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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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Screening of ISGs for the ability to inhibit CSFV infection. (A) Characterization of ISG expression in PK-ISG cells. Lysates of PK-ISG or PK-EGFP cells were analyzed by Western blotting (WB) using a mouse anti-GFP (1:1,000) or anti-GAPDH (1:1,000) antibody. (B) Effects of ISG expression on rCSFV-Fluc infection. PK-ISG and PK-EGFP cells were seeded into 48-well plates at a density of 2 × 105 per well. At 24 h postseeding, cells were infected with rCSFV-Fluc at a multiplicity of infection of 0.1, cultured for an additional 48 h, and assayed for luciferase activity using the luciferase reporter assay system (Promega). RLU, relative light units. As controls, parental PK-15 cells were either left untreated or pretreated with the indicated concentrations of IFN-β for 24 h, infected with rCSFV-Fluc, and assayed for luciferase activity at 48 h postinfection as described above. An RLU below the dashed line indicates that the candidate is a potential anti-CSFV ISG. Error bars represent standard deviations. Each sample was run in triplicate.*, P < 0.05; **, P < 0.01. (C) A cell viability assay was performed on cell lines stably overexpressing ISGs.
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Figure 1: Screening of ISGs for the ability to inhibit CSFV infection. (A) Characterization of ISG expression in PK-ISG cells. Lysates of PK-ISG or PK-EGFP cells were analyzed by Western blotting (WB) using a mouse anti-GFP (1:1,000) or anti-GAPDH (1:1,000) antibody. (B) Effects of ISG expression on rCSFV-Fluc infection. PK-ISG and PK-EGFP cells were seeded into 48-well plates at a density of 2 × 105 per well. At 24 h postseeding, cells were infected with rCSFV-Fluc at a multiplicity of infection of 0.1, cultured for an additional 48 h, and assayed for luciferase activity using the luciferase reporter assay system (Promega). RLU, relative light units. As controls, parental PK-15 cells were either left untreated or pretreated with the indicated concentrations of IFN-β for 24 h, infected with rCSFV-Fluc, and assayed for luciferase activity at 48 h postinfection as described above. An RLU below the dashed line indicates that the candidate is a potential anti-CSFV ISG. Error bars represent standard deviations. Each sample was run in triplicate.*, P < 0.05; **, P < 0.01. (C) A cell viability assay was performed on cell lines stably overexpressing ISGs.

Mentions: To screen ISGs for the ability to inhibit CSFV replication, we initially established stable PK-EGFP or PK-ISG cell lines. Twenty ISGs that are commonly induced by IFN-α/β were chosen for screening (Table 2). The expression of the ISGs with the EGFP tag was detected in the cell lines (Fig. 1A).


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)

Screening of ISGs for the ability to inhibit CSFV infection. (A) Characterization of ISG expression in PK-ISG cells. Lysates of PK-ISG or PK-EGFP cells were analyzed by Western blotting (WB) using a mouse anti-GFP (1:1,000) or anti-GAPDH (1:1,000) antibody. (B) Effects of ISG expression on rCSFV-Fluc infection. PK-ISG and PK-EGFP cells were seeded into 48-well plates at a density of 2 × 105 per well. At 24 h postseeding, cells were infected with rCSFV-Fluc at a multiplicity of infection of 0.1, cultured for an additional 48 h, and assayed for luciferase activity using the luciferase reporter assay system (Promega). RLU, relative light units. As controls, parental PK-15 cells were either left untreated or pretreated with the indicated concentrations of IFN-β for 24 h, infected with rCSFV-Fluc, and assayed for luciferase activity at 48 h postinfection as described above. An RLU below the dashed line indicates that the candidate is a potential anti-CSFV ISG. Error bars represent standard deviations. Each sample was run in triplicate.*, P < 0.05; **, P < 0.01. (C) A cell viability assay was performed on cell lines stably overexpressing ISGs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4836331&req=5

Figure 1: Screening of ISGs for the ability to inhibit CSFV infection. (A) Characterization of ISG expression in PK-ISG cells. Lysates of PK-ISG or PK-EGFP cells were analyzed by Western blotting (WB) using a mouse anti-GFP (1:1,000) or anti-GAPDH (1:1,000) antibody. (B) Effects of ISG expression on rCSFV-Fluc infection. PK-ISG and PK-EGFP cells were seeded into 48-well plates at a density of 2 × 105 per well. At 24 h postseeding, cells were infected with rCSFV-Fluc at a multiplicity of infection of 0.1, cultured for an additional 48 h, and assayed for luciferase activity using the luciferase reporter assay system (Promega). RLU, relative light units. As controls, parental PK-15 cells were either left untreated or pretreated with the indicated concentrations of IFN-β for 24 h, infected with rCSFV-Fluc, and assayed for luciferase activity at 48 h postinfection as described above. An RLU below the dashed line indicates that the candidate is a potential anti-CSFV ISG. Error bars represent standard deviations. Each sample was run in triplicate.*, P < 0.05; **, P < 0.01. (C) A cell viability assay was performed on cell lines stably overexpressing ISGs.
Mentions: To screen ISGs for the ability to inhibit CSFV replication, we initially established stable PK-EGFP or PK-ISG cell lines. Twenty ISGs that are commonly induced by IFN-α/β were chosen for screening (Table 2). The expression of the ISGs with the EGFP tag was detected in the cell lines (Fig. 1A).

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