Lacorte Cristiano, Lohuis Hendrikus, Goldbach Rob, Prins Marcel
Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands.
Virus Res. 2007 Nov;129(2):80-6. doi: 10.1016/j.virusres.2007.06.020. Epub 2007 Aug 14.
Chicken anemia virus (CAV) is an important pathogen of chicken worldwide, causing severe anemia and immunodeficiency. Its small single-stranded DNA genome (2.3kb) encodes three proteins: VP1, the only structural protein, VP2, a protein phosphatase, and VP3, also known as apoptin, which induces apoptosis. In this study, CAV proteins were expressed in plants as an alternative for recombinant protein production in animal cells. Additionally, the effect of VP3 expression was tested to evaluate possible involvement in programmed cell death in plants. The CAV genes were cloned in binary vectors with the Green fluorescent protein (GFP) as N terminal fusion, and into a Potato virus X (PVX) and Tobacco Mosaic Virus (TMV)-based vectors. Nicotiana benthamiana plants were inoculated with Agrobacterium tumefaciens containing the binary vector constructs or the PVX and TMV constructs. Upon transient expression GFP:VP1 and GFP:VP2 were observed throughout the nucleoplasm, whereas VP3 formed compact aggregates within the nucleus, indicating functional nuclear localization signals in all three proteins. An intense fluorescence was observed for VP2 and VP3 fusions, whereas GFP:VP1 fluorescence remained faint and was only detected in a limited number of cells. Co-expression of GFP:VP1 and VP2 had a marked alteration on the distribution of GFP:VP1, forming large VP1 aggregates throughout the nucleus, indicating an interaction of the two CAV proteins. No visible alteration on GFP pattern was detected upon co-expression of GFP:VP1 and VP3, or with GFP:VP2 and VP3. Plants infected with PVX or TMV-based vectors expressing VP3 displayed strong necrosis and wilting, however, a direct association with VP3 expression and programmed cell death in plants, could not be established. Overall, our results show that all CAV proteins can be expressed in plant cells, though expression level of VP1 needs to be further optimized before testing its potential as (edible) subunit vaccine.
鸡贫血病毒（CAV）是一种在全球范围内对鸡具有重要影响的病原体，可导致严重贫血和免疫缺陷。其小的单链DNA基因组（2.3kb）编码三种蛋白质：唯一的结构蛋白VP1、一种蛋白磷酸酶VP2以及也被称为凋亡素的VP3，后者可诱导细胞凋亡。在本研究中，CAV蛋白在植物中表达，作为在动物细胞中生产重组蛋白的一种替代方法。此外，测试了VP3表达的效果，以评估其是否可能参与植物中的程序性细胞死亡。CAV基因被克隆到以绿色荧光蛋白（GFP）作为N端融合的双元载体中，并克隆到基于马铃薯X病毒（PVX）和烟草花叶病毒（TMV）的载体中。用含有双元载体构建体或PVX和TMV构建体的根癌农杆菌接种本氏烟草植株。瞬时表达后，在整个核质中观察到GFP:VP1和GFP:VP2，而VP3在细胞核内形成紧密聚集体，表明这三种蛋白质中都存在功能性核定位信号。观察到VP2和VP3融合蛋白有强烈荧光，而GFP:VP1荧光较弱，仅在有限数量的细胞中检测到。GFP:VP1和VP2的共表达对GFP:VP1的分布有显著改变，在整个细胞核中形成大的VP1聚集体，表明这两种CAV蛋白之间存在相互作用。GFP:VP1和VP3或GFP:VP2和VP3共表达时，未检测到GFP模式有明显改变。感染表达VP3的基于PVX或TMV的载体的植物表现出强烈的坏死和萎蔫，然而，无法确定其与植物中VP3表达和程序性细胞死亡之间的直接关联。总体而言，我们的结果表明，所有CAV蛋白都可以在植物细胞中表达，不过在测试其作为（可食用）亚单位疫苗的潜力之前，VP1的表达水平需要进一步优化。
