CRISPR-Cas– induced IRF3 and MAVS knockouts in a salmonid cell line disrupt PRR signaling and affect viral replication

Abstract

BackgroundInterferon (IFN) responses are critical in the resolution of viral infections and are actively targeted by many viruses. They also play a role in inducing protective responses after vaccination and have been successfully tested as vaccine adjuvants. IFN responses are well conserved and function very similar in teleosts and mammals. Like in mammals, IFN responses in piscine cells are initiated by intracellular detection of the viral infection by different pattern recognition receptors. Upon the recognition of viral components, IFN responses are rapidly induced to combat the infection. However, many viruses may still replicate and be able to inhibit or circumvent the IFN response by different means.MethodsBy employing CRISPR Cas9 technology, we have disrupted proteins that are central for IFN signaling in the salmonid cell line CHSE-214. We successfully generated KO clones for the mitochondrial antiviral signaling protein MAVS, the transcription factors IRF3 and IRF7-1, as well as a double KO for IRF7-1/3 using an optimized protocol for delivery of CRISPR-Cas ribonucleoproteins through nucleofection.ResultsWe found that MAVS and IRF3 KOs inhibited IFN and IFN-stimulated gene induction after intracellular poly I:C stimulation as determined through gene expression and promoter activation assays. In contrast, the IRF7-1 KO had no clear effect. This shows that MAVS and IRF3 are essential for initiation of intracellular RNA-induced IFN responses in CHSE-214 cells. To elucidate viral interference with IFN induction pathways, the KOs were infected with Salmon alphavirus 3 (SAV3) and infectious pancreatic necrosis virus (IPNV). SAV3 infection in control and IRF7-1 KO cells yielded similar titers and no cytopathic effect, while IRF3 and MAVS KOs presented with severe cytopathic effect and increased titers 6 days after SAV 3 infection. In contrast, IPNV yields were reduced in IRF3 and MAVS KOs, suggesting a dependency on interactions between viral proteins and pattern recognition receptor signaling components during viral replication.ConclusionAside from more insight in this signaling in salmonids, our results indicate a possible method to increase viral titers in salmonid cells.

MAVS disruption impairs downstream signaling and results in higher virus replication levels of salmonid alphavirus subtype 3 but not infectious pancreatic necrosis virus in vitro

“…Farmed and wild teleost fish are infected with many RNA viruses where (+) RNA and dsRNA virus infections cause high losses, particularly in farmed salmon ( 14 – 17 ). Hence, understanding MAVS’ contribution to innate responses to SAV-3 (+RNA virus) and infectious pancreatic necrosis (IPN) virus (dsRNA) will shed light on crucial innate and adaptive defense mechanisms of salmonid teleost, and a recent study has shown that MAVS-knock out in vitro impairs PRR-signaling in fish cells ( 18 ).…”

Effect of CRISPR/Cas9 Targets Associated with Iron Metabolism and Its Variation on Transcriptional Regulation of SHK-1 Cell Line as a Model for Iron Metabolism

“…This cell line has proven to be useful to better understand the host-pathogen interaction between salmon and the infectious salmon anemia virus (ISAv) [25]. In our study, this cell line also has proven to be an excellent model for performing genome editing based on the CRISPR-Cas9 system, showing high levels of efficiency for a salmon cell line and relatively high KO efficiency compared to other fish cell lines (23-76%) [26], or in vivo induced mutations in embryos of S. salar by the CRISPR-Cas9 system (13-50%) [27,28], providing a useful initial step for research in salmonid species to better understand the specific role of gene regulation on infectious diseases and immune responses.…”

“…A study on the CHSE-214 cell line used the CRISPR-Cas9 system to evaluate the effect of a knock-out of the genes mavs, irf3, irf7-1, and irf3/7-1 (double KO) on PRR signaling [26]. The evaluation of Interferons (IFNs) and IFN-stimulated genes by qPCR after Poly I:C stimulation showed an inhibition of the expression in the mavs and irf3 edited groups, showing the advantages of using CRISPR edited cell lines in fish for understanding the immune response and the host-pathogen interaction.…”