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2025年4月4日 星期五
农业生物技术学报  2021, Vol. 29 Issue (2): 340-351    DOI: 10.3969/j.issn.1674-7968.2021.02.014
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
杆状病毒Ac78 N端保守氨基酸位点的功能分析
李赛男*, 吕怡娜, 甘田, 区炳明, 赵海洲, 刘文华
肇庆学院 生命科学学院,肇庆 526061
Functional Analysis of the N-terminal Conserved Amino Acids in Baculovirus Ac78
LI Sai-Nan*, LV Yi-Na, GAN Tian, OU Bing-Ming, ZHAO Hai-Zhou, LIU Wen-Hua
Department of Biology, Zhaoqing University, Zhaoqing 526061, China
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摘要 苜蓿丫纹夜蛾核多角体病毒(Autographa californica multiple nucleopolyhedrovirus, AcMNPV)的orf78 (即ac78)是杆状病毒的核心基因,在AcMNPV的生活周期中具有重要作用,作用机制未知,其编码蛋白Ac78的保守氨基酸2~25和64~88区域是Ac78行使功能的关键区域。本研究通过生物信息学分析发现,Ac78 N端第9位精氨酸残基(R9)和第22位赖氨酸残基(K22)在Ac78旁系同源物中高度保守,提示该保守位点可能在Ac78的功能中具有重要作用。通过定点突变技术分别将Ac78 R9和K22突变为丙氨酸(A),构建了重组杆状病毒vAc78R9A:HA和vAc78K22A:HA,将病毒DNA分别转染草地贪夜蛾(Spodoptera frugiperda)细胞Sf9。经荧光显微镜观察和病毒生长曲线分析发现,在vAc78R9A:HA转染的Sf9细胞中产生的感染性芽生型病毒粒子(budded virion, BV)与ac78全长补回型重组病毒(vAc78:HA)相当,而vAc78K22A:HA转染的Sf9细胞中产生的感染性BV的产量与vAc78:HA相比下降约100倍;电镜观察实验表明,vAc78R9A:HA转染的Sf9细胞中能正常形成多粒包埋型病毒粒子(multiple nucleocapsid-enveloped occlusion-derived virion, M-ODV)和包埋有M-ODV的多角体,与vAc78:HA的现象一致,而vAc78K22A:HA转染的Sf9细胞中产生的M-ODV显著减少,单粒包埋型病毒粒子显著增加。综上所述,Ac78的N端保守位点R9对于Ac78在BV产生和M-ODV形成中的功能非必需,而K22在Ac78的功能中具有重要作用。本研究丰富了Ac78的信息,为进一步解剖Ac78的结构及探明Ac78的作用机理提供了实验和理论基础。
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李赛男
吕怡娜
甘田
区炳明
赵海洲
刘文华
关键词 苜蓿丫纹夜蛾核多角体病毒(AcMNPV)ac78N端保守氨基酸位点芽生型病毒粒子(BV)多粒包埋型病毒粒子(M-ODV)    
Abstract:Baculoviruses are a family of enveloped, rod-shaped, double-stranded DNA viruses which are specifically pathogenic to arthropods, mainly belonging to the orders Lepidoptera, Hymenoptera, and Diptera. ac78 of the archetype of the Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was one of the 38 core genes of baculoviruses. Previous studies showed that Ac78 played an important role in the baculovirus life cycle, and Ac78 conserved amino acid (aa) 2~25 and 64~88 were essential for Ac78 functions during baculovirus life cycle, but the mechanism by which Ac78 employs that affected the budded virion (BV) production and multiple nucleocapsid-enveloped occlusion-derived virion (M-ODV) formation was still unknown. An aa alignment of the Ac78 homologs showed that Ac78 N-terminal aa 9 (R9) and 22 (K22) were highly conserved in baculoviruses, suggesting that R9 and K22 might be important in the functions of Ac78. In the present study, the ac78 with a HA tag prior to the stop codon, in which the codon of R9 or K22 was mutated into the codon of alanine (A), together with the enhanced green fluorescence protein gene (egfp; referred to as gfp in the present study) and the AcMNPV polyhedrin (polh) gene, was inserted into the polh locus of the ac78-knockout AcMNPV bacmid (bAc78KO) to construct the recombinant viruses, vAc78R9A:HA or vAc78K22A:HA. All recombinant viruses were confirmed by PCR analysis and further DNA sequencing. The ac78-knockout virus (vAc78KO), ac78-repaired virus (vAc78:HA), vAc78R9A:HA, or vAc78K22A:HA bacmid DNA was transfected into Sf9 (Spodoptera frugiperda IPLB-Sf21-AE clonal isolate 