FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2117084310> ?p ?o ?g. }

• W2117084310 endingPage "5025" @default.
• W2117084310 startingPage "5015" @default.
• W2117084310 abstract "Article5 October 2006free access Hepatitis C virus RNA replication is regulated by FKBP8 and Hsp90 Toru Okamoto Toru Okamoto Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Search for more papers by this author Yorihiro Nishimura Yorihiro Nishimura Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan Search for more papers by this author Tohru Ichimura Tohru Ichimura Department of Chemistry, Graduate School of Sciences and Engineering, Tokyo Metropolitan University, Tokyo, Japan Search for more papers by this author Kensuke Suzuki Kensuke Suzuki Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Search for more papers by this author Tatsuo Miyamura Tatsuo Miyamura Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan Search for more papers by this author Tetsuro Suzuki Tetsuro Suzuki Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan Search for more papers by this author Kohji Moriishi Kohji Moriishi Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Search for more papers by this author Yoshiharu Matsuura Corresponding Author Yoshiharu Matsuura Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Search for more papers by this author Toru Okamoto Toru Okamoto Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Search for more papers by this author Yorihiro Nishimura Yorihiro Nishimura Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan Search for more papers by this author Tohru Ichimura Tohru Ichimura Department of Chemistry, Graduate School of Sciences and Engineering, Tokyo Metropolitan University, Tokyo, Japan Search for more papers by this author Kensuke Suzuki Kensuke Suzuki Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Search for more papers by this author Tatsuo Miyamura Tatsuo Miyamura Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan Search for more papers by this author Tetsuro Suzuki Tetsuro Suzuki Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan Search for more papers by this author Kohji Moriishi Kohji Moriishi Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Search for more papers by this author Yoshiharu Matsuura Corresponding Author Yoshiharu Matsuura Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Search for more papers by this author Author Information Toru Okamoto1, Yorihiro Nishimura2, Tohru Ichimura3, Kensuke Suzuki1, Tatsuo Miyamura2, Tetsuro Suzuki2, Kohji Moriishi1 and Yoshiharu Matsuura 1 1Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan 2Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan 3Department of Chemistry, Graduate School of Sciences and Engineering, Tokyo Metropolitan University, Tokyo, Japan *Corresponding author. Department of Molecular Virology, Research Centre for Emerging Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita-chi, Osaka 565-0871, Japan. Tel.: +81 6 6879 8340; Fax: +81 6 6879 8269; E-mail: [email protected] The EMBO Journal (2006)25:5015-5025https://doi.org/10.1038/sj.emboj.7601367 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a component of viral replicase and is well known to modulate the functions of several host proteins. Here, we show that NS5A specifically interacts with FKBP8, a member of the FK506-binding protein family, but not with other homologous immunophilins. Three sets of tetratricopeptide repeats in FKBP8 are responsible for interactions with NS5A. The siRNA-mediated knockdown of FKBP8 in a human hepatoma cell line harboring an HCV RNA replicon suppressed HCV RNA replication, and this reduction was reversed by the expression of an siRNA-resistant FKBP8 mutant. Furthermore, immunoprecipitation analyses revealed that FKBP8 forms a complex with Hsp90 and NS5A. Treatment of HCV replicon cells with geldanamycin, an inhibitor of Hsp90, suppressed RNA replication in a dose-dependent manner. These results suggest that the complex consisting of NS5A, FKBP8, and Hsp90 plays an important role in HCV RNA replication. Introduction Hepatitis C virus (HCV) persistently infects approximately 170 million people worldwide, and it is responsible for most cases of severe