activation of transcriptional factors - c-myc -
AP-1 - NF-KB
Impaired apoptosis
Genetic instability
Defective cell cycle
PROMOTION OF ONCOGENESIS
22
Hepatitis C virus

• Genome cloned in 1989

• Member of the Flaviviridae family

• Leading cause of liver transplantation in the US

• Sustained responses in only 50 of treated
  patients

• No vaccine available

• High sequence divergence - 6 major genotypes

23
HCV genome organization

• 9.6 kb postive strand RNA genome

• Encodes a single ORF 3010-3033 aa

Virion components
RNA replication machinery
structural
nonstructural
3
5
(U/UC)
NS3
C
E1
E2
NS2
4B
5A
5B
4A
p7

Signal peptidase
NS3-4A Ser protease
24
HCV replicates exclusively in the cytoplasm via
an RNA intermediate
Viral entry uncoating
()
Translation processing
()
(-)
HCV RNA replication
Virus particle assembly
Replicative intermediate
()
Nucleus
25
In vivo replication and experimental systems

• Difficult to detect HCV RNA and protein in
  HCV-infected
• liver tissue

• Mathematical Models (measure viral production
  during therapy)
• - Virion half-life 3 hr
• - 1012 virions produced per day

• Chimpanzees is the only animal permissive for
  HCV infection
• ---gt Minimal liver disease develops

• No infection system in tissue culture cells

26
Models for HCV-associated HCC

• Indirect model - HCV infection initiates liver
  damage and
• hepatocyte
  regeneration?

• No genome integration

• Direct action of viral protein products on
  cellular growth control?

• Oxidative stress induced by viral protein
  expression?

27
HCV gene products implicated in oncogenesis
Core
NS3
NS5A
Nucleocapsid protein
Protease/helicase
?
Viral
Interacts and activates p53
Interacts with p53
Host
Modulates cell growth
Weak transforming potential
Transforms NIH3T3 cells
Transforms cells
Modulates apoptosis
Inhibits apoptosis
Suppresses apoptosis
Induces HCC in some transgenic mice
28
Oxidative stress, steatosis and HCC
Mitochondrial injury
29
HCV transgenic mice develop HCC
Essential cis-acting RNA sequences missing -- NO
HCV replication
30
Liver pathology in HCV transgenic mice
Steatosis Transgenic - 13 months
Non-transgenic - 12 months
Well-differentiated HCC Transgenic - 18 months
Steatosis Transgenic - 13 months
31
Fig. 1. Kinetics of serum HBsAg (A) and ALT (B)
in HBV transgenic mice.
32
Fig. 2. Histopathological features of prolonged
chronic immune-mediated hepatitis in HBV
transgenic mice
33
Table 1. Biochemical, Histological, and
Immunological features of HBV transgenic mice as
a consequence of chronic immune-mediated
hepatitis.
1
2
3
34
Fig. 3. Intrahepatic T cell and cytokine profiles
in HBV transgenic mice.
35
Fig. 4.  (A) In vitro cytotoxic activity of
splenocytes from group 1, 2, and 3 mice at the
time of autopsy. (B) Characteristics of in
vitro cytotoxic activity of splenocytes from
group 1 mice and HBs-vac-primed nontransgenic
mice.