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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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CSFV NS5A interacts with GBP1. (A to D) CSFV NS5A interacts with GBP1. HEK293T cells were cotransfected with pFlag-GBP1 and either pMyc-NS5A, pMyc-NS5B, or pCMV-Myc (pMyc-EV). The cell lysate was harvested. (A and B) Co-IP was performed using an anti-Flag MAb (1:1,000). The precipitated proteins were analyzed by Western blotting (WB) using antibodies against the Myc and Flag tags (A) and the HA and Flag tags (B). (C) For the GST pulldown assay, GST and the GST-GBP1 fusion protein expressed in E. coli BL21 were purified with glutathione resin. The resin was incubated with Myc-NS5A. The bound proteins were determined by Western blotting using a mouse anti-GST PAb (1:2,000) and an anti-Myc MAb (1:1,000). (D) For the endogenous co-IP assay, PK-15 cells were pretreated with IFN-β, infected with CSFV strain Shimen, and subjected to co-IP using an anti-GBP1 MAb (1:1,000). (E) The NS5A-GBP1 interaction is independent of RNA. HEK293T cells were cotransfected with pFlag-GBP1 and pMyc-NS5A. The cell lysate was collected and treated with RNase A. Co-IP was performed using an anti-Flag MAb (1:1,000). (F and G) Colocalization of GBP1 with NS5A. (F) Expression plasmids pFlag-GBP1 and pMyc-NS5A were cotransfected into BHK-21 cells and subjected to a confocal assay. (G) PK-15 cells were pretreated with IFN-β, infected with CSFV strain Shimen, and subjected to a confocal assay. The distribution and colocalization of GBP1 and NS5A were examined using a Leica SP2 confocal system.
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Figure 6: CSFV NS5A interacts with GBP1. (A to D) CSFV NS5A interacts with GBP1. HEK293T cells were cotransfected with pFlag-GBP1 and either pMyc-NS5A, pMyc-NS5B, or pCMV-Myc (pMyc-EV). The cell lysate was harvested. (A and B) Co-IP was performed using an anti-Flag MAb (1:1,000). The precipitated proteins were analyzed by Western blotting (WB) using antibodies against the Myc and Flag tags (A) and the HA and Flag tags (B). (C) For the GST pulldown assay, GST and the GST-GBP1 fusion protein expressed in E. coli BL21 were purified with glutathione resin. The resin was incubated with Myc-NS5A. The bound proteins were determined by Western blotting using a mouse anti-GST PAb (1:2,000) and an anti-Myc MAb (1:1,000). (D) For the endogenous co-IP assay, PK-15 cells were pretreated with IFN-β, infected with CSFV strain Shimen, and subjected to co-IP using an anti-GBP1 MAb (1:1,000). (E) The NS5A-GBP1 interaction is independent of RNA. HEK293T cells were cotransfected with pFlag-GBP1 and pMyc-NS5A. The cell lysate was collected and treated with RNase A. Co-IP was performed using an anti-Flag MAb (1:1,000). (F and G) Colocalization of GBP1 with NS5A. (F) Expression plasmids pFlag-GBP1 and pMyc-NS5A were cotransfected into BHK-21 cells and subjected to a confocal assay. (G) PK-15 cells were pretreated with IFN-β, infected with CSFV strain Shimen, and subjected to a confocal assay. The distribution and colocalization of GBP1 and NS5A were examined using a Leica SP2 confocal system.

Mentions: It has been reported that various viral nonstructural proteins interact with cellular proteins to evade immune responses. For example, the replicase proteins NS5B of HCV (20) and NS1 of IAV (36) interact with hGBP1. CSFV NS5A and NS5B are main components of the viral replicase complex. Hence, the question of whether CSFV NS5A or NS5B protein interacts with GBP1 to evade its antiviral activity was investigated using co-IP assays. The results showed that Flag-tagged GBP1 interacted with Myc-tagged NS5A but not with Myc-tagged NS5B after incubation with an anti-Flag MAb and protein G-agarose (Fig. 6A). Furthermore, Flag-tagged NS5A was shown to coimmunoprecipitate with hemagglutinin (HA)-tagged GBP1 after incubation with an anti-Flag MAb and protein G-agarose (Fig. 6B). To further confirm the interaction between NS5A and GBP1, endogenous co-IP and GST pulldown assays were performed. The results showed that GST-GBP1 but not GST alone interacted with NS5A (Fig. 6C) and that endogenous GBP1 interacted with CSFV-produced NS5A (Fig. 6D). To preclude nonspecific interaction mediated by RNA, the cell lysates were treated with RNase A prior to co-IP. The co-IP results confirmed that the interaction of GBP1 and NS5A was independent of RNA (Fig. 6E).


