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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 required for the inhibition of CSFV replication. (A) HEK293T cells were transfected with pFlag-GBP1, pFlag-GBP1(R48P), pFlag-GBP1(K51A), or pCMV-Flag (Flag-EV) and were harvested at 36 h posttransfection. GTPase activity was measured by an enzyme-linked inorganic phosphate assay (ELIPA). (B) Influence of GBP1(K51A) on rCSFV-Fluc replication. PK-GBP1, PK-GBP1(R48P), PK-GBP1(K51A), and PK-EGFP cells were first infected with rCSFV-Fluc at a multiplicity of infection of 0.1 for 48 h and then assayed for luciferase activity using the dual-luciferase reporter assay system (Promega). Error bars represent standard deviations. NS, not significant; *, P < 0.05; **, P < 0.01. RLU, relative light units. (C and D) Influence of GBP1(K51A) on CSFV strain Shimen replication. The cell lines were infected with Shimen at an MOI of 0.1 for 48 h. (C) The viral titers in the supernatants collected at 24 and 48 h postinfection were examined by an immunofluorescence assay and are presented as median tissue culture infective doses (TCID50) per milliliter. (D) The number of genomic copies of CSFV in PK-GBP1(K51A) cells was determined using a quantitative real-time reverse transcription-PCR assay. Each sample was run in triplicate. (E) Cell viability assay of cell lines stably overexpressing wild-type or mutant GBP1.
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Figure 5: The K51 of GBP1 is required for the inhibition of CSFV replication. (A) HEK293T cells were transfected with pFlag-GBP1, pFlag-GBP1(R48P), pFlag-GBP1(K51A), or pCMV-Flag (Flag-EV) and were harvested at 36 h posttransfection. GTPase activity was measured by an enzyme-linked inorganic phosphate assay (ELIPA). (B) Influence of GBP1(K51A) on rCSFV-Fluc replication. PK-GBP1, PK-GBP1(R48P), PK-GBP1(K51A), and PK-EGFP cells were first infected with rCSFV-Fluc at a multiplicity of infection of 0.1 for 48 h and then assayed for luciferase activity using the dual-luciferase reporter assay system (Promega). Error bars represent standard deviations. NS, not significant; *, P < 0.05; **, P < 0.01. RLU, relative light units. (C and D) Influence of GBP1(K51A) on CSFV strain Shimen replication. The cell lines were infected with Shimen at an MOI of 0.1 for 48 h. (C) The viral titers in the supernatants collected at 24 and 48 h postinfection were examined by an immunofluorescence assay and are presented as median tissue culture infective doses (TCID50) per milliliter. (D) The number of genomic copies of CSFV in PK-GBP1(K51A) cells was determined using a quantitative real-time reverse transcription-PCR assay. Each sample was run in triplicate. (E) Cell viability assay of cell lines stably overexpressing wild-type or mutant GBP1.

Mentions: It has been reported that the GTPase activity of GBP1 is necessary for its antiviral actions against some viruses, including HCV (20) and IAV (36). Considering that GBP1 exerts an antiviral activity independent of the type I IFN signaling pathway, we hypothesized that its anti-CSFV activity probably depends on its GTPase activity. Since the K51 and R48 of hGBP1 are essential for its GTPase activity (20, 36), we constructed two plasmids expressing two mutants of GBP1, i.e., GBP1(R48P) and GBP1(K51A). To analyze the enzymatic functions of GBP1(K51A) and GBP1(R48P), GTPase activity was examined using ELIPA. GTPase activity was significantly higher in GBP1-expressing cells than in empty vector-transfected cells. The GBP1(K51A) mutant had no GTPase activity, while GBP1(R48P) showed a lower level of GTPase activity than wild-type GBP1 (Fig. 5A). These data suggest that the K51 of GBP1 is crucial for its GTPase activity.


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 required for the inhibition of CSFV replication. (A) HEK293T cells were transfected with pFlag-GBP1, pFlag-GBP1(R48P), pFlag-GBP1(K51A), or pCMV-Flag (Flag-EV) and were harvested at 36 h posttransfection. GTPase activity was measured by an enzyme-linked inorganic phosphate assay (ELIPA). (B) Influence of GBP1(K51A) on rCSFV-Fluc replication. PK-GBP1, PK-GBP1(R48P), PK-GBP1(K51A), and PK-EGFP cells were first infected with rCSFV-Fluc at a multiplicity of infection of 0.1 for 48 h and then assayed for luciferase activity using the dual-luciferase reporter assay system (Promega). Error bars represent standard deviations. NS, not significant; *, P < 0.05; **, P < 0.01. RLU, relative light units. (C and D) Influence of GBP1(K51A) on CSFV strain Shimen replication. The cell lines were infected with Shimen at an MOI of 0.1 for 48 h. (C) The viral titers in the supernatants collected at 24 and 48 h postinfection were examined by an immunofluorescence assay and are presented as median tissue culture infective doses (TCID50) per milliliter. (D) The number of genomic copies of CSFV in PK-GBP1(K51A) cells was determined using a quantitative real-time reverse transcription-PCR assay. Each sample was run in triplicate. (E) Cell viability assay of cell lines stably overexpressing wild-type or mutant GBP1.
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Figure 5: The K51 of GBP1 is required for the inhibition of CSFV replication. (A) HEK293T cells were transfected with pFlag-GBP1, pFlag-GBP1(R48P), pFlag-GBP1(K51A), or pCMV-Flag (Flag-EV) and were harvested at 36 h posttransfection. GTPase activity was measured by an enzyme-linked inorganic phosphate assay (ELIPA). (B) Influence of GBP1(K51A) on rCSFV-Fluc replication. PK-GBP1, PK-GBP1(R48P), PK-GBP1(K51A), and PK-EGFP cells were first infected with rCSFV-Fluc at a multiplicity of infection of 0.1 for 48 h and then assayed for luciferase activity using the dual-luciferase reporter assay system (Promega). Error bars represent standard deviations. NS, not significant; *, P < 0.05; **, P < 0.01. RLU, relative light units. (C and D) Influence of GBP1(K51A) on CSFV strain Shimen replication. The cell lines were infected with Shimen at an MOI of 0.1 for 48 h. (C) The viral titers in the supernatants collected at 24 and 48 h postinfection were examined by an immunofluorescence assay and are presented as median tissue culture infective doses (TCID50) per milliliter. (D) The number of genomic copies of CSFV in PK-GBP1(K51A) cells was determined using a quantitative real-time reverse transcription-PCR assay. Each sample was run in triplicate. (E) Cell viability assay of cell lines stably overexpressing wild-type or mutant GBP1.
Mentions: It has been reported that the GTPase activity of GBP1 is necessary for its antiviral actions against some viruses, including HCV (20) and IAV (36). Considering that GBP1 exerts an antiviral activity independent of the type I IFN signaling pathway, we hypothesized that its anti-CSFV activity probably depends on its GTPase activity. Since the K51 and R48 of hGBP1 are essential for its GTPase activity (20, 36), we constructed two plasmids expressing two mutants of GBP1, i.e., GBP1(R48P) and GBP1(K51A). To analyze the enzymatic functions of GBP1(K51A) and GBP1(R48P), GTPase activity was examined using ELIPA. GTPase activity was significantly higher in GBP1-expressing cells than in empty vector-transfected cells. The GBP1(K51A) mutant had no GTPase activity, while GBP1(R48P) showed a lower level of GTPase activity than wild-type GBP1 (Fig. 5A). These data suggest that the K51 of GBP1 is crucial for its GTPase activity.

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