Functional Dissection Of The Plutella Xylostella Granulovirus Iap Gene

The diamondback moth, Plutella xylostella, is a major pest of cruciferous plants and is globally distributed. Plutella xylostella granulovirus (PlxyGV) is a natural control agent of diamondback moth. The complete genome sequence of PlxyGV was published in 2000.Iaps are a large evolutionarily conserved family of cellular genes bearing homology to the prototype baculovirus inhibitor of apoptosis. Two prominent structural features of IAPs baculovirus IAP repeat (BIR) and RING domain. Baculovirus infection can often induce the host cells apoptosis. Some IAPs from baculoviruses have been shown being able to block apoptosis of host cells, maintaining host cell survival, that allow continuing reproduction the virus. According to PlxyGV genome sequence analysis, PlxyGV contains an iap homologue gene, which is located PlxyGV Genome nt80675-81520. PlxyGV iap encodes a protein showing high similarities with IAPs of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Cydia pomonella granulovirus (CpGV). CpGV IAP3 was identified by its ability to functionally replace p35 in a genetic complementation assay. However the function of PlxyGV IAP has not been entirely resolved.In the study, experiments were performed to explore function of PlxyGV IAP, by transient assays. The PlxyGV iap gene was cloned into a transient expression vector and transfected into Sf9 cells. Then the cells tranfected were treated with actinomycin D or infected with AcMNPV p35 deficient mutant (AcMNPV△P35) to induce apoptosis. Apoptosis was detected by microscopy, flow cytometry and DNA ladder methods. It was shown that apoptosis rate in the cell culture transfected with PlxyGV lap and infected by AcMNPV△P35 was significantly lower than the ones mock transfected and infected by AcMNPV△P35. In the cell culture treated with actinomycin D, apoptosis occurred in both the cells transfected with PlxyGV iap and the ones mock transfected, no significant difference in apoptosis rate. This is the evidence that PlxyGV IAP can effectively inhibit apoptosis induced by infection of AcMNPV△P35 in Sf9 cells; but it can not effectively inhibit apoptosis induced by actinomycin D.To further determine the function domain of PlxyGV IAP. truncated mutants of the gene were constructed and tested apoptosis inhibition. It was shown that mutations involving C-terminal RING motif and N-termina BIR motifs knocked down apoptosis-inhibiting activity strikingly.