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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 FKBP8 knockdown on the replication of replicons derived from O and N strains.(a) HCV replicon cell lines were transfected with siFKBP8 (closed squares, solid lines) or non-targeted siRNA siControl (open circles, broken lines) at a final concentration of 10 nM. Transfected cells were incubated in medium lacking G418 and then collected at the indicated time. Luciferase activity was evaluated and then normalized with the cell number (upper panels). Cell viability was measured by the method described in the Materials and Methods section (lower panels). Asterisks indicate a significant difference from the control value (*P < 0.05). (b) Cell lysates prepared from cells harvested 72 h post-transfection were then subjected to Western blotting. Original blots are shown in Supplementary Figure 8. (c) The cells were transfected with siRNAs at 10 nM each and then incubated for 72 h. Luciferase activity and cell viability were measured in the O replicon cell line (upper) and N replicon cell line (lower) transfected with siFKBP6 and/or siFKBP8. The values obtained were standardized with that of the control and are presented as a percentage of the control (siControl). (d) The transcriptional levels of FKBP8 (upper) and FKBP6 (lower) were measured by qRT-PCR. The values of FKBP mRNAs were normalized with that of GAPDH mRNA and are presented as a percent of the control of the N replicon cells. Asterisks indicate a significant difference from the control value (*P < 0.05).
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f5: Effects of FKBP8 knockdown on the replication of replicons derived from O and N strains.(a) HCV replicon cell lines were transfected with siFKBP8 (closed squares, solid lines) or non-targeted siRNA siControl (open circles, broken lines) at a final concentration of 10 nM. Transfected cells were incubated in medium lacking G418 and then collected at the indicated time. Luciferase activity was evaluated and then normalized with the cell number (upper panels). Cell viability was measured by the method described in the Materials and Methods section (lower panels). Asterisks indicate a significant difference from the control value (*P < 0.05). (b) Cell lysates prepared from cells harvested 72 h post-transfection were then subjected to Western blotting. Original blots are shown in Supplementary Figure 8. (c) The cells were transfected with siRNAs at 10 nM each and then incubated for 72 h. Luciferase activity and cell viability were measured in the O replicon cell line (upper) and N replicon cell line (lower) transfected with siFKBP6 and/or siFKBP8. The values obtained were standardized with that of the control and are presented as a percentage of the control (siControl). (d) The transcriptional levels of FKBP8 (upper) and FKBP6 (lower) were measured by qRT-PCR. The values of FKBP mRNAs were normalized with that of GAPDH mRNA and are presented as a percent of the control of the N replicon cells. Asterisks indicate a significant difference from the control value (*P < 0.05).

Mentions: The siRNA targeting FKBP8 (siFKBP8) was transfected into two subgenomic replicon cell lines derived from the O and N strains, which had not been examined for the influence of the knockdown of FKBP8. The transfected replicon cell lines were harvested 24 h, 48 h, and 96 h post-transfection. Since both replicon RNAs encode a chimeric protein consisting of luciferase and neomycin phosphotransferase1718, luciferase activity corresponds to HCV replication. Luciferase activity decreased in a time-dependent manner in the cell line harboring subgenomic replicon RNA derived from strain O, designated the O replicon cell line in this study (Fig. 5a, upper left). However, luciferase activity was not impaired by the knockdown of FKBP8 in the cell line harboring subgenomic replicon RNA derived from strain N, designated as the N replicon cell line (Fig. 5a, upper right). The knockdown of FKBP8 did not alter the cell viability of either cell line from that of cells transfected with control siRNA (Fig. 5a, lower panels). Endogenous FKBP8 was reduced by the knockdown of FKBP8 in both cell lines (Fig. 5b). The knockdown of FKBP8 reduced the HCV proteins, NS3 and NS5B, in the O replicon cell line, but not in the N replicon cell line (Fig. 5b), suggesting that the down-regulation of luciferase activity is due to impaired viral replication. The viral RNA region encoding NS5A was amplified from the total cDNA of the N replicon cell line, and we determined the nucleotide sequence by direct sequencing. The K20, V34, N272, I306, V375, and S405 of NS5A deduced from the original database of the viral genome of strain N (GenBank accession No. AF139594) were replaced with R, A, T, V, A, and P, respectively, in NS5A amplified from the N replicon cell line. Val121 was conserved in NS5A proteins derived from the N replicon cell line as well as the original genome sequence. FLAG-NS5A derived from the N replicon cell line was immunoprecipitated with HA-FKBP8 using an anti-FLAG antibody, and HA-FKBP8 was also precipitated with FLAG-NS5A using an anti-HA antibody (Fig. 1b).


