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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 CSFV NS5A protein antagonizes the antiviral activity of GBP1 by inhibiting its GTPase activity. (A and B) Expression levels of GBP1 in transfected cells. HEK293T cells were transfected with the indicated plasmids and collected at 48 hpt. The expression level of GBP1 was detected by Western blotting (WB) (A) or quantitative real-time reverse transcription-PCR (B). Error bars represent standard deviations. (C) Effects of NS5A on the GTPase activity of GBP1. HEK293T cells were cotransfected with the indicated plasmids and harvested at 36 hpt. GTPase activity was measured by an enzyme-linked inorganic phosphate assay (ELIPA), and the expression of the indicated proteins was verified by Western blotting using antibodies against the Myc tag, the Flag tag, or GAPDH. (D and E) Influence of NS5A overexpression on CSFV in PK-GBP1 cells. PK-GBP1 or PK-EGFP cells were transfected with pMyc-NS5A, pMyc-NS5A(1-268), pMyc-NS5A(269-497), or pCMV-Myc (pMyc-EV). At 24 hpt, the transfected cells were infected with rCSFV-Fluc or CSFV strain Shimen at a multiplicity of infection of 0.1. The cells were collected at 48 h postinfection for analysis of luciferase activity using a luciferase reporter assay system (Promega) (D) and for virus titration using an immunofluorescence assay (E). RLU, relative light units. Each sample was run in triplicate. *, P < 0.05; **, P < 0.01.
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Figure 9: The CSFV NS5A protein antagonizes the antiviral activity of GBP1 by inhibiting its GTPase activity. (A and B) Expression levels of GBP1 in transfected cells. HEK293T cells were transfected with the indicated plasmids and collected at 48 hpt. The expression level of GBP1 was detected by Western blotting (WB) (A) or quantitative real-time reverse transcription-PCR (B). Error bars represent standard deviations. (C) Effects of NS5A on the GTPase activity of GBP1. HEK293T cells were cotransfected with the indicated plasmids and harvested at 36 hpt. GTPase activity was measured by an enzyme-linked inorganic phosphate assay (ELIPA), and the expression of the indicated proteins was verified by Western blotting using antibodies against the Myc tag, the Flag tag, or GAPDH. (D and E) Influence of NS5A overexpression on CSFV in PK-GBP1 cells. PK-GBP1 or PK-EGFP cells were transfected with pMyc-NS5A, pMyc-NS5A(1-268), pMyc-NS5A(269-497), or pCMV-Myc (pMyc-EV). At 24 hpt, the transfected cells were infected with rCSFV-Fluc or CSFV strain Shimen at a multiplicity of infection of 0.1. The cells were collected at 48 h postinfection for analysis of luciferase activity using a luciferase reporter assay system (Promega) (D) and for virus titration using an immunofluorescence assay (E). RLU, relative light units. Each sample was run in triplicate. *, P < 0.05; **, P < 0.01.

Mentions: To determine whether NS5A affects GBP1 expression, pFlag-GBP1 and different amounts of pMyc-NS5A were cotransfected into HEK293T cells. The results showed that NS5A did not influence the expression of GBP1 (Fig. 9A and B).


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 CSFV NS5A protein antagonizes the antiviral activity of GBP1 by inhibiting its GTPase activity. (A and B) Expression levels of GBP1 in transfected cells. HEK293T cells were transfected with the indicated plasmids and collected at 48 hpt. The expression level of GBP1 was detected by Western blotting (WB) (A) or quantitative real-time reverse transcription-PCR (B). Error bars represent standard deviations. (C) Effects of NS5A on the GTPase activity of GBP1. HEK293T cells were cotransfected with the indicated plasmids and harvested at 36 hpt. GTPase activity was measured by an enzyme-linked inorganic phosphate assay (ELIPA), and the expression of the indicated proteins was verified by Western blotting using antibodies against the Myc tag, the Flag tag, or GAPDH. (D and E) Influence of NS5A overexpression on CSFV in PK-GBP1 cells. PK-GBP1 or PK-EGFP cells were transfected with pMyc-NS5A, pMyc-NS5A(1-268), pMyc-NS5A(269-497), or pCMV-Myc (pMyc-EV). At 24 hpt, the transfected cells were infected with rCSFV-Fluc or CSFV strain Shimen at a multiplicity of infection of 0.1. The cells were collected at 48 h postinfection for analysis of luciferase activity using a luciferase reporter assay system (Promega) (D) and for virus titration using an immunofluorescence assay (E). RLU, relative light units. Each sample was run in triplicate. *, P < 0.05; **, P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 9: The CSFV NS5A protein antagonizes the antiviral activity of GBP1 by inhibiting its GTPase activity. (A and B) Expression levels of GBP1 in transfected cells. HEK293T cells were transfected with the indicated plasmids and collected at 48 hpt. The expression level of GBP1 was detected by Western blotting (WB) (A) or quantitative real-time reverse transcription-PCR (B). Error bars represent standard deviations. (C) Effects of NS5A on the GTPase activity of GBP1. HEK293T cells were cotransfected with the indicated plasmids and harvested at 36 hpt. GTPase activity was measured by an enzyme-linked inorganic phosphate assay (ELIPA), and the expression of the indicated proteins was verified by Western blotting using antibodies against the Myc tag, the Flag tag, or GAPDH. (D and E) Influence of NS5A overexpression on CSFV in PK-GBP1 cells. PK-GBP1 or PK-EGFP cells were transfected with pMyc-NS5A, pMyc-NS5A(1-268), pMyc-NS5A(269-497), or pCMV-Myc (pMyc-EV). At 24 hpt, the transfected cells were infected with rCSFV-Fluc or CSFV strain Shimen at a multiplicity of infection of 0.1. The cells were collected at 48 h postinfection for analysis of luciferase activity using a luciferase reporter assay system (Promega) (D) and for virus titration using an immunofluorescence assay (E). RLU, relative light units. Each sample was run in triplicate. *, P < 0.05; **, P < 0.01.
Mentions: To determine whether NS5A affects GBP1 expression, pFlag-GBP1 and different amounts of pMyc-NS5A were cotransfected into HEK293T cells. The results showed that NS5A did not influence the expression of GBP1 (Fig. 9A and B).

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