International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi -110067, India.
Virol J. 2010 Jun 14;7:128. doi: 10.1186/1743-422X-7-128.
Geminiviruses are emerging plant viruses that infect a wide variety of vegetable crops, ornamental plants and cereal crops. They undergo recombination during co-infections by different species of geminiviruses and give rise to more virulent species. Antiviral strategies targeting a broad range of viruses necessitate a detailed understanding of the basic biology of the viruses. ToLCKeV, a virus prevalent in the tomato crop of Kerala state of India and a member of genus Begomovirus has been used as a model system in this study.
AC3 is a geminiviral protein conserved across all the begomoviral species and is postulated to enhance viral DNA replication. In this work we have successfully expressed and purified the AC3 fusion proteins from E. coli. We demonstrated the higher order oligomerization of AC3 using sucrose gradient ultra-centrifugation and gel-filtration experiments. In addition we also established that ToLCKeV AC3 protein interacted with cognate AC1 protein and enhanced the AC1-mediated ATPase activity in vitro.
Highly hydrophobic viral protein AC3 can be purified as a fusion protein with either MBP or GST. The purification method of AC3 protein improves scope for the biochemical characterization of the viral protein. The enhancement of AC1-mediated ATPase activity might lead to increased viral DNA replication.
双生病毒是新兴的植物病毒，可感染多种蔬菜作物、观赏植物和谷类作物。它们在不同种双生病毒的共同感染过程中发生重组，从而产生更具毒性的物种。针对广泛病毒的抗病毒策略需要对病毒的基本生物学有详细的了解。ToLCKeV 是一种在印度喀拉拉邦番茄作物中流行的病毒，是 Begomovirus 属的成员，已被用作本研究的模型系统。
AC3 是一种在所有双生病毒物种中保守的双生病毒蛋白，据推测可增强病毒 DNA 复制。在这项工作中，我们已成功地从大肠杆菌中表达和纯化了 AC3 融合蛋白。我们使用蔗糖梯度超速离心和凝胶过滤实验证明了 AC3 的高级别寡聚化。此外，我们还证实了 ToLCKeV AC3 蛋白与同源的 AC1 蛋白相互作用，并在体外增强了 AC1 介导的 ATP 酶活性。
高度疏水性的病毒蛋白 AC3 可以作为与 MBP 或 GST 融合的融合蛋白进行纯化。AC3 蛋白的纯化方法提高了对病毒蛋白进行生化特性分析的范围。AC1 介导的 ATP 酶活性的增强可能导致病毒 DNA 复制增加。
