Analysis of molecular mechanisms for apoptosis in insects infected with baculoviruses

杆状病毒感染昆虫细胞凋亡的分子机制分析

基本信息

批准号：
14206007
负责人：
KOBAYASHI Michihiro
金额：
$ 34.28万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14206007/
关键词：
nucleopolyhedrovirus Ld652Y cell fall webworm gypsy moth apoptosis global protein synthesis shutdown anti-viral responce バキュロウイルス組換えウイルス gp64 培養細胞 sf9細胞 High Five細胞昆虫

项目摘要

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

Ld652Y 细胞被七种不同的核多角体病毒 (NPV) 感染，包括来自苜蓿银纹夜蛾 (AcMNPV)、家蚕 (BmNPV)、美国白蛾 (HycuNPV)、甜菜夜蛾 (SeMNPV)、舞毒蛾 (Lymantria dispar)、伪蠼螋 (OpMNPV) 和斜纹夜蛾 (Spodoptera litura) 的病毒（SpltMNPV）。结果表明，感染 BmNPV、HycuNPV、SeMNPV、OpMNPV 和 SpltMNPV 的 Ld652Y 细胞发生凋亡。在感染 HycuNPV 的 Ld652Y 细胞中，虽然没有检测到出芽病毒粒子和多角体蛋白，但合成了大量病毒 DNA。分析表明，HycuNPV 凋亡抑制剂 3 (Hycu-IAP3) 已被证明具有功能在 Sf9 细胞中，HycuNPV 感染的 Ld652Y 细胞中的表达水平与 HycuNPV 感染的 SpIm 细胞中的表达水平相当，后者产生了高滴度的子代病毒粒子，而没有任何凋亡反应。这些结果意味着 NPV 中细胞凋亡诱导相对容易。 - 感染的 Ld652Y 细胞很大程度上依赖于固有的细胞特性，而不是各自 NPV 的功能，并表明子代病毒粒子的产生不仅仅是由于HycuNPV感染的Ld652Y细胞中病毒诱导的细胞凋亡。HycuNPV感染早在感染后4小时就保护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基因组具有三个成员。因其氨基酸序列而被指定为 hycu-iap1、hycu-iap2 和 hycu-iap3 的 iap 基因通过在 Sf9 细胞中进行瞬时表达测定，对 Hycu-IAP 与其他杆状病毒中鉴定的 IAP 具有同源性，结果表明 Hycu-IAP3 可以阻断放线菌素 D 诱导的细胞凋亡，并挽救 p35 缺陷突变型 AcMNPV 的复制，而 Hycu-IAP1 和 Hycu-在 HycuNPV 感染期间，IAP2 没有表现出任何抗凋亡功能。这些结果表明，Hycu-IAP3 对于阻断 HycuNPV 感染 SpIm 细胞的细胞凋亡至关重要。