| Duck circovirus (DuCV), ovalently closed, circular, single-stranded DNA (ssDNA) genomes encapsidated within nonenveloped icosahedral virions is listed as a tentative member of the Genus circovirus. Ducks infected by DuCV exhibit disordered feather, retarded growth, reduced body weight in few weeks after out of their shells. Two major open reading frames (ORFs), encoding the replicase (ORF1/rep) and the capsid protein (ORF2/cap), have been recognized for DuCV. Sequence analysis show that another major conserved ORF (named ORFS) is located in the complementary strand of OKF1/rep of DuCV, and its function remains to be investigated. In the absence of available cell culture for the isolation of DuCV, the molecular technologies were often used for study on DuCV. The major contents in this study are as follows:1. Identification of Duck circovirus ORF3encoding a novel protein with apoptotic activity. In this study, the ORFS of DuCV was expressed in recombinant baculovirus-infected Sf9cells. By IFA and Western blot analysis, the ORFS protein was positive for the sera from ducks infected with DuCV. The percentages of apoptotic cells of the Sf9cells infected with the recombinant baculovirus encoding ORFS of DuCV were significantly higher than (P<0.05) that of the Sf9cells infected with wild-type baculovirus at24,48and72hours postinfection. Based on our knowledge, we deduced that the ORFS protein of DuCV might play an important role in viral pathogenesis via its apoptotic activity.2. Study of biological characteristics of ORF2of DuCV. The capsid protein of DuCV which was presumed to be associated with immunosuppression contains a high proportion of basic amino acids at N termini. Two potential bipartite nuclear localization signals (NLSs) with homology to others, located in this part of the protein, were analyzed using the online computer program PROSITE. In this study, a series of truncated capsid proteins (the intact CP, the CP knocked out the first NLS, the CP knocked out the second NLS, the CP knocked out the both NLSs, the CP knocked out36amino acids in the C-terminal) ligated with green fluorescence proteins (GFP) were tested for their subcellular distribution in Sf9cells. The results shows that the intact CP is localized to the nuclei as well as the CP knocked out the first NLS, the CP knocked out the second NLS, the CP knocked out36amino acids in the C-terminal. However the CP knocked out the both NLSs completely abolished the translocation of the protein into the nucleus. It significantly suggested that the both putative NLSs are functional NLSs and both of them can translocate the protein to the nucleus lonely.In the study of DNA binding assay, the intact CP, the CP knocked out the first NLS, the CP knocked out the second NLS, the CP knocked out the both NLSs and the CP knocked out36amino acids in the C-terminal were expressed to draw assistance from Bac to Bac baculovirus expression system, followed by the proteins were purified by CsCl gradient centrifugation. The interaction between the genome of DuCV and CP was explored using EMSA approach. The result showed that the purified intact CP, the CP knocked out the first NLS, the CP knocked out the second NLS, the CP knocked out36amino acids in the C-terminal showed a strongly retarded interaction to the movement of the genome of DuCV, while the CP knocked out the both NLSs and the intact CP treated with proteinase K completely abolished the retardation. Using this approach, we identified and mapped two potential nuclear protein target sequences which fell within the region containing the two potential NLSs. It significantly suggested that the both nuclear protein target sequences have the retardation and both of them can bind the genome of DuCV.When the putative CP expressed in Sf9purified by the sucrose cushion and CsCl gradient ultracentrifugation was observed into electron microscope, images indicated that subunits self-assembled to form VLPs were approximately17nm in diameter very similar in size and appearance to the DuCV virion... |
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