miRNA expression profiles in productively HIV-1 infected and bystander primary human CD4+ T-cells. 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细胞是HIV-1的主要靶标，多种宿主因子可对该类细胞的HIV-1感染产生正向或负向调控作用。微小RNA（miRNAs）是一类小型调控RNA，参与细胞基础功能的调控，如今也逐渐被证实为调控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感染的微小RNA（miRNAs），我们对产毒性感染以及暴露于HIV-1但未感染的旁观者CD4+ T细胞开展了全局转录组分析，并将其miRNA表达谱与未感染细胞进行比对。本分析使用从3名健康献血者体内分离得到的CD4+ T细胞完成，同时对比了miRNA与信使RNA（mRNA）的表达谱。
研究结果显示，尽管旁观者细胞与未感染细胞的信使RNA（mRNA）表达谱存在显著差异，但二者的miRNA表达谱并无明显差异。与之相反，产毒性感染的CD4+ T细胞的信使RNA（mRNA）与miRNA表达谱均与未感染细胞存在显著差异。总体而言，上述结果表明HIV-1感染会影响原代CD4+ T细胞的miRNA表达谱。
总体实验设计：从3名健康献血者的外周血单个核细胞（PBMCs）中分离得到人CD4+ T细胞，在添加抗CD3/抗CD28抗体、白细胞介素-2（IL-2）以及含10%胎牛血清（FBS）的RPMI培养基中活化3天。将活化后的T细胞用表达绿色荧光蛋白（GFP）标记的HIV-1毒株NL4.3-ADA-IRES-GFP以感染复数（MOI）=2的比例感染48小时。通过荧光激活细胞分选术（FACS）分选出产毒性感染的（GFP阳性）与旁观者细胞（GFP阴性），并使用QIAGEN公司的RNeasy试剂盒提取总RNA。