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Hepatitis C virus (HCV) is an enveloped positive RNA virus and belongs to the family Flaviviridae[8]. HCV causes persistent infection in 50 %-80 % of infected persons and may lead to the development of fibrosis, cirrhosis, and hepatocellular carcinoma[2, 33, 37]. The establishment of a HCV subgenomic replicon system[21] was a major breakthrough in research on HCV. Furthermore, an HCV infection system was established based on the HCV JFH-1 molecular clone and Huh-7 derived cell lines[3, 20, 39, 41]. These systems facilitate studies on the HCV life cycle and HCV-host cell interaction.
Since the viral replication is dependent on the host cell machinery, activation or suppression of cell signalling pathways may modulate virus replication. In the case of HCV, a number of intracellular signalling pathways, such as PI3K/Akt, IRF3, and the JAK-STAT pathway, have been shown to influence HCV replication[4, 7, 10, 11, 13, 15, 26, 34]. HCV has developed different strategies to interfere with host cell signalling pathways. It has been demonstrated that HCV NS3/4A blocks retinoic acid-inducible I gene (RIG-I) mediated signalling by cleaving the signalling adaptor Cardif and TLR3 mediated IRF3 activation through cleavage of the adaptor Toll/interleukin receptor domain-containing adapter-inducing interferon β (TRIF), thereby interfering with the induction of innate responses[12, 19, 28, 35]. The blockage of the RIG-I mediated activation of innate responses and subsequent interferon (IFN) production is essential for the maintenance of HCV replication, as HCV is highly sensitive to the antiviral action of IFN.
The mitogen activated protein kinases (MAPKs) are widely expressed serine/threonine kinases and mediate signals for the regulation of important cellular functions such as gene transcription, post transcriptional regulation, and cell cycle progression[32]. There are three main groups of MAPKs: extracellular signal regulated kinases (ERK), the p38 family kinases, and the JUN amino terminal kinases. The activation of the MAPK-ERK pathway is pivotal for the cell cycle progression[22, 27]. Mitogenic stimulation of cells causes phosphorylation of ERK and translocation of active ERK to the nucleus[9]. This translocation is necessary for ERK-dependent activation of DNA synthesis and progression from G1 into S phase[6]. It has been shown that the HCV NS5A protein may interfere with the activation of the MAPK-ERK pathway by altering the trafficking of epithelial growth factor (EGF) receptor[23, 25], thus attenuating the cellular response to EGF.
However, the significance of the MAPK pathway activation in HCV replication is not clear. In a previous study, HCV translation mediated by the internal ribosome binding site (IRES) was shown to be enhanced by inhibitors of the MAPK pathway[29]. However, this study did not clarify the influence of the MAPK pathway on HCV replication. Huang et al . examined the role of the MAPK pathway in the antiviral action of interferon-γ against HCV[16]. In a luciferase-based reporter replicon system, a blockage of the MAPK pathway activation could partially interfere with the inhibition of the reporter replicon by IFN-γ. It was suggested that the phosphorylation of HCV NS5A plays an important role in the HCV replication and is influenced by the MAPK activity.
In the current study, we examined the influence of the MAPK-ERK pathway on HCV replication. Using the HCV replicon system, we examined whether HCV replication and HCV IRES-dependent translation could be modulated by the inhibition or activation of the MAPK-ERK pathway. Furthermore, we investigated how the modulation of the MAPK-ERK pathway influences the IFN signalling and the inhibition of HCV replication by IFN. As the MAPK-ERK pathway is able to directly influence cell progression by activation of cyclin-dependent kinase 2 (CDK2), an important key protein of cell cycle control, we tested whether a blockage of cdk2 by an inhibitor roscovitine would also modulate HCV replication. A cell cycle inhibitor aphidicolin and an siRNA of a cellular negative regulator CDKN2B were used to modulate the cell cycles and to test the influence on HCV replication.
Regulation of Hepatitis C Virus Replication and Gene Expression by the MAPK-ERK Pathway
- Received Date: 26 April 2012
- Accepted Date: 17 August 2012
Abstract: The mitogen activated protein kinases-extracellular signal regulated kinases (MAPK-ERK) pathway is involved in regulation of multiple cellular processes including the cell cycle. In the present study using a Huh7 cell line Con1 with an HCV replicon, we have shown that the MAPK-ERK pathway plays a significant role in the modulation of HCV replication and protein expression and might influence IFN-? signalling. Epithelial growth factor (EGF) was able to stimulate ERK activation and decreased HCV RNA load while a MAPK-ERK pathway inhibitor U0126 led to an elevated HCV RNA load and higher NS5A protein amounts in Con1 cells. It could be demonstrated that the inhibition of the MAPK-ERK pathway facilitated the translation directed by the HCV internal ribosome entry site. Consistently, a U0126 treatment enhanced activity of the HCV reporter replicon in transient transfection assays. Thus, the MAPK-ERK pathway plays an important role in the regulation of HCV gene expression and replication. In addition, cyclin-dependent kinases (CDKs) downstream of ERK may be involved in the modulation of HCV replication since roscovitine, an inhibitor of CDKs had a similar effect to that of U0126. Modulation of the cell cycle progression by cell cycle inhibitor or RNAi resulted consistently in changes of HCV RNA levels. Further, the replication of HCV replicon in Con1 cells was inhibited by IFN-?. The inhibitory effect of IFN-? could be partly reversed by pre-incubation of Con-1 cells with inhibitors of the MAPK-ERK pathway and CDKs. It could be shown that the MAPK-ERK inhibitors are able to partially modulate the expression of interferon-stimulated genes.