番茄黃化捲葉病毒複製相關蛋白 (Rep) 與病毒基因體 DNA 之交互作用

Abstract

Tomato Yellow Leaf Curl Virus (TYLCV), a member of the family Geminiviridae, genus Begomovirus, harbors single-stranded circular DNA genome. The intergenic region of TYLCV genomic DNA contains bidirectional promoter elements interspersed within the origin of replication. The replication-associated protein (Rep) is essential for viral DNA replication, in which the Rep protein initiates rolling circle replication by binding to specific repeated sequences (iterons) within the origin of replication and acting as a topoisomerase. The aim of this study is to explore the underlying mechanism for the specific interaction between TYLCV Rep protein and the cognate viral genomic DNA by identifying the sequence and location of TYLCV iterons and DNA-binding domains of TYLCV Rep protein. The TYLCV genome was amplified from Bemisia tabacci (whiteflies) by Rolling Circle Amplification (RCA), and cloned into the pUC119 vector. The full length Rep open reading frame, N-terminus (Rep1-181) and C-terminus (Rep177-361) of Rep were cloned in pET21d and pET29a vectors and expressed in Escherichia coli. The target proteins were purified by electro-elution from polyacrylamide gels, and used to raise Rep-specific antibodies in rabbits. The interactions between Rep proteins and TYLCV genomic DNA were analyzed by Southwestern blot with Rep-specific antibodies. Preliminary results showed that Rep protein specifically interacted with double-strand circular geminivirus DNAs, instead of the linear form DNAs, suggesting that the binding specificity is partially determined by DNA topology. Both full-length Rep and Rep1-181 preferentially interact with IR and the coding regions of Rep and C4 protein, indicating that the main DNA-interacting domain locates at the N-terminus of Rep protein, while Rep177-361 might play the role of specificity determinant. The Type I topoisomerase function of Rep protein were analyzed by a mimicry RCA in vitro by using Φ29 DNA polymerase. The result confirmed that Rep protein possesses nicking / re-ligation activity, providing evidence that the native activity of the Rep protein expressed and purified from prokaryotic cells was still preserved. Through this study, it is expected that practical methods to disrupt the DNA replication cycles could be developed as the effective therapeutics for viral diseases caused by TYLCV and other geminiviruses.