tailieunhanh - Báo cáo khoa học: Expression, post-translational modification and biochemical characterization of proteins encoded by subgenomic mRNA8 of the severe acute respiratory syndrome coronavirus

The most striking difference between the subgenomic mRNA8 of severe acute respiratory syndrome coronavirus isolated from human and some animal species is the deletion of 29 nucleotides, resulting in splitting of a single ORF (ORF8) into two ORFs (ORF8a and ORF8b). ORF8a and ORF8b are predicted to encode two small proteins, 8a and 8b, and ORF8 a single protein, 8ab (a fusion form of 8a and 8b). | ỊFEBS Journal Expression post-translational modification and biochemical characterization of proteins encoded by subgenomic mRNA8 of the severe acute respiratory syndrome coronavirus Tra M. Le1 Hui H. Wong2 Felicia P. L. Tay2 Shouguo Fang2 Choong-Tat Keng2 Yee J. Tan2 and Ding X. Liu1 2 1 Department of BiologicalSciences National university of Singapore Singapore 2 Institute of Molecular and Cell Biology Proteos Singapore Keywords glycosylation mRNA8 rapid degradation SARS-CoV ubiquitination Correspondence D. X. Liu Institute of Molecular and Cell Biology 61 Biopolis Drive Proteos Singapore 138673 Fax 65 67791117 Tel 65 65869575 E-mail dxliu@ Received 22 March 2007 revised 15 June 2007 accepted 19 June 2007 doi The most striking difference between the subgenomic mRNA8 of severe acute respiratory syndrome coronavirus isolated from human and some animal species is the deletion of 29 nucleotides resulting in splitting of a single oRf ORF8 into two ORFs ORF8a and ORF8b . ORF8a and ORF8b are predicted to encode two small proteins 8a and 8b and ORF8 a single protein 8ab a fusion form of 8a and 8b . To understand the functions of these proteins we cloned cDNA fragments covering these ORFs into expression plasmids and expressed the constructs in both in vitro and in vivo systems. Expression of a construct containing ORF8a and ORF8b generated only a single protein 8a no 8b protein expression was obtained. Expression of a construct containing ORF8 generated the 8ab fusion protein. Site-directed mutagenesis and enzymatic treatment revealed that protein 8ab is modified by N-linked glycosylation on the N81 residue and by ubiquitination. In the absence of the 8a region protein 8b undergoes rapid degradation by proteasomes and addition of proteasome inhibitors inhibits the degradation of protein 8b as well as the protein 8b-induced rapid degradation of the severe acute respiratory syndrome coronavirus E protein. Glycosylation .