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)

CSFV NS5A interacts with GBP1. (A to D) CSFV NS5A interacts with GBP1. HEK293T cells were cotransfected with pFlag-GBP1 and either pMyc-NS5A, pMyc-NS5B, or pCMV-Myc (pMyc-EV). The cell lysate was harvested. (A and B) Co-IP was performed using an anti-Flag MAb (1:1,000). The precipitated proteins were analyzed by Western blotting (WB) using antibodies against the Myc and Flag tags (A) and the HA and Flag tags (B). (C) For the GST pulldown assay, GST and the GST-GBP1 fusion protein expressed in E. coli BL21 were purified with glutathione resin. The resin was incubated with Myc-NS5A. The bound proteins were determined by Western blotting using a mouse anti-GST PAb (1:2,000) and an anti-Myc MAb (1:1,000). (D) For the endogenous co-IP assay, PK-15 cells were pretreated with IFN-β, infected with CSFV strain Shimen, and subjected to co-IP using an anti-GBP1 MAb (1:1,000). (E) The NS5A-GBP1 interaction is independent of RNA. HEK293T cells were cotransfected with pFlag-GBP1 and pMyc-NS5A. The cell lysate was collected and treated with RNase A. Co-IP was performed using an anti-Flag MAb (1:1,000). (F and G) Colocalization of GBP1 with NS5A. (F) Expression plasmids pFlag-GBP1 and pMyc-NS5A were cotransfected into BHK-21 cells and subjected to a confocal assay. (G) PK-15 cells were pretreated with IFN-β, infected with CSFV strain Shimen, and subjected to a confocal assay. The distribution and colocalization of GBP1 and NS5A were examined using a Leica SP2 confocal system.
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Figure 6: CSFV NS5A interacts with GBP1. (A to D) CSFV NS5A interacts with GBP1. HEK293T cells were cotransfected with pFlag-GBP1 and either pMyc-NS5A, pMyc-NS5B, or pCMV-Myc (pMyc-EV). The cell lysate was harvested. (A and B) Co-IP was performed using an anti-Flag MAb (1:1,000). The precipitated proteins were analyzed by Western blotting (WB) using antibodies against the Myc and Flag tags (A) and the HA and Flag tags (B). (C) For the GST pulldown assay, GST and the GST-GBP1 fusion protein expressed in E. coli BL21 were purified with glutathione resin. The resin was incubated with Myc-NS5A. The bound proteins were determined by Western blotting using a mouse anti-GST PAb (1:2,000) and an anti-Myc MAb (1:1,000). (D) For the endogenous co-IP assay, PK-15 cells were pretreated with IFN-β, infected with CSFV strain Shimen, and subjected to co-IP using an anti-GBP1 MAb (1:1,000). (E) The NS5A-GBP1 interaction is independent of RNA. HEK293T cells were cotransfected with pFlag-GBP1 and pMyc-NS5A. The cell lysate was collected and treated with RNase A. Co-IP was performed using an anti-Flag MAb (1:1,000). (F and G) Colocalization of GBP1 with NS5A. (F) Expression plasmids pFlag-GBP1 and pMyc-NS5A were cotransfected into BHK-21 cells and subjected to a confocal assay. (G) PK-15 cells were pretreated with IFN-β, infected with CSFV strain Shimen, and subjected to a confocal assay. The distribution and colocalization of GBP1 and NS5A were examined using a Leica SP2 confocal system.
Mentions: It has been reported that various viral nonstructural proteins interact with cellular proteins to evade immune responses. For example, the replicase proteins NS5B of HCV (20) and NS1 of IAV (36) interact with hGBP1. CSFV NS5A and NS5B are main components of the viral replicase complex. Hence, the question of whether CSFV NS5A or NS5B protein interacts with GBP1 to evade its antiviral activity was investigated using co-IP assays. The results showed that Flag-tagged GBP1 interacted with Myc-tagged NS5A but not with Myc-tagged NS5B after incubation with an anti-Flag MAb and protein G-agarose (Fig. 6A). Furthermore, Flag-tagged NS5A was shown to coimmunoprecipitate with hemagglutinin (HA)-tagged GBP1 after incubation with an anti-Flag MAb and protein G-agarose (Fig. 6B). To further confirm the interaction between NS5A and GBP1, endogenous co-IP and GST pulldown assays were performed. The results showed that GST-GBP1 but not GST alone interacted with NS5A (Fig. 6C) and that endogenous GBP1 interacted with CSFV-produced NS5A (Fig. 6D). To preclude nonspecific interaction mediated by RNA, the cell lysates were treated with RNase A prior to co-IP. The co-IP results confirmed that the interaction of GBP1 and NS5A was independent of RNA (Fig. 6E).

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