人巨细胞病毒耐药相关蛋白PUL97局部结构模拟和突变分析

Modelling of partial structure of human cytomegalovirus PUL97 protein and drugresistant analysis of new non-synonymous mutations

目的初步探索人巨细胞病毒耐药相关蛋白PUL97局部序列(329～572aa)的三维模拟结构并进行突变的耐药分析。方法采用巢式PCR方法扩增UL97基因776 bp片段并测序,以此发现非同义突变;然后利用SWISS-MODEL结构模拟综合服务器和其他相关软件比对模式对野生型PUL97蛋白片段进行同源模建并优化,通过各种指标或服务器来评估模拟结构的优劣并作筛选。将筛选所得的最佳模拟结构用于非同义新突变型的初步耐药分析。结果最终筛选出的模拟结构是以1YDTE为模板,以L1.FASTA为比对文件所得的Model A。整个分子的能量最低,ProQ的预测分数最高。分子对接分析也发现了潜在的ATP和GCV结合口袋。利用已知的阴阳性耐药点突变验证了该结构的合理性。同时根据突变前后结构信息的改变,提示C518Y单点突变以及联合突变(E517G和C518Y)呈现高度可疑的耐药特性。结论 PUL97野生型蛋白计算机结构模拟和突变分析法是初步评估新突变型耐药特性的又一方便快捷的方法 ,可用于实验验证前的筛选;但模拟结构本身的准确性是成功运用该方法的前提。

Objective To preliminarily explore the 3-dimensional modelling of partial structure （329-572aa） of cytomegalovirus PUL97 protein and elementarily analyse the possibility of drug-resistance of new non-synonymous mutations. Methods Nested PCR was employed to amplify 776 bp fragment of UL97 gene. And some non-synonymous mutations in it were found by sequencing. After that, the online server of SWISS-MODEL and other related softwares were used for modelling and optimizing the partial structure of wildtype PUL97 protein based on the alignment mode. Then, the modelled structures were evaluated and screened by various classical indicators or online servers. Finally, the best structure was obtained and employed for preliminarily analysis of potential drug-resistant possibility of non-synonymous mutations. Results The finally-screened model was Model A based upon the template of 1YDTE and the alignment file of LI. FASTA. The whole energy of the structure was the lowest, while the prediction result of ProQ was on the top compared with the rest. Using the method of molecular docking, the potential binding sites of ATP and GCV were found in it. And most important, the mutation analysis based upon the modelled structure and positive/negative drug-resistant mutations confirmed that this structure was reasonable. Finally, the newly-found mutations （ namely, point mutation C518Y and combined mutation E517G/C518Y） showed some evidences of having the possibility of drug-resistance under the benefit of the above-established system. Such evidences were the significant differences in structural information before and after mutagenesis. Conclusion The above-described system of PUL97 structure modelling and mutation analysis is the valuable and alternative method for preliminarily predicting the possibility of drug-resistance of new mutations. It is more convenient and quick, and can be regarded as the screener before experimental verification. However, the accuracy of modelled structure itself is the big prerequisite for successful application of this system.