9) cells, viral replication and infection were monitored by a fluorescence microscope. At 24 h post transfection (h p.t.), no obvious differences in the amounts of GFP-positive cells were observed among all 4 groups, indicating comparable transfection efficiencies. At 72 h p.t., almost all Sf9 cells transfected with vAc78R9A:HA or vAc78:HA bacmid DNA showed GFP fluorescence, the number of fluorescent cells increased slightly onwards in the vAc78KO- or vAc78K22A: HA-transfected cells, but the amount of GFP-positive cells in Sf9 cells transfected with vAc78K22A:HA was a little more than that in Sf9 cells transfected with vAc78KO. Using virus supernatants harvested from vAc78KO-, vAc78:HA-, vAc78R9A:HA-, or vAc78K22A:HA-transfected Sf9 cells, viral growth curve analysis was performed by fifty percent tissue culture infective dose endpoint dilution assay to further evaluate the ability of vAc78R9A:HA and vAc78K22A:HA to produce infectious BVs. There was no significant difference between vAc78R9A:HA and vAc78:HA in capabilities to generate infectious BVs, while the titer of vAc78K22A:HA was approximately 100-fold lower than that of vAc78:HA at 120 h p.t. The results showed that the R9 mutation had no effect on infectious BV production, but the K22 mutation significantly reduced the production of the infectious BVs. Thin sections of vAc78KO-, vAc78:HA-, vAc78R9A:HA-, or vAc78K22A:HA-transfected Sf9 cells at 72 h p.t. were observed using an electron microscope. In vAc78:HA- or vAc78R9A:HA-transfected Sf9 cells, numerous M-ODVs and occlusion bodies (OBs) embedding the numerous M-ODVs emerged in the ring zone, but in the vAc78KO- or vAc78K22A:HA-transfected cells, the M-ODVs in the ring zone and OBs were significantly fewer than those in the vAc78:HA-transfected Sf9 cells, showing that the R9 mutation did not affect the M-ODV formation, or the embedding of M-ODVs into the OBs, while the K22 was important in the formation of M-ODVs and the embedding of M-ODVs into the OBs. Taken together, the results demonstrated that Ac78 R9 was not essential for the infectious BV production and M-ODV formation, but K22 played an important role in the Ac78 functions. This study enriches the information of Ac78, and would lay a foundation for uncovering the mechanism employed by Ac78.
Key wordsAutographa californica multiple nucleopolyhedrovirus (AcMNPV)    ac78    N-terminal conserved amino acid    Budded virion (BV)    Multiple nucleocapsid-enveloped occlusion-derived virion (M-ODV)
收稿日期: 2020-05-28      出版日期: 2021-02-01
ZTFLH:  S182  
基金资助:国家自然科学基金(31501705); 广东高校省级重点平台和科研项目(2018KTSCX248); 广东省自然科学基金(2014A030313664)
通讯作者: *lisainan2001@sina.com.cn   
引用本文:   
李赛男, 吕怡娜, 甘田, 区炳明, 赵海洲, 刘文华. 杆状病毒Ac78 N端保守氨基酸位点的功能分析[J]. 农业生物技术学报, 2021, 29(2): 340-351.
LI Sai-Nan, LV Yi-Na, GAN Tian, OU Bing-Ming, ZHAO Hai-Zhou, LIU Wen-Hua. Functional Analysis of the N-terminal Conserved Amino Acids in Baculovirus Ac78. 农业生物技术学报, 2021, 29(2): 340-351.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2021.02.014     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2021/V29/I2/340
 
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