登革熱病毒之第三非結構蛋白對於基因體5端及3端非轉譯區的解旋活性以及黏著活性

Abstract

登革熱病毒基因體上並位於開放閱讀框外之5端與3端的非轉譯區有著高度複雜的結構性。兩端的非轉譯區擁有自己局部的二級結構，除此之外有文獻指出它們還有著能跟另一端構成長距離交互作用的互補序列，因此認為登革熱病毒的基因體在不同複製階段有著兩種構型：１）一個線型結構於５端及３端擁有著各自的二級結構 或２）一個由５端及３端交互作用形成雙股結構進而形成的環型結構。登革熱的非結構性蛋白３（ＮＳ３）的Ｎ端有著一個類胰蛋白酶絲氨酸蛋白酶功能區，而Ｃ端有著一個屬於ＤＥｘＨ家族的解旋酶負責將剛複製好的基因體從模板上分離。除此之外，有文獻指出ＮＳ３解旋酶有著另一個功能，就是藉由它對５端及３端結構的調整進而控制整個基因體所處的型態（線型或環型）。在此篇研究中，我複製且純化了登革熱的ＮＳ３解旋酶。我同時也表現了ＮＳ２Ｂ－ＮＳ３以及ＮＳ２Ｂ－ＮＳ3protease，但是表現及純化結果並不理想。對於純化的ＮＳ３解旋酶則是做了初步的黏著實驗，可是並未發現有所希望的黏著活性，原因為何則有待進一步分析。

Outside of the long ORF of dengue virus, highly structured untranslated regions are found at both the 5’ and 3’ ends. In addition to local secondary structures, complementary sequences were found in 5’ and 3’ UTR which are able to induce long range interactions. It is believed that dengue genome exist in two forms during different replication stages: a linear form with local secondary structures on each ends of the genome or a circular form with the 5’ and 3’ UTR forming a duplex. Non-structural protein 3, NS3, contains a trypsin-like serine protease domain within the N-terminal domain. Moreover, NS3, on its C-terminal region, has a DExH family helicase which is responsible for separating nascent genome strand from the template. In addition, NS3Helicase have been suggested a novel role, which is the modulator of genome structures as it could unwind secondary structures within the UTR to promote genome circularization and vice versa. In our study, we expressed and purified dengue NS3-Helicase. We also constructed expression vector for NS2B-NS3 and NS2B-NS3protease, however, the results of expression and purification of these recombinant proteins were undesirable. Simple DNA annealing reactions were carried out using purified NS3-Helicase, yet, no sign of any annealing activity was observed. Further investigation will be needed to uncover the cause.