Krzywkowski Tomasz, Ciftci Sibel, Assadian Farzaneh, Nilsson Mats, Punga Tanel
Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.
Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
J Virol. 2017 May 12;91(11). doi: 10.1128/JVI.00166-17. Print 2017 Jun 1.
An efficient adenovirus infection results in high-level accumulation of viral DNA and mRNAs in the infected cell population. However, the average viral DNA and mRNA content in a heterogeneous cell population does not necessarily reflect the same abundance in individual cells. Here, we describe a novel padlock probe-based rolling-circle amplification technique that enables simultaneous detection and analysis of human adenovirus type 5 (HAdV-5) genomic DNA and virus-encoded mRNAs in individual infected cells. We demonstrate that the method is applicable for detection and quantification of HAdV-5 DNA and mRNAs in short-term infections in human epithelial cells and in long-term infections in human B lymphocytes. Single-cell evaluation of these infections revealed high heterogeneity and unique cell subpopulations defined by differential viral DNA content and mRNA expression. Further, our single-cell analysis shows that the specific expression pattern of viral and mRNA splice variants is linked to HAdV-5 DNA content in the individual cells. Furthermore, we show that expression of a mature form of the HAdV-5 histone-like protein VII affects virus genome detection in HAdV-5-infected cells. Collectively, padlock probes combined with rolling-circle amplification should be a welcome addition to the method repertoire for the characterization of the molecular details of the HAdV life cycle in individual infected cells. Human adenoviruses (HAdVs) have been extensively used as model systems to study various aspects of eukaryotic gene expression and genome organization. The vast majority of the HAdV studies are based on standard experimental procedures carried out using heterogeneous cell populations, where data averaging often masks biological differences. As every cell is unique, characteristics and efficiency of an HAdV infection can vary from cell to cell. Therefore, the analysis of HAdV gene expression and genome organization would benefit from a method that permits analysis of individual infected cells in the heterogeneous cell population. Here, we show that the padlock probe-based rolling-circle amplification method can be used to study concurrent viral DNA accumulation and mRNA expression patterns in individual HAdV-5-infected cells. Hence, this versatile method can be applied to detect the extent of infection and virus gene expression changes in different HAdV-5 infections.
高效的腺病毒感染会导致病毒DNA和mRNA在受感染细胞群体中高水平积累。然而，异质细胞群体中病毒DNA和mRNA的平均含量不一定反映单个细胞中的相同丰度。在此，我们描述了一种基于新型锁式探针的滚环扩增技术，该技术能够同时检测和分析单个受感染细胞中的人5型腺病毒（HAdV-5）基因组DNA和病毒编码的mRNA。我们证明该方法适用于检测和定量人上皮细胞短期感染以及人B淋巴细胞长期感染中的HAdV-5 DNA和mRNA。对这些感染的单细胞评估揭示了高度的异质性以及由不同病毒DNA含量和mRNA表达所定义的独特细胞亚群。此外，我们的单细胞分析表明，病毒 和 mRNA剪接变体的特定表达模式与单个细胞中的HAdV-5 DNA含量相关。此外，我们表明HAdV-5组蛋白样蛋白VII成熟形式的表达会影响HAdV-5感染细胞中的病毒基因组检测。总体而言，锁式探针与滚环扩增相结合应是用于表征单个受感染细胞中HAdV生命周期分子细节的方法库中的一个受欢迎的补充。人腺病毒（HAdV）已被广泛用作模型系统来研究真核基因表达和基因组组织的各个方面。绝大多数HAdV研究基于使用异质细胞群体进行的标准实验程序，其中数据平均往往掩盖了生物学差异。由于每个细胞都是独特的，HAdV感染的特征和效率可能因细胞而异。因此，HAdV基因表达和基因组组织的分析将受益于一种允许对异质细胞群体中的单个受感染细胞进行分析的方法。在此，我们表明基于锁式探针的滚环扩增方法可用于研究单个HAdV-5感染细胞中病毒DNA积累和mRNA表达模式的同时情况。因此，这种通用方法可用于检测不同HAdV-5感染中的感染程度和病毒基因表达变化。