chronic liver diseases, including cirrhosis and hepatocellular carcinoma (Wasley and Alter, 2000). Although treatment with interferon (IFN) alpha and ribavirin is available for about half of the population of HCV patients (Manns et al, 2001), therapeutic and preventative vaccines are still necessary for more effective treatment; however, such vaccines have not yet been developed. HCV belongs to the Flaviviridae family and possesses a positive-sense single-stranded RNA with a nucleotide length of 9.6 kb. The HCV genome encodes a single large precursor polyprotein composed of about 3000 amino acids, and the polyprotein is processed by cellular and viral proteases into at least 10 structural and nonstructural (NS) proteins (Moriishi and Matsuura, 2003). The development of efficient therapies for hepatitis C has been hampered by the lack of a reliable cell-culture system, as well as by the absence of a non-primate animal model. The HCV replicon consists of an antibiotic selection marker and a genotype 1b HCV RNA, which replicates autonomously in the intracellular compartments in a human hepatoma cell line, Huh7 (Lohmann et al, 1999). This replicon system has functioned as an important tool in the investigation of HCV replication and it has served as a cell-based assay system for the evaluation of antiviral compounds. Recently, cell culture systems for in vitro replication and infectious viral production were established based on the full-length HCV genome of genotype 2a, which was isolated from an HCV-infected patient who developed fulminant hepatitis (Lindenbach et al, 2005; Wakita et al, 2005; Zhong et al, 2005). However, no robust in vitro culture systems for the 1a and 1b genotypes, which are the most prevalent HCV genotypes in the world, have been established to date. Several viruses require viral and host molecular chaperones for entry, replication, and assembly, as well as for other steps in viral production (Maggioni and Braakman, 2005; Mayer, 2005). Cyclosporine A has been found to effectively inhibit viral replication in hepatitis C patients and in HCV replicon cells (Inoue et al, 2003; Watashi et al, 2003). Recently, it was shown that cyclophilin (Cyp) B specifically binds to NS5B and promotes association with the genomic RNA; furthermore, cyclosporine A was shown to disrupt interactions between NS5B and CypB (Watashi et al, 2005). CypB belongs to the immunophilin family, which shares peptidyl propyl cis/trans isomerase (PPIase) activity and an affinity for the immunosuppressive drug (Fischer and Aumuller, 2003). Furthermore, blockades of CypA, CypB, and CypC, as well as the induction of cellular stress responses, have been suggested to be involved in cyclosporine A-induced reduction of HCV RNA replication (Nakagawa et al, 2005). However, the involvement of other immunophilins in HCV RNA replication is not yet well understood. HCV nonstructural protein 5A (NS5A) is a membrane-anchored phosphoprotein that possesses multiple functions in viral replication, IFN resistance, and pathogenesis (Macdonald and Harris, 2004). NS5A contains a zinc metal-binding motif within the N-terminal domain, and this zinc-binding ability is known to be essential for HCV replication (Tellinghuisen et al, 2004, 2005). Adaptive mutations frequently mapped in the coding region of NS5A have been shown to increase RNA replication (Yi and Lemon, 2004; Appel et al, 2005) and they are known to affect the hyperphosphorylation of NS5A by an unknown host kinase (Koch and Bartenschlager, 1999; Neddermann et al, 1999; Pietschmann et al, 2001). RNA replication in HCV replicon cells has been shown to be inhibited by treatment with lovastatin, a drug that decreases the production of mevalonate by inhibiting 3-hydroxy-3-methylglutaryl CoA reductase; this inhibition of RNA replication was reversed by the addition of geranylgeraniol, which suggests that HCV RNA replication requires geranylgeranylated proteins (Ye et al, 2003; Kapadia and Chisari, 2005). A NS5A-pull-down assay identified a geranylgeranylated protein, FBL2, as a NS5A-binding protein (Wang et al, 2005). Although several host proteins could potentially interact with NS5A, little is known about NS5A function. To gain a better understanding