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. 2018 Oct 23;15(1):162.
doi: 10.1186/s12985-018-1080-x.

RIG-I is responsible for activation of type I interferon pathway in Seneca Valley virus-infected porcine cells to suppress viral replication

Pengfei Li  1 , Xiangle Zhang  1 , Weijun Cao  1 , Fan Yang  1 , Xiaoli Du  1 , Zhengwang Shi  1 , Miaotao Zhang  2 , Xiangtao Liu  1 , Zixiang Zhu  3 , Haixue Zheng  4
Affiliations

RIG-I is responsible for activation of type I interferon pathway in Seneca Valley virus-infected porcine cells to suppress viral replication

Pengfei Li et al. Virol J. .

Abstract

Background: Retinoic 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.

Methods: In 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.

Results: Gene 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.

Conclusion: Altogether, 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.

Keywords: Immune response; Interferon; RIG-I; SVV; Viral replication.

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Conflict of interest statement

Ethics approval and consent to participate

The requirement for individual written informed consent was waived since the study was cell experiment.

Consent for publication

This manuscript is approved for publication by Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

Competing interests

The authors declare that they have no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Generation of a RIG-I KO PK-15 cell line by CRISPR/Cas9 system. a The electrophoresis results of the T7E1 assay. Red arrow showed PCR amplicons and green arrow indicated the cleaved fragments. b The sgRNA3 targeted sequences in the RIG-I gene. GGG (green) was the PAM locus. A bi-allelic modifications were presented in KO-1# and KO-2#. The dashed line (red) respresented deletion and highlighted bases indicated indels. c Sanger sequencing results showing the indels into the two alleles. d Western bloting analysis of RIG-I expression in the RIG-I KO and WT PK-15 cells. β-actin was used to normalize the protein loading quantities
Fig. 2
Fig. 2
RIG-I KO PK-15 cells showed higher levels of IFN-β and ISGs than that in the WT PK-15 cells upon Sev stimulation. RIG-I KO and WT PK-15 cells were mock-infected or infected with SeV (100 HAU/ml) for 12 h. The expression levels of IFN-β (a), ISG15 (b), MxA (c)), GBP1 (d) were evaluated by qPCR
Fig. 3
Fig. 3
Knockout of RIG-I enhanced SVV replication in PK-15 cells. a RIG-I KO and WT PK-15 cells were infected with SVV (MOI of 0.1) respectively, and the cellular samples were collected at 10 hpi. Viral RNA levels were detected by qPCR, (b) viral protein levels were detected by western bloting, and (c) viral protein levels were detected by IFA. d RIG-I KO and WT PK-15 cells were infected with equal amounts of SVV for 6, 10 or 14 h, the viral titers were detected by TCID50 assay
Fig. 4
Fig. 4
RIG-I was essential for type I IFN signal pathway activation during SVV infection. RIG-I KO and WT PK-15 cells were mock-infected or infected with SVV at an MOI of 0.1 for 10 h. The expression levels of IFN-β (a), MxA (b), ISG15 (c), GBP1 (d) were measured by qPCR
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