| Summary: | 碩士 === 國立陽明大學 === 生物藥學研究所 === 97 === SARS-CoV, a coronavirus, was identified as the etiologic agent for severe acute respiratory syndrome. It is established that human SARS-CoV originated from animals, such as palm civets and raccoon dogs. SARS-CoV genome shares about 99.8% homology with its animal counterpart. The major difference of the two viruses is the 29 nucleotide deletion found in the corresponding open reading frame (ORF) 8 of human SARS-CoV, given rise to ORF8a and ORF8b. Our previous data had shown that ORF8a localizes to mitochondria. Using yeast two-hybrid screening, ORF8a, but not ORF8 and ORF8b, was shown to interact with calcium-modulating cyclophilin ligand (CAML), a protein had been known to regulate the intracellular Ca2+ concentration. Using Hela cell Tet/on system, ORF8a can promote the apoptosis initiated by staurosporine. The main goal of this dissertation is to determine the biological functions of ORF8 and ORF8a that might give better understandings to the pathogenesis of both viruses.
With immunofluorescence assay and subcellular fractionation, ORF8a and ORF8 are shown to be located in mitochondria and ER, separately, in the presence of endogenous CAML. However, when we ectopically expressed CAML, both ORF 8a and ORF8 co-localized with exogenous CAML and displayed a similar pattern of distribution, which differed from that in the presence of endogenous CAML. ORF8a was shown to have higher binding capacity for CAML than that of ORF8 in an immunoprecipitation assay, which likely contributed to the redistribution of ORF8a. In light of reports indicating that in SARS patient lung cells showed readily apoptosis than that of enterocytes, although both tissues are permissive for viral infection and growth. We proceeded to evaluate the apoptotic effect of either ORF8a or ORR8 on an alveolar epithelium cell, A549 and an enterocyte, Caco-2. A549 was showed to have DNA fragementation in the presence of ORF8a or ORF8 by propidium iodide staining, while both proteins also facilitated staurosporine-induced apoptosis using TMRM staining assay that targeting at changes in mitochondrial membrane potential. A549 was also showed low grade of apoptosis in the DMSO control group in the presence of ORF8a or ORF8, which coincided with the observed induction of DNA fragmentation by both proteins. Interestingly, Caco-2 with ectopically expressed ORF8a or ORF8 failed to exhibit apoptotic phenotype enhancement regardless of the presence or absence of staurosporine.
Currently, the contribution of interaction between CAML and ORF8a or even ORF8 in apoptosis remains unclear. In the future, we will re-examine the above stated observations in both A549 and Caco-2 cells with CAML knockdown for the evaluation of the contribution of the interactions between CAML and ORF8a or CAML and ORF8.
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