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 FKBP8 knockdown on the replication of replicons derived from O and N strains.(a) HCV replicon cell lines were transfected with siFKBP8 (closed squares, solid lines) or non-targeted siRNA siControl (open circles, broken lines) at a final concentration of 10 nM. Transfected cells were incubated in medium lacking G418 and then collected at the indicated time. Luciferase activity was evaluated and then normalized with the cell number (upper panels). Cell viability was measured by the method described in the Materials and Methods section (lower panels). Asterisks indicate a significant difference from the control value (*P < 0.05). (b) Cell lysates prepared from cells harvested 72 h post-transfection were then subjected to Western blotting. Original blots are shown in Supplementary Figure 8. (c) The cells were transfected with siRNAs at 10 nM each and then incubated for 72 h. Luciferase activity and cell viability were measured in the O replicon cell line (upper) and N replicon cell line (lower) transfected with siFKBP6 and/or siFKBP8. The values obtained were standardized with that of the control and are presented as a percentage of the control (siControl). (d) The transcriptional levels of FKBP8 (upper) and FKBP6 (lower) were measured by qRT-PCR. The values of FKBP mRNAs were normalized with that of GAPDH mRNA and are presented as a percent of the control of the N replicon cells. Asterisks indicate a significant difference from the control value (*P < 0.05).
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f5: Effects of FKBP8 knockdown on the replication of replicons derived from O and N strains.(a) HCV replicon cell lines were transfected with siFKBP8 (closed squares, solid lines) or non-targeted siRNA siControl (open circles, broken lines) at a final concentration of 10 nM. Transfected cells were incubated in medium lacking G418 and then collected at the indicated time. Luciferase activity was evaluated and then normalized with the cell number (upper panels). Cell viability was measured by the method described in the Materials and Methods section (lower panels). Asterisks indicate a significant difference from the control value (*P < 0.05). (b) Cell lysates prepared from cells harvested 72 h post-transfection were then subjected to Western blotting. Original blots are shown in Supplementary Figure 8. (c) The cells were transfected with siRNAs at 10 nM each and then incubated for 72 h. Luciferase activity and cell viability were measured in the O replicon cell line (upper) and N replicon cell line (lower) transfected with siFKBP6 and/or siFKBP8. The values obtained were standardized with that of the control and are presented as a percentage of the control (siControl). (d) The transcriptional levels of FKBP8 (upper) and FKBP6 (lower) were measured by qRT-PCR. The values of FKBP mRNAs were normalized with that of GAPDH mRNA and are presented as a percent of the control of the N replicon cells. Asterisks indicate a significant difference from the control value (*P < 0.05).
Mentions: The siRNA targeting FKBP8 (siFKBP8) was transfected into two subgenomic replicon cell lines derived from the O and N strains, which had not been examined for the influence of the knockdown of FKBP8. The transfected replicon cell lines were harvested 24 h, 48 h, and 96 h post-transfection. Since both replicon RNAs encode a chimeric protein consisting of luciferase and neomycin phosphotransferase1718, luciferase activity corresponds to HCV replication. Luciferase activity decreased in a time-dependent manner in the cell line harboring subgenomic replicon RNA derived from strain O, designated the O replicon cell line in this study (Fig. 5a, upper left). However, luciferase activity was not impaired by the knockdown of FKBP8 in the cell line harboring subgenomic replicon RNA derived from strain N, designated as the N replicon cell line (Fig. 5a, upper right). The knockdown of FKBP8 did not alter the cell viability of either cell line from that of cells transfected with control siRNA (Fig. 5a, lower panels). Endogenous FKBP8 was reduced by the knockdown of FKBP8 in both cell lines (Fig. 5b). The knockdown of FKBP8 reduced the HCV proteins, NS3 and NS5B, in the O replicon cell line, but not in the N replicon cell line (Fig. 5b), suggesting that the down-regulation of luciferase activity is due to impaired viral replication. The viral RNA region encoding NS5A was amplified from the total cDNA of the N replicon cell line, and we determined the nucleotide sequence by direct sequencing. The K20, V34, N272, I306, V375, and S405 of NS5A deduced from the original database of the viral genome of strain N (GenBank accession No. AF139594) were replaced with R, A, T, V, A, and P, respectively, in NS5A amplified from the N replicon cell line. Val121 was conserved in NS5A proteins derived from the N replicon cell line as well as the original genome sequence. FLAG-NS5A derived from the N replicon cell line was immunoprecipitated with HA-FKBP8 using an anti-FLAG antibody, and HA-FKBP8 was also precipitated with FLAG-NS5A using an anti-HA antibody (Fig. 1b).

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