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Involvement of FKBP6 in hepatitis C virus replication.

Kasai H, Kawakami K, Yokoe H, Yoshimura K, Matsuda M, Yasumoto J, Maekawa S, Yamashita A, Tanaka T, Ikeda M, Kato N, Okamoto T, Matsuura Y, Sakamoto N, Enomoto N, Takeda S, Fujii H, Tsubuki M, Kusunoki M, Moriishi K - Sci Rep (2015)

Bottom Line: A treatment with the FKBP8 inhibitor N-(N', N'-dimethylcarboxamidomethyl)cycloheximide impaired the formation of a homo- or hetero-complex consisting of FKBP6 and/or FKBP8, and suppressed HCV replication.HCV infection promoted the expression of FKBP6, but not that of FKBP8, in cultured cells and human liver tissue.These results indicate that FKBP6 is an HCV-induced host factor that supports viral replication in cooperation with NS5A.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Medicine, University of Yamanashi, Chuo-shi, Yamanashi 409-3898, Japan.

ABSTRACT
The chaperone system is known to be exploited by viruses for their replication. In the present study, we identified the cochaperone FKBP6 as a host factor required for hepatitis C virus (HCV) replication. FKBP6 is a peptidyl prolyl cis-trans isomerase with three domains of the tetratricopeptide repeat (TPR), but lacks FK-506 binding ability. FKBP6 interacted with HCV nonstructural protein 5A (NS5A) and also formed a complex with FKBP6 itself or FKBP8, which is known to be critical for HCV replication. The Val(121) of NS5A and TPR domains of FKBP6 were responsible for the interaction between NS5A and FKBP6. FKBP6 was colocalized with NS5A, FKBP8, and double-stranded RNA in HCV-infected cells. HCV replication was completely suppressed in FKBP6-knockout hepatoma cell lines, while the expression of FKBP6 restored HCV replication in FKBP6-knockout cells. A treatment with the FKBP8 inhibitor N-(N', N'-dimethylcarboxamidomethyl)cycloheximide impaired the formation of a homo- or hetero-complex consisting of FKBP6 and/or FKBP8, and suppressed HCV replication. HCV infection promoted the expression of FKBP6, but not that of FKBP8, in cultured cells and human liver tissue. These results indicate that FKBP6 is an HCV-induced host factor that supports viral replication in cooperation with NS5A.

No MeSH data available.


Related in: MedlinePlus

Effects of HCV replication on FKBP6 expression.(a) The stained cells used in Fig. 2e were observed at 200 times magnification using the fluorescence microscope BZ-9000 (Keyence, Osaka, Japan), which is not a confocal microscope. (b) Naïve and HCVcc-infected Huh7OK1 cells were harvested and then subjected to immunoblotting using antibodies to FKBP6, FKBP8, NS5A, and beta-actin. (c) FKBP6, FKBP8, and GAPDH mRNAs were estimated by qRT-PCR in naïve and HCVcc-infected cells, as described for the cells used in (b). The values obtained for FKBP6 and FKBP8 mRNAs were normalized with that of GAPDH mRNA and are presented as levels relative to the control (mock). Asterisks indicate a significant difference from the control value (*P < 0.05). (d) Human non-cancerous liver tissues (no. 1, 2, and 3) were obtained from three different liver samples of one donor. The HCV RNAs and mRNAs of FKBP6 and FKBP8 were estimated by qRT-PCR, normalized with GAPDH mRNA, and are presented as values relative to tissue no. 1. ND means “not detected”. The data shown in this figure are representative of three independent experiments.
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f6: Effects of HCV replication on FKBP6 expression.(a) The stained cells used in Fig. 2e were observed at 200 times magnification using the fluorescence microscope BZ-9000 (Keyence, Osaka, Japan), which is not a confocal microscope. (b) Naïve and HCVcc-infected Huh7OK1 cells were harvested and then subjected to immunoblotting using antibodies to FKBP6, FKBP8, NS5A, and beta-actin. (c) FKBP6, FKBP8, and GAPDH mRNAs were estimated by qRT-PCR in naïve and HCVcc-infected cells, as described for the cells used in (b). The values obtained for FKBP6 and FKBP8 mRNAs were normalized with that of GAPDH mRNA and are presented as levels relative to the control (mock). Asterisks indicate a significant difference from the control value (*P < 0.05). (d) Human non-cancerous liver tissues (no. 1, 2, and 3) were obtained from three different liver samples of one donor. The HCV RNAs and mRNAs of FKBP6 and FKBP8 were estimated by qRT-PCR, normalized with GAPDH mRNA, and are presented as values relative to tissue no. 1. ND means “not detected”. The data shown in this figure are representative of three independent experiments.

