博士 / 國立臺灣大學 / 生化科學研究所 / 96 / White spot syndrome virus (WSSV) is a pathogen responsible for population collapse in intensive white shrimp (Litopenaeus vannamei) culture. This research was initiated to better understand WSSV pathogenesis and cell pathways affected in the infection process. Initially two-dimensional gel electrophoresis (2-DE) was used to create protein expression profiles for specific pathogen free and infected shrimp at 48 hours post-infection (48 hpi). Seventy-five protein spots consistently showed either a marked change in accumulated levels (>50%) or else were highly expressed throughout the course of WSSV infection. These were considered to be important in pathogenesis and were selected for further study. Bioinformatics databases were searched for matches with the results of LC-nanoESI-MS/MS. Of the 75 proteins, 53 were identified in this way. Of these, all but one had widely varying known functions, including energy production, calcium homeostasis, nucleic acid synthesis, signaling/communication, oxygen carrier/transportation, and SUMO related modification. The remaining one was an altered and highly expressed nonstructural protein which we named ICP11. We then studied this protein in detail to determine its function. The crystal structure was determined using the MAD phasing method. ICP11 is dimer. It has two rows of negatively-charged spots. Interestingly, in crystal, ICP11 dimer forms a polymer where the distribution of negative charges approximates the duplex arrangement of the phosphate groups in DNA. This suggests that ICP11 is a DNA mimic protein. By using far western analysis we found that ICP11 can interact with histone proteins (H2A, H2B, H3 and H2A.x). ICP11 out-competed DNA in binding to these proteins. Further, ICP11 was found to colocalize with histone H3 and activated H2A.x in the hemocytes of WSSV infected shrimp. This suggests that ICP11 may interfere with DNA packaging and prevent H2A.x from fulfilling its critical function of repairing DNA double strand breaks (DSBs). In addition, ICP11 exhibited an unexpected endonuclease activity in the presence of Mg2+ ions. ICP11 therefore demonstrates a multifunctional anti-host strategy never before reported.
| Identifer | oai:union.ndltd.org:TW/096NTU05103024 |
| Date | January 2008 |
| Creators | Hao-Ching Wang, 王皓青 |
| Contributors | 王惠鈞 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 34 |
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