of the functional role of NS5A in HCV replication, we screened human libraries by employing a yeast two-hybrid system and using NS5A as bait. We thereby successfully identified FKBP8 as an NS5A-binding protein. FKBP8 is classified as a member of the FK506-binding protein family, but it lacks several amino-acid residues thought to be important for PPIase activity and FK506 binding (Lam et al, 1995). We demonstrated here that FKBP8 forms a complex with Hsp90 and NS5A, and that this complex is critical for HCV replication, as based on the finding that treatment of the HCV replicon cells with geldanamycin, an inhibitor of Hsp90, suppressed RNA replication. These results therefore suggest that protein complex formation with NS5A, FKBP8, and Hsp90 plays a crucial role in HCV RNA replication. Results Identification of human FKBP8 as an HCV NS5A-binding partner To identify host proteins that specifically interact with NS5A, we screened human brain and liver libraries using a yeast two-hybrid system that employs NS5A as bait. One positive clone was isolated from among 2 million colonies of the human fetal brain library, and the nucleotide sequence of this clone was determined. Several positive clones were isolated from the human liver library, but most of these clones included exon fragments of other than FKBP and/or noncoding regions. A BLAST search revealed that the positive clone encodes a full-length coding region of FKBP38, human FK506-binding protein 38 kDa. Although FKBP38 has been isolated from human and mouse mRNA (Lam et al, 1995), an additional sequence at the N-terminus of FKBP38 was revealed based on an analysis of the transcriptional start site in the genomic sequences of FKBP38 (Nielsen et al, 2004). The isoforms of FKBP38 were designated as FKBP8, which includes splicing variants of 44 and 46 kDa in mice, and 45 kDa in humans corresponds to the 44 kDa of the mouse FKBP8 (Nielsen et al, 2004). Human FKBP8 is identical to FKBP38 except for the extra 58 amino-acid residues at the N-terminus, and the FK506-binding domain in the N-terminal half, followed by three sets of tetratricopeptide repeats (TPRs), a calmodulin binding site, and a transmembrane domain (Figure 1A). Because the levels of expression of FKBP8 and FKBP38 have not been well characterized in human cell lines, we generated a mouse monoclonal antibody against human FKBP8, and we designated it as clone KDM19. This antibody recognizes a 50-kDa of endogenous FKBP8 in 293T cells, as well as exogenous HA-tagged FKBP8 (HA-FKBP8), which has slightly greater molecular weight (Figure 1B). Although the KDM19 antibody detected an exogenous HA-tagged FKBP38 (HA-FKBP38) in 293T cells, no protein band corresponding to endogenous FKBP38 was detected. Similar results were obtained in human liver tissue and in the hepatoma cell lines Huh7, HepG2, and FLC-4 (data not shown). These findings suggest that FKBP8, but not FKBP38, is a major product in human cells. In order to examine whether or not FKBP8 binds to NS5A protein in mammalian cells, Flag-tagged NS5A (Flag-NS5A) was expressed together with HA-FKBP8 in 293T cells. Cells transfected with the expression plasmids were harvested at 48-h post-transfection, lysed, and subjected to immunoprecipitation. Flag-NS5A was co-precipitated with HA-FKBP8 by anti-HA antibody (Figure 1C). Flag-NS5A was also immunoprecipitated together with HA-FKBP38, suggesting that the extra N-terminal sequence of FKBP8 is not critical for NS5A binding (data not shown). To further confirm the specific interaction of HCV NS5A with endogenous FKBP8, this interaction was examined in Huh7(9–13) cells harboring subgenomic HCV RNA replicon. Endogenous FKBP8 was co-precipitated with HCV NS5A by anti-FKBP8 antibody (Figure 1D). To determine the direct interaction between FKBP8 and NS5A, His6-tagged FKBP8 (His-FKBP8) and thioredoxin-fused domain 1 of NS5A (Trx-NS5A) prepared in Escherichia coli were examined by pull-down analysis. Trx-NS5A was co-precipitated with His-FKBP8 by anti-FKBP8 antibody (Supplementary Figure 1), suggesting that FKBP8 can directly bind to NS5A domain I. Figure 1.Expression of FKBP8 and FKBP38 in mammalian cells. (A) Schematic representation of FKBP8 and FKBP38. The FK506-binding