Screening Of Host Genes Affecting The Replication Of Herpes Simplex Virus Type ? And Type ?

Research Background and Significance:Herpes si mplex virus type ? and type ? are important pathogens causing many diseases,which usually have the characteristics of repeated attacks and can not be cured.The high infection rate of herpes simplex virus(HS V)in human population is largely due to its characteristic of latent infection.When the underlying cellular molecular biological environment of the virus changes,it will reactivate the proliferation of the virus and cause the recurrence of the disease.In the complex mechanism of latency and activation of HSV,many key factors play a role,including LATS from HSV,microRNAs and their own expressed proteins,as well as host influencing factors,including neuron inhibitors,epigenetic regulation and immune system control.Our team found a neuron-specific microRNA,miR-138,which can promote viral latency by targeting the immediate early gene ICPO of HSV-1.In follow-up studies,we found that miR-138 can also inhibit HSV-1 replication by not relying on targeting ICPO.At the same time,PAR-CLIP and RNAseq were used to analyze the target sites of miR-138 binding.No viral genes other than ICPO were found to bind to miR-138.Whether this suggests that miR-138 can also play a role in promoting viral latency through non-viral pathway,i.e.host pathway.Based on this basic idea,we have carried out the research on the target host gene of miR-138.Finally,based on some highly expressed host genes of HSV-1 latent in mouse neurons,candidate genes that can play a key role in the latent period of HSV were screened out.Methods:Based on the host gene information that has been acquired in the laboratory and can bind to miR-138.We use the specific siRNA of these genes to knock down the expression of these genes,so as to observe whether the down-regulation of these genes can cause the decrease of viral replication level.At the same time,we constructed the overexpression vectors of these genes to observe whether the protein of these genes can promote the level of vims at the same time·From these two perspectives,we screened out genes that have a great impact on viral replication level,and served as key candidate genes for the regulation of viral latency by the gene pathway targeting the host of miR-138.Furthermore,the real regulatory effect of miR-138 on these genes was further determined by double luciferase reporter gene analysis and Western blot experiments.Then we used the CRISPR/cas9 gene editing system to knock out the candidate genes selected from the cell line to verify whether the miR-138 could affect the replication level of the virus in the absence of candidate genes.Finally,we used overexpression to screen candidate genes that play a key role in the latency of HSV.Results:We observed the effect of siRNA knockdown on HSV-1 replication.We found that knockdown of the genes of Ccdc-6,Daam-2,Mllt6,Nfyc,Aridla,Oct-1 and Foxcl could inhibit HSV-1 replication more than twice.We also overexpressed the host gene in the cell line.Only Oct-1 and Foxcl could promote the replication of the virus in Neuro-ZA cells.The promoting effect of Foxcl was about 12 times.Subsequently,the results of double luciferase reporter gene analysis and Western blot showed that both the CDS region of Oct-1 gene and the 3'UTR region of Foxcl gene had two sites of miR-138,which could inhibit the expression of protein.Moreover,in Foxcl knockout Neuro-2A cell line,the inhibition of HSV-1 replication level by miR-138 showed a strong functional deficiency.Overexpression experiments showed that Egrl and Gprc5a host genes had strong inhibitory effects on viral replication.Conclusion:Foxcl and Oct-1 play key roles in the pathway of promoting viral latency in the study of the mechanism of miR-138,in which Foxc1 gene plays a leading role.miR-138 has a strong conservative effect on HSV-2 virus ICP0 3'UTR.Egrl and Gprc5a are key candidate genes for host to maintain viral latency environment.