我国HIV-1优势毒株RRE基因多态性与病毒复制能力的相关性研究

The Rev Response element of HIV-1 is a 351 nucleotide, highly structured, cis-acting RNA element. By binding to the viral protein Rev, RRE can help the unspliced viral mRNA export from the nucleus to the cytoplasm to and express. RRE plays an important role in the nuclear export of the full-length mRNA of the virus and is an RNA transport mode unique to the retrovirus life cycle. Studies on RRE of subtype B show that a small amount of point mutations on the RRE can significantly affect the virus replication. However, A large number of RRE related studies aimed at HIV-1 subtype B. While the strains circulating in China are mainly non-B subtypes and strains of B subtype only accounting for 9.6%. And the RRE sequences of China’s dominant recombination strains have significant differences compared with the RRE of B strains. Therefore, Researches on the RRE of China’s dominant strains need to be carried out urgently. This study intends to analyze the influence of RRE gene polymorphism of the four dominant strains in China (CRF07_BC, CRF01_AE, CRF08_BC and B ') on virus replication from the perspective of population and virus. First, through cross-sectional analysis of different populations, we screened the polymorphism sites of RRE in each subtypes to identify their effect on the structure and activity of RRE, and then introduced the mutations into the corresponding infectious clones to verify their impact on virus replication. Finally, we determined the key functional sites of RRE in our dominant strains. The results will not only help us to understand the mechanism of Rev-RRE, explain the differences in the pathogenicity of different strains. It will also lay a foundation for the development of a more targeted inhibitor or gene therapy for the HIV-1 strains circulating in China.

HIV-1的Rev反应元件（Rev response element, RRE）是位于病毒基因组env编码区的全长351nt且高度结构化的RNA顺式作用元件。RRE在病毒全长mRNA的出核转运中发挥重要作用，为逆转录病毒生命周期所特有。B亚型相关研究显示，RRE上少量点突变即可显著影响病毒复制。目前大量RRE研究聚焦B亚型，但在我国B亚型仅占9.6%，而占比83.2%的优势重组型毒株的RRE与B亚型有显著差异。本课题拟从人群和病毒两个角度分析我国四种优势毒株（CRF07_BC、CRF01_AE、CRF08_BC和B’）的RRE基因多态性对于病毒复制的影响。先通过横断面分析筛选各亚型RRE的多态性位点，鉴定其对RRE结构和活性的影响，再将突变引入对应的感染性克隆，验证其对病毒复制的影响，最终确定我国优势毒株的RRE关键功能位点。这将为研发对我国HIV-1更有针对性的抑制剂或基因疗法奠定基础。

HIV-1的Rev反应元件（Rev response element, RRE）是位于病毒基因组env编码区的全长351nt的高度结构化的RNA顺式作用元件。RRE在病毒mRNA的出核转运中发挥重要作用，为逆转录病毒生命周期所特有。B亚型毒株相关研究显示，RRE上少量点突变即可显著影响病毒复制。目前大量RRE研究聚焦B亚型，但在我国B亚型仅占9.6%，而占比83.2%的优势重组型毒株的RRE与B亚型有显著差异。.本课题从人群和病毒两个角度分析我国四种优势毒株（CRF07_BC、CRF01_AE、CRF08_BC和B’）的RRE基因多态性对于病毒复制的影响。我们从河北、四川、广西的感染者血液样本中选取4种优势毒株样本进行扩增，获得RRE全长序列共711条（CRF07_BC：215条，CRF01_AE：250条，CRF08_BC：118条；B’：128条）。将各亚型毒株RRE序列与欧美B亚型使用碱基不同，且在该亚型中出现频率大于 90%的位点作为本研究关注的RRE亚型特征性位点。将包括Rev蛋白第一和第二结合位点及茎环结构可变区的第82-266位作为关注重点，筛选出关键功能区域亚型特征性位点。使用RNA fold软件对这4种毒株的RRE共有序列和关键亚型特征性位点突变前后的RRE二级结构进行预测，鉴定出对RRE结构有影响的亚型特异性位点。分别构建4种亚型Rev蛋白表达质粒和GagPol-RRE报告基因质粒及其突变体，完成亚型特征性位点对于Rev-RRE功能活性影响的鉴定。分别在B亚型和CRF01_AE亚型感染性克隆上引入点突变，完成亚型特征性位点对病毒复制能力影响的鉴定。.结果显示，第243和262位（HXB2 RRE）对4种亚型的RRE二级结构均有影响；186和262+264位点对我国HIV-1主要流行毒株Rev-RRE活性均有显著影响；B’亚型RRE第186位点对于复制有明显增强作用；CRF01_AE的RRE第243和262位对于复制有下调作用， 而第70+73、298和348+351位对于复制有明显增强作用。.本研究发现了我国HIV-1优势毒株区别于欧美流行的B亚型毒株对病毒复制有显著影响的RRE功能活性位点，这将为HIV-1新型抑制剂的设计提供新靶标，为研发对我国HIV-1更有针对性的新型抑制剂或基因疗法奠定基础。.