Mlotshwa Sizolwenkosi, Verver Jan, Sithole-Niang Idah, Gopinath Kodetham, Carette Jan, van Kammen Ab, Wellink Joan
Laboratory of Molecular Biology, Wageningen University, The Netherlands.
Virus Genes. 2002 Oct;25(2):207-16. doi: 10.1023/a:1020122104651.
The helper component-proteinase (HC-Pro) of Cowpea aphid-borne mosaic virus (CABMV) was expressed in Escherichia coli and used to obtain HC-Pro antiserum that was used as an analytical tool for HC-Pro studies. The antiserum was used in immunofluorescence assays to study the subcellular location of HC-Pro expressed with other viral proteins in cowpea protoplasts in a natural CABMV infection, or in protoplasts transfected with a transient expression construct expressing HC-Pro separately from other viral proteins under the control of the 35S promoter. In both cases the protein showed a diffuse cytoplasmic location. Similar localisation patterns were shown in live protoplasts when the transient expression system was used to express HC-Pro as a fusion with the green fluorescent protein as a reporter. In an alternative expression system, the HC-Pro coding region was subcloned in-frame between the movement protein and large coat protein genes of RNA2 of Cowpea mosaic virus (CPMV). Upon transfection of protoplasts with this construct, HC-Pro was expressed as part of the RNA2 encoded polyprotein from which it was fully processed. In this case, the protein localised in broad cytoplasmic patches reminiscent of the typical CPMV induced cytopathic structures in which CPMV replication occurs, suggesting an interaction of HC-Pro with CPMV proteins or host factors in these structures. Finally, recombinant CPMV expressing HC-Pro showed a strongly enhanced virulence on cowpea and Nicotiana benthamiana consistent with the role of HC-Pro as a pathogenicity determinant, a phenomenon now known to be linked to its role as a suppressor of host defense responses based on post-transcriptional gene silencing.
豇豆蚜传花叶病毒（CABMV）的辅助成分蛋白酶（HC-Pro）在大肠杆菌中表达，并用于获得HC-Pro抗血清，该抗血清用作HC-Pro研究的分析工具。该抗血清用于免疫荧光分析，以研究在自然CABMV感染的豇豆原生质体中，或在用在35S启动子控制下与其他病毒蛋白分开表达HC-Pro的瞬时表达构建体转染的原生质体中，与其他病毒蛋白一起表达的HC-Pro的亚细胞定位。在这两种情况下，该蛋白均显示出弥漫性的细胞质定位。当使用瞬时表达系统将HC-Pro作为与绿色荧光蛋白融合的报告基因表达时，在活原生质体中也显示出类似的定位模式。在另一种表达系统中，HC-Pro编码区被框内亚克隆到豇豆花叶病毒（CPMV）RNA2的运动蛋白和大外壳蛋白基因之间。用该构建体转染原生质体后，HC-Pro作为RNA2编码的多蛋白的一部分表达，并从中完全加工。在这种情况下，该蛋白定位于广泛的细胞质斑块中，让人联想到典型的CPMV诱导的细胞病变结构，CPMV在其中进行复制，这表明HC-Pro与这些结构中的CPMV蛋白或宿主因子相互作用。最后，表达HC-Pro的重组CPMV对豇豆和本氏烟草表现出强烈增强的毒力，这与HC-Pro作为致病性决定因素的作用一致，现在已知这种现象与其作为基于转录后基因沉默的宿主防御反应抑制剂的作用有关。
