Strengthen The Pcbp2 Interferons Alpha, The Molecular Mechanisms Of Hcv Antiviral Activity In Research

Hepatitis C virus (HCV) is a positive RNA virus. HCV infection leads to chronic hepatitis in up to60-80%of infected adults and then progresses to liver cirrhosis and hepatocellular carcinoma. Interferon-a (IFN-a) was shown to have beneficial effects in patients with chronic hepatitis C. IFN-a therapy lead to a rapid decline in HCV-RNA levels in serum, and long-term responses are marked by sustained loss of HCV RNA from the serum and liver and provide resolution of the chronic Infection.Host cells are exclusive carriers for the life procession of HCV, a number of the cellular proteins have been reported to participate the life cycle of HCV. As HCV is a positive-strand RNA virus, chief among those cellular factors are RNA binding proteins (RBPs), which have been shown to interact with the viral ciselements. Poly(C) binding protein2(PCBP2, hnRNP E2or a-CP2) belongs to an hnRNP E family of RNA binding proteins that interact in a sequence-specific motif with single-stranded poly(C). In its cellular capacity, PCBP2is involved in posttranscriptional controls, protein interactions and decoy of microRNA. Apart from its roles in maintaining mRNA stability and regulating translation, PCBP2can also participate in the replication or translation of many RNA viruses, including poliovirus, coxsackievirus, rhinovirus, which are positive RNA viruses structurally similar to HCV, belonging to members of the family Picornaviridae. Investigations have demonstrated that PCBP2can bind to the5’-UTR and3’-UTR regions of HCV genome, which suggests some relation between PCBP2and HCV. However, how PCBP2act on HCV life processing is still to be elucidated.HCV replicon cell model greatly contributes to the study of HCV molecular mechanism in vivo. This system allowed for the efficient propagation of genetically modified HCV RNAs (Replicons) in a human hepatoma cell line which reflects the life procession of virus in cells. In our initial study, we find significant down-regulation of PCBP2in HCV subgenome replicon containing Huh7.5.1cells (Rib) compared to naive Huh7.5.1cells, which differe from other RBPs interacting with HCV. Ectopic expression of NS proteins in naive Huh7.5.1cells showed that NS4B and NS5A significantly inhibit the expression of PCBP2. Realtime PCR indicated that NS4B may posttranscriptionally regulate PCBP2while NS5A may function a transcriptional regulation. IFN-a restores the expression level of PCBP2in R1b cells after inhibiting the expression of both HCV replicon RNA and proteins. The PCBP2expression level gradually restored after treatment of IFN-a which showed a dose-dependent manner, and the protein level of PCBP2did not retrieve to low level in IFN-a cured R1b cells. There was no such restore in other three hepatocyte-derived cell lines, which demonstrated that the inhibition of PCBP2in R1b ascribed to the HCV NS proteins.To make clear the relationship between PCBP2and HCV, we performed over-expression and knock-down of PCBP2in R1b cells. The results indicated that PCBP2had no direct effect on HCV replication. Interestingly, we found that PCBP2facilitated the antiviral activity of IFN-a against HCV, and IFN-a combined with ribavirin presented an accordant tendency similar to IFN-a, while no effect on ribavirin alone. The effect of PCBP2on antiviral activity of IFN-a against HCV was confirmed in PCBP2knockdown cells. These results verified that PCBP2plays an important role in the effect of IFN-a against HCV.In order to illuminate the mechanism of PCBP2on IFN-a, we applied RNA immunoprecipitation technology to investigate whether the IFN-a signal pathway molecular were involved in PCBP2substrates. The results showed that mRNA of STAT1and STAT2enriched in PCBP2RNP complex which were corresponded to that of positive substrate, a-globin. These findings indicated that mRNA of STAT1and STAT2might serve as substrate of PCBP2. Besides, binding of mRNA of STAT1and STAT2with PCBP2led to the up-regulation of totle protein and consistent increase of phosphorylated level. RNA pull down technology further displayed that the PCBP2binding sites in the3’-UTR of mRNA of STAT1and STAT2, which contained polyC rich regions and core sequences of PCBP2recognition.Realtime PCR and RNase Protection Assay (RPA) demonstrated that PCBP2upregulated mRNA level of STAT1and STAT2. The mechanism is that the binding of these mRNA with PCBP2enhances the stability of mRNA and elongate their half-life. The enhancement of mRNA stability of STAT1and STAT2contributed to the totle protein up-regulation and thus enhance the antiviral activity of IFN-a against HCV.The paper reveals that PCBP2plays an important role in the effect of IFN-a against HCV, which may benefit the clinically individual therapy of HCV infection. Meanwhile, we disclose a new mechanism of interaction between RBPs and HCV, which enhances the effect of IFN-a other than exerting a direct effect on virus genome. These findings enlighten us preferably understanding HCV mechanism in vivo.