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Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on Its GTPase Activity.

Li LF, Yu J, Li Y, Wang J, Li S, Zhang L, Xia SL, Yang Q, Wang X, Yu S, Luo Y, Sun Y, Zhu Y, Munir M, Qiu HJ - J. Virol. (2016)

Bottom Line: In addition, we found that GBP1 was upregulated at the transcriptional level in CSFV-infected PK-15 cells and in various organs of CSFV-infected pigs.We showed further that the NS5A-GBP1 interaction inhibited GTPase activity, which was critical for its antiviral effect.The K51 of GBP1, critical for its GTPase activity, is essential for the antiviral action of GBP1 against CSFV replication, and the binding of the NS5A protein to GBP1 antagonizes the GTPase activity and thus the antiviral effect.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.

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The K51 of GBP1 is critical for the NS5A-GBP1 interaction. (A) Schematic representation of the protein domains of GBP1. The GBP1 protein and two deletion mutants of GBP1 are diagramed. (B) Expression of full-length or truncated forms of GBP1. The indicated expression plasmids were transfected into HEK293T cells, and cell lysates were analyzed by Western blotting (WB) using antibodies against the Flag tag and GAPDH. (C) The N-terminal region of GBP1 is required for its binding to NS5A. HEK293T cells were cotransfected with the indicated expression plasmids expressing Flag-tagged full-length or truncated GBP1 constructs. Cell lysates were collected and subjected to co-IP analysis using an anti-Flag MAb (1:1,000). (D) The K51 of GBP1 is required for binding to NS5A. The interaction of NS5A with Flag-tagged wild-type or mutant GBP1 was examined by co-IP using an anti-Myc MAb (1:1,000). (E) NS5A interacts with GBP1, GBP1(1-308), and GBP1(R48P) but not with GBP1(309-591) or GBP1(K51A). HEK293T cells were cotransfected with the indicated expression plasmids, and co-IP was performed using an anti-Myc MAb (1:1,000). The precipitated proteins were analyzed by Western blotting using antibodies against the Flag and Myc tags. (F) Colocalization of GBP1(K51A) with NS5A. Expression plasmids pFlag-GBP1(K51A) and pMyc-NS5A were cotransfected into BHK-21 cells, and a confocal assay was performed.
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Figure 7: The K51 of GBP1 is critical for the NS5A-GBP1 interaction. (A) Schematic representation of the protein domains of GBP1. The GBP1 protein and two deletion mutants of GBP1 are diagramed. (B) Expression of full-length or truncated forms of GBP1. The indicated expression plasmids were transfected into HEK293T cells, and cell lysates were analyzed by Western blotting (WB) using antibodies against the Flag tag and GAPDH. (C) The N-terminal region of GBP1 is required for its binding to NS5A. HEK293T cells were cotransfected with the indicated expression plasmids expressing Flag-tagged full-length or truncated GBP1 constructs. Cell lysates were collected and subjected to co-IP analysis using an anti-Flag MAb (1:1,000). (D) The K51 of GBP1 is required for binding to NS5A. The interaction of NS5A with Flag-tagged wild-type or mutant GBP1 was examined by co-IP using an anti-Myc MAb (1:1,000). (E) NS5A interacts with GBP1, GBP1(1-308), and GBP1(R48P) but not with GBP1(309-591) or GBP1(K51A). HEK293T cells were cotransfected with the indicated expression plasmids, and co-IP was performed using an anti-Myc MAb (1:1,000). The precipitated proteins were analyzed by Western blotting using antibodies against the Flag and Myc tags. (F) Colocalization of GBP1(K51A) with NS5A. Expression plasmids pFlag-GBP1(K51A) and pMyc-NS5A were cotransfected into BHK-21 cells, and a confocal assay was performed.

Mentions: To map the region of GBP1 required for binding to NS5A, we constructed two plasmids expressing Flag-tagged truncated mutants of GBP1, i.e., GBP1(1-308) and GBP1(309-591) (20) (Fig. 7A and B). These plasmids were cotransfected with pMyc-NS5A into HEK293T cells and subjected to the co-IP assay. The results showed that NS5A interacted with GBP1 and GBP1(1-308) but not with GBP1(309-591) (Fig. 7C). These findings indicate that the N-terminal globular GTPase domain of GBP1 is critical for the NS5A-GBP1 interaction.


Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on Its GTPase Activity.