domain (FBD), tetratricopeptide repeat (TPR), putative calmodulin binding motif (CaM), and transmembrane domain (TM) are shown. (B) N-terminally HA-tagged FKBP8 and FKBP38 were expressed in 293T cells and visualized by immunoblotting using mouse monoclonal antibody to FKBP8 or the HA tag. (C) HA-FKBP8 was expressed together with Flag-NS5A of genotype 1b (J1) in 293T cells and was immunoprecipitated with anti-HA antibody. Immunoprecipitated proteins were subjected to immunoblot with anti-Flag or HA antibody. (D) Endogenous FKBP8 in HCV replicon (9–13) cells was immunoprecipitated with isotype control (lane 1) or anti-FKBP8 antibody, KDM-11 (lane 2). Endogenous FKBP8 was co-immunoprecipitated with HCV NS5A. The data shown in each panel are representative of three independent experiments. Download figure Download PowerPoint In order to investigate the interaction of FKBP8 with the NS5A of other HCV genotypes, HA-tagged NS5A (HA-NS5A) proteins of genotype 1a (H77C), 1b (Con1 and J1), or 2a (JFH1) were expressed together with Flag-tagged FKBP8 (Flag-FKBP8) in 293T cells (Figure 2A). Flag-FKBP8 was co-immunoprecipitated with the HA-NS5As of all of the genotypes examined here by anti-HA antibody, although it should be noted that the interaction between Flag-FKBP8 and the HA-NS5A of genotype 2a was weaker than that of the other genotypes tested. Furthermore, the HA-NS5As were co-precipitated with Flag-FKBP8 by anti-Flag antibody (Figure 2A, bottom panel). The TPR domain of FKBP8 is known to be responsible for protein–protein interactions. Among the immunophilins, FKBP8 shares high homology with CypD and FKBP52, both of which contain three tandem repeats of TPR, as does FKBP8 (Boguski et al, 1990; Hirano et al, 1990). However, co-immunoprecipitation of Flag-NS5A with HA-FKBP52 and HA-CypD by anti-Flag or anti-HA antibody was not successful (Figure 2B). These results indicate that FKBP8 specifically interacts with NS5A. Figure 2.Specific interaction between FKBP8 and NS5A. (A) HA-NS5As were obtained from several genotypes of HCV and were expressed with Flag-FKBP8 in 293T cells. Proteins immunoprecipitated with anti-HA or Flag antibody were subjected to Western blotting. (B) Flag-NS5A was coexpressed with HA-FKBP8, -CypD, or -FKBP52 in 293T cells. Proteins immunoprecipitated with anti-HA or -Flag tag antibody were subjected to Western blotting. The data shown in each panel are representative of three independent experiments. Download figure Download PowerPoint The TPR domain is required for the interaction between NS5A and FKBP8 FKBP8, CypD, and FKBP52 have high similarity and identity to each other within the TPR domain (Lam et al, 1995). Several FKBP8 mutants lacking the transmembrane region, the calmodulin-binding region, the TPR domains, and/or the FK506-binding domain were generated in order to identify the region responsible for the interaction with NS5A (Figure 3A). HA-tagged FKBP8 mutants were coexpressed with Flag-NS5A in 293T cells and were immunoprecipitated with anti-HA antibody. Flag-NS5A was co-immunoprecipitated with the FKBP8 mutants, except in the case of a dTPR mutant lacking the transmembrane, calmodulin binding, and TPR domains (Figure 3B). Although the level of expression of dFBD, an FKBP8 mutant with a deletion in the N-terminal region containing the FK506-binding domain, was lower than that of dTPR, co-immunoprecipitated NA5A was clearly detected. These findings suggested that the lack of an association of dTPR with NS5A was not due to the relatively low level of expression of dTPR, as compared to those of the other FKBP8 mutants. A specific interaction of NS5A with the TPR domain, but not with the transmembrane, calmodulin binding, or FK506-binding domains of FKBP8, was also observed using the yeast two-hybrid system (data not shown). These results indicated that FKBP8 interacts with HCV NS5A through the TPR domain. Figure 3.Determination of the NS5A-binding region in FKBP8. (A) Schematic representation of FKBP8 and deleted mutants. (B) Flag-NS5A was coexpressed with HA-FKBP8 and its mutants in 293T cells. Proteins immunoprecipitated with anti-HA antibody were subjected to Western blotting. The data shown in each panel are representative of three independent