Mentions: Infected cells (NS5A-positive cells) were more potently stained with an anti-FKBP6 antibody than HCV-negative cells in the same field of view (Fig. 6a). Thus, we examined the effects of HCV infection on the expression of FKBP6 in cultured cells and the human livers. FKBP6 was more highly expressed at the protein and mRNA levels in HCVcc-infected cells than in non-infected cells (mock), whereas the expression of FKBP8 remained unchanged in HCV-infected cells and mock controls (Fig. 6a,c). Furthermore, a treatment with daclatasvir successfully remove HCV form HCV-infected cells and decreased the amount of FKBP6, corresponding to downregulation of HCV (Supplementary Figure 13). In human non-cancerous liver, FKBP6 was expressed at a significantly higher level in HCV-positive tissue than in HCV-negative tissue (Fig. 6d), whereas FKBP8 was not promoted more in HCV-positive tissue than in HCV-negative tissue (Fig. 6d). The data using additional liver samples also showed HCV-dependent upregulation of FKBP6 (Supplementary Figure 14). These results suggest that the expression of FKBP6 is promoted ex vivo and in vivo by HCV infection.


Involvement of FKBP6 in hepatitis C virus replication.

Kasai H, Kawakami K, Yokoe H, Yoshimura K, Matsuda M, Yasumoto J, Maekawa S, Yamashita A, Tanaka T, Ikeda M, Kato N, Okamoto T, Matsuura Y, Sakamoto N, Enomoto N, Takeda S, Fujii H, Tsubuki M, Kusunoki M, Moriishi K - Sci Rep (2015)

Effects of HCV replication on FKBP6 expression.(a) The stained cells used in Fig. 2e were observed at 200 times magnification using the fluorescence microscope BZ-9000 (Keyence, Osaka, Japan), which is not a confocal microscope. (b) Naïve and HCVcc-infected Huh7OK1 cells were harvested and then subjected to immunoblotting using antibodies to FKBP6, FKBP8, NS5A, and beta-actin. (c) FKBP6, FKBP8, and GAPDH mRNAs were estimated by qRT-PCR in naïve and HCVcc-infected cells, as described for the cells used in (b). The values obtained for FKBP6 and FKBP8 mRNAs were normalized with that of GAPDH mRNA and are presented as levels relative to the control (mock). Asterisks indicate a significant difference from the control value (*P < 0.05). (d) Human non-cancerous liver tissues (no. 1, 2, and 3) were obtained from three different liver samples of one donor. The HCV RNAs and mRNAs of FKBP6 and FKBP8 were estimated by qRT-PCR, normalized with GAPDH mRNA, and are presented as values relative to tissue no. 1. ND means “not detected”. The data shown in this figure are representative of three independent experiments.
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Related In: Results  -  Collection

