Hepatitis C Virus
Casey McGrath
BIO 360
Outline
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Epidemiology
Introduction to Hepatitis C Virus
Immune response
Novel drug therapies
Conclusions
Epidemiology
Hepatitis C Virus (HCV):
• ~170 million people worldwide
• Chronic hepatitis, liver cirrhosis,
hepatocellular carcinoma (HCC)
• Transmitted via blood-transfusions, intravenous drug use
Prevalence of HCV by world region
HEPATITIS C ESTIMATED WORLD INFECTION
PREVALENCE
(BY WHO REGION)
Total
Population
(Millions)
Hepatitis C
Prevalence
(Rate %)
Infected
Population
(Millions)
Number of
Countries
(by WHO
Region)
Where Data
Unavailable
Africa
602
5.3
31.9
12
Americas
785
1.7
13.1
7
Eastern
Mediterrane
an
466
4.6
21.3
7
Europe
858
1.03
8.9
19
South-East
Asia
1500
2.15
32.3
3
Western
Pacific
1600
3.9
62.2
11
Total
5811
3.1
169.7
59
WHO
Region
Data Source: Weekly Epidemiological Record No. 49 / Dec, 1999 / WHO
Transmission sources
Disease statistics
Infected Individuals
85%
Persistent Infection
Most patients are asymptomatic and unaware they’re infected
30%
Liver Disease
1-5%
Death
HCV research
• Unknowns
• No cell culture system
• No small animal model
Outline
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Epidemiology
Introduction to Hepatitis C Virus
Immune response
Novel drug therapies
Conclusions
HCV
• Genus Hepacivirus
• Family Flaviviridae, with classical
flaviviruses and animal pestiviruses
• 6 genotypes worldwide, many
subtypes and isolates based on
nucleotide diversity
• Quasispecies within individual
Distribution of Hepatitis C genotypes
From Forns and Bukh, 1999.
HCV virion structure
Hypothesized structure:
• Icosahedral lipid membrane with
E1/E2 glycoproteins
• Icosahedral nucleocapsid
HCV Genome
• 9.6 kb positive strand RNA genome
• 5’ (with IRES) and 3’ noncoding
regions
• Open reading frame encoding
polyprotein of ~3000 amino acids
5’ UTR
C
Core protein (nucleocapsid)
E1
Envelope glycoprotein-1
HVR-1
E2
Envelope glycoprotein-2
HVR-2
p7
ORF
Viroporin ?
NS2
Zn-dependent proteinase
NS3
Zn-dependent proteinase, serine protease, helicase
NS4A
NS3 cofactor
NS4B
ER-derived membranous web formation
NS5A
Unknown function; component of replicase?
NS5B
RNA dependent RNA polymerase
3’ UTR
Protein F
• Newly discovered protein F
• Produced by ribosomal frameshift
mutation around codon 11 of Core
protein
• Infected individuals contain
antibodies
• Function unknown
Hepatitis C Life Cycle
CD81?
www.rockefeller.edu/pubinfo/hepc.jpg
Outline
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Epidemiology
Introduction to Hepatitis C Virus
Immune response
Novel drug therapies
Conclusions
Patterns of Viremia
1. Drop after peak  successful
control
2. Drop followed by rebound 
chronic infection
3. Consistent HCV  chronic
infection
Innate Immune Response
2 days after infection:
• Protein kinase R (PKR)
• Interferon regulatory factors (IRFs)
• Antiviral gene products (type I IFNinducible genes and immune TFs)
IRFs
dsRNA
PKR
PKR
PKR activated
phosphorylation of IRFs
IRFs act as transcription factors
to upregulate antiviral gene products
Gene products degrade viral RNA
and prohibit protein translation
Innate Immune Response
• Regardless of infection outcome
• Viral resistance
• Targeting by HCV proteins?
