Screening Of Interferon-stimulated Genes Against Classical Swine Fever Virus And Uncovering Of The Antiviral Mechanism Of Guanylate-binding Protein 1

Many viruses trigger the type I interferon(IFN) signaling pathway upon infection, which relies on the ability of host to recognize pathogen associated molecular patterns through specific pattern recognition receptors, resulting in the transcription of hundreds of interferon-stimulated genes(ISGs), and define the antiviral state of the host. At same time, many viruses have evolved numerous strategies to counter and evade host’s antiviral responses, such as inhibiting IFN synthesis, blocking IFN-activated signaling pathway and binding and inactivating secreted IFN molecular. ISGs are considered as the important interferon-inducible antiviral effectors, which play critical roles in defense and clearance of exogenous pathogens. Therefore, characterization of antiviral ISGs is necessary for the control and treatment of viral infections.Classical swine fever(CSF) is a highly contagious viral disease endangering the pig industry in China and other regions of world. Classical swine fever virus(CSFV), the causative agent of CSF, belongs to the genus Pestivirus of the family Flaviviridae. The type I IFN pathway is triggered and the expression of ISGs is induced upon CSFV infection. However, compared with other members of Flaviviridae, the anti-CSFV ISGs are poorly documented, especially on the antiviral molecular mechanisms of ISGs. Here, we screened 20 swine ISGs that are commonly induced by type I IFNs against CSFV in lentivirus-delivered PK-15 cell lines, and successfully screened 6 ISGs that significantly inhibited rCSFV-Fluc replication, which are GBP1, GBP2, CD47, ZNF313, OASL and OAS1. Overexpression of GBP1, CD47, ZNF313 and OASL inhibited CSFV Shimen strain replication, which indicates that those four ISGs are potent anti-CSFV ISGs. We selected the guanylate-binding protein 1(GBP1) for further study. We observed that overexpression of GBP1 remarkably suppressed CSFV replication, whereas knocking down the endogenous GBP1 expression by small interfering RNAs significantly promoted CSFV growth. Furthermore, we demonstrated that GBP1 acted mainly upon the early phase of CSFV replication and inhibited the translation efficiency of the internal ribosome entry site of CSFV in a dose-dependent manner. 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. For the molecular mechanism of GBP1 against CSFV, we showed that GBP1 did not activate IFN-β and NF-κB signaling pathway, which was dependent on its GTPase activity. However, we also found that the CSFV NS5 A protein interacted with GBP1, the NS5A-GBP1 interaction inhibited GTPase activity of GBP1, which was critical for its antiviral effect. Interestingly, the K51 of GBP1, which is crucial for its GTPase activity, was essential for the inhibition of CSFV replication. Coimmunoprecipitation and GST pulldown assays revealed that the NS5A-GBP1 interaction was mapped in the N-terminal globular GTPase domain of GBP1 and the C-terminal domain of NS5 A.In conclusion, six anti-CSFV candidates were identified by the high-throughput screening method based on the reporter-viruses bind to overexpressing cell lines in this study, and GBP1 is an anti-CSFV ISG, of which antiviral activity depends on its GTPase activity, while binding of the CSFV-NS5 A to GBP1 counters the antiviral effect through inhibition of GTPase activity.