Ubiquitin protein ligase activity of the anti-apoptotic baculovirus protein Op-IAP3

The baculovirus inhibitor of apoptosis protein (IAP) Op-IAP3 is required to prevent apoptosis during infection of insect cells by Orgyia pseudotsugata M nucleopolyhedrovirus (OpMNPV) and inhibits apoptosis when overexpressed in insect and mammalian cells. Although previous reports have demonstrated that the RING domain is important for the anti-apoptotic function of Op-IAP3, the function of this domain in Op-IAP3 has not been studied. Here, the ability of Op-IAP3 to function as an E3 ubiquitin protein ligase was examined. Op-IAP3 expressed in the insect cell line Spodoptera frugiperda (Sf21) was ubiquitinated, but only if the RING domain was intact. In addition, co-expression of Op-IAP3 and the pro-apoptotic Drosophila protein HID resulted in the ubiquitination of HID. Recombinant Op-IAP3 protein also promoted the ubiquitination of both itself and recombinant HID protein in vitro, and the ubiquitination of HID required both the RING and BIR2 of Op-IAP3. Thus, we conclude that Op-IAP3 is a functional E3 ubiquitin ligase, and the ability to ubiquitinate pro-apoptotic cellular proteins such as HID may play an important role in the anti-apoptotic function of Op-IAP3.