近日，來自美國(guó)密蘇里州立大學(xué)的研究人員闡明了SARS病毒解旋酶NSP13的作用機(jī)制,。相關(guān)研究成果于5月15日在線發(fā)表在Plos ONE上。
研究發(fā)現(xiàn),，嚴(yán)重急性呼吸道綜合征冠狀病毒(SARS-CoV)的非結(jié)構(gòu)蛋白13(NSP13)是一種解旋酶,，利用能量水解核苷酸，分開雙鏈DNA(dsRNA)或者是具有5′→3′極性的DNA(dsDNA),。
他們發(fā)現(xiàn),，解旋的延遲伴隨著雙鏈區(qū)域核苷酸的長(zhǎng)度增加，這表明在解旋過程中存在中間體,。為了闡明中間體的本質(zhì),，他們利用瞬變動(dòng)力學(xué)來分析nsp13的解旋酶活性。結(jié)果發(fā)現(xiàn),，解旋酶是以一系列不連續(xù)的步驟來參與解旋作用,，每一步解旋9.3個(gè)堿基對(duì)(bp),，其催化速率為每秒30步。因此,，nsp13的凈解旋率約是每秒280bp,。
SARS-CoV內(nèi)RNA依賴的RNA聚合酶(RdRp)是另一種非結(jié)構(gòu)蛋白nsp12。研究發(fā)現(xiàn),，它能夠提高核苷酸(RNA/RNA或DNA/DNA)解旋的步長(zhǎng),，增強(qiáng)nsp13的催化效率。使用口蹄疫病毒RdRp代替nsp12后,，nsp13的活性無明顯變化,，表明這種增強(qiáng)作用對(duì)SARS-CoV的nsp12是特異性的。
研究人員Stefan G. Sarafianos表示,，nsp13及nsp12能夠以一個(gè)協(xié)調(diào)的模式來發(fā)揮作用,，提高病毒復(fù)制的效率。該研究對(duì)理解SARS-CoV RNA合成期間nsp13的作用具有重要意義,。（生物谷Deepblue編譯）
doi: 10.1371/journal.pone.0036521
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Mechanism of Nucleic Acid Unwinding by SARS-CoV Helicase
Adeyemi O. Adedeji, Bruno Marchand, Aartjan J. W. te Velthuis, Eric J. Snijder, Susan Weiss, Robert L. Eoff, Kamalendra Singh, Stefan G. Sarafianos.
The non-structural protein 13 (nsp13) of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a helicase that separates double-stranded RNA (dsRNA) or DNA (dsDNA) with a 5′→3′ polarity, using the energy of nucleotide hydrolysis.We determined the minimal mechanism of helicase function by nsp13. We showed a clear unwinding lag with increasing length of the double-stranded region of the nucleic acid, suggesting the presence of intermediates in the unwinding process.To elucidate the nature of the intermediates we carried out transient kinetic analysis of the nsp13 helicase activity. We demonstrated that the enzyme unwinds nucleic acid in discrete steps of 9.3 base-pairs (bp) each, with a catalytic rate of 30 steps per second.Therefore the net unwinding rate is ~280 base-pairs per second. We also showed that nsp12, the SARS-CoV RNA-dependent RNA polymerase (RdRp), enhances (2-fold) the catalytic efficiency of nsp13 by increasing the step size of nucleic acid (RNA/RNA or DNA/DNA) unwinding.This effect is specific for SARS-CoV nsp12, as no change in nsp13 activity was observed when foot-and-mouth-disease virus RdRp was used in place of nsp12.Our data provide experimental evidence that nsp13 and nsp12 can function in a concerted manner to improve the efficiency of viral replication and enhance our understanding of nsp13 function during SARS-CoV RNA synthesis.
