In depth analysis of the interaction of HIV-1 with cellular microRNA biogenesis and effector mechanisms. Homo sapiens

The question of how HIV-1 interfaces with cellular microRNA (miRNA) biogenesis and effector mechanisms has been highly controversial. Here, we first used deep sequencing of small RNAs present in two different infected cell lines (TZM-bl and C8166) and two types of primary human cells (CD4+ PBMCs and macrophages) to unequivocally demonstrate that HIV-1 does not encode any viral miRNAs. Perhaps surprisingly, we also observed that infection of T cells by HIV-1 has only a modest effect on the expression of cellular miRNAs at early times after infection. Comprehensive analysis of miRNA binding to the HIV-1 genome using the photoactivatable ribonucleoside-induced crosslinking and immunoprecipitation (PAR-CLIP) technique revealed several binding sites for cellular miRNAs, a subset of which were shown to be capable of mediating miRNA-mediated repression of gene expression. However, the main finding from this analysis is that HIV-1 transcripts are largely refractory to miRNA binding, most probably due to extensive viral RNA secondary structure. Together, these data demonstrate that HIV-1 neither encodes viral miRNAs nor strongly influences cellular miRNA expression, at least early after infection, and imply that HIV-1 transcripts have evolved to avoid inhibition by pre-existing cellular miRNAs by adopting extensive RNA secondary structures that occlude most potential miRNA binding sites. Overall design: Small RNA profiling of HIV-1 infected cells through total small RNA deep sequencing and identification of RNA-induced silencing complex (RISC)-associated HIV-1 sequences through the RNP-immunoprecipitation technique of PAR-CLIP on RISC

关于HIV-1如何与细胞微小RNA（microRNA, miRNA）的生物发生及效应机制相互作用的问题，长期以来极具争议。本研究首先对两种不同的感染细胞系（TZM-bl与C8166）以及两种原代人细胞（CD4+外周血单个核细胞（peripheral blood mononuclear cell, PBMC）和巨噬细胞）中的小RNA开展深度测序，确凿证实HIV-1并未编码任何病毒源性miRNA。颇为意外的是，我们还观察到HIV-1感染T细胞后，在感染早期仅对细胞miRNA的表达产生轻微影响。利用光活化核糖核苷交联免疫沉淀（photoactivatable ribonucleoside-induced crosslinking and immunoprecipitation, PAR-CLIP）技术，对细胞miRNA结合HIV-1基因组的情况进行全面分析，结果发现了多个细胞miRNA的结合位点，其中部分位点可介导miRNA对基因表达的抑制作用。不过，该分析的核心发现是，HIV-1转录本在很大程度上难以与miRNA结合，这一现象大概率源于病毒RNA具有广泛的二级结构。综上，上述数据表明，至少在感染早期，HIV-1既不编码病毒miRNA，也不会显著影响细胞miRNA的表达；同时提示HIV-1转录本通过形成广泛的RNA二级结构，遮蔽绝大多数潜在的miRNA结合位点，从而进化出逃避现有细胞miRNA抑制的能力。整体实验设计：通过全小RNA深度测序对HIV-1感染细胞进行小RNA表达谱分析，并利用针对RNA诱导沉默复合物（RNA-induced silencing complex, RISC）的PAR-CLIP核糖核蛋白免疫沉淀技术，鉴定与RISC结合的HIV-1序列。