The Investigation of the Interaction between the Coat Protein and the Helicase-Like Domain of Bamboo Mosaic Virus Replicase

• Main Authors: Yuan-Ning Ho, 何苑寧
• Other Authors: Meng-Hsiao Meng
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/74736917239148959200

碩士 === 國立中興大學 === 生物科技學研究所 === 96 === Bamboo mosaic virus (BaMV) has a single-strand RNA genome which consists of five open reading frames (ORFs). ORF 1 encodes a ~155 kDa replicase that contains a capping enzyme domain, a helicase-like domain, and a RNA dependent RNA polymerase. ORF5 encodes the viral coat protein. Previous studies showed a interaction between the helicase-like domain and the coat protein of BaMV by yeast-two-hybrid screening. Hence, I focused on investigating the biological significance of the interaction between the helicase-like domain and the coat protein of this virus in this study. First, I investigated the effect of the helicas-like domain on coat protein accumulation by overexpressing extra helicase-like domain in protoplast assay. Preliminary results showed that the coat protein accumulation was reduced when the helicase-like domain was additionally expressed. To verify the reduction of BaMV replication, I will perform real-time PCR to assay the RNA accumulation of BaMV. I also randomly mutated amino acids on the coat protein by the error-prone PCR to find out the interaction sites with the helicase-like domain in a bacteria-two-hybrid screening. The screening identified two mutant coat proteins which one with 4 amino acid substitutions (I130M, D170N, A209G and N218K) and another with 1 amino acid substitution (N210S) that has weaken interaction with the helicase-like domain . Furthermore, I confirmed that two mutant coat proteins reduce the interaction ability with the helicase-like domain by pull down assay. In the future, I will continue to search for more interaction sites between the helicase-like domain and the coat protein. In order to elucidate more biological significance of the interaction, we will mutate these amino acids respectively which I have found to apply in N. benthamiana.