Study On Functions And Molecular Mechanism Of AcMNPV Iap Genes

As is known, apoptosis plays an indispensable role in protecting host cells from virusinfection as an important defense mechanism. In turn, many viruses have evolved somecorresponding mechanisms to regulate host cell suicide and promote virus production.Baculoviruses were one of the first viruses found to regulate host apoptosis. So far, manystudies on baculoviral genes involving in host apoptosis have provided important insightsinto the molecular mechanisms of apoptosis and interaction between viruses and host cells.Baculoviruses encode three different antiapoptotic genes, namely p35, p49 and theinhibitors of apoptosis(iap). P35 is a suicide substrate inhibitor of many caspasesfunctioning to block various stimuli-induced apoptosis in phylogenetically diverseorganisms among nematodes, flies and mammals, whlie P49 is a homolog of P35. Unlikep35 and p49, many members of iap gene family have been found in diverse organismsincluding viruses, invertibrates and vertibrates from yeast to human. However, only a fewbaculoviral IAPs were identified as functional inhibitors of apoptosis and the mechanismsby which IAPs block apoptoic ceil death remain unclear.Autographa california nucleopolyhedrovirus (AcMNPV), registered as a verysuccessful insecticide, serves as a primary model on studying regulation of host apoptosisby baculoviruses. AcMNPV encodes a p35 gene, the firstly found potential inhibitor ofapoptosis, and two homologies of lap genes, iap1 and iap2. However, whether these twoIAPs are functional in inhibiting apoptosis remains to be identified, hitherto. Aspreviously mentioned, the mutant of AcMNPV p35 null (AcMNPVAp35) was usually usedto screen and identify fucntional antiapoptosis genes as an inducer of apoptosis inSpodoptera frugiperda cells and the identification of Cp-iap and Op-iap gene is theobvious instance. Whereas, to be surprising, it was shown here that the mutant AcMNPVΔp35 completely inhibited the apoptosis of Helicoverpa armigeranucleopolyhedrovirus(HearNPV)-induced Tn-Hi5 cells as well as wild type AcMNPV inour study. Further investigations were carded out and a hypothesis was put forward tospeculate that iap1 or/and iap2 could function to inhibit the apoptosis here.based on the study above-mentioned, transiently expressing plaimids of iap1/2 andrecombinant HearNPV overexpressing iap1/2 were constructed. It was shown that bothIAP1 and IAP2 were functional inhibitor to HearNPV-induced apoptosis of Tn-Hi5 cellsresulting from transient transfection assay and recombinant virus infection. Furthermore, toprobe into the mechanism by which IAPs functioned, IAP2 and two insect effector caspaseswere expressed and purificated in E. coli. The result of activity assay in vitro suggested thatactive Bm-caspase-1 could activate the nonactive pro-Tn-caspase-1 and IAP2 inhibited theactivity of Bm-caspase-1.In this study, AcMNPV IAP1 and IAP2 have been confirmed to function to blockapoptosis. In addition, this study further reinforces the diversity and cell-specificity onfunctional IAP homologs and lays a foundation for elucidating the mechanism of IAPinhibiting apoptosis.