experiments. Download figure Download PowerPoint FKBP8 forms a homomultimer and a heteromultimer with NS5A FKBP8 is similar to FKBP52 and CypD with respect to their amino-acid sequences and functional domains. In order to examine the interactions among FKBP8, FKBP52, and CypD, Flag-FKBP8 was coexpressed with HA-FKBP52, HA-CypD, or HA-FKBP8 in 293T cells and it was immunoprecipitated with anti-Flag or anti-HA antibody. Flag-FKBP8 and HA-FKBP8 were co-immunoprecipitated with each antibody, but not with HA-FKBP52 or HA-CypD. It is known that Hsp90 forms a homodimer and also interacts with FKBP52 through TPR domain as FKBP8 (Chadli et al, 2000). If homodimer of FKBP8 is due to intermediating of Hsp90 as FKBP8-Hsp90-Hsp90-FKBP8 complex, FKBP52 would be co-precipitated with FKBP8 as FKBP8-Hsp90-Hsp90-FKBP52. However, we could not detect any association of FKBP8 and FKBP52 in the immunoprecipitation analysis (Figure 4A). These data suggest that FKBP8 can form a homomultimer without Hsp90 and associate with neither FKBP52 nor CypD through Hsp90. To examine the effects of the interaction with NS5A on the homomultimerization of FKBP8, HA-NS5A was coexpressed with Flag-FKBP8 and Glu-Glu-tagged FKBP8 (EE-FKBP8) in 293T cells, and was then immunoprecipitated with anti-Flag or anti-EE antibody. HA-NS5A was co-immunoprecipitated with Flag-FKBP8 and EE-FKBP8 by anti-Flag or anti-EE antibody (Figure 4B). Although multimerization of EE-FKBP8 and Flag-FKBP8 was increased about 2 times in the presence of HA-NS5A, but no further increase of the multimerization of FKBP8 was observed by the increase of HA-NS5A expression (Figure 4C). These results further support the notion that NS5A binds to FKBP8 via the TPR domain and slightly influence homomultimerization exerted by the FK506-binding domain. Figure 4.Homomultimerization of FKBP8. (A) Flag-FKBP8 was coexpressed with HA-FKBP52, -CypD, or -FKBP8 in 293T cells, and was immunoprecipitated with anti-HA or Flag antibody. Precipitates were analyzed by Western blotting. (B) Flag- or EE-tagged FKBP8 was coexpressed with HA-NS5A in 293T cells and was immunoprecipitated with anti-EE or Flag antibody. Precipitates were analyzed by Western blotting. (C) Flag- and EE-tagged FKBP8 were coexpressed with increasing amounts of HA-NS5A (0.1, 0.2, and 0.4 μg of expression plasmid/well) in 293T cells. Immunoprecipitates with anti-Flag antibody were analyzed by Western blotting. The data shown in each panel are representative of three independent experiments. Download figure Download PowerPoint Knockdown of FKBP8 decreases RNA replication in HCV replicon cells In order to determine the role of endogenous FKBP8 on HCV RNA replication, 80 nM of small interfering RNA (siRNA) targeted to FKBP8 or control siRNA was transfected into Huh7 (9–13) cells harboring subgenomic HCV replicon RNA. To verify the specificity of the knockdown of FKBP mRNA, we synthesized three siRNAs targeted to different regions of FKBP8 (Targets 1–3). The total RNA was extracted from the transfected cells, and HCV RNA and FKBP8 mRNA levels were determined by real-time polymerase chain reaction (PCR). HCV subgenomic RNA and FKBP8 mRNA levels in the cells transfected with each of the FKBP8 siRNAs were reduced by more than 60%, as compared to the levels in cells treated with the control siRNA at 72 h post-transfection (Figure 5A). The levels of expression of FKBP8 and the HCV proteins (i.e., NS4B, NS5A, and NS5B) decreased in HCV replicon cells transfected with 80 or 160 nM of the FKBP8 siRNA (Target 1), but this was not observed in the cells with the control siRNA (Figure 5B). To further confirm the specificity of the reduction in HCV RNA replication in the replicon cells putatively achieved by the knockdown of FKBP8, a plasmid encoding Flag-FKBP8 containing either a silent mutation within the siRNA target sequence (Flag-rFKBP8) or empty plasmid was transfected into the HCV replicon cells and then selection was carried out with the appropriate antibiotics. The remaining cells, that is, Huh7rFKBP8 and Huh7c cells, harboring the Flag-rFKBP8 and empty plasmid, respectively, were pooled and then transfected with the FKBP8 siRNA (Target 1) or control siRNA. Although transfection of the FKBP8 