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0022-538X/09/$08.00⫹0 doi:10.1128/JVI.00751-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Hepatitis C virus (HCV) utilizes strategies to suppress or evade the host immune response for establishment
of persistent infection. We have shown previously that HCV nonstructural protein 5A (NS5A) impairs tumor
necrosis factor alpha (TNF-␣)-mediated apoptosis. In this study, we have examined the immunomodulatory
role of HCV NS5A protein in transgenic mouse (NS5A-Tg) liver when mice were challenged with an unrelated
hepatotropic adenovirus as a nonspecific stimulus. Hepatotropic adenovirus was introduced intravenously into
NS5A-Tg mice and control mice, and virus clearance from liver was compared over a time course of 3 weeks.
Hepatitis C virus (HCV), a member of Flaviviridae, causes transient viral clearance in the context of an intrahepatic HCV-
chronic infection associated with cirrhosis and hepatocellular specific T-cell response (26, 41, 47, 52). An increase in the
carcinoma. The HCV genome containing positive-strand RNA IFN-␥ gene was not detected by microarray or by quantitative
is approximately 9.6 kb and encodes a polyprotein precursor of PCR in the liver of chimpanzees chronically infected with
about 3,000 amino acids, which is cleaved by both viral and HCV compared with HCV-uninfected chimpanzees (4, 16).
host proteases into structural (core, E1, E2, and p7) and non- IFN-␥ mRNA expression levels in peripheral blood mononu-
structural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) pro- clear cells of patients are significantly lower in nonresponders
teins. HCV NS5A exists as two phosphoproteins, p56 and p58, (15). During the first 6 months of onset of HCV infection, the
phosphorylated at serine residues after the mature protein is number of IFN-␥-producing HCV-specific CD8⫹ T cells is
released from the polyprotein (46). HCV NS5A has an anti- associated with virus eradication (13). IFN-␥ also inhibits HCV
apoptotic effect (11, 27) and modulates immune responses genome replication in vitro (9). On the other hand, tumor
(1, 39). necrosis factor alpha (TNF-␣), a primary initiator of innate
Interferons (IFNs) are key players of the innate immune immune response, determines the magnitude and course of
response to virus infection. alpha IFN (IFN-␣) and IFN- acquired immune responses. TNF-␣-induced apoptosis is im-
(type I IFNs) are secreted by almost all virus-infected cells and portant for clearance of virus-infected cells, and IFN-␥ accel-
by specialized blood lymphocytes. In contrast, the production erates the killing of virus-infected cells (17, 32, 48, 52).
of IFN-␥ (type II IFN) is restricted to cells of the immune A number of viruses have been shown to encode proteins
system, such as natural killer (NK) cells, macrophages, and T that have the potential to inhibit antiviral activity of innate and
cells. Although the antiviral effect of type I IFN on the repli- adaptive immune responses. Inflammatory cytokines contrib-
cation of HCV has been the subject of extensive research and
ute to viral clearance during acute viral hepatitis in humans,
although the endogenous type I IFN antiviral effector pathway
and the induction of these cytokines in the liver and other
is known to be broadly activated during chronic HCV disease
tissues of chronically infected patients may have therapeutic
(25), little is known about the role of other cytokines in cellular
value (20). Disson et al. (8) have shown that transgenic mice
defense against HCV. Further, how HCV manages to evade
expressing HCV polyprotein failed to clear an adenoviral in-
the host defense is poorly understood.
fection. However, transgenic mice expressing either core or
In the liver of chimpanzees acutely infected with HCV,
structural proteins do not appear to be immunologically
IFN-␥ was detected only in animals displaying sustained or
impaired as they are fully competent to eliminate hepato-
cytes infected with adenovirus (Ad) (24, 42). Together,
* Corresponding author. Mailing address: Department of Pathology, these results suggest a role of HCV nonstructural protein(s)
Saint Louis University, 1100 S. Grand Blvd., DRC-207, St. Louis, MO in the modulation of the immune system. In this present
63104. Phone: (314) 977-7822. Fax: (314) 771-3816. E-mail: rayrb@slu study, we have examined the role of HCV NS5A protein on
.edu.
