Imaging poliovirus entry in live cells.
Imaging poliovirus entry in live cells.
复制标题
DOI：
10.1371/journal.pbio.0050183
复制
发表时间：
2007-07
期刊：
影响因子：
9.8
通讯作者：
Hogle JM
中科院分区：
文献类型：
--
作者：
Brandenburg B;Lee LY;Lakadamyali M;Rust MJ;Zhuang X;Hogle JM
病毒通过将其遗传物质穿过细胞膜并进入细胞的适当区域来引发感染。动物病毒，尤其是无包膜病毒，传递其基因组的机制了解甚少。这部分是由于直接观察病毒基因传递存在技术困难，以及大多数动物病毒的高颗粒数与空斑形成单位比率导致难以区分有效和无效途径。在此，我们将一种在活细胞中同时追踪病毒衣壳和基因组的成像检测方法与一种基于感染性的RNA释放检测方法相结合，以描述脊髓灰质炎病毒（PV）感染的早期事件。通过这两种方法系统地探究了细胞运输途径抑制剂对RNA基因组传递的影响。令人惊讶的是，我们观察到PV的基因组释放高效且迅速，因此不会限制整体感染性或感染速率。研究结果确定了一种途径，即PV与细胞表面的受体结合，并通过一种不依赖网格蛋白、窖蛋白、浮舰蛋白和微管，但依赖酪氨酸激酶和肌动蛋白的内吞机制进入细胞。在病毒颗粒内化后，基因组立即从位于质膜100 - 200nm范围内的囊泡或紧密密封的膜内陷处释放。这些结果解决了关于PV是直接突破质膜屏障还是依靠内吞作用将其基因组传递到细胞内的长期争论。我们期望这种成像检测方法能广泛应用于无包膜病毒进入机制的研究。
在宿主之间传播时，病毒的基因组受到病毒衣壳和/或包膜的良好保护。在与靶细胞特异性结合后，病毒颗粒通过劫持细胞运输途径进入细胞，然后将病毒基因组传递到细胞的适当区域，在那里它指导子代病毒颗粒的产生。像脊髓灰质炎病毒这样的无包膜病毒如何进入靶细胞还不太清楚。在此，我们制备了基因组和衣壳都被荧光染料特异性标记的完全具有感染性的脊髓灰质炎病毒。然后我们可以使用实时荧光显微镜在感染过程中追踪单个病毒颗粒，以确定它们如何进入细胞以及确定基因组在细胞内何时何地释放。我们用病毒学检测方法对显微镜研究进行了补充，这些检测方法表明通过显微镜观察到的途径是有效的。我们表明脊髓灰质炎病毒进入活细胞的过程需要能量、完整的肌动蛋白细胞骨架和细胞信号通路，但不依赖于内吞途径的知名标志物。我们表明在内化之后，基因组释放出奇地高效，并且从非常接近细胞表面的囊泡中发生。我们的实验为脊髓灰质炎病毒感染的早期步骤提供了新的见解，并描述了可用于多种其他病毒的方法。
作者将一种在活细胞中同时追踪病毒衣壳和基因组的成像检测方法与一种基于感染性的RNA释放生物学检测方法相结合，解决了关于脊髓灰质炎病毒进入宿主细胞性质的长期争论。
Viruses initiate infection by transferring their genetic material across a cellular membrane and into the appropriate compartment of the cell. The mechanisms by which animal viruses, especially nonenveloped viruses, deliver their genomes are only poorly understood. This is due in part to technical difficulties involved in direct visualization of viral gene delivery and to uncertainties in distinguishing productive and nonproductive pathways caused by the high particle-to–plaque forming unit ratio of most animal viruses. Here, we combine an imaging assay that simultaneously tracks the viral capsid and genome in live cells with an infectivity-based assay for RNA release to characterize the early events in the poliovirus (PV) infection. Effects on RNA genome delivery from inhibitors of cell trafficking pathways were probed systematically by both methods. Surprisingly, we observe that genome release by PV is highly efficient and rapid, and thus does not limit the overall infectivity or the infection rate. The results define a pathway in which PV binds to receptors on the cell surface and enters the cell by a clathrin-, caveolin-, flotillin-, and microtubule-independent, but tyrosine kinase- and actin-dependent, endocytic mechanism. Immediately after the internalization of the virus particle, genome release takes place from vesicles or tightly sealed membrane invaginations located within 100–200 nm of the plasma membrane. These results settle a long-lasting debate of whether PV directly breaks the plasma membrane barrier or relies on endocytosis to deliver its genome into the cell. We expect this imaging assay to be broadly applicable to the investigation of entry mechanisms for nonenveloped viruses. During travel between hosts, the genome of a virus is well protected by the viral capsid and/or envelope. After binding specifically to target cells, the virus particles enter cells by hijacking cell trafficking pathways and then deliver the viral genome into the appropriate compartment of the cell where it directs the production of progeny virus particles. How nonenveloped viruses, such as poliovirus, enter target cells is not well understood. Here, we produced fully infectious poliovirus with both genome and capsid specifically labeled by fluorescent dyes. We could then use real-time fluorescent microscopy to follow single virus particles during infection, to define how they enter cells and to determine when and where in the cell the genome gets released. We have complemented the microscopic studies with virological assays, which demonstrate that the pathways observed by microscopy are productive. We show that poliovirus enters live cells in a process that requires energy, an intact actin cytoskeleton, and cell signaling pathways, but does not depend on the well-known markers of endocytic pathways. We show that after internalization, the genome release is surprisingly efficient and occurs from vesicles that are very close to the cell surface. Our experiments offer new insights into the early steps of poliovirus infection, and describe methods that can be used for a wide variety of other viruses. Combining an imaging assay that simultaneously tracks the viral capsid and genome in live cells with an infectivity-based biological assay for RNA release, the authors settle a long-lasting debate on the nature of poliovirus entry into the host cell.
登录
查看更多内容
影响因子：
5.4
作者：
FRICKS, CE;HOGLE, JM
通讯作者：
HOGLE, JM
影响因子：
5.4
作者：
GROMEIER, M;WETZ, K
通讯作者：
WETZ, K
影响因子：
2.9
作者：
Irurzun, A;Carrasco, L
通讯作者：
Carrasco, L
影响因子：
3.7
作者：
CROWTHER, D;MELNICK, JL
通讯作者：
MELNICK, JL
影响因子：
5.4
作者：
Danthi, P;Chow, M
通讯作者：
Chow, M