Bmapaf-1 is Involved in the Response against BmNPV Infection by the Mitochondrial Apoptosis Pathway.
Bmapaf-1 is Involved in the Response against BmNPV Infection by the Mitochondrial Apoptosis Pathway.
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Bmapaf-1 参与通过线粒体凋亡途径对抗 BmNPV 感染的反应
DOI：
10.3390/insects11090647
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发表时间：
2020-09-22
期刊：
影响因子：
3
通讯作者：
Li MW
中科院分区：
文献类型：
--
作者：
Wang XY;Ding XY;Chen QY;Zhang KX;Zhao CX;Tang XD;Wu YC;Li MW
简单总结 Apaf-1 参与细胞凋亡途径，并且 Bmapaf-1 在我们之前的转录组数据中显示出对 BmNPV 感染的显着反应。在本研究中，研究了 Bmapaf-1 响应 BmNPV 感染的潜在机制。为了初步确定Bmapaf-1与BmNPV的关系，分析了病毒感染后差异抗性家蚕品系不同组织中Bmapaf-1的表达模式。为了进一步明确 Bmapaf-1 在 BmNPV 感染中的作用，在使用 siRNA 和 pIZT/V5-His-mCherry 昆虫载体敲低和过表达 Bmapaf-1 后，分别分析了 BmN 细胞中 BmNPV 感染的变化以及 Bmcas-Nc 和 Bmcas-1 的表达模式。此外，为了分析Bmapaf-1是否通过细胞凋亡参与BmNPV感染，使用诱导剂NSC348884和抑制剂Z-DEVD-FMK。摘要 抗BmNPV（家蚕核型多角体病毒）家蚕品系的发现表明，某种抗病毒分子机制确实存在，但仍不清楚。细胞凋亡作为免疫系统的固有部分，在抵抗病原体感染的反应中发挥着重要作用，并可能参与抗BmNPV感染。我们之前的研究鉴定了几个参与线粒体凋亡途径的候选基因。家蚕凋亡蛋白酶激活因子-1（Bmapaf-1）就是其中之一，但其抗病毒机制尚不清楚。在本研究中，对 BmApaf-1 的序列进行了表征。研究发现它含有独特的转座酶_1 功能域，并与其他物种共享高 CARD 和 NB-ARC 域。 Bmapaf-1在胚胎发育、变态和生殖发育的关键时刻有相对较高的表达水平。此外，病毒感染后不同组织中Bmapaf-1表达的显着差异表明其与BmNPV密切相关，这通过RNAi和BmN细胞中的过表达进一步得到验证。简而言之，Bmapaf-1 过表达后 72 小时，增强型绿色荧光蛋白 (BV-EGFP) 芽生病毒的感染受到显着抑制，这在用 siRNA 敲低 Bmapaf-1 后得到证实。此外，在BmN细胞中过表达Bmapaf-1后，Bmapaf-1的下游基因，包括BmNc）和Bmcaspase-1（Bmcas-1）上调，这与RNAi结果一致。此外，使用凋亡诱导剂 NSC348884 和抑制剂 Z-DEVD-FMK 确定 Bmapaf-1 响应 BmNPV 感染的现象与细胞凋亡有关。因此，Bmapaf-1 通过线粒体凋亡途径参与针对 BmNPV 感染的反应。该结果为阐明家蚕抗BmNPV机制及抗性家蚕品系选育提供了有价值的数据。
Simple Summary Apaf-1 is involved in the apoptosis pathway and Bmapaf-1 showed a significant response to BmNPV infection in our previous transcriptome data. In this study, the underlying mechanism of Bmapaf-1 in response to BmNPV infection was studied. To preliminarily determine the relationship of Bmapaf-1 with BmNPV, the expression pattern of Bmapaf-1 was analyzed in different tissues of differentially resistant silkworm strains following virus infection. To further define the role of Bmapaf-1 in BmNPV infection, the alteration of BmNPV infection in BmN cells and the expression patterns of Bmcas-Nc and Bmcas-1 were analyzed following knockdown and overexpression of Bmapaf-1 using siRNA and the pIZT/V5-His-mCherry insect vector, respectively. Furthermore, to analyze whether Bmapaf-1 is involved in BmNPV infection by apoptosis, the inducer NSC348884 and inhibitor Z-DEVD-FMK were used. Abstract Discovery of the anti-BmNPV (Bombyx mori nuclearpolyhedrovirus) silkworm strain suggests that some kind of antiviral molecular mechanism does exist but is still unclear. Apoptosis, as an innate part of the immune system, plays an important role in the response against pathogen infections and may be involved in the anti-BmNPV infection. Several candidate genes involved in the mitochondrial apoptosis pathway were identified from our previous study. Bombyx mori apoptosis protease-activating factor-1 (Bmapaf-1) was one of them, but the antiviral mechanism is still unclear. In this study, sequences of BmApaf-1 were characterized. It was found to contain a unique transposase_1 functional domain and share high CARD and NB-ARC domains with other species. Relatively high expression levels of Bmapaf-1 were found at key moments of embryonic development, metamorphosis, and reproductive development. Further, the significant difference in expression of Bmapaf-1 in different tissues following virus infection indicated its close relationship with BmNPV, which was further validated by RNAi and overexpression in BmN cells. Briefly, infection of budded virus with enhanced green fluorescent protein (BV-EGFP) was significantly inhibited at 72 h after overexpression of Bmapaf-1, which was confirmed after knockdown of Bmapaf-1 with siRNA. Moreover, the downstream genes of Bmapaf-1, including Bmnedd2-like caspase (BmNc) and Bmcaspase-1 (Bmcas-1), were upregulated after overexpression of Bmapaf-1 in BmN cells, which was consistent with the RNAi results. Furthermore, the phenomenon of Bmapaf-1 in response to BmNPV infection was determined to be related to apoptosis using the apoptosis inducer NSC348884 and inhibitor Z-DEVD-FMK. Therefore, Bmapaf-1 is involved in the response against BmNPV infection by the mitochondrial apoptosis pathway. This result provides valuable data for clarifying the anti-BmNPV mechanism of silkworms and breeding of resistant silkworm strains.
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