美洲棉铃虫多核多角体病毒 ORF11 对于芽生病毒的产生和包埋型病毒的包膜至关重要。
Autographa californica multiple nucleopolyhedrovirus ORF11 is essential for budded-virus production and occlusion-derived-virus envelopment.
机构信息
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, China Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang, China.
Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, South Korea.
出版信息
J Virol. 2015 Jan;89(1):373-83. doi: 10.1128/JVI.01742-14. Epub 2014 Oct 15.
UNLABELLED
ORF11 (ac11) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene with unknown function. To determine the role of ac11 in the baculovirus life cycle, an ac11 knockout mutant of AcMNPV, Ac11KO, was constructed. Northern blot and 5' rapid amplification of cDNA ends (RACE) analyses revealed that ac11 is an early gene in the life cycle. Microscopy, titration assays, and Western blot analysis revealed that budded viruses (BVs) were not produced in Ac11KO-transfected Sf9 cells. However, quantitative PCR (qPCR) analysis demonstrated that the deletion of ac11 did not affect viral DNA replication. Furthermore, electron microscopy revealed that there was no nucleocapsid in the cytoplasm or plasma membrane of Ac11KO-transfected cells, which demonstrates that the defect in BV production in Ac11KO-transfected cells is due to the inefficient egress of nucleocapsids from the nucleus to the cytoplasm. In addition, electron microscopy observations showed that the nucleocapsids in the nucleus were not enveloped to form occlusion-derived viruses (ODVs) and that their subsequent embedding into occlusion bodies (OBs) was also blocked in Ac11KO-transfected cells, demonstrating that ac11 is required for ODV envelopment. These results therefore demonstrate that ac11 is an early gene that is essential for BV production and ODV envelopment.
IMPORTANCE
Baculoviruses have been extensively used not only as specific, environmentally benign insecticides but also as helper-independent protein expression vectors. Although the function of baculovirus genes in viral replication has been studied by using gene knockout technology, the functions of more than one-third of viral genes, which include some highly conserved genes, are still unknown. In this study, ac11 was proven to play a crucial role in BV production and ODV envelopment. These results will lead to a better understanding of baculovirus infection cycles.
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美洲棉铃虫多角体病毒（AcMNPV）的 ORF11（ac11）是一个高度保守的基因，其功能未知。为了确定 ac11 在杆状病毒生命周期中的作用，构建了 AcMNPV 的 ac11 敲除突变体 Ac11KO。Northern blot 和 5'快速扩增 cDNA 末端（RACE）分析显示，ac11 是生命周期中的早期基因。显微镜、滴定测定和 Western blot 分析显示，ac11KO 转染的 Sf9 细胞中未产生出芽病毒（BVs）。然而，定量 PCR（qPCR）分析表明，ac11 的缺失并不影响病毒 DNA 复制。此外，电子显微镜显示，ac11KO 转染细胞的细胞质或质膜中没有核衣壳，这表明 ac11KO 转染细胞中 BV 产量的缺陷是由于核衣壳从细胞核到细胞质的出核效率低下所致。此外，电子显微镜观察表明，细胞核中的核衣壳未被包裹形成出芽病毒（ODV），并且它们随后嵌入空斑形成病毒（OB）中也在 ac11KO 转染细胞中受阻，这表明 ac11 是 ODV 包膜所必需的。这些结果表明，ac11 是一个早期基因，对于 BV 产生和 ODV 包膜至关重要。
重要性
杆状病毒不仅被广泛用作特异性、环境友好型杀虫剂，还被用作无辅助蛋白表达载体。尽管已经使用基因敲除技术研究了杆状病毒基因在病毒复制中的功能，但仍有超过三分之一的病毒基因（包括一些高度保守的基因）的功能未知。在这项研究中，ac11 被证明在 BV 产生和 ODV 包膜中发挥关键作用。这些结果将有助于更好地了解杆状病毒感染周期。
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