Visualizing the replicating HSV-1 virus using STED super-resolution microscopy.
Visualizing the replicating HSV-1 virus using STED super-resolution microscopy.
复制标题
使用 STED 超分辨率显微镜观察复制的 HSV-1 病毒
DOI：
10.1186/s12985-016-0521-7
复制
发表时间：
2016-04-09
期刊：
影响因子：
4.8
通讯作者：
Zhou J
中科院分区：
文献类型：
--
作者：
Li Z;Fang C;Su Y;Liu H;Lang F;Li X;Chen G;Lu D;Zhou J
研究背景病毒基因组复制是单纯疱疹病毒1型（HSV-1）裂解感染周期的中心环节。然而，HSV-1复制过程的细节仍然是难以捉摸的，由于目前的分子和常规荧光显微镜方法的限制。受激发射耗尽（STED）显微镜是最近可用的超分辨率技术之一，其允许在亚衍射分辨率下观察。用荧光原位杂交（FISH）和免疫荧光（IF）技术观察HSV-1的复制过程。1基因组，这两个探针高度相关的复制前和复制基因组。相比之下，当使用来自不同区域的探针时，在病毒进入复制后，两个探针之间的平均距离增加，这表明HSV-1基因组经历了从紧凑到松弛的动态结构变化，并且当它进入复制时占据了更大的空间。FISH和IF结果显示，病毒单链结合蛋白ICP 8与HSV-1基因组定位紧密。相反，ICP 8和宿主RNA聚合酶II的相关性较低。这一结果表明，ICP 8标记的DNA复制区域在空间上与活性转录区域分离，活性转录区域由病毒复制区室内RNA聚合酶II的延伸形式表示。比较HSV-1复制早期和后期的基因组，我们也注意到不同值之间的总体增加。结论1）超分辨率显微镜可以观察到HSV-1基因组的复制过程; 2）病毒基因组在复制过程中发生空间扩展; 3）病毒复制和转录在复制区室中被划分为不同的亚结构。
BackgroundReplication of viral genome is the central event during the lytic infectious cycle of herpes simplex virus 1 (HSV-1). However, the details of HSV-1 replication process are still elusive due to the limitations of current molecular and conventional fluorescent microscopy methods. Stimulated emission depletion (STED) microscopy is one of the recently available super-resolution techniques allowing observation at sub-diffraction resolution.MethodsTo gain new insight into HSV-1 replication, we used a combination of stimulated emission depletion microscopy, fluorescence in situ hybridization (FISH) and immunofluorescence (IF) to observe the HSV-1 replication process.ResultsUsing two colored probes labeling the same region of HSV-1 genome, the two probes highly correlated in both pre-replication and replicating genomes. In comparison, when probes from different regions were used, the average distance between the two probes increased after the virus enters replication, suggesting that the HSV-1 genome undergoes dynamic structure changes from a compact to a relaxed formation and occupies larger space as it enters replication. Using FISH and IF, viral single strand binding protein ICP8 was seen closely positioned with HSV-1 genome. In contrast, ICP8 and host RNA polymerase II were less related. This result suggests that ICP8 marked regions of DNA replication are spatially separated from regions of active transcription, represented by the elongating form of RNA polymerase II within the viral replication compartments. Comparing HSV-1 genomes at early stage of replication with that in later stage, we also noted overall increases among different values. These results suggest stimulated emission depletion microscopy is capable of investigating events during HSV-1 replication.Conclusion1) Replicating HSV-1 genome could be observed by super-resolution microscopy; 2) Viral genome expands spatially during replication; 3) Viral replication and transcription are partitioned into different sub-structures within the replication compartments.