miRNA expression profiles in productively HIV-1 infected and bystander primary human CD4+ T-cells

CD4+ T-cells are the main target of HIV-1 and several host factors can positively or negatively modulate HIV-1 infection of these cells. MiRNAs aresmall regulatory RNAs that are involved in the regulation of basic cellular functions. They are also increasingly recognized as host factors regulatingHIV-1 infection, replication and persistence. In order to identify miRNAs involved in HIV-1 infection of CD4+ T-cells, we performed globaltranscriptomic analyses of productively infected and HIV-1 exposed but non infected bystander CD4+ T-cells and compared their MIRNA profiles with uninfected cells. Theseanalyses were performed in CD4+T-cells isolated from 3 different healthy blood donors. Both miRNA and mRNA expression profiles were compared. Our results show that bystander and uninfected CD4+ T-cells do not display important differences in their miRNA expression profiles even though their respectivemRNA expression profiles were markedly different. In contrast, both mRNA and miRNA expressionprofiles from productively infected CD4+ T-cells were significantly different from those of uninfected cells. Overall, these results suggest that HIV-1infection impacts the miRNA expression profile of primary CD4+ T-cells. Overall design: Human CD4+ T-cells from 3 different healthy blood donors were isolated from peripheral blood mononuclear cells (PBMCs) and activated for 3 days in the presence of anti-CD3/anti-CD28 and IL-2 in RPMI+10% fetal bovine serum (FBS). Activated T-cells were infected with GFP-marked HIV-1 (strain NL4.3-ADA-IRES-GFP) for 48h at a multiplicity of infection (MOI)=2. Productively infected (GFP+) and bystander cells (GFP-) were sorted by FACS and their total RNA extracted using the RNeasy kit from QIAGEN.

CD4+ T细胞(CD4+ T-cells)是人类免疫缺陷病毒1型(HIV-1, human immunodeficiency virus type 1)的主要靶标，多种宿主因子可对该类细胞的HIV-1感染过程发挥正向或负向调控作用。微小RNA(miRNAs, microRNAs)是一类小型调控RNA，参与细胞基本功能的调控过程，同时也逐渐被证实是调控HIV-1感染、复制与持续存在的宿主因子。为鉴定参与CD4+ T细胞HIV-1感染过程的miRNAs，本研究对有效感染的、暴露于HIV-1但未受感染的旁观者CD4+ T细胞开展了全转录组分析，并将三者的miRNA表达谱与未感染细胞进行了比对。本次分析的样本均来自3名健康献血者分离得到的CD4+ T细胞，研究同时比对了miRNA与信使RNA(mRNA, messenger RNA)的表达谱。研究结果显示，尽管旁观者CD4+ T细胞与未感染CD4+ T细胞的mRNA表达谱存在显著差异，但二者的miRNA表达谱并无明显区别。与之相反，有效感染的CD4+ T细胞的mRNA与miRNA表达谱均与未感染细胞存在显著差异。综上，本研究结果提示HIV-1感染可影响原代CD4+ T细胞的miRNA表达谱。整体实验设计：从3名健康献血者的外周血单个核细胞(PBMCs, peripheral blood mononuclear cells)中分离得到CD4+ T细胞，在抗CD3/抗CD28抗体、白细胞介素2(IL-2, interleukin 2)存在的RPMI培养基+10%胎牛血清(FBS, fetal bovine serum)中活化3天。将活化后的T细胞以感染复数(MOI, multiplicity of infection)=2的剂量感染携带绿色荧光蛋白(GFP, green fluorescent protein)标记的HIV-1毒株NL4.3-ADA-IRES-GFP，感染时长为48小时。通过荧光激活细胞分选术(FACS, fluorescence-activated cell sorting)分选出有效感染的(GFP阳性)与旁观者细胞(GFP阴性)，并使用凯杰(QIAGEN)公司的RNeasy试剂盒提取总RNA。