Screening Host Genes That Regulate The Replication Of Foot-and-mouth Disease Virus Based On ID1 And ID3 Knockout Cell Lines

Foot-and-mouth disease(FMD)is an acute,febrile and highly transmissible disease caused by footand-mouth disease virus(FMDV)that mainly infects cloven-hoofed animals such as cattle,goats,pigs and deer.FMD spreads rapidly and in many ways.It has been spreading worldwide for many times,which has caused serious economic losses to the livestock production and breeding industry.FMDV which belongs to the genus Aphthovirus of Picornaviridae family has 7 main serotypes.And there is almost no cross-protection among the serotypes,which brings severe challenges to the prevention,control and eradication of FMD.Inhibitor of DNA binding(ID)is a family of proteins that widely found in mammals.Its family members include ID1,ID2,ID3 and ID4 proteins.As negative regulators of nuclear transcription factors,ID proteins play an important role in cell growth,development,differentiation,senescence and tumorigenesis.Among them,family members ID1 and ID3 proteins have been proved to be functionally redundant,and they may play a synergistic role in maintaining cell growth.At present,there are few studies on the involvement of ID family proteins in virus infection and replication.The results of previous studies in our laboratory showed that ID1 protein was involved in regulating the replication of FMDV.In order to reveal the regulatory mechanism between host gene and ID gene during foot-and-mouth disease virus infection at the molecular level,ID1 gene knockout cell line,ID3 knockout cell line and ID1 and ID3 genes co-knockout cell line were screened by CRISPR/Cas9 technique to explore the effect of ID genes knockout on virus replication.The results showed that when the control(Negative control,NC)cell line and each gene knockout cell lines were infected with FMDV for the same time,ID gene-knockout could promote the replication of FMDV compared with the control cell line.In order to further study the changes of host genes in gene-knockout cell lines after FMDV infection,transcriptome sequencing(RNA-Seq)was used to analyze the gene expression of each cell line.The results showed that compared with the control cell line,there were 1773 differentially expressed genes(DEGs)in ID1 KO cell line;2761 DEGs in ID3 KO cell line and 2376 DEGs in ID1/3 KO cell line.There were 208 up-regulated genes and 571 down-regulated genes in the three knockout cell lines.In order to verify the reliability of RNA-Seq results,we selected 17 differentially expressed genes(11 downregulated genes and 6 up-regulated genes)from knockout cell lines for RT-q PCR verification.The results showed that the RT-q PCR and RNA-Seq results had similar trends,indicating the reproducibility of the RNA-Seq results.These findings provide clues for the further study of the regulatory mechanism between host genes and ID1 and ID3 genes in the process of FMDV infection.