Mohammad Abdul-Aleem, Costa Helena, Landázuri Natalia, Lui Weng-Onn, Hultenby Kjell, Rahbar Afsar, Yaiw Koon-Chu, Söderberg-Nauclér Cecilia
Department of Medicine, Solna, Experimental Cardiovascular Unit, Department of Neurology, Center for Molecular Medicine, Karolinska Institute, Stockholm, Sweden.
Department of Oncology-Pathology, Karolinska Institutet, Cancer Center Karolinska, Karolinska University Hospital, Stockholm, Sweden.
J Gen Virol. 2017 May;98(5):1058-1072. doi: 10.1099/jgv.0.000736.
Human cytomegalovirus (HCMV) infection results in the production of virions, dense bodies (DBs) and non-infectious enveloped particles, all of which incorporate proteins and RNAs that can be transferred to host cells. Here, we investigated whether virions and DBs also carry microRNAs (miRNAs) and assessed their delivery and functionality in cells. Human lung fibroblasts (MRC-5) were infected with the HCMV strain AD169, and conditioned cell culture medium was collected and centrifuged. The pellets were treated with RNase-ONE, and the virions and DBs were purified with a potassium tartrate-glycerol gradient and dialysed. The virions and DBs were incubated with micrococcal nuclease, DNA and RNA were extracted and then analysed with TaqMan PCR assays, while the proteins were examined with Western blots. To assess the delivery of miRNAs to cells and their functionality, virions and DBs were irradiated with UV light. The purity of the virions and DBs was confirmed by typical morphology, the presence of the structural protein pp65 and the HCMV genome, the ability to infect MRC-5 cells and the absence of the host genome. RNA analysis revealed the presence of 14 HCMV-encoded miRNAs (UL22A-5p, US25-1-5p, UL22A-3p, US5-2-3p, UL112-3p, US25-2-3p, US25-2-5p, US33-3p, US5-1, UL36-5p, US4-5p, UL36-3p, UL70-5p and US25-1-3p), HCMV immediate-early mRNA and long non-coding RNA2.7, moreover, two host-encoded miRNAs (hsa-miR-218-5p and hsa-miR-21-5p) and beta-2-microglobulin RNA. UV-irradiated virions and DBs delivered viral miRNAs (US25-1-5p and UL112-3p) to the host cells, and miR-US25-1-5p was functional in a luciferase reporter assay. We conclude that virions and DBs carry miRNAs that are biologically functional and can be delivered to cells, which may affect cellular processes.
人巨细胞病毒（HCMV）感染会产生病毒粒子、致密体（DBs）和无感染性的包膜颗粒，所有这些都包含可转移至宿主细胞的蛋白质和RNA。在此，我们研究了病毒粒子和致密体是否也携带微小RNA（miRNA），并评估了它们在细胞中的传递及功能。用人巨细胞病毒AD169株感染人肺成纤维细胞（MRC-5），收集条件性细胞培养基并离心。将沉淀用核糖核酸酶-ONE处理，然后用酒石酸钾-甘油梯度纯化病毒粒子和致密体并进行透析。将病毒粒子和致密体与微球菌核酸酶一起孵育，提取DNA和RNA，然后用TaqMan PCR分析，同时用蛋白质印迹法检测蛋白质。为了评估miRNA向细胞的传递及其功能，用紫外线照射病毒粒子和致密体。通过典型形态、结构蛋白pp65和人巨细胞病毒基因组的存在、感染MRC-5细胞的能力以及宿主基因组的缺失来确认病毒粒子和致密体的纯度。RNA分析显示存在14种人巨细胞病毒编码的miRNA（UL22A-5p、US25-1-5p、UL22A-3p、US5-2-3p、UL112-3p、US25-2-3p、US25-2-5p、US33-3p、US5-1、UL36-5p、US4-5p、UL36-3p、UL70-5p和US25-1-3p）、人巨细胞病毒立即早期mRNA和长链非编码RNA2.7，此外，还有两种宿主编码的miRNA（hsa-miR-218-5p和hsa-miR-21-5p）以及β2-微球蛋白RNA。紫外线照射的病毒粒子和致密体将病毒miRNA（US25-1-5p和UL112-3p）传递至宿主细胞，并且miR-US25-1-5p在荧光素酶报告基因检测中具有功能。我们得出结论，病毒粒子和致密体携带具有生物学功能且可传递至细胞的miRNA，这可能会影响细胞过程。
