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

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.