Molecular Mechanism Underlying IBDV VP2 Induced Cell Apoptosis Via Interacting With ORAOV1

Infectious bursal disease (IBD) is a highly infectious disease caused by IBD virus (IBDV),characterized by rapidly massive depletion of lymphocytes in the bursa of Fabricius (BF) which trigger immunosuppression in young chickens. The genome of IBDV has three open reading frames(ORF) encoding VP1, VP2, VP3 VP4 and VP5. Structural protein VP2 is the major components of the viral capsids, and performs multiple roles in virulence, genetic variation, IBDV-induced autophagy and so on. Above all, VP2 is the first published viral protein of IBDV as an apoptotic inducer in a various cell lines. However, the exact molecular mechanism for VP2 induced apoptosis remains unclear.The present study is focused on searching for the VP2-host interactions that participate in IBDV-induced apoptosis. The yeast-two hybrids assay was employed to screen the targets of VP2 in host cell, employing VP2 of IBDV as the bait and cDNA library of chicken BF as the prey. Oral cancer overexpressed 1 (ORAOV1), a potential oncoprotein with anti-apoptotic activity, was identified as the target of IBDV VP2. The VP2-ORAOV1 interaction was further confirmed by Co-IP,Pull-down assay and laser confocal assays. Notably, we found that transient expression of VP2 or infection of IBDV induced reduction of ORAOV1 and led to cell death accompanied by the cleavages of Caspase-3 and PARP, so did knockdown of ORAOV1. Furthermore, we found that the VP2-induced ORAOV1 degradation was not blocked with MG132, indicating that the degradation of ORAOV1 by VP2 was independent of proteasome-mediated protein degradation pathway and that the overexpression of ORAOV1 could counteract VP2 transfection- or IBDV infection-induced apoptosis,which restricts the virus release early after infection. Importantly, we determined the 331-452 residues of C-terminal of VP2 as the functional domain for apoptosis and ORAOV1 degradation. Moreover,we mixed the purified recombinant VP2 with cell lysates or purified ORAOV1, and found that VP2 could degrade ORAOV1, acting as a viral protease.In summary, our findings show that IBDV VP2, possibly acting as a viral protease in host cells,induces apoptosis via the interaction and degradation of cellular ORAOV1,which will help to elucidate the pathogenesis of IBDV infection, and shed lights on the development of VP2 related vaccines against IBDV.