Subcellular Localization Analysis Of SARS Coronavirus ORF9b

The ORF9b of SARS-CoV is a unique gene and the function of which wasunknown. In this thesis, the subcellular localization of ORF9b was investigated byusing transcient expression system.Chapter 1 is a general introduction. It contained a description of the biologyand molecular biology of SARS-CoV. The recent researches on SARS-CoV ORF9bwere introduced and the aim of the thesis was presented.In chapter 2, the ORF9b was cloned and expressed in E. coli fused withGST-tag in frame. The GST-tagged fusion protein was purified and used toimmunize rabbits to generate antiserum against ORF9b. In addition, two potentialsorting signals of ORF9b, that of tyrosine-dependent sorting signal Yxxφandnuclear export signal (NES), were characterized by bioinformatics analysis.In chapter 3, the subcellular localization of ORF9b was investigated. Theplasmid containing GFP-tagged ORF9b was transfected into different mammaliancells and observed under confocal microscopy or live image. The results indicatedthat 9b was mainly located in the cytoplasmic and had different patterns includingdiffusion in cytoplasmic, perinuclear stacks and formation vesicle-like structure.Alanine substitution was employed to study the potential tyrosine-dependent sortingsignal of 9b. Our results showed that trafficking of 9b may be altered to some extendwhen the tyrosine residue at 43 and isoleucine residue at 46 were mutated. Finally, thealanine substitution and deletion mutagenesis were performed to investigate theindividual contribution of leucine residues within NES to the nuclear export process in 9b. Our results indicated that Leu49 and Leu55 were required for efficient nuclearexport of 9b.In chapter 4, the co-localization of SARS-CoV ORF9b and ORF7a wereinvestigated. Different fluorescent tagged fusion expression vectors were constructedto label ORF7a and ORF9b. Then the vectors were co-transfected into differentmammalian cells and the distribution of fluorescence were observed. Our resultsshowed that GFP tagged 7a or RFP tagged 9b mainly concentrated on perinuclearand could form punctuate loci in cytoplasm when expressed separately. Theirlocalization patterns were not apparently altered and could co-localize in sometranfected cells. These indicated that 9b may localize in different organelles to 7aand might interact with 7a on secretory pathway.