Specific interaction between SARS-CoVnucleocapsid protein and cellular factor Daxx

• Main Authors: Yiu-Hua Cheng, 鄭又華
• Other Authors: 張明富
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/79212918060616558435

碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 93 === In November 2002, an atypical pneumonia, Severe Acute Respiratory Syndrome (SARS), emerged in Guangdong Province, Southern China, and spread worldwide in 2003. SARS infection exhibits a wide clinical course, mainly characterized by fever, dyspnea, lymphopenia, lower tract respiratory infection, and diarrhea, and causes death in approximately 10% of cases. A novel coronavirus termed SARS-CoV was isolated from SARS patients and Vero E6 cells inoculated with clinical specimens, and identified to be the causative agent of SARS in monkey model experiment. SARS-CoV is an enveloped, positive-sense single-stranded RNA virus with about 30,000 nucleotides in length. The virus particle consists of four structural proteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N). The SARS-CoV N protein possesses RNA-binding activity and is capable of undergoing self-dimerization. The N protein was also demonstrated to induce apoptosis and actin reorganization in mammalian cells under stress conditions. In this study, Daxx (Death-associated protein) was identified to be an N-interacting protein by performing functional yeast array analysis. Yeast two hybrid system further demonstrated that the N protein specifically bound to the C-terminal domain of Daxx protein from 570 to 740 amino acid residues. The C-terminal region is also involved in binding of Daxx to many proteins such as, SUMO, Fas, PML, and Ets-1. It is possible that the SARS-CoV N protein can be modified by SUMO proteins. To test this hypothesis, 293T cells were cotransfected with plasmids encoding the N protein and various SUMO proteins including SUMO-1, SUMO-2, and SUMO-3. Western blot analysis indicated that the N protein may be preferentially modified by SUMO-1 and SUMO-2 proteins. In addition, in vitro sumoylation assay identified two sumoylation sites in the N-terminal 235 amino acid residues of the N protein. Functional significances of the SUMO-modified N protein involved in the life cycle and pathogenesis of SARS-CoV need to be further elucidated. This study suggests a new link between host cell machinery and a SARS-CoV structural component, and will help us to understand the role of N protein .