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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 FKBP6 knockdown on HCV replication.(a) Huh7OK1 cells infected with HCVcc (JFH-1) were transfected with siFKBP8 and/or siFKBP6 at 10 nM. Culture supernatants were harvested from HCVcc-infected cells at the indicated times after transfection with 10 nM siRNAs. Viral RNA was estimated by qRT-PCR, normalized with the value of GAPDH mRNA and is presented as a percent of the control. (b) Replicon O cells were transfected with siFKBP6 or siFKBP8 at a final concentration of 10 nM and incubated for 16 h. The resulting cells were transfected again with an empty plasmid or a plasmid encoding FKBP8 or FKBP6, further incubated for 56 h and then harvested in order to measure luciferase activity and cell viability. The values obtained were standardized to those of the control cells. The amount of total transfected siRNA was adjusted with siControl in the absence of one of the siRNAs targeting FKBPs. Asterisks indicate a significant difference versus the control value (*P < 0.05). The data shown in this figure are representative of three independent experiments. (c) Cell lysates were prepared from Huh7OK1 cells (WT), FKBP6KO cells and FKBP6KO Huh7OK1 cells expressing FLAG-FKBP6 (FKBP6 + FKBP6KO), and were subjected to immunoblotting. (d) A colony formation assay was performed. Colony formation efficiency (colony number per μg of transfected subgenomic RNA) is shown under each panel. (e) Endogenous FKBP6 and NS5A in the infected cells were stained with the first antibodies and the secondary fluorescent antibodies, and observed under a confocal microscope at 400 times magnification. (f) Endogenous FKBP6 and double-stranded RNA (dsRNA) in the infected cells were stained with a goat anti-FKBP6 antibody and mouse anti-dsRNA antibody, followed by staining with an AF594-conjugated donkey antibody to goat IgG and an AF488-conjugated goat antibody to mouse IgG, respectively. The secondary antibody to goat IgG was reacted first in order to avoid a cross-reaction. The localization of the stained proteins was analyzed by confocal laser scanning microscopy. The stained samples were observed under a confocal microscope at 600 times magnification.
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f2: Effects of FKBP6 knockdown on HCV replication.(a) Huh7OK1 cells infected with HCVcc (JFH-1) were transfected with siFKBP8 and/or siFKBP6 at 10 nM. Culture supernatants were harvested from HCVcc-infected cells at the indicated times after transfection with 10 nM siRNAs. Viral RNA was estimated by qRT-PCR, normalized with the value of GAPDH mRNA and is presented as a percent of the control. (b) Replicon O cells were transfected with siFKBP6 or siFKBP8 at a final concentration of 10 nM and incubated for 16 h. The resulting cells were transfected again with an empty plasmid or a plasmid encoding FKBP8 or FKBP6, further incubated for 56 h and then harvested in order to measure luciferase activity and cell viability. The values obtained were standardized to those of the control cells. The amount of total transfected siRNA was adjusted with siControl in the absence of one of the siRNAs targeting FKBPs. Asterisks indicate a significant difference versus the control value (*P < 0.05). The data shown in this figure are representative of three independent experiments. (c) Cell lysates were prepared from Huh7OK1 cells (WT), FKBP6KO cells and FKBP6KO Huh7OK1 cells expressing FLAG-FKBP6 (FKBP6 + FKBP6KO), and were subjected to immunoblotting. (d) A colony formation assay was performed. Colony formation efficiency (colony number per μg of transfected subgenomic RNA) is shown under each panel. (e) Endogenous FKBP6 and NS5A in the infected cells were stained with the first antibodies and the secondary fluorescent antibodies, and observed under a confocal microscope at 400 times magnification. (f) Endogenous FKBP6 and double-stranded RNA (dsRNA) in the infected cells were stained with a goat anti-FKBP6 antibody and mouse anti-dsRNA antibody, followed by staining with an AF594-conjugated donkey antibody to goat IgG and an AF488-conjugated goat antibody to mouse IgG, respectively. The secondary antibody to goat IgG was reacted first in order to avoid a cross-reaction. The localization of the stained proteins was analyzed by confocal laser scanning microscopy. The stained samples were observed under a confocal microscope at 600 times magnification.

