Hyphantria cunea nucleopolyhedrovirus (HycuNPV) infection protected SpIm cells from actinomycin D (ActD)-induced apoptosis as early as 4 h postinfection. Analysis by Southern hybridization revealed that the HycuNPV genome possessed three members of inhibitor of apoptosis genes (iaps) that were designated as hycu-iap1, hycu-iap2, and hycu-iap3 because of their amino acid sequence homology with iaps identified in other baculoviruses. Functional analysis of Hycu-IAPs by transient expression assay in Sf9 cells revealed that Hycu-IAP3 blocked apoptosis induced by actinomycin D and rescued replication of p35 deficient-mutant AcMNPV, while Hycu-IAP1 and Hycu-IAP2 did not show any anti-apoptotic functions. Knockdown of hycu-iap3 expression by RNAi during HycuNPV infection in SpIm cells induced apoptosis. These results indicate that Hycu-IAP3 is essential for blockage of apoptosis during HycuNPV infection of permissive SpIm cells.
Ld652Y cells derived from the gypsy moth, Lymantria dispar, were infected with seven different nucleopolyhedroviruses (NPVs) including those from Autographa californica, Bombyx mori (BmNPV), Hyphantria cunea (HycuNPV), Spodoptera exigua (SeMNPV), L. dispar, Orgyia pseudotsugata (OpMNPV) and Spodoptera litura (SpltMNPV). The results showed that Ld652Y cells infected with BmNPV, HycuNPV, SeMNPV, OpMNPV and SpltMNPV underwent apoptosis, displaying apoptotic bodies, characteristic DNA fragmentation and increased caspase-3-like protease activity; HycuNPV induced the most severe apoptosis. In HycuNPV-infected Ld652Y cells, a considerable amount of viral DNA was synthesized although there was no detectable yield of budded virions and polyhedrin. Northern blot and immunoblot analyses revealed that HycuNPV inhibitor of apoptosis 3 (IAP3), which has been shown to function in Sf9 cells, was expressed in HycuNPV-infected Ld652Y cells at a level higher than or comparable with that in HycuNPVinfected SpIm cells, which produced a high titre of progeny virions without any apoptotic response. These results imply that the relative ease of apoptosis induction in NPV-infected Ld652Y cells is largely dependent on inherent cellular properties rather than functions of the respective NPVs, and indicate that the defect in progeny virion production is not merely due to the virus-induced apoptosis in HycuNPV-infected Ld652Y cells.
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