特定MHC 基因型食蟹猴microRNA 抗SIV 病毒感染的分子机制研究

The interaction mechanism of HIV with the host is now become a hot and cutting-edge research on effective AIDS prevention pathway. SIV-infected cynomolgus macaques are preferable models for studies on AIDS pathogenesis and testing of novel vaccines. Results from recent studies show that microRNA may exert regulatory effects towards host and/or viral genes and influence viral replication and host’s response to viral infection, which could be the vital step to understand pathogenesis of SIV. However, few microRNAs has been identified in HIV-infected cells or individuals, which makes comprehensive understanding of the pathogenesis of SIV impossible, and to some extent impedes development of HIV vaccines and drugs. In this study, 30 cynomolgus macaques with Mafa-B*008 MHC genotype will be screened for SIV infection test. At first, the immortalized cell lines will be constructed using TERT vector transfection method and then be carried on SIV infection test. In the next, microRNA will be identified in each samples using small RNA sequencing technology before and after immortalization and SIV infection. According to the level of viral load, these cell lines will be divided into two groups: SIV elite controllers (ECs) and progressors. At a series of timestamps after infection, each samples will be obtained for dynamic monitoring viral RNA copies and the expression profiling of host microRNAs. Based on analyzing the relationship between microRNA expression profiling and the viral RNA copies, microRNA molecules involved in controlling of SIV replication will be identified. Finally, In order to explore the regulation mechanisms of microRNA controlling SIV replication, the microRNA candidate target will be test by EGFP expression systems. These results will be able to provide insights into both understanding of HIV Pathogenesis and development of safe and effective AIDS vaccines and drugs.

研究食蟹猴抗SIV复制的机制是揭示宿主与艾滋病毒相互作用必不可少的补充。microRNA在病毒与宿主相互作用中承担重要角色，但有关食蟹猴microRNA调控SIV复制的研究鲜见报道。本课题前期研究中发现越南来源食蟹猴含多种抗SIV的保护性MHC基因型，适合用于抗SIV复制机理研究。本课题拟先挑出30只具保护性MHC Mafa-B*008的食蟹猴，采用端粒酶转染外周血细胞的方法，构建永生化细胞系，在此基础上进行细胞水平SIV感染实验。采用高通量Solexa测序技术对永生化前后、感染前后各样品进行小RNA测序，结合细胞感染前后SIV RNA拷贝与microRNA表达变化的关联分析，鉴定调控SIV复制的microRNA。并采用EGFP表达系统检验microRNA与靶标的相互作用，探讨microRNA调控SIV病毒复制的作用机制，为阐明艾滋病发病机理提供线索，并为开发有效艾滋疫苗和药物提供新思路。

研究食蟹猴抗SIV复制的机制是揭示宿主与艾滋病毒相互作用必不可少的补充。microRNA在病毒与宿主相互作用中承担重要角色，但有关食蟹猴microRNA调控SIV复制的研究鲜有报道。前期研究中发现越南来源食蟹猴含有多种抗SIV的保护性MHC基因型，适合进行艾滋病相关研究。本项目以该群体为样本，进一步分析了该猴群中保护性MHC基因型的分布频率；同时进行体内攻毒实验，在感染SIV病毒后0天、7天和28天，用磁珠分选方法分离B细胞，采用高通量Solexa技术进行小RNA测序，共鉴定了66种与调控SIV复制相关的microRNA，采用GO，KEGG等分析，探讨了microRNA调控SIV复制的可能作用机制，为阐明艾滋病发病机理提供了线索。