Mentions: We examined the effects of FKBP6 on HCV infection. siRNA targeting FKBP6 (siFKBP6) or FKBP8 (siFKBP8) was transfected to Huh7 OK1 infected with HCVcc derived from the genotype 2a JFH1 strain. Culture supernatants of the transfected cells were harvested 24, 48, and 72 h post-transfection. Viral RNA in the culture supernatants was estimated by qRT-PCR. Viral production was impaired in JFH-1-infected Huh7OK1 by transfection with siFKBP6, siFKBP8, or both (Fig. 2a, and Supplementary Figure 9). The knockdown of FKBP6 also impaired replication of the subgenomic replicon at a level similar to the knockdown of FKBP8 (Fig. 2b). Knockout of the FKBP6 gene in the Huh7OK1 cell line was achieved using the CRISPR/Cas9 system for the colony formation assay. The deleted region in each allele was determined by direct sequencing as described in the Materials and Methods section. FKBP6 was weakly expressed in Huh7OK1 cells, but not in FKBP-knockout (FKBP6KO) Huh7OK1 cells, while FKBP8 was expressed at a similar level in Huh7OK1 and FKBP6KO cells (Fig. 2c). FLAG-FKBP6 was highly expressed in FKBP6KO Huh7OK1 cells expressing FLAG-FKBP6, but did not affect the expression of FKBP8 (Fig. 2c). The synthesized replicon RNA, carrying a neomycin resistance gene, was introduced into Huh-7OK1, FKBP6KO, or FLAG-FKBP6-expressing FKBP6KO cells. The resulting transfected cells were cultivated in the presence of 0.4 mg/ml G418 for 4 weeks. The remaining cell colonies were fixed and stained with crystal violet. The replicon RNA yielded 2.5 × 105 colonies per μg of the transfected RNA in the Huh7OK1 cell line, but no colony in the FKBP6KO cell line (Fig. 2d). Furthermore, the colony formation efficiency of FKBP6KO cells expressing FLAG-FKBP6 was 1.4 × 105 colonies per μg of the transfected RNA (Fig. 2d). Huh7 OK1 and FKBPKO Huh7OK1 cells were infected with HCVcc and then harvested at 4 days post-infections. Intracellular HCV RNA was 100 times decreased by the knockout of FKBP6 gene (Supplementary Figure 10). Endogenous FKBP6 was mainly localized in the cytoplasm as dot-like structures and co-localized with NS5A in Huh7 OK1 cells infected with HCVcc (Fig. 2e). FKBP6 was also co-localized with double-stranded RNA (dsRNA), the detection of which is a hallmark of viral replication, in the infected cells as dot-like structures (Fig. 2f). These findings suggest that FKBP6 is essential for HCV replication.


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 FKBP6 knockdown on HCV replication.(a) Huh7OK1 cells infected with HCVcc (JFH-1) were transfected with siFKBP8 and/or siFKBP6 at 10 nM. Culture supernatants were harvested from HCVcc-infected cells at the indicated times after transfection with 10 nM siRNAs. Viral RNA was estimated by qRT-PCR, normalized with the value of GAPDH mRNA and is presented as a percent of the control. (b) Replicon O cells were transfected with siFKBP6 or siFKBP8 at a final concentration of 10 nM and incubated for 16 h. The resulting cells were transfected again with an empty plasmid or a plasmid encoding FKBP8 or FKBP6, further incubated for 56 h and then harvested in order to measure luciferase activity and cell viability. The values obtained were standardized to those of the control cells. The amount of total transfected siRNA was adjusted with siControl in the absence of one of the siRNAs targeting FKBPs. Asterisks indicate a significant difference versus the control value (*P < 0.05). The data shown in this figure are representative of three independent experiments. (c) Cell lysates were prepared from Huh7OK1 cells (WT), FKBP6KO cells and FKBP6KO Huh7OK1 cells expressing FLAG-FKBP6 (FKBP6 + FKBP6KO), and were subjected to immunoblotting. (d) A colony formation assay was performed. Colony formation efficiency (colony number per μg of transfected subgenomic RNA) is shown under each panel. (e) Endogenous FKBP6 and NS5A in the infected cells were stained with the first antibodies and the secondary fluorescent antibodies, and observed under a confocal microscope at 400 times magnification. (f) Endogenous FKBP6 and double-stranded RNA (dsRNA) in the infected cells were stained with a goat anti-FKBP6 antibody and mouse anti-dsRNA antibody, followed by staining with an AF594-conjugated donkey antibody to goat IgG and an AF488-conjugated goat antibody to mouse IgG, respectively. The secondary antibody to goat IgG was reacted first in order to avoid a cross-reaction. The localization of the stained proteins was analyzed by confocal laser scanning microscopy. The stained samples were observed under a confocal microscope at 600 times magnification.
