2016年4月9日，国际病毒学领域专业期刊《Virology Journal》杂志上在线发表中国科学院昆明动物研究所周巨民课题组与上海徕卡显微镜公司合作利用超分辨显微镜研究HSV-1病毒复制的研究论文，周巨民课题组的博士研究生李卓然为第一作者，周巨民研究员为通讯作者。

单纯疱疹病毒(Herpes Simplex Virus, HSV)是影响人类健康的重要病毒之一。HSV-1潜伏感染近90%的人群，重激活时会引起一系列临床症状，轻者有口腔疱疹，重者会导致单纯疱疹病毒脑炎。DNA复制是HSV-1裂解感染阶段的核心事件，涉及到HSV-1基因组、病毒蛋白和宿主蛋白等众多方面的动态变化和有序调控，但是受到现有分子生物学手段和传统荧光显微镜的限制，病毒复制的细节一直难以进行观察。受激发射损耗显微镜(stimulated emission depletion, STED)的分辨率远低于衍射极限，将STED显微技术与传统的免疫荧光(immunofluorescence, IF)和荧光原位杂交(fluorescence in situ hybridization, FISH)技术相结合，能够在亚衍射极限水平获得HSV-1复制过程中的更多细节和信息。

研究组对不同复制阶段的HSV-1基因组、病毒蛋白和宿主蛋白进行超高分辨率图像采集，通过分析发现：(1)STED显微镜能够更可靠地分辨HSV-1基因组的不同区域;(2)HSV-1基因组进入宿主细胞核后，逐渐由压缩状态转变为松散状态，并占据更大的空间位置;(3)病毒蛋白ICP8与复制中的HSV-1基因组密切相关;(4)虽然HSV-1的复制和转录都在病毒复制区内进行，但是这两种生物学过程发生在病毒复制区不同的亚结构内。

原文摘要：

Background

Replication 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 currentmolecular 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.

Methods

To 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.

Results

Using 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.

Conclusion

1) 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.