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Biochemical Characterization of Evtropis Oblique Picorna-like Virus Nonstructural Protein2C

Author: ChengZhenYun
Tutor: HuYuanYang
School: Wuhan University
Course: Microbiology
Keywords: Ectropis obliqua picorna-like virus (EoPV) MBP-2C ATPaseactivity helix-destabilizing activity of NTP-independent RNA chaperone
CLC: Q936
Type: PhD thesis
Year: 2012
Downloads: 3
Quote: 0
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Ectropis obliqua picorna-like virus(EoPV)is an insect RNA virus which belongs to the family Iflaviridae first identified by our country. As now, the nonstructural protein3C and3D have been studied by other people of our lab, but the protein2C, a putative helicase has not been studied by anyone. Here, we will study the activity of it.In the benginning, we expressed the protein using E.coli, but we didn’t get the soluble protein and we also didn’t detect the NTPase activity and helix-destabilizing activity. Then, we adopt Bac-to-Bac baculovirus expresstion system to express the protein in sf9.In order to increase the soluble protein, we add the MBP to N-terminal of2C.Using the purified protein, we detected the helix-destabilizing activity and NTPase activity.We found that MBP-2C possessed NTPase activity. It could hydrolyze all four NTPs and dNTPs.With different divalent cations, the protein was able to hydrolyze ATP, but the efficiency was not the same. Then, we optimized the reaction conditions to get the result that the ATPase activity of MBP-2C could reach the maximum at pH8.0,25mM NaCl, and2.5mM Mg2+.MBP-2C possessed the helix-destabilizing activity. We acknowledged that it could disrupt all three kinds of duplexes including RNA-RNA, DNA-RNA and DNA-DNA duplexes all with sigle-stranded overhangs. It displayed no polarity in destabilizing a partial duplex but it couldn’t destabilize the duplex with blunt end. We also chose defferent concentrations of Mg2+and Zn2+to examine the inhibition action on helix-destabilizing activity and the result showed that the inhibition action of Zn2+was sensitive, which could completely inhibit the helix-destabiling activity in the concentration of2mM. The helix-destabiling activity could also be inhibited at a high concentration of Mg2+, but the inhibition action was not sensitive. There was still a large number of duplex could be unwound at a concentration of15mM.We detected the ATPase activity and the helix-destabilizing activity of MBP-2C mutants and the results also proved that the ATPase activity and helix-destabilizing activity were independent. When the ATPase activity decreased to half of wild protein, there was little difference in the helix-destabilizing activity, while the activity of ATPase of H1562didn’t reduce, but the helix-destabilizing activity reduced a lot.We also found that helix-destabilizing activity was NTP and Mg2+independent. According to the definition of helicase,it is NTP-dependent to unwinding helix.so we did some experiments correlated to chaperon and obtained the result that MBP-2C displayed nucleic acid chaperon activity in promoting the hybridization of complementary nucleic acids.According all the results obtained we considered that2C is a nucleic acid chaperon.This is the first time that the function of2C protein was clearly studied in the picornavirales.

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