Baculoviral IAP2 and IAP3 encoded by Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV) suppress insect cell apoptosis in a transient expression assay

Baculoviral anti-apoptotic genes, p35 and iap (inhibitor of apoptosis), play important roles in the initiation stage of viral infection. While some iap genes in baculoviruses are not directly involved in anti-apoptotic activity, two iap genes (ly-iap2 and ly-iap3) were found and cloned from Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV) and used to investigate the roles in LyxyMNPV infection. From the transcriptional start site analysis, ly-iap2 consisted of a late promoter motif (TAAG) located upstream at nt −17 bp, and ly-iap3 had a potential baculoviral promoter consensus sequence (GCTTGTT) located upstream at nt −44 bp. Both the ly-iap2 and ly-iap3 genes were initially transcribed in the LyxyMNPV-infected Ld652Y cells (IPLB-LD652Y cell line) at 3 h post infection (h p.i.), increased dramatically at 12 h p.i. and reached the peak at 72 h p.i. according to a quantitative RT-PCR (qRT-PCR) assay. Functional assays of these iap genes were performed using an overexpression method in Sf9 cells. Full-length LY-IAP2, LY-IAP3 and LY-IAP2-BIR (baculoviral IAP repeat) could inhibit varying degrees of apoptosis induced by Drosophila RPR protein (DRPR). Interestingly, Ly-IAP2-BIR showed higher inhibitory activity than full-length Ly-IAP2, and the protein expression level was dramatically increased while the Ly-IAP2-RING domain was deleted. The effect of MG-132 on the overexpression of Ly-IAP2 was investigated. Under MG-132 treatments, high-molecular-weight signals were also detected. Taken together, this study suggested that both Ly-IAP2 and Ly-IAP3 were anti-apoptotic protein through the BIR functional domain. The Ly-IAP2-RING domain was possibly involved in ubiquitin E3 ligase activity, leading to the degradation of Ly-IAP2 protein, whereas Ly-IAP3-RING might be working as a “helper domain” to inhibit DRPR-induced apoptosis.