Mechanism Analysis Of Transcription Regulation By HSV1 ICP22 And The Co-regulation With HSV1 VP16 And ICP0 In Viral Gene Transcription

Herpes simplex virus type I(HSV1) is a double-stranded DNA virus. After its infection, expression of viral genes are in an ordered cascade process. Viral genes are classified into immediate early gene(a gene),early gene(βgene),late gene(r gene) by its cascade expression. The immediate early genes encode five infected cell polypeptides: ICPO,ICP4,ICP22,ICP27,ICP47, which play major roles in virus replication and direct expression of viralβand r genes.Study of proteins encoded by a gene is a major point in biology research of HSV1 infection, one of which the ICP22 protein, little is known about it in infected cells as it was firstly classified as non essential for viral replication and diffucult to express in isolation. Recently found that hard to express ICP22 in transfected cell is due to it repress its expression by some expression vectors. We previously found ICP22 represses transcription of some viral immediate early gene promoters, including HSV1,SV40,ADV, and repression by ICP22 are not affected by upstream cis-acting elements of specific viral and cellular promoters.We also found ICP22 does not repress transcription of CMV IE promoter, and in the presence of viral VP16 protein, repression of HSV1 a4 gene transcription by ICP22 could be overcame.Base on our previous study, effects on transcription of HSV1 a,β,r genes by ICP22 were further carried in this research, results indicated that ICP22 generally represses HSV1 gene transcription. It's well known that HSV1 VP16 and ICPO proteins possess activation and promotion ability to HSV1 gene expression, studies on co-regulation of viral gene transcription by ICP22,VP16,ICPO revealed VP16 could disarm transcription repression of a4 gene by ICP22, but notβand r genes; ICPO could not disarm transcription repression of viral a,β,r genes by ICP22, on the contrary ICP22 could repress transcription promotion of viralβand r genes by ICPO.Study of ICP22- virus suggested it triggers loss of a specific phosphorylated form of RNA polymeraseⅡ, and this effect might affect transcription elongation of RNA polymeraseⅡ. In our research, we found ICP22 interacts with CDK9 and CyclinTl, which compose P-TEFb, a cellular positive transcription elongation factor, play major role in phosphorylating RNA polymerase II nad promoting transcription elongation. We also found VP16 interacts with CDK9 and CyclinTl. Besides, ICP22,VP16 and cellular CDK9,CyclinTl interact with each other while co-expression ICP22 and VP16 in CHO-k1 cells. We didn't detect interaction between ICPO and P-TEFb proteins in transfected CHO-kl cells. Further studies using up-regulating(over-expression) and down-regulating(RNAi) for changing cellular P-TEFb expression level failed to affect transcription repression of viral gene by ICP22. However we found ICP22 represses recruit of P-TEFb to the viral promoter by ChIP, suggested transcription repression effect of ICP22 might related to it interferes recruit of P-TEFb to promoter and transcription elongation. Furthermore, ChIP assays demonstrated VP16 promotes recruit of P-TEFb to viral a4 gene promoter, and could overcome recruit repression of P-TEFb to a4 promoter by ICP22. Besides, we didn't found ICPO could affect recruit of P-TEFb to viral promoters. Collectively, Our results suggested ICP22 and VP 16 may co-regulate viral gene transcription by affecting recruit of P-TEFb to viral gene promoter. We also measured some cellular housekeeping genes and cyclins if ICP22 affects recruit of P-TEFb to these genes, but the results are negative and we believe its due to the complex of cellular P-TEFb's recruit.