Full length articleIncrease of viral hemorrhagic septicemia virus growth by knockout of IRF9 gene in Epithelioma papulosum cyprini cells
Introduction
Viral hemorrhagic septicemia virus (VHSV) is a member of the genus Novirhabdovirus in the family Rhabdoviridae, and has been a cause of mass mortalities in cultured fish worldwide [1,2]. The protective efficacy of various types of VHSV vaccines including DNA vaccines, inactivated vaccines, and attenuated vaccines have been reported [1,3]. Although, lately, European Union (EU) decided to treat DNA vaccinated fish as non-genetically modified organism (non-GMO), there are still many struggles on the GMO problems related to DNA vaccines in fish in many countries including Korea. Thus, whole virus-based vaccines would be more favorable for the permission of field vaccines against VHSV in countries that treat DNA vaccinated fishes as GMO. Furthermore, considering economical aspects of aquaculture vaccines, methods that can produce high-titered viruses are needed to develop cost-effective vaccines.
Type I interferon (IFN) responses are the key elements of vertebrates’ antiviral activities, and viruses should equip with countermeasures that can suppress or utilize type I IFN responses for the successful replication in host cells [4,5]. The high sensitivity of VHSV to type I IFN responses has been shown, and most of the studies were conducted to demonstrate the effects of pre-established type I IFN responses on VHSV replication [[6], [7], [8]], but reversely, in this paper, we tried to increase VHSV titers by the inhibition of type I IFN responses.
Type I IFNs produced by several pathways bind to the IFN receptor (INFAR), and initiate Janus kinase/Signal transducers and activators of transcription (JAK1-STAT) pathway that leads to the formation of the heterotrimeric complex, IFN-stimulated gene factor 3 (ISGF3), which is composed of STAT1, STAT2, and interferon regulatory factor 9 (IRF9). The ISGF3 complex regulates expression of IFN-stimulated genes by binding to the IFN-stimulated response elements (ISRE) in the promoter region [[9], [10], [11]]. Considering the critical role of IRF9 in the formation of ISGF3, the inhibition of IRF9 would compromise host's type I IFN responses, which would weaken host antiviral activity.
Recently, the clustered regulatory interspaced short palindromic repeat (CRISPR)-associated 9 (Cas9)-mediated gene editing has provided a convenient way to produce a specific gene knockout cells or organisms [12,13]. In the present study, to increase VHSV titer through the inhibition of type I IFN responses, we produced IRF9 knockout Epithelioma papulosum cyprini (EPC) cells using a CRISPR/Cas9 vector that contain an EPC cell's U6 promoter-driven single-guide RNA cassette (targeting IRF9 gene) and a Cas9 expressing cassette, and evaluated the effect of IRF9 knockout on type I IFN response and VHSV replication.
Section snippets
Cells and virus
EPC cells (ATCC no. CRL-2872) subcultured within 10 times (from purchase) were grown in Leibovitz medium (L-15, Sigma) supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml) and 10% fetal bovine serum (FBS, Sigma). VHSV (VHSV KJ2008) isolated from a diseased olive flounder (Paralichthys olivaceus) was propagated in a monolayer of EPC cells at 15 °C in the presence of 2% FBS and antibiotics.
IRF9 gene knockout
Using the CRISPR/Cas9 single-guide RNA (sgRNA) design tools (//www.rgenome.net/cas-offinder/
Generation of IRF9 knockout EPC cells
To knockout IRF9 gene in EPC cells by Cas9, sgRNA targeting IRF9 gene was expressed through EPC cell's U6 polymerase III promoter (Fig. 1). Selected clones of cells transfected with pCRISPR/Cas9-U6-IRF9 vector (Fig. 1) showed homozygous or heterozygous insertion/deletion (indel) mutations (Fig. 2), which led to the generation of premature stop codons resulting in the knockout of IRF9 gene (Fig. 2).
In the analysis of IRF9 gene transcript level using semi-quantitative RT-PCR, IRF9 mRNA level in
Discussion
Although type I IFNs can be expressed through several pathways such as RIG-I, TRIF, and IRF7 pathways [[14], [15], [16]], the expression of type I IFN-stimulated genes can be severely compromised if IRF9 (a critical component for the formation of ISGF3) is not functional. Thus, the knockout of IRF9 gene can lead to the unresponsiveness of cells in type I IFN responses to type I IFN inducers. In the present study, we produced an IRF9 knockout EPC cells using a CRISPR/Cas9 vector and finally got
Acknowledgements
This research was a part of the project titled ‘Development of Fish Vaccines and Human Resource Training’(20130291), funded by the Ministry of Oceans and Fisheries, Republic of Korea.
References (19)
- et al.
A comparative review on European-farmed finfish RNA viruses and their vaccines
Vaccine
(2011) - et al.
A hybrid IRF9-STAT2 protein recapitulates interferon-stimulated gene expression and antiviral response
J. Biol. Chem.
(2003) - et al.
Type I interferon gene induction by the interferon regulatory factor family of transcription factors
Immunity
(2006) - et al.
Development and applications of CRISPR-Cas9 for genome engineering
Cell
(2014) - et al.
Cell type-specific involvement of RIG-I in antiviral response
Immunity
(2005) - et al.
Poly I: C induces Mx transcription and promotes an antiviral state against sole aquabirnavirus in the flatfish Senegalese sole (Solea senegalensis Kaup)
Fish Shellfish Immunol.
(2008) - et al.
Live vaccine of viral hemorrhagic septicemia virus (VHSV) for Japanese flounder at fish rearing temperature of 21°C instead of Poly(I:C) administration
Vaccine
(2011) - et al.
Viral hemorrhagic septicemia virus in marine fish and its implications for fish farming - a review
J. Fish. Dis.
(2005) - et al.
Family Rhabdoviridae
Cited by (23)
Two IFNa3s mediate the regulation of IRF9 in the process of infection with Streptococcus iniae in yellowfin seabream, Acanthopagrus latus (Hottuyn, 1782)
2024, Developmental and Comparative ImmunologyIRF9 inhibits CyHV-3 replication by regulating the PI3K-AKT signalling pathway in common carp (Cyprinus carpio) epithelial cells
2023, Developmental and Comparative ImmunologyEffect of temperature and IRF-9 gene-knockout on dynamics of vRNA, cRNA, and mRNA of viral hemorrhagic septicemia virus (VHSV)
2023, Fish and Shellfish ImmunologyEffect of CRISPR/Cas9-mediated knockout of either IRF-3 or IRF-5 gene in Epithelioma papulosum cyprini cells on type I interferon response and NF-κB activity
2023, Fish and Shellfish ImmunologyCitation Excerpt :One of the ways to know the functional role of certain genes is the production of target gene(s) knockout cells or organisms. Previously, we had reported the role of IRF9 in the induction of type I IFN responses in Epithelioma papulosum cyprini (EPC) cells through the knockout of the IRF gene using a CRISPR/Cas9 system [34]. In this study, a CRISPR-Cas9 vector that contains both a single guide RNA cassette (targeting either IRF3 or IRF5) driven by EPC U6 promoter and a Cas9 expressing cassette was constructed and used for the transfection of EPC cells to generate IRF3 gene and IRF5 gene knockout EPC cells.