siRNA led to a 60% reduction of HCV RNA and FKBP8 mRNA in Huh7c cells, in comparison with levels in cells transfected with the control siRNA, no reduction in HCV RNA, and only a slight reduction in FKBP8 mRNA levels were observed in Huh7rFKBP8 cells (Figure 5C). Flag-rFKBP8 expression was clearly detected in Huh7rFKBP8 cells after transfection with the FKBP8 siRNA or control siRNA, whereas the endogenous FKBP8 decreased in both Huh7rFKBP8 and Huh7c cells with the FKBP8 siRNA (Figure 5D). These findings suggest that the slight reduction of FKBP8 mRNA in the Huh7rFKBP8 cells was due to a loss of endogenous FKBP8. Knockdown of FKBP8 by siRNA induce no apoptosis in a hepatoma cell line (Supplementary Figure 2). These results therefore confirmed that the inhibition of HCVRNA replication by FKBP8 siRNA was due to a specific reduction in the mRNA of FKBP8, but was not due to a nonspecific reduction of any other host mRNA. Figure 5.Decrease in HCV RNA by FKBP8-targeted siRNA. (A) HCV replicon cells (9–13 cells) were transfected with each of three kinds of siRNA targeted to FKBP8 or nontargeted siRNA at a final concentration of 80 nM. Transfected cells were collected at 72 h post-transfection, and FKBP8 mRNA and HCV RNA levels were determined by real-time PCR after being normalized with β-actin mRNA. (B) HCV replicon cells transfected with 80 and 160 nM of Target 1 or nontargeted siRNA were harvested at 72 h post-transfection, and the samples were analyzed by immunoblotting. (C) HCV replicon cells expressing Flag-rFKBP8 mutant (Huh7rFKBP8) or control cells (Huh7c) were transfected with Target 1 (gray bars) or nontargeted (white bars) siRNA at a concentration of 80 nM. Transfected cells were harvested at 72 h post-transfection, and HCV RNA (left) and FKBP8 mRNA (right) were measured by real-time PCR and expressed as % increase after being normalized with the expression of β-actin mRNA. (D) Levels of expression of endogenous FKBP8, exogenous Flag-rFKBP8, and β-actin in the replicon cells after transfection of the siRNAs were determined by immunoblotting using specific antibodies. The data shown in each panel are representative of three independent experiments. Download figure Download PowerPoint To further examine the involvement of FKBP8 on HCV replication, we established a line of Huh7 cells that stably expresses shRNA targeted to FKBP8. Huh7 was transfected with pSilencer 2.1 U6 hygro containing the cDNA of shRNA to FKBP8, and then selection was carried out with hygromycin. FKBP8 was detected in Huh7 cells harboring a control plasmid (Huh7N), whereas decreased expression of FKBP8 was clearly observed in cells expressing the shRNA to FKBP8 (Huh7FKBP8KD) (Figure 6A). In order to examine the effects of the knockdown of FKBP8 on HCV RNA replication, a chimeric HCV RNA containing the Renilla luciferase gene was transfected into these cell lines. Although the chimeric HCV RNA exhibited 5.5 times higher replication than a replication deficient GND mutant RNA in Huh7N, only a doubling of the levels of replication was observed in Huh7FKBP8KD (Figure 6B). Furthermore, HCV RNA containing a neomycin-resistant gene was transfected into the cell lines in order to examine the role played by FKBP8 in HCV RNA replication. The efficiency of colony formation in Huh7N and Huh7FKBP8KD cells with the HCV RNA were 1700 and 23 colonies/μg RNA, respectively (Figure 6C). We also examined the role of FKBP8 on the cell culture system for HCV infection. The siRNA-mediated knockdown of FKBP8 impaired both intracellular viral RNA replication and release of HCV core protein into the culture supernatants (Figure 6D). These results further confirmed that FKBP8 plays a crucial role in the efficient replication of HCV RNA. Figure 6.Effect of knockdown of FKBP8 on the transient replication, colony formation, and viral infection. (A) Levels of expression of FKBP8 and β-actin in Huh7N and Huh7 FKBP8KD cell lines bearing plasmids encoding shRNA for control mRNA (lane 1) and for FKBP8 mRNA (lane 2), respectively. (B) Each cell line was transfected with in vitro-transcribed HCV replicon RNA, pFK-I389 hRL/NS3-3′/NK5.1 (HCVRNA), or a replication-negative mutant, pFK-I389 hRL/NS3-3′/NK5.1GND (HCV/GND). The fold increase