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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

The TPR domain of FKBP6 and Val121 of NS5A are critical for their interaction.(a) The region spanning from the 1st to 125th positions of NS5A (HA-NS5A125wt) or a mutant in which Val was replaced with Ala at the 121st position (HA-NS5A125V121A) was expressed with FLAG-FKBP6 and then subjected to immunoprecipitation. Original blots are shown in Supplementary Figure 4. (b) Schematic diagram of the C-terminal deletion mutants of FKBP6 used in (c). (c) HA-tagged C-terminal deletion mutants of FKBP6 were expressed with FLAG-NS5A in 293T cells. The transfected cells were subjected to immunoprecipitation. Original blots are shown in Supplementary Figure 5. Asterisks indicate nonspecific bands. The results are representative of three independent assays.
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f3: The TPR domain of FKBP6 and Val121 of NS5A are critical for their interaction.(a) The region spanning from the 1st to 125th positions of NS5A (HA-NS5A125wt) or a mutant in which Val was replaced with Ala at the 121st position (HA-NS5A125V121A) was expressed with FLAG-FKBP6 and then subjected to immunoprecipitation. Original blots are shown in Supplementary Figure 4. (b) Schematic diagram of the C-terminal deletion mutants of FKBP6 used in (c). (c) HA-tagged C-terminal deletion mutants of FKBP6 were expressed with FLAG-NS5A in 293T cells. The transfected cells were subjected to immunoprecipitation. Original blots are shown in Supplementary Figure 5. Asterisks indicate nonspecific bands. The results are representative of three independent assays.

Mentions: Our previous findings suggested that domain I of NS5A (1–213) interacts with FKBP88,12. Further mutational analyses of NS5A in our previous study suggest that the Val121 of NS5A is critical for the binding of NS5A’s N-terminal portion to FKBP8 and for viral replication12. We then investigated the role of Val121 of NS5A in binding to FKBP6. FLAG-FKBP6 was expressed with an N-terminally HA-tagged NS5A region spanning from amino acid residue 1 to 125 (HA-NS5A125wt) or with HA-NS5A125, the Val of which was replaced with Ala at the 121st position (HA-NS5AV121A). FLAG-FKBP6 was precipitated with HA-NS5A125wt, but not HA-NS5AV121A using an anti-FLAG antibody (Fig. 3a). Our previous findings suggest that the TPR domains of FKBP8 are required for the interaction between FKBP8 and NS5A8. C-terminal deletion mutants of FKBP6 were used in the present study (Fig. 3b) as follows: HA-dTPR1 was deficient in the C-terminal last TPR domain, HA-dTPR2 lacked the last two C-terminal TPR domains, and HA-TPR3 lacked all TPR domains. FLAG-NS5A was precipitated with wild-type FKBP6, dTPR1, and dTPR2 using an anti-FLAG antibody (Fig. 3c), while HA-dTPR2 was co-precipitated at a lower level than the wild type and HA-dTPR1 (Fig. 3c). In the reverse experiment, HA-FKBP6 wild type, HA-dTPR1, and HA-dTPR2 were each precipitated with FLAG-NS5A using an anti-HA antibody. HA-dTPR1 mainly precipitated FLAG-NS5A with a greater molecular weight than FLAG-NS5A precipitated with HA-FKBP6 wild-type, while FLAG-NS5A was precipitated with HA-dTPR2 at a lower level than with the wild type and dTPR1 (Fig. 3c). FKBP6 may regulate the phosphorylation state of NS5A in cooperation with Hsp90. FLAG-NS5A was not precipitated with HA-dTPR3 using an anti-FLAG antibody, and the interaction between FLAG-NS5A and HA-dTPR3 was not found in the reverse immunoprecipitation test (Fig. 3c). Furthermore, the overexpression of HA-FKBP6, but not that of HA-dTPR3, partially recovered HCV replication in FKBP6-knockdown replicon cells (Supplementary Figure 11). These results suggest that the Val121 of NS5A and complete TPR domains of FKBP6 are required for the full interaction between NS5A and FKBP6.


