摘要
Pea enation mosaic virus (PEMV: family Luteoviridae) is transmitted in a persistent, circulative manner by aphids. We inserted cDNAs encoding the structural proteins of PEMV, the coat protein (CP) and coat protein-read through domain (CPRT) into the genome of the baculovirus Autographa californica multiple nucleopolyhedrovirus with and without a histidine tag or an upstream Kozak consensus sequence. The Sf21 cell line provided the highest level of CP expression of the cell lines tested and resulted in production of virus-like particles (VLPs). The CPRT was not detected in recombinant baculovirus-infected cells by Western blot. Addition of a Kozak sequence increased the yield of baculovirus produced VLPs. Baculovirus-expressed VLPs purified on a nickel NTA column were of variable size (13–30 nm) and contained CP mRNA. The purified VLPs were also shown by RT-PCR to contain 70% of 154 baculovirus mRNAs, indicative of non-specific RNA encapsidation in the absence of viral RNA replication. When fed to the pea aphid, Acyrthosiphon pisum (Harris), the VLPs entered the aphid hemocoel, demonstrating that CPRT is not required for uptake of PEMV from the aphid gut. Baculovirus expression of PEMV VLPs provides a useful tool for future analysis of RNA encapsidation requirements and molecular aphid–virus interactions.
摘要译文
豌豆enation花叶病毒（PEMV：家庭Luteoviridae）是由蚜虫持续，循环地传播。我们插入编码PEMV结构蛋白的cDNA，将通过蛋白质结构域（CPRT）的外壳蛋白读取到具有或不具有组氨酸标签或上游Kozak共有序列的杆状病毒苜蓿银纹夜蛾多核型多角体病毒的基因组中。Sf21细胞系提供了测试的细胞系的最高水平的CP表达，并导致产生病毒样颗粒（VLP）。通过蛋白质印迹在重组杆状病毒感染的细胞中未检测到CPRT。添加Kozak序列增加了杆状病毒产生的VLP的产量。在镍NTA柱上纯化的杆状病毒表达的VLP具有不同的大小（13-30nm）并含有CP mRNA。通过RT-PCR也显示纯化的VLP含有70％的154种杆状病毒mRNA，指示在不存在病毒RNA复制的情况下的非特异性RNA衣壳化。当喂养豌豆蚜Acyrthosiphon pisum（Harris）时，VLPs进入蚜虫hemocoel，表明CPRT不需要从蚜虫肠道吸收PEMV。PEMV VLPs的杆状病毒表达为RNA衣壳需求和分子蚜虫 - 病毒相互作用的未来分析提供了有用的工具。
S.Sivakumar[a];ZhaohuiWang[b];Robert L.Harrison[a][1];SijunLiu[a];W. AllenMiller[b];Bryony C.Bonning[a];. Baculovirus-expressed virus-like particles of Pea enation mosaic virus vary in size and encapsidate baculovirus mRNAs[J]. Virus Research, 2009,139(1): 54-63