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。它是痘病毒科中痘病毒属的一个成员。它以非持久性的方式由蚜虫传播，自然感染主要是十字花科植物。TuMV的基因组由一个大约10kb的单链阳性RNA分子组成，具有聚腺苷化的3'端。这表现为一个大的多蛋白，它被蛋白酶裂解以产生几种功能蛋白。功能蛋白有；P1，辅助成分-蛋白酶（HC-Pro）， P3, 6K1，细胞质包涵体，6K2， nia基因组连接病毒，nia蛋白酶，nib聚合酶和外壳蛋白（CP）。已知HG-Pro蛋白参与蚜虫传播，并在受感染的完整植物中为二聚体。然而，该蛋白在完整植物中的积累情况以及HG-Pro蛋白基因中负责蚜虫传播的区域仍不清楚。从侵染植物中纯化HG-Pro蛋白是一个相当困难的过程。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表达。纯化后的蛋白注射到家兔体内。用抗hc - pro蛋白血清作为探针，追踪感染TuMV的完整植株中HG-Pro蛋白的积累。HG-Pro蛋白和CP积累的时间过程表明，CP是逐渐积累的，而HG-Pro蛋白是先积累后逐渐减少的，说明HC-Pro蛋白比CP在完整植株中更不稳定。为了研究HC-Pro蛋白基因在蚜虫传播中的作用区域，将完整的HC-Pro蛋白基因克隆到烟草花叶病毒侵染cDNA中。然后转录嵌合病毒的rna，将其接种到本菌烟草植株上。结果表明，HG-Pro蛋白的n端区有30个氨基酸和c端区有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.