Mapping the Functional Domains of Pseudorabies Virus Early Protein UL54

• Main Authors: Ya-Ju Huang, 黃雅如
• Other Authors: Chienjin Huang
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/51130438783178877608

碩士 === 國立中興大學 === 獸醫微生物學研究所 === 93 === Pseudorabies virus (PRV) belongs to the family Herpesviridae, and is the causative agent of Aujesky’s disease. PRV gene organization and regulation are similar to well characterized human herpes simplex virus (HSV-1), and the PRV UL54 gene is an early gene with homology to the essential immediate-early protein ICP27 of HSV-1. The major function of ICP27 regulatoy protein is to regulate the expression of early and late genes at post- transcription level and inhibit the expression of cellular genes during infection. The N-terminal half of ICP27 has three important function domains: nuclear export signal (NES), nuclear localization signal (NLS) and RGG box RNA binding domain. The purpose of this study was to identify the functional regions conferring for nuclear location and RNA-binding activity. Several recombinant expression plasmids containing various coding regions of UL54 gene were constructed for producing a series of C-terminally truncated or internally deleted forms of UL54 mutants in E. coli or porcine kidney (PK-15) cells. RNA binding activity of E. coli-expressed UL54 mutants was characterized by the binding ability to poly(G) RNA homopolymer. The result of dot blotting hybridization was shown that N-terminal 83 residues of UL54 are responsible for RNA binding and the region of residues 35-82 containing an RGG box is necessary for its function. Furthermore, the region responsible for nuclear localization was investigated by transient expression of various deletion mutants in PK-15 cells followed by detection of their subcellular distribution. The results showed that C-terminal deletion beyond the amino acid residue 83 or internal deletion containing the RGG box sequence could restrict UL54 mutants in the cytoplasm. The ability of the N-terminal 83 residues to target the green fluorescence protein to the nucleus confirmed further its role as a functional nuclear localization signal (NLS). The utmost N-terminal 83 residues portion of UL54 contains two important functional domains, NLS and RNA binding, and thus it wound play an indispensable role in UL54 regulatory function.