in replication was determined by the increase in luciferase activity at 48 h compared with that observed 4 h after standardization, as based on the activity of the replication-deficient HCV/GND replicon. (C) Huh7N and Huh7 FKBP8KD cell lines were transfected with in vitro-transcribed replicon RNA (pFK-I389 neo/NS3-3′/NK5.1) and the cells were incubated for 4 weeks. The remaining cells were fixed with 4% paraformaldehyde and then were stained. (D) Huh7.5.1 cells were transfected with either of siRNA targeted to FKBP8 (Target 1) or nontarget control at a concentration of 80 nM. The cells were inoculated with HCVcc at 24 h after transfection and cells and culture supernatants were harvested every day. Intracellular viral RNA (upper) and HCV core protein in the supernatant (lower) were determined. The data shown in each panel are representative of three independent experiments. Download figure Download PowerPoint FKBP8 forms a multicomplex with NS5A and Hsp90 To identify the cellular proteins that associate with FKBP8, we employed a purification strategy using an MEF affinity tag composed of myc and FLAG tags fused in tandem and separated by a spacer sequence containing a TEV protease cleavage site (myc-TEV-FLAG) (Ichimura et al, 2005). The MEF expression cassette fused with FKBP8 was transfected into 293T cells and the cells were immunoprecipitated. The endogenous FKBP8-binding proteins bound to the Flag beads were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and were then visualized by silver staining. The visible protein bands were excised and determined by a nanoflow LC-MS/MS system. Major protein bands with a molecular size of 94 and 53 kDa were identified as Hsp90 and FKBP8, respectively, although it should be noted that the remaining bands detected in the samples could not be reliably identified (Figure 7A). Figure 7.FKBP8 forms complex with NS5A and Hsp90. (A) An N-terminally myc-TEV-Flag-tagged FKBP8 was expressed in 293T cells and immunoprecipitated. The precipitated proteins were applied to SDS–PAGE and then stained with silver staining. Hsp90 and FKBP8 were identified by LC-MS/MS. (B) HA-Hsp90 was coexpressed with Flag-FKBP8 or Flag-NS5A in 293T cells, and was immunoprecipitated by anti-HA or anti-Flag antibody. Precipitates were analyzed by Western blotting. (C) HA-Hsp90 was coexpressed with EE-FKBP8 and/or Flag-NS5A in 293T cells and was immunoprecipitated with anti-HA antibody. Precipitates were analyzed by Western blotting by anti-EE, -HA or -Flag antibody. Download figure Download PowerPoint In order to elucidate the interaction of Hsp90 with FKBP8 in mammalian cells, Flag-FKBP8 was coexpressed with HA-Hsp90 and immunoprecipitated by anti-Flag or anti-HA antibody. HA-Hsp90 and Flag-FKBP8 were co-precipitated with each other by either of the antibodies but no interaction was observed between HA-Hsp90 and Flag-NS5A (Figure 7B). To examine the interplay among NS5A, FKBP8, and Hsp90, HA-Hsp90 was coexpressed with EE-FKBP8 and/or Flag-NS5A (Figure 7C). Co-immunoprecipitation of Hsp90 and NS5A was clearly detected in the presence but not in the absence of FKBP8. The increase in NS5A expression had no effect on the interaction between FKBP8 and Hsp90 (Supplementary Figure 3). These results suggest that Hsp90 does not directly bind to NS5A but forms complex with NS5A through the interaction with FKBP8. FKBP8 interacts with NS5A and Hsp90 via different sites in the TP" @default.
• W2117084310 created "2016-06-24" @default.
• W2117084310 creator A5013296031 @default.
• W2117084310 creator A5038192407 @default.
• W2117084310 creator A5065367444 @default.
• W2117084310 creator A5075892037 @default.
• W2117084310 creator A5077385450 @default.
• W2117084310 creator A5086372970 @default.
• W2117084310 creator A5087837717 @default.
• W2117084310 creator A5089303528 @default.
• W2117084310 date "2006-10-05" @default.
• W2117084310 modified "2023-10-11" @default.
• W2117084310 title "Hepatitis C virus RNA replication is regulated by FKBP8 and Hsp90" @default.
• W2117084310 cites W1595559221 @default.
• W2117084310 cites W1764690254 @default.
• W2117084310 cites W1781220540 @default.
• W2117084310 cites W1955276614 @default.
• W2117084310 cites W1970605987 @default.
