Transcriptional Regulation And Anti-cervical Cancer Oncolytic Engineering Of Herpes Simplex Virus UL54 Gene

An oncolytic herpes simplex virus(o HSV)has proven amenable in oncolytic virotherapy and was approved to treat melanoma.The immediate-early(IE)protein ICP27 encoded by gene UL54 is essential for HSV infection.Transcriptional modification of UL54 would increase tumor targeting of o HSVs.ICP27 is a 512 aa IE protein,which is modified by phosphorylation and arginine methylation.ICP27 was phosphorylated by serine/threonine,kinases.c AMP-dependent protein kinase A,calmodulin-dependent protein kinase I/II,mitogen and stress-activated protein kinase and cyclin-dependent kinase 3(cdk-3)in the serine kinase inducing region.Its phosphorylation enhances its activity.However,the transcriptional regulatory sequence and transcription factors of UL54 gene have not been reported.It is important to study the transcriptional regulation sequences of UL54 gene for HSV infection,which will provide experimental basis for the study of novel oncolytic virus anticervical cancer drug.Objective: To discover and engineer the transcriptional regulation sequences of HSV UL54 gene(ICP27)and laydown the basis of o HSV-1 oncolytic virotherapy against cervical cancer.Methods: 1.Isolation of HSV and DNA sequencing.2.CRISPR/Cas9 gRNA design and synthesis.3.Cloning into vectors and sequencing 4.Analysis of the transcriptional regulation sequences of HSV UL54 gene.Results: We isolated a Chinese strain of HSV-1 and named LXMW.We then identified the possible transcriptional regulatory sequence and factors of HSV UL54.Our study found that HSV-1-LXMW is closely related to HSV-1 strain Patton and H129 in the US.Through multiple sequence analysis,we found that UL54 transcriptional regulatorysequences in HSV-1-LXMW bind to transcription factors Oct-1,v-Myb and Pax-6,while in HSV-2 strains they only bind to Oct-1 and Hairy.Further validation showed that HSV-1 and HSV-2 had the same binding sequence with Oct-1,but they had unique binding sequences with v-Myb and Pax-6 or Hairy,respectively.The results showed that the expression of transcription factors was consistent with the tissue tropism of HSV-1 and HSV-2.Our findings highlight a new understanding of the principles of transcriptional regulation in HSV biology and oncolytic Meanwhile,I designed an oncolytic HSV with engineered transcriptional regulatory region of the UL54 gene,constructed the recombinant plasmids,and laid down the basis for the construction of oncolytic HSV against cervical cancer.Conclusions:1.I isolated and characterized a Chinese strain of HSV-1 and named HSV-1-LXMW,which was found to be closely related to HSV-1 strain Patton and H129 in the US.2.For the first time,I found HSV UL54 transcriptional regulatory sequences and transcription factors Oct-1,v-Myb,Pax-6 and Hairy,and predicted they may tedermine tissue tropism of HSV.3.Using our new HSV-1-LXMW,I,for the first time,designed an unique oncolytic HSV with engineered transcriptional regulatory region of the UL54 gene,constructed the recombinant plasmids,and laid down the basis for the construction of oncolytic HSV against cervical cancer.