Study On The Role Of DNMT1 During VSV Replication In Mouse Macrophages

Vesicular stomatitis virus(VSV)and rabies virus belong to the Rhabdoviridae family,and the virus is the prototype of Vesiculvirus.Some livestock,such as pigs,cattle,and horses,are infected with the vesicular stomatitis virus,and their clinical manifestations are very similar to foot-and-mouth disease and swine vesicular herpes.They have symptoms of blisters and ulcers on the tongue,lips,and nipples.Therefore,the disease Misdiagnosis is also prone to occur in veterinary clinical diagnosis.In addition,the disease is a zoonotic infectious disease.Humans are susceptible to VSV.Direct contact and aerosols are all modes of transmission of the disease.Its clinical manifestations are flu-like symptoms such as headache,fever,nausea,and weakness.At present,there is no effective and reliable treatment for vesicular stomatitis,so the research on this disease has important public health significance.Although vesicular stomatitis caused by VSV infection has a low lethality to herds,it has a serious impact on the growth and development of individual herds and serious economic losses to the livestock industry.The disease has occurred in various countries,and has been listed as a key animal disease that must be reported by many countries including the United States.China has listed vesicular stomatitis as 13 in the national mid-to-long-term animal disease prevention plan from 2012 to 2020.This is one of the important animal epidemic diseases to prevent.DNA methylation is the most mature epigenetic modification currently studied,which plays a vital role in regulating gene expression and maintaining chromosome conformation.Its principle is through DNA methyltransferase(DNA,methylthansferase,DNMT)binds the methyl group to the 5th carbon position of the cytosine of the genomic CpG dinucleotide,which causes changes in chromosome structure,DNA conformation,and DNA stability,and thus controls gene expression.Among them,DNA methyltransferase 1(DNMT1)is a key enzyme to maintain cellular gene methylation.A large number of experiments have proved that VSV infection can affect the expression of various host protein genes,and there are few reports about the effects of VSV on cell infection through the regulation of host protein gene expression.Therefore,this experiment transferred from DNA methylation Starting with enzyme 1(DNMT1),the mechanism of DNMT1 in VSV infection and replication was studied to provide new ideas for the treatment of VSV.In this experiment,DNMT1 in host cell RAW264.7 was pharmacologically inactivated with drugs.The expression of a variety of interferon-related genes in the cells was significantly increased through real-time quantitative PCR experiments,and the expression of the P protein gene in VSV was significantly reduced;The TCID50 experiment showed that after inactivation of DNMT1 in the host cell,the virulence of VSV was significantly reduced.The lentiviral packaging technology was used to construct a Dnmt1 gene silencing RAW cell line,which was verified at the gene and protein levels.The bisulfite method was used to detect the degree of methylation of CpG island of IRF3 gene to determine the effect of blocking DNMT1 on IRF3 gene expression.The above results indicate that DNMT1 participates in the VSV replication process.And inactivation of DNMT1 and reduction of gene expression in host cells can significantly increase the expression of multiple interferon-related genes,thereby inhibiting VSV infection and replication.In summary,this experiment used DNMT1 as the research object and mouse macrophages(RAW264.7)as a model to study the effect and mechanism of DNMT1 on vesicular stomatitis virus infection and replication.