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

芜菁花叶病毒（Turnipmosaicvirus，TuMV）在世界范围内均有发生。它是Potyviridoe家族中Potyvirus属的成员。它通过蚜虫以非持久性方式传播，主要自然感染十字花科植物。TuMV的基因组由一个约10 kb的单链正义RNA分子组成，其3'端具有聚腺苷酸化。这被表达为一个大的多聚蛋白，其被蛋白酶切割以产生几种功能蛋白。功能蛋白质有：P1，辅助组分-蛋白酶（HC-Pro），P3，6 K1，细胞质内含物，6 K2，NIa-基因组连锁病毒，NIa-蛋白酶，Nib-聚合酶和外壳蛋白（CP）。已知HG-Pro蛋白参与蚜虫的传播，并且在受感染的完整植物中是二聚体。然而，HG-Pro蛋白在完整植株中的积累情况以及HG-Pro蛋白基因中与蚜虫传播有关的区域尚不清楚，因此从受感染的植株中纯化HG-Pro蛋白是一个相当困难的问题。将HC-Pro基因克隆到表达载体中，并在大肠杆菌中进行表达。纯化蛋白后注射家兔体内。抗HC-Pro蛋白血清用作探针，用于追踪在TuMV感染的完整植物中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蛋白则是先积累后逐渐减少，说明HC-Pro蛋白在完整植株中比CP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性cDNA中，并将其转录成嵌合病毒RNA，接种本氏烟草（Nicotianabenthamiana）。结果表明，HG-Pro蛋白N端30个氨基酸和C端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.