Timm Andrea C, Warrick Jay W, Yin John
Systems Biology Theme, Wisconsin Institute for Discovery, Department of Chemical and Biological Engineering, University of Wisconsin, Madison, WI, USA.
Integr Biol (Camb). 2017 Sep 18;9(9):782-791. doi: 10.1039/c7ib00082k.
Cells infected by viruses can exhibit diverse patterns of viral and cellular gene expression. The patterns arise in part from the stochastic or noisy reaction kinetics associated with the small number of genomes, enzymes, and other molecules that typically initiate virus replication and activate cellular anti-viral defenses. It is not known what features, if any, of the early viral or cellular gene expression correlate with later processes of viral replication or cell survival. Here we used two fluorescent reporters to visualize innate immune activation of human prostate cancer (PC3) cells against infection by vesicular stomatitis virus. The cells were engineered to express green-fluorescent protein under control of the promoter for IFIT2, an interferon-sensitive component of the anti-viral response, while red-fluorescent protein was expressed as a byproduct of virus infection. To isolate and quantitatively analyze single-cells, we used a unique microwell array device and open-source image processing software. Kinetic analysis of viral and cellular reporter profiles from hundreds of cells revealed novel relationships between gene expression and the outcome of infection. Specifically, the relative timing rather than the magnitude of the viral gene expression and innate immune activation correlated with the infection outcome. Earlier viral or anti-viral gene expression favored or hindered virus growth, respectively. Further, analysis of kinetic parameters estimated from these data suggests a trade-off between robust antiviral signaling and cell death, as indicated by a higher rate of detectable cell lysis in infected cells with a detectable immune response. In short, cells that activate an immune response lyse at a higher rate. More broadly, we demonstrate how the intrinsic heterogeneity of individual cell behaviors can be exploited to discover features of viral and host gene expression that correlate with single-cell outcomes, which will ultimately impact whether or not infections spread.
被病毒感染的细胞会呈现出多样的病毒和细胞基因表达模式。这些模式部分源于与少量基因组、酶及其他通常启动病毒复制并激活细胞抗病毒防御的分子相关的随机或有噪声的反应动力学。目前尚不清楚早期病毒或细胞基因表达的哪些特征（如果有的话）与病毒复制或细胞存活的后期过程相关。在此，我们使用了两种荧光报告基因来可视化人前列腺癌（PC3）细胞针对水疱性口炎病毒感染的先天免疫激活。这些细胞经过工程改造，可在抗病毒反应中对干扰素敏感的成分IFIT2的启动子控制下表达绿色荧光蛋白，而红色荧光蛋白则作为病毒感染的副产物表达。为了分离并定量分析单细胞，我们使用了一种独特的微孔阵列装置和开源图像处理软件。对数百个细胞的病毒和细胞报告基因图谱进行动力学分析，揭示了基因表达与感染结果之间的新关系。具体而言，病毒基因表达和先天免疫激活的相对时间而非幅度与感染结果相关。较早的病毒或抗病毒基因表达分别有利于或阻碍病毒生长。此外，对从这些数据估计的动力学参数进行分析表明，在强大的抗病毒信号传导和细胞死亡之间存在权衡，这表现为在具有可检测免疫反应的感染细胞中可检测到的细胞裂解率更高。简而言之，激活免疫反应的细胞裂解率更高。更广泛地说，我们展示了如何利用单个细胞行为的内在异质性来发现与单细胞结果相关的病毒和宿主基因表达特征，这最终将影响感染是否会传播。
