Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on 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.
Affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.Show MeSH
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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).
Affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.