Abstract
BackgroundRetinoic acid-inducible gene I (RIG-I) is a key cytosolic receptor of the innate immune system. Seneca valley virus (SVV) is a newly emerging RNA virus that infects pigs causing significant economic losses in pig industry. RIG-I plays different roles during different viruses infections. The role of RIG-I in SVV-infected cells remains unknown. Understanding of the role of RIG-I during SVV infection will help to clarify the infection process of SVV in the infected cells.MethodsIn this study, we generated a RIG-I knockout (KO) porcine kidney PK-15 cell line using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome editing tool. The RIG-I gene sequence of RIG-I KO cells were determined by Sanger sequencing method, and the expression of RIG-I protein in the RIG-I KO cells were detected by Western bloting. The activation status of type I interferon pathway in Sendai virus (SeV)- or SVV-infected RIG-I KO cells was investigated by measuring the mRNA expression levels of interferon (IFN)-β and IFN-stimulated genes (ISGs). The replicative state of SVV in the RIG-I KO cells was evaluated by qPCR, Western bloting, TCID50 assay and indirect immunofluorescence assay.ResultsGene editing of RIG-I in PK-15 cells successfully resulted in the destruction of RIG-I expression. RIG-I KO PK-15 cells had a lower expression of IFN-β and ISGs compared with wildtype (WT) PK-15 cells when stimulated by the model RNA virus SeV. The amounts of viral RNA and viral protein as well as viral yields in SVV-infected RIG-I WT and KO cells were determined and compared, which showed that knockout of RIG-I significantly increased SVV replication and propagation. Meanwhile, the expression of IFN-β and ISGs were considerably decreased in RIG-I KO cells compared with that in RIG-I WT cells during SVV infection.ConclusionAltogether, this study indicated that RIG-I showed an antiviral role against SVV and was essential for activation of type I IFN signaling during SVV infection. In addition, this study suggested that the CRISPR/Cas9 system can be used as an effective tool to modify cell lines to increase viral yields during SVV vaccine development.
Highlights
Retinoic acid-inducible gene I (RIG-I) is a key cytosolic receptor of the innate immune system
Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) system has a series of advantages such as simple to design, efficient, cheap, and relatively accurate genome editing in host cells comparing with the others genome editing technologies, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) [10]
We successfully acquired one homozygous RIG-I KO cell line with 11 nucleotides deletion in one allele and one nucleotide insertion in another allele of RIG-I genome (Fig. 1b)
Summary
Retinoic acid-inducible gene I (RIG-I) is a key cytosolic receptor of the innate immune system. The role of RIG-I in SVV-infected cells remains unknown. Understanding of the role of RIG-I during SVV infection will help to clarify the infection process of SVV in the infected cells. Cas genes encode an allogenetic and more complex family of proteins that perform similar functions as helicases, nuclease, and RNA-binding proteins [5]. The type CRISPR system includes the Cas nuclease, a noncoding trans-activating crRNA (tracerRNA) and a precursor crRNA [7].The crRNA fuses to the tracerRNA guiding the Cas protein to bind with the target DNA sequences causing a strand-specific cleavage [8]. CRISPR-Cas system has a series of advantages such as simple to design, efficient, cheap, and relatively accurate genome editing in host cells comparing with the others genome editing technologies, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) [10]
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