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)

The TPR domain of FKBP6 and Val121 of NS5A are critical for their interaction.(a) The region spanning from the 1st to 125th positions of NS5A (HA-NS5A125wt) or a mutant in which Val was replaced with Ala at the 121st position (HA-NS5A125V121A) was expressed with FLAG-FKBP6 and then subjected to immunoprecipitation. Original blots are shown in Supplementary Figure 4. (b) Schematic diagram of the C-terminal deletion mutants of FKBP6 used in (c). (c) HA-tagged C-terminal deletion mutants of FKBP6 were expressed with FLAG-NS5A in 293T cells. The transfected cells were subjected to immunoprecipitation. Original blots are shown in Supplementary Figure 5. Asterisks indicate nonspecific bands. The results are representative of three independent assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f3: The TPR domain of FKBP6 and Val121 of NS5A are critical for their interaction.(a) The region spanning from the 1st to 125th positions of NS5A (HA-NS5A125wt) or a mutant in which Val was replaced with Ala at the 121st position (HA-NS5A125V121A) was expressed with FLAG-FKBP6 and then subjected to immunoprecipitation. Original blots are shown in Supplementary Figure 4. (b) Schematic diagram of the C-terminal deletion mutants of FKBP6 used in (c). (c) HA-tagged C-terminal deletion mutants of FKBP6 were expressed with FLAG-NS5A in 293T cells. The transfected cells were subjected to immunoprecipitation. Original blots are shown in Supplementary Figure 5. Asterisks indicate nonspecific bands. The results are representative of three independent assays.
Mentions: Our previous findings suggested that domain I of NS5A (1–213) interacts with FKBP88,12. Further mutational analyses of NS5A in our previous study suggest that the Val121 of NS5A is critical for the binding of NS5A’s N-terminal portion to FKBP8 and for viral replication12. We then investigated the role of Val121 of NS5A in binding to FKBP6. FLAG-FKBP6 was expressed with an N-terminally HA-tagged NS5A region spanning from amino acid residue 1 to 125 (HA-NS5A125wt) or with HA-NS5A125, the Val of which was replaced with Ala at the 121st position (HA-NS5AV121A). FLAG-FKBP6 was precipitated with HA-NS5A125wt, but not HA-NS5AV121A using an anti-FLAG antibody (Fig. 3a). Our previous findings suggest that the TPR domains of FKBP8 are required for the interaction between FKBP8 and NS5A8. C-terminal deletion mutants of FKBP6 were used in the present study (Fig. 3b) as follows: HA-dTPR1 was deficient in the C-terminal last TPR domain, HA-dTPR2 lacked the last two C-terminal TPR domains, and HA-TPR3 lacked all TPR domains. FLAG-NS5A was precipitated with wild-type FKBP6, dTPR1, and dTPR2 using an anti-FLAG antibody (Fig. 3c), while HA-dTPR2 was co-precipitated at a lower level than the wild type and HA-dTPR1 (Fig. 3c). In the reverse experiment, HA-FKBP6 wild type, HA-dTPR1, and HA-dTPR2 were each precipitated with FLAG-NS5A using an anti-HA antibody. HA-dTPR1 mainly precipitated FLAG-NS5A with a greater molecular weight than FLAG-NS5A precipitated with HA-FKBP6 wild-type, while FLAG-NS5A was precipitated with HA-dTPR2 at a lower level than with the wild type and dTPR1 (Fig. 3c). FKBP6 may regulate the phosphorylation state of NS5A in cooperation with Hsp90. FLAG-NS5A was not precipitated with HA-dTPR3 using an anti-FLAG antibody, and the interaction between FLAG-NS5A and HA-dTPR3 was not found in the reverse immunoprecipitation test (Fig. 3c). Furthermore, the overexpression of HA-FKBP6, but not that of HA-dTPR3, partially recovered HCV replication in FKBP6-knockdown replicon cells (Supplementary Figure 11). These results suggest that the Val121 of NS5A and complete TPR domains of FKBP6 are required for the full interaction between NS5A and FKBP6.

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