– NS5A and E2 (PKR)
– Core (JAK-STAT pathway)
– NS3/4A (phosphorylated IRF-3)
Adaptive Immune Response
Individuals who control virus:
• IFN-γ preferentially expressed in
liver
• Induces expression of
– genes encoding chemokines that
attract T cells into inflamed tissues
– proteins associated with antigen
processing and presentation
CD8+ and CD4+ T cells
• More vigorous CD8+ and CD4+ T
cell responses in all individuals that
controlled infection
• Chronic infections occur when
– unable to mount HCV-specific T cell
responses
– strong response that results in viral
RNA clearance, followed by
contraction in CD8+/CD4+ and
rebound in viremia
Chronic HCV infection
• Low frequencies and reduced capacity
of HCV-specific CD8+ cells
• Dendritic cells do not mature normally
and have impaired stimulatory activity
• CD4+ cells have
reduced IL-2
production and
proliferation
http://www.lbl.gov/Publications/Currents/Archive/Oct-03-2003.html
Chronic HCV Infection
• Impairment of Natural Killer (NK)
cell cytotoxic activity
– Reversible in patients responsive to
IFN-α drug therapy
• Frequency of NKT cells decreased
NKT cells (orange) attacking an infected cell (pink)
Natural Killer cell
http://www.spectroscopynow.com/ftp_images/killertcells.jpg
http://www.wasatchhealth.com/images/NK-Picture.jpg
Antibodies
• Role of antibodies unclear and
poorly studied
• Virus can be cleared in absence of
detectable antibody responses
• Neutralizing antibodies target E2,
which is highly variable and able to
evade
Immune-mediated liver
injury
• Mechanisms responsible for liver
injury poorly understood
• Host immune response and not
viral replication
• High CD8+ in liver 
immunopathogenesis and liver
injury
Liver Environment
Normal liver:
• “Immuno-silent” state
• CD8+ T cells trapped  apoptosis
• Prevents unnecessary immune
response to thousands of antigens
liver is exposed to
Liver Environment
HCV-infected liver:
• Type I IFN production
• Release of chemokines that
promote infiltration of NK cells
• Induced IFN-γ production in NK
cells
• Expression of chemokines that
recruit activated T cells to liver
Liver Environment
Depletion of NK cells before
hepatotropic viral infection leads to
inhibition of virus-specific T cell
response and liver injury
Immune-mediated liver
injury
• HCV infects only 1-10% of
hepatocytes
• IFN-γ and TNF-α from CD8+
destroy nearby non-infected
hepatocytes (“bystander killing”)
• HCC occurs mainly
due to high turnover
rate in hepatocytes
http://medicalimages.allrefer.com/large/hepatocellular-cancer-ct-scan.jpg
Outline
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Epidemiology
Introduction to Hepatitis C Virus
Immune response
Novel drug therapies
Conclusions
Current therapy
• Combination pegylated interferon-α
and ribavirin (nucleoside analog)
• Mechanism poorly understood
• Protein synthesis suppression;
degradation of plus strand RNA
• 50-80% effective
Current therapy
Side effects:
• Flu-like symptoms, tiredness, hair loss,
trouble with thinking, moodiness, and
depression
• Hematologic
– Anemia
– bone marrow suppression by IFN 
neutropenia, thrombocytopenia
– ribavirin directly toxic to red blood cells 
hemolysis
• Worsening of liver disease
Novel drug therapies
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Non-nucleoside inhibitors (NNIs)
Protease inhibitor
TGF-β
Cyclosporin A
Arsenic trioxide
RNA therapieis
Non-nucleoside Inhibitors
• Target RdRp
• Discovery method
• Structurally distinct:
– Benzothiadiazine
– Disubstituted phenylalanine
– 2 benzimidazole derivatives
• Allosteric inhibition
• Distinct binding sites
http://www.replizyme.com/images/rev_rna_hep_c.gif
Protease Inhibitor
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BILN 2061—NS3 protease inhibitor
Peptidomimetic
Oral ingestion
Clinical trial:
– Rapid decline in viral load
– Rebound
4-11 days
after
treatment
http://web.chemistry.gatech.edu/~williams/bCourse_Information/6521/protein/images/hcvmac1.gif
Transforming growth factor-β
• Naturally occurring cytokine
induced by core protein
• Direct effect on HCV replication
unknown
• Decreased viral load
• Increased fibrosis and cirrhosis
Cyclosporin A
• Immunosuppressive drug
• Mechanism unknown
• FK506 does not suppress HCV
replication
• CsA binds to cyclophilins and blocks
calcineurin 
inhibits stimulation
of genes essential
for T cell activation
• Combination with IFN
http://www.alexis-corp.com/files/formula/lkt-c9611.gif
Arsenic Trioxide
• Inhibits HCV replication at
submicromolar concentrations
• Non-toxic
• Combination with IFN
• Mechanism unknown
RNA treatments
• Treatments that use RNA to halt
viral replication
• Three treatments in development:
– RNA interference (RNAi) to degrade
viral RNA
– Small RNAs to bind to viral proteins
– RNAs to outcompete viral proteins for
binding to cellular proteins
RNAi
http://www.life.uiuc.edu/shapiro/RNAipathway.jpg
RNAi
http://www.life.uiuc.edu/shapiro/RNAipathway.jpg
RNAi
• Inhibits HCV replication
• Highly sequence specific (to 1 nt)
• Multiple siRNAs to target different
sites of viral genome
• Short hairpin RNAs targeting
conserved motifs encoded by
retroviruses
Small RNAs
• Overexpression of viral RNA
elements
• Bind to viral regulatory proteins and
prevent binding of viral RNA 
inhibits gene expression
• RNAs analogous to 5’ UTR
inhibited IRES-mediated translation
• Combats sequence specificity
problem
siRNAs
• siRNAs targeted to cellular
cofactors for HCV
– La, PTB, hVAP-33
• Blocks HCV replication
• Combats sequence specificity
problem
• Adenoviral-mediated expression
Conclusions
• HCV is a major worldwide health
concern
• Much remains unknown about HCV
• Current drug therapy is inadequate
and insufficient
• Novel therapies offer IFN-resistant
patients and those with serious
side effects hope of elimination of
hepatitis C infection
http://www.english.bayerconosur.com/noticias/tema008-1.asp
References
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Ahmad, A. and Alvarez, F. (2004). Role of NK and NKT cells in the
immunopathogenesis of HCV-induced hepatitis. Journal of Leukocyte
Biology 76: 743-759.
