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Knockout of HDAC9 Gene Enhances Foot-and-Mouth Disease Virus Replication
Shitong Hou1, Xiangwei Wang1, Shanhui Ren1
1State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Diseases Reference Laboratory, Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China.
Abstract:
Foot-and-mouth disease virus (FMDV) is a highly contagious viral disease that mainly infects cloven-hoofed animals. Propagation of FMDV by cell culture is an important method to preserve viral biological and antigenic characteristics, which is crucial in FMD monitoring and vaccine production. However, only a few cell lines are sensitive to FMDV, and there is still a lot of room for improvement. Acetylation is an important post-translational modification, which is dynamically regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). However, the study of the relationship between FMDV and HDACs is still unclear. HDAC9 belongs to the class II of HDACs family; in this study, HDAC9 knockout (KO) BHK-21 cells were successfully established using CRISPR/cas9 technology. The results of karyotype analysis, growth curve analysis, and morphological observation showed that the HDAC9 knockout cell line was stable in growth and morphological characteristics. After infection with FMDV, the expression of viral RNA and protein, viral titers, and the copies of viral RNA in HDAC9-KO cells were significantly higher than those in NC cells. Meanwhile, RNA-seq technology was used to sequence HDAC9-KO cells and NC cells infected and uninfected with FMDV. It was found that the differentially expressed innate immune factors containing NFKBIA, SOD2, IL2RG, BCL2L1, CXCL1/2/3, and IL1RAP have significantly enriched in the Jak-STAT, NOD-like receptor, Toll-like receptor, NF-κB, and MAPK signaling pathway. RT-qPCR was performed to detect the expression level of differentially expressed genes and showed consistency with the RNA-seq data. These results preliminarily reveal the role of HDAC9 in host antiviral innate immune response, and the HDAC9-KO cell line could also serve as a useful tool for FMDV research.
Insights
Histone deacetylase 9 (HDAC9) knockout cells enhance foot-and-mouth disease virus (FMDV) replication by impacting innate immunity. This HDAC9-deficient cell line offers a valuable tool for FMDV research and vaccine development.
Area of Science:
- Virology
- Immunology
- Molecular Biology
Background:
- Foot-and-mouth disease virus (FMDV) poses a significant threat to cloven-hoofed animals, necessitating effective cell culture methods for monitoring and vaccine production.
- Existing FMDV-susceptible cell lines have limitations, highlighting the need for improved cell-based research tools.
- Histone deacetylases (HDACs), including HDAC9, play a role in post-translational modifications, but their specific involvement in FMDV infection remains largely unexplored.
Purpose of the Study:
- To investigate the role of HDAC9 in the host's innate immune response to FMDV infection.
- To establish and characterize a HDAC9 knockout (KO) BHK-21 cell line for FMDV research.
Main Methods:
- CRISPR/cas9 technology was employed to generate HDAC9-KO BHK-21 cells.
- Karyotype analysis, growth curve analysis, and morphological observation were performed to confirm cell line stability.
- FMDV infection studies involved quantifying viral RNA and protein expression, viral titers, and RNA sequencing (RNA-seq) to analyze host gene expression changes in HDAC9-KO versus control (NC) cells.
Main Results:
- The established HDAC9-KO cell line exhibited stable growth and morphological characteristics.
- HDAC9-KO cells showed significantly higher FMDV RNA and protein expression, increased viral titers, and more viral RNA copies compared to NC cells post-infection.
- RNA-seq analysis revealed significant enrichment of differentially expressed innate immune factors (e.g., NFKBIA, SOD2, IL2RG) in key signaling pathways (Jak-STAT, NOD-like receptor, Toll-like receptor, NF-κB, MAPK) in HDAC9-KO cells.
Conclusions:
- HDAC9 plays a crucial role in regulating the host's antiviral innate immune response against FMDV.
- The developed HDAC9-KO cell line serves as a valuable and enhanced tool for studying FMDV replication and host-pathogen interactions.
- These findings contribute to a better understanding of FMDV pathogenesis and may inform future strategies for FMD control and vaccine development.
