Screening And Functional Study Of Proteins Binding With Bombyx Mori Baculovirus Inhibitor Of Apoptosis Gene Iap2 Promoter

Insect baculovirus is a kind of double-stranded DNA virus,specific infect and widely exist in lepidoptera insect,and generally contain the inhibitor of apoptosis(iap)gene.Based on the studies on insect baculovirus gene functions,iap deprived from different virus genome have showed different biological functions.Although some iap genes' function of apoptotic inhibition has been confirmed,anti-apoptotic function of some iap genes were still unclear,and even showed the opposite function.In addition,the transcription regulatory mechanism of iap gene is still not clear.At present,it has been confirmed that iap2 gene of Bombyx mori baculovirus(BmNPV)exerted the inhibition function of apoptosis,so it was used as the subject in this study.Firstly,using the yeast one-hybrid system,13 kinds of binding proteins were screened out which could bind with iap2 gene promoter.According to the results of previous studies and bioinformatics analysis,these binding proteins were divided into host source genes and virus source genes,including structure genes,the late expression factors,regulate genes and unknown function genes.Then lef-6 and bro-d were selected for further studies.The lef-6 and bro-d deficient viruses were constructed by Red recombinant system,respectively,and were used to study the effects of deletion on virus replication and transcription.Fluorescence quantitative PCR(qRT-PCR)results show that the lef-6 and bro-d deficiency not only led to iap2 gene transcription level decreased significantly(p<0.05),but also influenced the level of the virus genome replication(p<0.05).Moreover the transcriptional levels of early gene lef-3,late gene vp39 and very late gene p10 were decreased significantly(p<0.05).In addition,in order to explore the effects of lef-6-null and bro-d-null virus on host cell apoptosis,the MTT(Thiazole blue)and Western blot were used to measure the cell viabilities and host cells apoptosis after transfected the cells with the viruses.The results showed that cell viabilities decreased significantly compared with wild type virus,although lef-6 or bro-d deficiency resulted in viral genome replication and transcription levels decreased significantly and accelerated host cell apoptosis process.IIIn conclusion,lef-6 and bro-d gene can promote the viral genome replication and transcription levels,but it's not an essential gene for viral progagation;lef-6 and bro-d gene deficiency will cause the host cells apoptosis severely,and they are likely to involve in the inhibition of host cell apoptosis by up-regulation of iap2 gene expression,therefore inhibiting mitochondrial apoptosis signaling pathway.