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f6: Effects of HCV replication on FKBP6 expression.(a) The stained cells used in Fig. 2e were observed at 200 times magnification using the fluorescence microscope BZ-9000 (Keyence, Osaka, Japan), which is not a confocal microscope. (b) Naïve and HCVcc-infected Huh7OK1 cells were harvested and then subjected to immunoblotting using antibodies to FKBP6, FKBP8, NS5A, and beta-actin. (c) FKBP6, FKBP8, and GAPDH mRNAs were estimated by qRT-PCR in naïve and HCVcc-infected cells, as described for the cells used in (b). The values obtained for FKBP6 and FKBP8 mRNAs were normalized with that of GAPDH mRNA and are presented as levels relative to the control (mock). Asterisks indicate a significant difference from the control value (*P < 0.05). (d) Human non-cancerous liver tissues (no. 1, 2, and 3) were obtained from three different liver samples of one donor. The HCV RNAs and mRNAs of FKBP6 and FKBP8 were estimated by qRT-PCR, normalized with GAPDH mRNA, and are presented as values relative to tissue no. 1. ND means “not detected”. The data shown in this figure are representative of three independent experiments.
Mentions: Infected cells (NS5A-positive cells) were more potently stained with an anti-FKBP6 antibody than HCV-negative cells in the same field of view (Fig. 6a). Thus, we examined the effects of HCV infection on the expression of FKBP6 in cultured cells and the human livers. FKBP6 was more highly expressed at the protein and mRNA levels in HCVcc-infected cells than in non-infected cells (mock), whereas the expression of FKBP8 remained unchanged in HCV-infected cells and mock controls (Fig. 6a,c). Furthermore, a treatment with daclatasvir successfully remove HCV form HCV-infected cells and decreased the amount of FKBP6, corresponding to downregulation of HCV (Supplementary Figure 13). In human non-cancerous liver, FKBP6 was expressed at a significantly higher level in HCV-positive tissue than in HCV-negative tissue (Fig. 6d), whereas FKBP8 was not promoted more in HCV-positive tissue than in HCV-negative tissue (Fig. 6d). The data using additional liver samples also showed HCV-dependent upregulation of FKBP6 (Supplementary Figure 14). These results suggest that the expression of FKBP6 is promoted ex vivo and in vivo by HCV infection.

Bottom Line: A treatment with the FKBP8 inhibitor N-(N', N'-dimethylcarboxamidomethyl)cycloheximide impaired the formation of a homo- or hetero-complex consisting of FKBP6 and/or FKBP8, and suppressed HCV replication.HCV infection promoted the expression of FKBP6, but not that of FKBP8, in cultured cells and human liver tissue.These results indicate that FKBP6 is an HCV-induced host factor that supports viral replication in cooperation with NS5A.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Medicine, University of Yamanashi, Chuo-shi, Yamanashi 409-3898, Japan.

ABSTRACT
The chaperone system is known to be exploited by viruses for their replication. In the present study, we identified the cochaperone FKBP6 as a host factor required for hepatitis C virus (HCV) replication. FKBP6 is a peptidyl prolyl cis-trans isomerase with three domains of the tetratricopeptide repeat (TPR), but lacks FK-506 binding ability. FKBP6 interacted with HCV nonstructural protein 5A (NS5A) and also formed a complex with FKBP6 itself or FKBP8, which is known to be critical for HCV replication. The Val(121) of NS5A and TPR domains of FKBP6 were responsible for the interaction between NS5A and FKBP6. FKBP6 was colocalized with NS5A, FKBP8, and double-stranded RNA in HCV-infected cells. HCV replication was completely suppressed in FKBP6-knockout hepatoma cell lines, while the expression of FKBP6 restored HCV replication in FKBP6-knockout cells. A treatment with the FKBP8 inhibitor N-(N', N'-dimethylcarboxamidomethyl)cycloheximide impaired the formation of a homo- or hetero-complex consisting of FKBP6 and/or FKBP8, and suppressed HCV replication. HCV infection promoted the expression of FKBP6, but not that of FKBP8, in cultured cells and human liver tissue. These results indicate that FKBP6 is an HCV-induced host factor that supports viral replication in cooperation with NS5A.

No MeSH data available.


Related in: MedlinePlus