Li LF, Yu J, Li Y, Wang J, Li S, Zhang L, Xia SL, Yang Q, Wang X, Yu S, Luo Y, Sun Y, Zhu Y, Munir M, Qiu HJ - J. Virol. (2016)

The K51 of GBP1 is critical for the NS5A-GBP1 interaction. (A) Schematic representation of the protein domains of GBP1. The GBP1 protein and two deletion mutants of GBP1 are diagramed. (B) Expression of full-length or truncated forms of GBP1. The indicated expression plasmids were transfected into HEK293T cells, and cell lysates were analyzed by Western blotting (WB) using antibodies against the Flag tag and GAPDH. (C) The N-terminal region of GBP1 is required for its binding to NS5A. HEK293T cells were cotransfected with the indicated expression plasmids expressing Flag-tagged full-length or truncated GBP1 constructs. Cell lysates were collected and subjected to co-IP analysis using an anti-Flag MAb (1:1,000). (D) The K51 of GBP1 is required for binding to NS5A. The interaction of NS5A with Flag-tagged wild-type or mutant GBP1 was examined by co-IP using an anti-Myc MAb (1:1,000). (E) NS5A interacts with GBP1, GBP1(1-308), and GBP1(R48P) but not with GBP1(309-591) or GBP1(K51A). HEK293T cells were cotransfected with the indicated expression plasmids, and co-IP was performed using an anti-Myc MAb (1:1,000). The precipitated proteins were analyzed by Western blotting using antibodies against the Flag and Myc tags. (F) Colocalization of GBP1(K51A) with NS5A. Expression plasmids pFlag-GBP1(K51A) and pMyc-NS5A were cotransfected into BHK-21 cells, and a confocal assay was performed.
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Figure 7: The K51 of GBP1 is critical for the NS5A-GBP1 interaction. (A) Schematic representation of the protein domains of GBP1. The GBP1 protein and two deletion mutants of GBP1 are diagramed. (B) Expression of full-length or truncated forms of GBP1. The indicated expression plasmids were transfected into HEK293T cells, and cell lysates were analyzed by Western blotting (WB) using antibodies against the Flag tag and GAPDH. (C) The N-terminal region of GBP1 is required for its binding to NS5A. HEK293T cells were cotransfected with the indicated expression plasmids expressing Flag-tagged full-length or truncated GBP1 constructs. Cell lysates were collected and subjected to co-IP analysis using an anti-Flag MAb (1:1,000). (D) The K51 of GBP1 is required for binding to NS5A. The interaction of NS5A with Flag-tagged wild-type or mutant GBP1 was examined by co-IP using an anti-Myc MAb (1:1,000). (E) NS5A interacts with GBP1, GBP1(1-308), and GBP1(R48P) but not with GBP1(309-591) or GBP1(K51A). HEK293T cells were cotransfected with the indicated expression plasmids, and co-IP was performed using an anti-Myc MAb (1:1,000). The precipitated proteins were analyzed by Western blotting using antibodies against the Flag and Myc tags. (F) Colocalization of GBP1(K51A) with NS5A. Expression plasmids pFlag-GBP1(K51A) and pMyc-NS5A were cotransfected into BHK-21 cells, and a confocal assay was performed.
Mentions: To map the region of GBP1 required for binding to NS5A, we constructed two plasmids expressing Flag-tagged truncated mutants of GBP1, i.e., GBP1(1-308) and GBP1(309-591) (20) (Fig. 7A and B). These plasmids were cotransfected with pMyc-NS5A into HEK293T cells and subjected to the co-IP assay. The results showed that NS5A interacted with GBP1 and GBP1(1-308) but not with GBP1(309-591) (Fig. 7C). These findings indicate that the N-terminal globular GTPase domain of GBP1 is critical for the NS5A-GBP1 interaction.

Bottom Line: In addition, we found that GBP1 was upregulated at the transcriptional level in CSFV-infected PK-15 cells and in various organs of CSFV-infected pigs.We showed further that the NS5A-GBP1 interaction inhibited GTPase activity, which was critical for its antiviral effect.The K51 of GBP1, critical for its GTPase activity, is essential for the antiviral action of GBP1 against CSFV replication, and the binding of the NS5A protein to GBP1 antagonizes the GTPase activity and thus the antiviral effect.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.

Show MeSH
Related in: MedlinePlus