Abstract
Cellular mechanisms that regulate the replication of hepatitis C virus (HCV) RNA are poorly understood. p21-activated kinase 1 (PAK1) is a serine/threonine kinase that has been suggested to participate in antiviral signaling. We studied its role in the cellular control of HCV replication. Transfection of PAK1-specific small interfering RNA enhanced viral RNA and protein abundance in established replicon cell lines as well as cells infected with chimeric genotype 1a/2a HCV, despite reducing cellular proliferation, suggesting specific regulation of HCV replication. PAK1 knockdown did not reduce interferon regulatory factor 3-dependent gene expression, indicating that this regulation is independent of the retinoic acid-inducible gene I/interferon regulatory factor 3 pathway. On the other hand, LY294002 and rapamycin abolished PAK1 phosphorylation and enhanced HCV abundance, suggesting that the mammalian target of rapamycin (mTOR) is involved in PAK1 regulation of HCV. Small interfering RNA knockdown of the mTOR substrate p70 S6 kinase abrogated PAK1 phosphorylation and enhanced HCV RNA abundance, whereas overexpression of a constitutively active alternate substrate, eukaryotic translation initiation factor 4E-binding protein 1, increased cap-independent viral translation and viral RNA abundance without influencing PAK1 phosphorylation. Similar data indicated that mTOR is regulated by both phosphatidylinositol 3-kinase/Akt and ERK. Taken together, the data indicate that p70 S6 kinase activates PAK1 and contributes to phosphatidylinositol 3-kinase- and ERK-mediated regulation of HCV RNA replication.
Highlights
2 The abbreviations used are: HCV, hepatitis C virus; IRF-3, interferon regulatory factor 3; retinoic acidinducible gene I (RIG-I), retinoic acid-inducible gene I; TLR3, Toll-like receptor 3; IRES, internal ribosome entry site; IFN, interferon; PI3K, phosphatidylinositol 3-kinase; JAK, Janus kinase; STAT, signal transducers and activators of transcription; IFN-stimulated genes (ISGs), IFN-stimulated gene; p21-activated kinases (PAKs), p21-activated kinase 1; Approximately 170 million people are estimated to be infected with the virus worldwide [2]
We could not demonstrate the difference in the basal activities of IRF-3-responsive promoters reported previously in these cells [6], these results demonstrated that p21-activated kinase 1 (PAK1) knockdown does not suppress basal activities of the ISG56 or IFN- promoters in either cell line (Fig. 2C)
We have shown here that HCV replication is suppressed by activation of PAK1
Summary
Cell Culture—The human hepatocellular carcinoma cell line, Huh, was cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, glucose (4.5 mg/ml) at 37 °C in a 5% CO2 atmosphere. To assess the role of PAK1 in IRF-3 activation induced by Sendai virus, Huh cells (5 ϫ 104 cells per well in 24-well plates) were transfected with PAK1 siRNA, followed by the transfection of reporter plasmids (350 ng) and pCMV--gal (50 ng) 24 h later. A7 cells (1 ϫ 104 cells/well) were seeded into 96-well flat-bottom plates, transfected with negative control or PAK1 siRNA as above, and cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. To confirm that the enhanced replication of HCV RNA was related to PAK1 knockdown and not a result of an off target effect of one of the siRNAs included in the pool, we transfected A7 replicon cells with each of the four individual. Previous experimental results demonstrated that IRF-3 is basally acti-
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