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f2: Effects of FKBP6 knockdown on HCV replication.(a) Huh7OK1 cells infected with HCVcc (JFH-1) were transfected with siFKBP8 and/or siFKBP6 at 10 nM. Culture supernatants were harvested from HCVcc-infected cells at the indicated times after transfection with 10 nM siRNAs. Viral RNA was estimated by qRT-PCR, normalized with the value of GAPDH mRNA and is presented as a percent of the control. (b) Replicon O cells were transfected with siFKBP6 or siFKBP8 at a final concentration of 10 nM and incubated for 16 h. The resulting cells were transfected again with an empty plasmid or a plasmid encoding FKBP8 or FKBP6, further incubated for 56 h and then harvested in order to measure luciferase activity and cell viability. The values obtained were standardized to those of the control cells. The amount of total transfected siRNA was adjusted with siControl in the absence of one of the siRNAs targeting FKBPs. Asterisks indicate a significant difference versus the control value (*P < 0.05). The data shown in this figure are representative of three independent experiments. (c) Cell lysates were prepared from Huh7OK1 cells (WT), FKBP6KO cells and FKBP6KO Huh7OK1 cells expressing FLAG-FKBP6 (FKBP6 + FKBP6KO), and were subjected to immunoblotting. (d) A colony formation assay was performed. Colony formation efficiency (colony number per μg of transfected subgenomic RNA) is shown under each panel. (e) Endogenous FKBP6 and NS5A in the infected cells were stained with the first antibodies and the secondary fluorescent antibodies, and observed under a confocal microscope at 400 times magnification. (f) Endogenous FKBP6 and double-stranded RNA (dsRNA) in the infected cells were stained with a goat anti-FKBP6 antibody and mouse anti-dsRNA antibody, followed by staining with an AF594-conjugated donkey antibody to goat IgG and an AF488-conjugated goat antibody to mouse IgG, respectively. The secondary antibody to goat IgG was reacted first in order to avoid a cross-reaction. The localization of the stained proteins was analyzed by confocal laser scanning microscopy. The stained samples were observed under a confocal microscope at 600 times magnification.
Mentions: We examined the effects of FKBP6 on HCV infection. siRNA targeting FKBP6 (siFKBP6) or FKBP8 (siFKBP8) was transfected to Huh7 OK1 infected with HCVcc derived from the genotype 2a JFH1 strain. Culture supernatants of the transfected cells were harvested 24, 48, and 72 h post-transfection. Viral RNA in the culture supernatants was estimated by qRT-PCR. Viral production was impaired in JFH-1-infected Huh7OK1 by transfection with siFKBP6, siFKBP8, or both (Fig. 2a, and Supplementary Figure 9). The knockdown of FKBP6 also impaired replication of the subgenomic replicon at a level similar to the knockdown of FKBP8 (Fig. 2b). Knockout of the FKBP6 gene in the Huh7OK1 cell line was achieved using the CRISPR/Cas9 system for the colony formation assay. The deleted region in each allele was determined by direct sequencing as described in the Materials and Methods section. FKBP6 was weakly expressed in Huh7OK1 cells, but not in FKBP-knockout (FKBP6KO) Huh7OK1 cells, while FKBP8 was expressed at a similar level in Huh7OK1 and FKBP6KO cells (Fig. 2c). FLAG-FKBP6 was highly expressed in FKBP6KO Huh7OK1 cells expressing FLAG-FKBP6, but did not affect the expression of FKBP8 (Fig. 2c). The synthesized replicon RNA, carrying a neomycin resistance gene, was introduced into Huh-7OK1, FKBP6KO, or FLAG-FKBP6-expressing FKBP6KO cells. The resulting transfected cells were cultivated in the presence of 0.4 mg/ml G418 for 4 weeks. The remaining cell colonies were fixed and stained with crystal violet. The replicon RNA yielded 2.5 × 105 colonies per μg of the transfected RNA in the Huh7OK1 cell line, but no colony in the FKBP6KO cell line (Fig. 2d). Furthermore, the colony formation efficiency of FKBP6KO cells expressing FLAG-FKBP6 was 1.4 × 105 colonies per μg of the transfected RNA (Fig. 2d). Huh7 OK1 and FKBPKO Huh7OK1 cells were infected with HCVcc and then harvested at 4 days post-infections. Intracellular HCV RNA was 100 times decreased by the knockout of FKBP6 gene (Supplementary Figure 10). Endogenous FKBP6 was mainly localized in the cytoplasm as dot-like structures and co-localized with NS5A in Huh7 OK1 cells infected with HCVcc (Fig. 2e). FKBP6 was also co-localized with double-stranded RNA (dsRNA), the detection of which is a hallmark of viral replication, in the infected cells as dot-like structures (Fig. 2f). These findings suggest that FKBP6 is essential for HCV replication.

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