| Summary: | 碩士 === 國立陽明大學 === 生物藥學研究所 === 96 === SARS-CoV coronavirus was identified as the cause of SARS (Severe acute respiratory syndrome), which is likely originated from SARS-like virus of civet cat. The reason why SARS-CoV causes more severe respiratory disease in human than SARS-like virus in animal is still unknown. Additionally, Genomic sequences of SARS-like virus and SARS-CoV share 99.8% identity with the major differences residing within the region of open-reading frame 8 (ORF8, 122 aa). In the ORF8-corresponding region of human SARS-CoV there is a 29-nuclecotide deletion that renders the generation of ORF8a (39 aa) and ORF8b (84 aa) expression units. The first 35 amino acid residues (aa# 1-35) of ORF8a are identical to the N-terminus of ORF8 (aa #1-35), and the last 77 amino acid residues (aa# 8-84) of ORF8b are identical to the C-terminus of ORF8 (aa #46-122). The biological significance of the differences, in term of functionality of these proteins and pathogenesis of virus in the host, between ORF8 and ORF8a and 8b is still unknown. Our previous data have shown that ORF8a interacts with calcium-modulating cyclophilin ligand (CAML) in both yeast-two hybrid assay and Vero E6 cells. ORF8a is located in the mitochondria, while others reported that CAML residing in the ER. The main goal of this dissertation is to determine the biological function of ORF8a, and to do characteristic comparison of ORF8 and ORF8a in Vero E6 cells.
Using HeLa cell Tet/on system, we demonstrated here that ORF8a can promote the apoptosis initiated by staurosporine. This observation is in sharp contrast to a report that indicates a K7 protein of KSHV promotes antiapoptosis through its interaction with CAML. However this apoptosis promotion of ORF8a is in agreement to the observation that at least 8 known SARS-CoV proteins promote apoptosis in host cells. In light of reports indicating that in SARS patients lung cells showed readily apoptosis than that of enterocytes, although both tissues are permissive for viral infection and growth, we are establishing a Tet/on system in both A549, a lung epithelium, and Caco-2, a colon cell line, to ascertain if ORF8a functions differently in the aforementioned cell lines. In this study, we also showed that ORF8 locates in mitochondria as that of ORF8a using mitochondria-fractionation assay. The next stage of experiment is to ascertain if ORF8 interacts with CAML, which at least will illustrate if ORF8 and ORF8a function differently in regarding to the modulation of CAML. Additionally, we have determined that CAML is located in the mitochondrial region, which is in disparity with others’ reports indicating ER as the target site for CAML. Since our data are derived from using different cell lines, Vero E6 and 293T, this discrepancy is likely cell line dependent. At least our data confirmed that both ORF8a and CAML colocalize in mitochondria.
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