† Present address: Chiba University, School of Medicine, Chiba,
intrahepatic cytokine production for clearance of an unre-
Japan. lated hepatotropic Ad using a transgenic mouse model. Our
䌤
Published ahead of print on 24 June 2009. results demonstrated that IFN-␥ production is inhibited and
8463
8464 KANDA ET AL. J. VIROL.
previously (28), and GFP expression was quantitated using a Intrahepatic cytokine gene expression during Ad infection in
fluorometer (Fig. 1B). GFP expression was normalized to that HCV NS5A-Tg mice. CD4⫹ T cells are a major player in the
from mouse livers on day 3. GFP expression was also examined induction of protective immune response to HCV infection.
by Western blot analysis (Fig. 1C). Together, our results re- Th17 is a newly defined subset of CD4⫹ T cells. The discovery
vealed that the NS5A-Tg mice fail to clear Ad from liver up to of the Th17 lineage has revealed additional complexity in the
21 days postinfection. fates chosen by helper T cells and has begun to reshape our
We have examined the T-cell infiltration in both NTg and view of how signaling and transcriptional networks generate
NS5A-Tg mice at day 7 following Ad infection. For this, the appropriate and inappropriate immunity (37). We have exam-
presence of CD3⫹ cells was examined in liver sections by ined cytokine-related gene expression profiles using real-time
immunohistochemistry. Slides were immunostained with anti- PCR-based focused microarrays for intrahepatic inflammatory
CD3 antibody and counterstained with hematoxylin. CD3⫹ genes related to the Th17 regulatory network. A comparison of
cells from at least five fields of each slide were counted in a intrahepatic cytokines between NS5A-Tg and NTg mice at day
blinded manner. We did not observe a significant difference in 14 of Ad infection is shown in Fig. 3A. Out of 84 cytokine-
the level of CD3 marker between the NS5A-Tg and NTg mice related genes examined, 63 (75.0%) were upregulated by 2.0-
(Fig. 2). The slides were also stained with hematoxylin and
eosin to examine inflammation. We have observed a slightly
higher level of inflammation in NTg mice than in HCV
NS5A-Tg mice following Ad infection. A representative illus- TABLE 1. Serum ALT level in NTg and NS5A-Tg mice
tration is shown in Fig. 2 (bottom panel). Our results are in Time point ALT level (U/liter)a
agreement with the earlier study of transgenic mice expressing (day) NTg mice NS5A-Tg mice
the entire HCV genome (8), suggesting that the presence of
the HCV transgene does not impair intrahepatic T-cell infil- 0 120 ⫾ 31 122 ⫾ 23
7 696 ⫾ 218 188 ⫾ 65
tration. We also examined the alanine aminotransferase 14 1071 ⫾ 206 298 ⫾ 104
(ALT) level from serum of NS5A-Tg and NTg mice at different 24 ND 144 ⫾ 56
time points. The ALT level was increased approximately five- a
Data for each time point represent the sera of six animals, and values are
to eightfold on day 7 and day 14 in NTg mice compared to means ⫾ standard deviations. ND, not done (AdGFP was cleared from mouse
levels in NS5A-Tg mice (Table 1). liver).
8466 KANDA ET AL. J. VIROL.
broad cellular Th1-dominant responses. Abundant evidence ential expression of toll-like receptor mRNA in treatment non-responders
and sustained virologic responders at baseline in patients with chronic hep-
indicates that the immune system fails to eliminate virus from atitis C. Liver Int. 26:1100–1110.
the liver in a large number of HCV-infected patients, and as a 16. Huang, Y., J. J. Feld, R. K. Sapp, S. Nanda, J. H. Lin, L. M. Blatt, M. W.
result, most acute infections lead to long-term viral persistence Fried, K. Murthy, and T. J. Liang. 2007. Defective hepatic response to
interferon and activation of suppressor of cytokine signaling 3 in chronic
(12, 36). The efficacies of therapeutic vaccination for chronic hepatitis C. Gastroenterology 132:733–744.
hepatitis C nonresponder patients are correlated with the in- 17. Kafrouni, M., G. R. Brown, and D. L. Thiele. 2003. The role of TNF-TNFR2
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that HCV NS5A affects Gata-3 and Tbx21 expression and 18. Klade, C. S., H. Wedemeyer, T. Berg, H. Hinrichsen, G. Cholewinska, S.
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and M. P. Manns. 2008. Therapeutic vaccination of chronic hepatitis C
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This work was supported by research grants AI45144 and AI065535 21. Kulkarni, A., D. S. Ravi, K. Singh, S. Rampalli, V. Parekh, D. Mitra, and S.
(R.B.R.) from the National Institutes of Health and by the Blue Rib- Chattopadhyay. 2005. HIV Tat modulates T-bet expression and induces Th1
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