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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 C-terminal region of CSFV NS5A is critical for the NS5A-GBP1 interaction. (A) Schematic representation of the protein domains of NS5A. Full-length NS5A and two deletion mutants were examined in this study. (B) Expression of full-length and truncated forms of NS5A. The indicated expression plasmids were transfected into HEK293T cells, and cell lysates were analyzed by Western blotting (WB) using antibodies against the Myc tag and GAPDH. (C and D) The C-terminal region of NS5A is required for its binding to GBP1 and GBP1(1-308). The full-length and truncated forms of NS5A were examined by co-IP analysis for interaction with GBP1 (C) or GBP1(1-308) (D). HEK293T cells were transfected with the indicated expression plasmids. Co-IP was performed using an anti-Flag monoclonal antibody (1:1,000). The precipitated proteins were analyzed by Western blotting using antibodies against the Myc and Flag tags.
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Figure 8: The C-terminal region of CSFV NS5A is critical for the NS5A-GBP1 interaction. (A) Schematic representation of the protein domains of NS5A. Full-length NS5A and two deletion mutants were examined in this study. (B) Expression of full-length and truncated forms of NS5A. The indicated expression plasmids were transfected into HEK293T cells, and cell lysates were analyzed by Western blotting (WB) using antibodies against the Myc tag and GAPDH. (C and D) The C-terminal region of NS5A is required for its binding to GBP1 and GBP1(1-308). The full-length and truncated forms of NS5A were examined by co-IP analysis for interaction with GBP1 (C) or GBP1(1-308) (D). HEK293T cells were transfected with the indicated expression plasmids. Co-IP was performed using an anti-Flag monoclonal antibody (1:1,000). The precipitated proteins were analyzed by Western blotting using antibodies against the Myc and Flag tags.

Mentions: To further investigate the region of NS5A required for binding to GBP1, we constructed two plasmids expressing Myc-tagged truncated mutants of NS5A (37) (Fig. 8A and B). HEK293T cells were transfected with expression plasmids, and the interaction of GBP1 with NS5A was determined using a co-IP assay. The results revealed that amino acids 269 to 497 at the C-terminal region of NS5A were essential for the interaction with GBP1 (Fig. 8C). As expected, the co-IP results confirmed that NS5A(269-497) coimmunoprecipitated with GBP1(1-308) (Fig. 8D).


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 C-terminal region of CSFV NS5A is critical for the NS5A-GBP1 interaction. (A) Schematic representation of the protein domains of NS5A. Full-length NS5A and two deletion mutants were examined in this study. (B) Expression of full-length and truncated forms of NS5A. The indicated expression plasmids were transfected into HEK293T cells, and cell lysates were analyzed by Western blotting (WB) using antibodies against the Myc tag and GAPDH. (C and D) The C-terminal region of NS5A is required for its binding to GBP1 and GBP1(1-308). The full-length and truncated forms of NS5A were examined by co-IP analysis for interaction with GBP1 (C) or GBP1(1-308) (D). HEK293T cells were transfected with the indicated expression plasmids. Co-IP was performed using an anti-Flag monoclonal antibody (1:1,000). The precipitated proteins were analyzed by Western blotting using antibodies against the Myc and Flag tags.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 8: The C-terminal region of CSFV NS5A is critical for the NS5A-GBP1 interaction. (A) Schematic representation of the protein domains of NS5A. Full-length NS5A and two deletion mutants were examined in this study. (B) Expression of full-length and truncated forms of NS5A. The indicated expression plasmids were transfected into HEK293T cells, and cell lysates were analyzed by Western blotting (WB) using antibodies against the Myc tag and GAPDH. (C and D) The C-terminal region of NS5A is required for its binding to GBP1 and GBP1(1-308). The full-length and truncated forms of NS5A were examined by co-IP analysis for interaction with GBP1 (C) or GBP1(1-308) (D). HEK293T cells were transfected with the indicated expression plasmids. Co-IP was performed using an anti-Flag monoclonal antibody (1:1,000). The precipitated proteins were analyzed by Western blotting using antibodies against the Myc and Flag tags.
Mentions: To further investigate the region of NS5A required for binding to GBP1, we constructed two plasmids expressing Myc-tagged truncated mutants of NS5A (37) (Fig. 8A and B). HEK293T cells were transfected with expression plasmids, and the interaction of GBP1 with NS5A was determined using a co-IP assay. The results revealed that amino acids 269 to 497 at the C-terminal region of NS5A were essential for the interaction with GBP1 (Fig. 8C). As expected, the co-IP results confirmed that NS5A(269-497) coimmunoprecipitated with GBP1(1-308) (Fig. 8D).

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