Studies on function and structure of plant viral proteins using chimeric virus

芜菁花叶病毒(TuMV)在世界范围内发生。它是马铃薯Y病毒属的一员，属于马铃薯Y病毒科。它以非持久性的方式由蚜虫传播，主要侵染十字花科植物。TuMV基因组由一个约10kb的单链正义RNA分子组成，3‘端为多聚腺苷酸化。它被表达为一种大的多聚蛋白，被蛋白酶裂解，产生几种功能蛋白。这些功能蛋白包括：P1、辅助成分-蛋白水解酶(HC-Pro)、P3、6K1、细胞质包涵体、6K2、NIA基因组连锁病毒、NIA-蛋白酶、NIB-聚合体和外壳蛋白(CP)。已知HG-Pro蛋白参与了蚜虫的传播，并在受感染的完整植物中是一种二聚体。然而，HG-Pro蛋白在完整植物中的积累情况以及导致蚜虫传播的HG-Pro蛋白基因的区域仍不清楚，从受感染的植物中分离纯化HG-Pro蛋白是一个相当困难的问题。因此，将HC-Pro蛋白基因克隆到表达载体中，并在大肠杆菌细胞中进行了表达。纯化后的蛋白注射到兔体内。以抗HC-Pro蛋白血清为探针，追踪HG-Pro蛋白在感染TuMV的完整植株中的积累。HG-Pro蛋白和CP的积累过程表明，CP是逐渐积累的，而HG-Pro蛋白是先积累后逐渐减少的，说明在完整植株中，HC-Pro蛋白比CP更不稳定。为研究HC-Pro蛋白基因在烟蚜传播中的作用区域，将完整的HC-Pro蛋白基因克隆到烟草花叶病毒侵染的cDNA中，然后将嵌合病毒的RNA转录并接种到烟草植株上。结果表明，HG-Pro蛋白N-末端的30个氨基酸和C-末端的67个氨基酸不是蚜虫传播所必需的。

Turnip mosaic virus (TuMV) occurs worldwidu. It is a member of the genus Potyvirus in the family Potyviridoe. It is transmitted in the non-persistent manner by aphids and naturally infects mainly Cruciferae plants. The genome of TuMV consists of one molecule of single-stranded, positive sense RNA of approximately 10kb with a polyadunylated 3' end. This is expressed as a large polyprotein which is cleaved by proteases to yield several functional proteins. The functional proteins are ; P1, helper component - proteinase (HC-Pro), P3, 6K1, cytoplasmic inclusion, 6K2, NIa-genome linked viral, NIa-proteinase, Nib-polymerse and coat proteins (CP). HG-Pro protein is known to be involved as aphid transmission and to be a dimer in infected intact plants. However, accumulation of the protein in intact plants and the region of HG-Pro protein gene responsible for aphid transmission are still remained unclear.It is a quite difficult to purify HG-Pro protein from infected plants. Thus, HC-Pro protein gene was cloned into expression vector and was expressed in Eschericlzia coli cells. The protein was purified and then injected to rabbit. Anti-HC-Pro protein serum was used as a probe for tracing accumulation of HG-Pro proteins in TuMV infected intact plants. The time course of accumulation of HG-Pro protein and CP showed that CP gradually accmulated, whereas HG-Pro protein accumulated initially and then gradually decreased The result indicates HC-Pro protein is much unstable than CP in intact plants. To investigate the responsible region of HC-Pro protein gene for aphid transmission, complete and duleted HC-Pro protein gene were cloned into tobacco mosaic virus infectious cDNA.Then the RNAs of chimeric virus were transcribed and inoculated to Nicotiana benthamiana plants. The results indicate that 30 amino acids of N-terminal region and 67 amino acids of C-terminal region of HG-Pro protein was not necessary for aphid transmission.