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標題: SARS冠狀病毒-1轉譯框架移轉機制之研究
Study of the mechanism of translational -1 frameshift of SARS-CoV
作者: 蘇美綺
Su, Mei-Chi
關鍵字: 1 frameshift;1轉譯框架移轉
出版社: 生物化學研究所
嚴重急性呼吸道症候群其致病原為SARS冠狀病毒,由基因序列得知其ORF1a與ORF1b序列之間有一個stop codon可以使轉譯停止,導致ORF1ab無法被轉譯以形成replicase complex proteins,進而使得病毒無法進行複製與轉錄,因此ribosome必需進行-1轉譯框架移轉,才能使ORF1ab的轉譯繼續進行。根據序列比對及RNA二級結構的預測顯示SARS冠狀病毒具有-1轉譯框架移轉所需的slippery sequence及可能的pseudoknot structure信號,且其pseudoknot structure在loop 2的區域可形成一額外的stem loop (stem 3)。
利用in vitro transcription coupled translation的分析,我們證實了SARS冠狀病毒核酸包含了slippery sequence及可能pseudoknot structure的序列能夠誘發-1轉譯框架移轉。若破壞這兩個信號皆會導致-1轉譯框架移轉無法進行,並且也觀察到破壞stem 3之間的鹼基配對會使得-1轉譯框架移轉效率下降,一旦回復stem 3之間的配對其-1轉譯框架移轉效率則又回升,顯示stem 3的鹼基配對能影響-1轉譯框架移轉效率,但非-1轉譯框架移轉所必需的信號。此外我們在slippery sequence的上游SARS13222-13369區域觀察到可能具有調節SARS冠狀病毒-1轉譯框架移轉效率的RNA信號,但由目前的實驗結果還無法確切的瞭解這個RNA信號是如何調節-1轉譯框架移轉的效率。最後已知可以提高及減低一些病毒系統中之-1轉譯框架移轉效率的Sparsomycin及Anisomycin,在我們的藥物測試實驗中似乎不太能影響SARS冠狀病毒-1轉譯框架移轉的效率。

The servere acute respiratory syndrome is caused by SARS coronavirus (SARS-CoV). The genomic sequence of SARS-CoV possesses a stop codon between the junction of ORF1a and ORF1b that can cause the translational termination, and thus block ORF1ab fusion protein synthesis crucial for replicase complex formation. Eventhally, it will lead to the inhibition of virus replication. The continuing translation of ORF1ab will need a —1 ribosomal frameshift, and sequence alignment as well as RNA secondary structure prediction indicate the existence of a set of putative frameshifting signal for SARS-CoV. It is formed by a slippery sequence and a downstream atypical pseudoknot structure with an extra stem loop (stem 3) in the loop 2 of the pseudoknot structure.
We have shown the putative SARS-CoV —1 frameshifting signal can induce —1 frameshift in vitro. Disruption of either signal can eliminates the —1 frameshift. Furthermore, the disruption of the base pairs forming stem 3 will reduce the efficiency of —1 frameshift while restoring the base pairs also restore the —1 frameshift efficiency. The result indicates the integrity of stem 3 is a factor for the —1 frameshift, but is not essential for —1 frameshift to occur. In addition, we locate an attenuation RNA signal (SARS13222-13369) upstream of the slippery sequence with activity of regulating the SARS-CoV —1 frameshifting efficiency. However, the mechanisms of this attenuation remain unknown. Finally, two antibiotics, sparsomycin and anisomycin, capable of modulating —1 frameshift efficiency in several viral systems fail to modulate the efficiency of —1 frameshift of SARS-CoV in our assay system.
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