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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 22, 2003 as doi:10.1096/fj.02-0664fje. |
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antagonistic activity of HCV core protein involves induction of suppressor of cytokine signaling-31
Klinik für Gastroenterologie, Hepatologie und Infektiologie, Medizinische Klinik der Heinrich-Heine Universität, 40255 Düsseldorf, Germany;
# Institut für Medizinische Strahlenkunde und Zellforschung, Universität Würzburg, 97078 Würzburg; and
Institut für Biochemie, Universitätsklinikum der Rheinisch-Westfälischen Technischen Hochschule Aachen, 52074 Aachen, Germany
2Correspondence: Klinik für Gastroenterologie, Hepatologie und Infektiologie, Moorenstrasse 05, D-40225 Düsseldorf, Germany. E-mail: Johannes.Bode{at}t-online.de.
SPECIFIC AIMS
> 80% of patients newly infected with the hepatitis C virus develop chronic infection, suggesting that the hepatitis C virus can develop effective strategies to escape the host’s unspecific and specific immune response. Since SOCS molecules have been recognized to be powerful inhibitors of cytokine signaling via the Jak/STAT pathway, virus-induced expression of these molecules should be an efficient instrument to counteract the cellular response toward interferon, an essential part of first-line antiviral immune response.
PRINCIPAL FINDINGS
HCV core induces SOCS3 expression and transcriptional activation of the SOCS3 promoter
To examine whether the HCV core protein interferes with the Jak/STAT signal transduction via induction of SOCS3, the virus protein was transiently transfected in HepG2 cells and its influence on SOCS3 mRNA expression and SOCS3 promoter/luciferase reporter gene construct was analyzed. Overexpression of HCV core protein in the human hepatoma cell line HepG2 was sufficient to induce expression of SOCS3 mRNA and enhanced transcriptional activation of SOCS3 promoter/luciferase reporter gene construct containing the SOCS3 promoter region from -2757 to + 929 (Fig. 1
).
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Activation and nuclear translocation of STAT1 is inhibited by overexpression of HCV core
SOCS3 interferes with activation of STAT molecules. Corresponding to findings that HCV core induces SOCS3 expression, interferon-
(IFN-)-induced STAT1 DNA binding was inhibited in HepG2 cells transiently transfected with HCV core protein when nuclear extracts were analyzed using a DNA probe derived from the IFN-
activation sequence site of the interferon regulatory factor-1 promoter, considered specific to STAT1 binding. Nuclear translocation of STAT1 upon stimulation with IFN- was inhibited by cotransfection of HCV core protein when nuclear translocation was studied using a STAT1 construct, where STAT1 is amino-terminal fused to enhanced yellow fluorescing protein. Accordingly, IFN--mediated STAT1 tyrosine phosphorylation is suppressed by transient overexpression of HCV core protein and transient overexpression SOCS3.
HCV core partially rescues growth of a genetically engineered influenza A virus lacking its own interferon antagonist
The data shown so far indicate that the HCV core protein inhibits IFN- signaling at the level of induction of SOCS3. To further analyze whether this inhibitory function of the viral protein enables the protein to suppress the antiviral activity of IFN- toward a type I IFN-sensitive virus, a test system developed by Basler et al. was applied. The procedure takes advantage of a genetically engineered influenza A virus, the 
NS1 virus, which lacks its own IFN antagonist, the NS1 protein. Consistent with an IFN antagonistic function of the HCV core protein, a significant increase in virus production could be detected after infection of MDCK cells expressing the HCV core protein with NS1 virus (Fig. 2
). This indicates that HCV is competent to partially replace some functions of the optimized genuine virus protein, most likely IFN antagonistic activity.
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CONCLUSIONS AND SIGNIFICANCE
The battle between viruses and the IFN-mediated innate immune response developed during evolution is a subject of intense research. A main focus of these studies is to understand the mechanisms by which some viruses may evade IFN antiviral surveillance to establish persistent infection. Here we report that HCV core protein induces the expression of SOCS3 mRNA in a human hepatoma cell line. To our knowledge, this is the first demonstration that a viral protein is able to induce expression of SOCS proteins in general. Expression of SOCS3 correlates closely with the inhibition of IFN--mediated tyrosin phosphorylation, nuclear translocation, and activation of STAT1 by expression of HCV core protein or SOCS3 itself, suggesting that inhibition of STAT1 activation and induction of SOCS3 are functionally linked (summarized in Fig. 3
). As to the functional relevance of these findings, it is shown that HCV core protein partially confers IFN resistance to a genetically engineered influenza A virus, the NS1 virus, which lacks its own IFN antagonist, the NS1 protein.
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Another HCV mechanism to overcome cellular antiviral strategies has been described for the NS5A protein of HCV: based on heterologous expression of NS5A in cell culture and in vitro systems, it was demonstrated that NS5A is capable of interacting with and inhibiting PKR. Moreover, viral protein synthesis of IFN-sensitive viruses was partially restored in HeLa cells stably expressing NS5A or cells infected with recombinant vaccinia virus expressing NS5A.These results correlated with the phosphorylation levels of PKR and eIF2a in the infected cells, indicating that NS5A expression alone confers partial resistance to the antiviral effects of IFN against IFN-sensitive virus, most probably via interference with the PKR activity. Thus, as summarized in Fig. 3
, HCV uses at least two different strategies to infiltrate host defense against viral infection: 1) inhibition of IFN signal transduction via Jak/STAT and 2) disturbance of PKR activation.
The complete HCV replicon as well as the HCV core protein were reported to inhibit STAT-1 activation by IFN-. However, the underlying mechanism of the inhibition of STAT-1 activation remained unclear. Basu et al. provided evidence that HCV core protein stably expressed in HeLa or HepG2 cells led to a reduction of IFN--dependent activation of STAT1 due to decreased expression of STAT1 proteins. Since neither the data presented here nor those described on induced expression of the complete HCV replicon in osteosarcoma cells provide evidence for impaired STAT1 expression, this decrease might reflect a long-term response to HCV core protein.
The mechanism involved in HCV core protein-induced SOCS3 expression remains to be established and is under investigation. The clinical relevance of our data concerning IFN resistance and improved virus survival remains to be demonstrated. In support of our findings, SOCS1 and SOCS3 but not SOCS2 were reported to inhibit IFN-- and IFN--mediated antiviral and antiproliferative activity in HeLa and MCF-7 cell lines. Thus, the HCV core protein-induced SOCS3 expression and inhibition of IFN--mediated activation of STAT1 reported here may contribute to the IFN- unresponsiveness found in about half of chronically infected HCV patients.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0664fje; to cite this article, use FASEB J. (January 22, 2003) 10.1096/fj.02-0664fje 
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