Analysis of molecular mechanisms for apoptosis in insects infected with baculoviruses

用7种不同的核型多角体病毒(NPVs)感染Ld652Y细胞，其中包括褐斑病病毒(AcMNPV)、家蚕病毒(BmNPV)、南美白蛾病毒(HycuNPV)、甜菜夜蛾病毒(SeMNPV)、毒蛾病毒、拟甜菜夜蛾病毒(OpMNPV)和斜纹夜蛾病毒(SpltMNPV)。结果表明，感染BmNPV、HycuNPV、SeMNPV、OpMNPV和SpltMNPV的Ld652Y细胞发生了凋亡。在HycuNPV感染的Ld652Y细胞中，虽然没有检测到萌发的病毒粒子和多角体蛋白，但仍合成了相当数量的病毒DNA。Northern杂交和免疫印迹分析表明，已被证明在Sf9细胞中发挥作用的HycuNPV凋亡抑制因子3(Hycu-IAP3)在感染HycuNPV的Ld652Y细胞中的表达水平与感染HycuNPV的SPIM细胞相当，后者产生了高滴度的子代病毒粒子，而没有任何凋亡反应。这些结果表明，在新城疫病毒感染的Ld652Y细胞中诱导凋亡的相对容易的是L…更多地依赖于固有的细胞特性而不是各自的NPV的功能，这表明后代病毒产生的缺陷不仅仅是由于病毒诱导Ld652Y细胞的凋亡。HycuNPV感染最早在感染后4h就保护SPIM细胞免受放线菌素D诱导的细胞凋亡。Southern杂交分析表明，HycuNPV基因组含有三个IAP基因，分别命名为hycu-iap1、hycu-iap2和hycu-iap3，因为它们的氨基酸序列与其他杆状病毒的IAP同源性。瞬时表达分析表明，Hycu-IAP3阻断了放线菌素D诱导的细胞凋亡，并挽救了p35缺陷突变株AcMNPV的复制，而Hycu-IAP1和Hycu-IAP2没有任何抗凋亡作用。在HycuNPV感染允许的SPIM细胞诱导凋亡过程中RNAi下调hycu-iap3的表达。这些结果表明，Hycu-IAP3在HycuNPV感染允许的SPIM细胞过程中对细胞凋亡的阻断是必不可少的。较少

Ld652Y cells were infected with seven different nucleopolyhedroviruses (NPVs), including those from Autographa californica (AcMNPV), Bombyx mori (BmNPV), Hyphantria cunea (HycuNPV), Spodoptera exigua (SeMNPV), Lymantria dispar, Orgyia pseudotsugata (OpMNPV) and Spodoptera litura (SpltMNPV). The results showed that Ld652Y cells infected with BmNPV, HycuNPV, SeMNPV, OpMNPV and SpltMNPV underwent 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 (Hycu-IAP3), which has been shown to function in Sf9 cells, was expressed in HycuNPV-infected Ld652Y cells at a level comparable with that in HycuNPV-infected SpIm cells, which produced a high titer of progeny virions without any apoptotic response. These results imply that the relative ease of apoptosis induction in NPV-infected Ld652Y cells is l … More argely 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.HycuNPV infection protected SpIm cells from actinomycin D-induced apoptosis as early as 4 h postinfection. Analysis by Southern hybridization revealed that the HycuNPV genome possessed three members of iap genes 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 of permissive SpIm cells induced apoptosis. These results indicate that Hycu-IAP3 is essential for blockage of apoptosis during HycuNPV infection of permissive SpIm cells. Less