• W2117084310 cites W1971983190 @default.
• W2117084310 cites W1974865581 @default.
• W2117084310 cites W1975356481 @default.
• W2117084310 cites W1980786000 @default.
• W2117084310 cites W1984150038 @default.
• W2117084310 cites W1985648234 @default.
• W2117084310 cites W1992212684 @default.
• W2117084310 cites W1993496957 @default.
• W2117084310 cites W1997198762 @default.
• W2117084310 cites W2003579943 @default.
• W2117084310 cites W2004792969 @default.
• W2117084310 cites W2012808888 @default.
• W2117084310 cites W2015236192 @default.
• W2117084310 cites W2022198259 @default.
• W2117084310 cites W2033125798 @default.
• W2117084310 cites W2041561339 @default.
• W2117084310 cites W2041765591 @default.
• W2117084310 cites W2042412060 @default.
• W2117084310 cites W2044193462 @default.
• W2117084310 cites W2048162805 @default.
• W2117084310 cites W2057318017 @default.
• W2117084310 cites W2062395204 @default.
• W2117084310 cites W2063839793 @default.
• W2117084310 cites W2068128206 @default.
• W2117084310 cites W2079724990 @default.
• W2117084310 cites W2088686584 @default.
• W2117084310 cites W2092106545 @default.
• W2117084310 cites W2097084266 @default.
• W2117084310 cites W2101177020 @default.
• W2117084310 cites W2105891409 @default.
• W2117084310 cites W2107085672 @default.
• W2117084310 cites W2109708832 @default.
• W2117084310 cites W2114579885 @default.
• W2117084310 cites W2115610036 @default.
• W2117084310 cites W2119148710 @default.
• W2117084310 cites W2127685726 @default.
• W2117084310 cites W2132879836 @default.
• W2117084310 cites W2136523601 @default.
• W2117084310 cites W2140990045 @default.
• W2117084310 cites W2141482826 @default.
• W2117084310 cites W2147126871 @default.
• W2117084310 cites W2150639096 @default.
• W2117084310 cites W2151898297 @default.
• W2117084310 cites W2167559643 @default.
• W2117084310 cites W2168307424 @default.
• W2117084310 cites W2745205534 @default.
• W2117084310 cites W404415962 @default.
• W2117084310 doi "https://doi.org/10.1038/sj.emboj.7601367" @default.
• W2117084310 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1679773" @default.
• W2117084310 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17024179" @default.
• W2117084310 hasPublicationYear "2006" @default.
• W2117084310 type Work @default.
• W2117084310 sameAs 2117084310 @default.
• W2117084310 citedByCount "224" @default.
• W2117084310 countsByYear W21170843102012 @default.
• W2117084310 countsByYear W21170843102013 @default.
• W2117084310 countsByYear W21170843102014 @default.
• W2117084310 countsByYear W21170843102015 @default.
• W2117084310 countsByYear W21170843102016 @default.
• W2117084310 countsByYear W21170843102017 @default.
• W2117084310 countsByYear W21170843102018 @default.
• W2117084310 countsByYear W21170843102019 @default.
• W2117084310 countsByYear W21170843102020 @default.
• W2117084310 countsByYear W21170843102021 @default.
• W2117084310 countsByYear W21170843102022 @default.
• W2117084310 countsByYear W21170843102023 @default.
• W2117084310 crossrefType "journal-article" @default.
• W2117084310 hasAuthorship W2117084310A5013296031 @default.
• W2117084310 hasAuthorship W2117084310A5038192407 @default.
• W2117084310 hasAuthorship W2117084310A5065367444 @default.
• W2117084310 hasAuthorship W2117084310A5075892037 @default.
• W2117084310 hasAuthorship W2117084310A5077385450 @default.
• W2117084310 hasAuthorship W2117084310A5086372970 @default.
• W2117084310 hasAuthorship W2117084310A5087837717 @default.
• W2117084310 hasAuthorship W2117084310A5089303528 @default.
• W2117084310 hasBestOaLocation W21170843101 @default.
• W2117084310 hasConcept C104317684 @default.
• W2117084310 hasConcept C12590798 @default.
• W2117084310 hasConcept C140704245 @default.
• W2117084310 hasConcept C159047783 @default.

• next