Hepatitis C Virus Non-Structural Protein NS5A Interacts With FKBP38 And Inhibits Apoptosis In Huh7 Hepatoma Cells

Hepatitis C Virus (HCV) is a major causative agent for public health problem worldwide. Infection of HCV often leads to chronic hepatitis, which may progress to liver cirrhosis, liver dysfunction and hepatocellular carcinoma. The understanding of the mechanism for HCV pathogenesis so far is still limited. A growing body of evidence has shown that the interactions between viral proteins and host cellular proteins may play an important role in HCV pathogenesis. Among the structural and non-structural proteins, the non-structural protein 5A (NS5A) has been paid extensive attention. A number of studies have demonstrated that NS5A plays various roles in cellular signal transduction, protein trafficking, anti-apoptosis and other cellular events, most of which may ultimately result in persistent viral infection. To gain further insight into the molecular mechanism, we screened a human fetal liver cDNA library to find out its cellar partners and their physiological functions.To uncover possible NS5A-interacting proteins, the Ga14 yeast two-hybrid system was employed to screen a human fetal liver cDNA library using NS5A as the bait. After the first-round screening, the putative positive clones were re-transformed into the yeast cells with NS5A to undergo the second-round screening, respectively. Twelve positive clones were obtained. After sequencing and searching the NCBI database, we found these clones encoded two putative NS5A-interacting proteins, a 38kD immunosuppressant FK506-binding protein (FKBP38) and an ADP-ribosylation-like factor-6 interacting protein 1 (Aip-1). As NS5A could inhibit apoptosis through different pathways and FKBP38 was reported to exert its anti-apoptotic function by targeting Bcl-2 into the outer membrane of mitochondria, it is proposed that the interaction of NS5A and FKBP38 may be involved inanti-apoptosis and worthy to be further investigated.To confirm the association between NS5A and FKBP38, the in vitro coimmunoprecipitation assay was first carried out. NS5A and tagged protein HA-FKBP38 were translated in vitro in the presence of [³⁵S] methionine and immunoprecipitation was performed by incubation with anti-HA antibody. Results showed that both proteins were found in the immuno-complex, indicating NS5A could physically associate with FKBP38 in vitro. To further verify such association in mammalian cells, NS5A-myc and HA-FKBP38 mammalian expressing plasmids were constructed and co-transfected into Cos7 cells. Immunoprecipitation results showed that each protein could be detected in the immuno-complex precipitated by anti-myc or anti-HA antibody. Additional experiments using HCV subgenomic replicon lysates also showed that NS5A could interact with endogenous FKBP38 and such interaction was FK506-independent. To study the cellular localization of NS5A and FKBP38, the immunofluorescence staining and confocal laser scanning microscopy were performed. Results showed that both transiently-expressing and endogenous NS5A and FKBP38 could localize in the cytoplasm. More importantly, both were found in the endoplasmic reticulum and mitochondria, although NS5A only partly resided in the mitochondria. To examine the region of NS5A responsible for interacting with FKBP38, a series of truncated mutants of NS5A were constructed and association was assayed by using both yeast two-hybrid system and confocal laser scanning microscopy. Results showed that the aa148-236 of NS5A was essential for interacting with FKBP38. In addition, a Bcl-2 homology domain (BH Domain) was also found in this region. These results above strongly confirm that NS5A could interact with FKBP38.Since both NS5A and FKBP38 were implicated in the anti-apoptosis process, to study whether such interaction could confer the anti-apoptotic capability to the cell, a hepatoma cell line Huh7 where Bcl-2 expression was deficient, was utilized to construct the Huh7-NS5A stablely expressing cells. Results showed that both Huh7-NS5A stable cells and HCV subgenomic replicon cells could inhibit apoptosis induced by a series of pro-apoptotic drugs. More importantly, with depletion of endogenous FKBP38, such inhibition of apoptosis could be abrogated in Huh7-NS5A stablely expressing cells whereas no differences were observed in control cells. In addition, the cleaved PARP product, a marker of intrinsic mitochondrial-related apoptosis, was also decreased in Huh7-NS5A stable cells compared to the controlcells. These results indicate that the anti-apoptotic function of NS5A may be mediated through the interaction with FKBP38 and NS5A may mimic the function of Bcl-2 in hepatoma cells.In summary, by using the yeast two-hybrid screening we find a novel interaction between HCV NS5A protein and cellular protein FKBP38 and such interaction confers the anti-apoptotic function to NS5A. These findings provide new insights into the mechanism for NS5A evading apoptosis and may be helpful to understand the detailed mechanisms for HCV persistence.