CDC FAQ: http://www.cdc.gov/ncidod/diseases/hepatitis/c/faq.htm
Forns, X. and Bukh, J. (1999). The Molecular Biology of Hepatitis C
Virus: Genotypes and Quasispecies. Clinics in Liver Disease 3.
Guo, J., Sohn, A., Zhu, Q. and Seeger, C. (2004). Mechanism of the
interferon alpha response against hepatitis C virus replicons. Virology
325: 71-81.
Hwang, D. et al (2004). Inhibition of hepatitis C virus replication by
arsenic trioxide. Antimicrobial Agents and Chemotherapy 48: 28762882.
Kowdley, K.V. (2005). Hematologic side effects of interferon and
ribavirin therapy. Journal of Clinical Gastroenterology 39, Suppl 1: S3S8.
Kronke, J., Kittler, R., Buchholz, F., Windisch, M.P., Pietschmann, T.,
Bartenschlager, R. and Fresei, M. (2004). Alternative approaches for
efficient inhibition of hepatitis C virus RNA replication by small
interfering RNAs. Journal of Virology 78: 3436-3446.
Slide template picture: http://www.english.bayerconosur.com/noticias/tema008-1.asp
References
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•
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Lamarre, D. et al (2003). An NS3 protease inhibitor with antiviral effects
in humans infected with hepatitis C virus. Nature 426: 186-189.
Liver Foundation: http://www.liverfoundation.org/db/articles/1028
Mercer D, Schiller D, Elliot J, Douglas DN, Hao C, Rinfret A, Addison
WR. (2001) Hepatitis C virus replication in mice with chimeric human
livers. Nat Med 7: 927-933.
Moradpour, D., Cerny, A., Heim, M.H. and Blum, H.E. (2001). Hepatitis
C: an update. Swiss Medical Weekly 131: 231-298.
Moradpour, D. and Blum, H.E. (2004). A primer on the molecular
virology of hepatitis C. Liver International 24: 519-525.
Murata, T., Ohshima, T., Yamaji, M., Hosaka, M., Miyanari, Y., Hijikata,
M. and Shimotohno, K. (2005). Suppression of hepatitis C virus
replicon by TGF-β. Virology 331: 407-417.
Nakagawa, M. et al (2004). Specific inhibition of hepatitis C virus
replication by cyclosporine A. Biochemical and Biophysical Research
Communications 313: 42-47.
Penin, F., Dubuisson, J., Rey, F.A., Moradpour, D. and Pawlotsky, J.
(2004). Structural Biology of Hepatitis C Virus. Hepatology 39: 5-19.
Puig, M., Major, M.E., Mihallik, K. and Feinstone, S.M. (2004).
Immunization of chimpanzees with an envelope protein-based vaccine
enhances specific humoral and cellular immune responses that delay
hepatitis C virus infection. Vaccine 22: 991-1000
References
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Ray, P.S. and Das, S. (2004). Inhibition of hepatitis C virus IRESmediated translation by small RNAs analogous to stem-loop structures
of the 5’-untranslated region. Nucleic Acids Research 32: 1678-1687.
Sarisky, R.T. (2004). Non-nucleoside inhibitors of the HCV polymerase.
Journal of Antimicrobial Chemotherapy 54: 14-16.
Shoukry, N.H., Cawthon, A.G. and Walker, C.M. (2004). Cell-mediated
immunity and the outcome of hepatitis C virus infection. Annual
Reviews in Microbiology 58: 391-424.
Sun, J., Li, K., Shata, M.T. and Chan, T. (2004). The immunologic basis
for hepatitis C infection. Current Opinions in Gastroenterology 20: 598602.
Trujillo-Murillo, et al. (2004). Experimental models for hepatitis C virus
(HCV): New opportunities for combating hepatitis C. Annals of
Hepatology 3: 54-62.
World Health Organization (WHO) (1999). Weekly Epidemiological
Record No. 49, December.
Zhang, J., Yamada, O., Sakamoto, T., Yoshida, H., Iwai, T., Matsushita,
Y., Shimamura, H., Araki, H. and Shimotohno, K. (2004). Downregulation of viral replication by adenoviral-mediated expression of
siRNA against cellular cofactors for hepatitis C virus. Virology 320:
135-143.