Researches Into The Nucleocapsid Proteins VP15 Of White Spot Syndrome Virus And Capsomers Assembly Mechanism

White spot syndrome virus (WSSV) is a large, rod-shaped, circular double-stranded DNA virus, belonging to the Nimaviridae family. With a wide range of hosts in crustaceans, WSSV is the major pathogen of cultured shrimp because of its high lethality.This thesis mainly focuses on the functions of the viral nucleocapsid protein VP15. In addition, the thesis is also concerned about the capsomeres assembly. This work mainly includes the following aspects:(1)VP15 is a major structural protein of viral nucleocapsid. Previous research showed that VP15 was able to interact with DNA, and thus considered as a DNA-binding protein. In our research, VP15 was isolated by using acid extraction. Gel-filtration chromatography suggested that VP15 formed dimmer in natural state. In addition, VP15 was shown to bind with DNA of minimum length of 10 bp by EMSA and dot spot. And there is no interaction of VP15 with other viral capsid proteins by using far-western blot and Co-binding precipitation assay. Furthermore, RNAi assay was carried out by using in vitro synthesized long length dsRNAs. RT-PCR analysis revealed that vp15-dsRNA could specifically knockout vp15 gene transcription, and real-time fluorescence quantitative PCR analysis showed that vp15-dsRNA can interfere with the VP15 gene expression and effectively inhibited WSSV proliferation. In addition, ultra thin section electron microscopy showed that vp15 dsRNA interference can significantly inhibit the viral multiplication and cause abnormal packaging.(2)We found an interesting phenomenon in the research of VP15, which was easily degradation in vitro but stable in virion or nucleocapsid suspension. It was found that FITC labeling,heat denaturation,adding nucleic acids, protease inhibitors or 50 mM EDTA into VP15 solution can effectively inhibit the degradation of VP15. In addition, we fonud that VP15 was stable when dialysis in the buffer of pH<7, while it was easily degradation in the buffer of pH≥7. Furthermore, VP15 was found degradation when it was mixed with other protein followed by dialysis. We hypothesized that VP15 may have a function of self-degradation.(3)Currently, WSSV structural proteins have been characterized by MALDI-TOF MS, including 8 nucleocapsid proteins. Until recently, a basic understanding of virion assembly is still lacking, especially for nucleocapsid. In this study, nucleocapsid was treated with GuHCl, urea, HCl (1M and 0.1M) and NaOH (1M and 0.1M). After extraction, all samples were analyzed by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing condition and transmission electron microscopy (TEM). The results showed that the stability of WSSV nucleocapsid is relative to the pH value of buffer. Comparing with bovine serum albumin (BSA), we found WSSV nucleocapsid is more tolerant to low pH (1M or 0.1 M HCl) and more sensitive to high pH (1M or 0.1M NaOH) than BSA. In addition, we found that 6M urea or GuHCl resulted in the dissociation of the capsids into small particles, whose size was consistent with the capsomeres. Furthermore , reducing/non-reducing SDS-PAGE analysis suggested that the intermolecular forces of the capsomeres were primarily disulfide bonds.