棉铃虫5型质型多角体病毒RNA聚合酶的确定与功能机制研究

棉铃虫5型质型多角体病毒属于呼肠孤病毒科质型多角体病毒属,以重要农业害虫棉铃虫为其天然宿主,对棉铃虫的生物控制具有重要意义.本文对棉铃虫5型质型多角体病毒第3片段编码的蛋白的功能进行了初步研究.首先通过同源性对比,推测其所编码的蛋白可能行使RNA依赖的RNA聚合酶(RdRP)的功能.通过体外活性研究确定了该蛋白的RdRP活性,并确定了其保守活性位点GDD.随后以病毒基因组RNA和3′-OH封闭的病毒基因组RNA为模板,利用Northern blot方法研究该蛋白起始病毒基因组RNA合成的分子机制.结果表明,该病毒的RdRP主要通过引物非依赖的方式起始病毒基因组RNA的合成,并且该RdRP蛋白并不具有末端转移酶活性.最后,对RdRP行使功能的生化条件进行探索,发现RdRP功能的发挥需要二价金属离子Mg2+的存在.

Identification and Characterization of Helicoverpa armigera Cypovirus RNA Dependent RNA Polymerase

The type 5 H. armigera cypovirus (HaCPV-5) was initially isolated by our laboratory in 2006 from a mixture of HaCPVs, and can quickly cause lethal disease in H. armigera that is one of the most serious agricultural pests in China. In this study, we expressed and purified recombinant HaCPV-5 RNA-dependent RNA polymerase (RdRP) in E. coli and characterized its RdRP activity in detail. Our study revealed that this protein can initiate RNA synthesis without a primer or other viral or cellular factors. The conserved motif GDD was also identified by mutagenesis assay. Further results showed that the RdRP does not possess the terminal nucleotidyl transferase (TNTase) activity. Furthermore, we examined its RdRP activity under varying concentrations of Mg²⁺ to characterize the biochemical properties of RdRP.