Nilaratanakul Voraphoj, Hauer Debra A, Griffin Diane E
W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.
Cellular and Molecular Medicine Graduate Program, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
J Gen Virol. 2017 May;98(5):992-1003. doi: 10.1099/jgv.0.000755. Epub 2017 May 30.
Viral RNA studies often rely on in situ hybridization and reverse transcriptase-PCR to provide snapshots of RNA dynamics in infected cells. To facilitate analysis of cellular RNAs, aptamers Spinach and Spinach2 that bind and activate the conditional fluorophore 3, 5-difluoro-4-hydroxybenzylidene imidazolinon have been developed. To determine the feasibility of applying this technology to viral RNA, we have used cDNA clones of the TE strain of Sindbis virus (SINV) to construct multiple viruses containing one or two copies of tRNA-scaffolded Spinach2 after a second subgenomic promoter, TEds-1Sp and TEds-2Sp within the 3'UTR, TE-1UTRSp, or after a second subgenomic promoter and in the 3'UTR, TEds-1Sp+1 UTRSp. TEds-1Sp+1 UTRSp gave the brightest signal and replicated well in cell culture, while TEds-2Sp was the dimmest and replicated poorly. Selection of baby hamster kidney cells infected with TEds-1Sp+1 UTRSp for improved signal intensity identified a virus with a stronger signal and point mutations in the tRNA scaffold. Imaging of SINV in BHK cells showed RNA to be concentrated in filopodia that contacted and transferred RNA to adjacent cells. The effect of treatment with anti-E2 antibody, which effects non-cytolytic clearance of SINV from neurons, on viral RNA was cell-type-dependent. In antibody-treated BHK cells, intracellular viral RNA increased and spread of infection continued. In undifferentiated and differentiated AP7 neuronal cells antibody treatment induced viral RNA clearance. Both viruses with two inserted aptamers were prone to deletion. These studies form the basis for further development of aptamer-labelled viral RNAs that will facilitate functional studies on the dynamics of infection and clearance.
病毒RNA研究通常依靠原位杂交和逆转录聚合酶链反应来提供受感染细胞中RNA动态变化的快照。为便于分析细胞RNA，已开发出与条件荧光团3,5-二氟-4-羟基苄叉咪唑啉酮结合并激活它的适体菠菜（Spinach）和菠菜2（Spinach2）。为确定将该技术应用于病毒RNA的可行性，我们利用辛德毕斯病毒（SINV）TE株的cDNA克隆构建了多种病毒，这些病毒在3'非翻译区（UTR）内的第二个亚基因组启动子之后含有一个或两个tRNA支架的菠菜2拷贝，即TEds-1Sp和TEds-2Sp、TE-1UTRSp，或者在第二个亚基因组启动子之后且在3'UTR内，即TEds-1Sp+1 UTRSp。TEds-1Sp+1 UTRSp发出的信号最亮，且在细胞培养中复制良好，而TEds-2Sp发出的信号最暗，且复制不佳。选择感染TEds-1Sp+1 UTRSp以提高信号强度的幼仓鼠肾细胞，鉴定出一种信号更强且tRNA支架存在点突变的病毒。在BHK细胞中对SINV进行成像显示，RNA集中在丝状伪足中，丝状伪足与相邻细胞接触并将RNA转移至相邻细胞。抗E2抗体处理对病毒RNA的影响具有细胞类型依赖性，抗E2抗体可实现SINV从神经元的非溶细胞性清除。在抗体处理的BHK细胞中，细胞内病毒RNA增加，感染继续扩散。在未分化和分化的AP7神经元细胞中，抗体处理可诱导病毒RNA清除。两种插入了两个适体的病毒都容易发生缺失。这些研究为进一步开发适体标记的病毒RNA奠定了基础，这将有助于对感染和清除动态进行功能研究。
