Tan Xi, Ravasio Andrea, Ong Hui T, Wu Jinlu, Hew Choy L
Mechanobiology Institute National University of Singapore Singapore Singapore.
Present address: School of Basic Medical Sciences Guizhou University of Traditional Chinese Medicine Guiyang Guizhou Province China.
FASEB Bioadv. 2020 Mar 17;2(4):264-279. doi: 10.1096/fba.2019-00086. eCollection 2020 Apr.
Viral protein 9 (VP9) is a non-structural protein of white spot syndrome virus (WSSV) highly expressed during the early stage of infection. The crystal structure of VP9 suggests that the polymers of VP9 dimers resemble a DNA mimic, but its function remains elusive. In this study, we demonstrated that VP9 impedes histones binding to DNA via single-molecule manipulation. We established VP9 expression in HeLa cells due to the lack of a WSSV-susceptible cell line, and observed abundant VP9 in the nucleus, which mirrors its distribution in the hemocytes of WSSV-infected shrimp. VP9 expression increased the dynamics and rotational mobility of histones in stable H3-GFP HeLa cells as revealed by fluorescent recovery after photobleaching and fluorescence anisotropy imaging, which suggested a loosened compaction of chromatin structure. Successive salt fractionation showed that a prominent population of histones was solubilized in high salt concentrations, which implies alterations of bulk chromatin structure. Southern blotting identified that VP9 alters juxtacentromeric chromatin structures to be more accessible to micrococcal nuclease digestion. RNA microarray revealed that VP9 expression also leads to significant changes of cellular gene expression. Our findings provide evidence that VP9 alters the cellular higher-order chromatin structure, uncovering a potential strategy adopted by WSSV to facilitate its replication.
病毒蛋白9（VP9）是白斑综合征病毒（WSSV）的一种非结构蛋白，在感染早期高度表达。VP9的晶体结构表明，VP9二聚体的聚合物类似于一种DNA模拟物，但其功能仍然难以捉摸。在本研究中，我们通过单分子操作证明VP9阻碍组蛋白与DNA结合。由于缺乏对WSSV敏感的细胞系，我们在HeLa细胞中建立了VP9表达，并观察到细胞核中有大量VP9，这反映了其在WSSV感染虾血细胞中的分布。如光漂白后荧光恢复和荧光各向异性成像所示，VP9表达增加了稳定表达H3-GFP的HeLa细胞中组蛋白的动力学和旋转流动性，这表明染色质结构的压缩程度有所松动。连续盐分级分离表明，大量组蛋白在高盐浓度下可溶解，这意味着整体染色质结构发生了改变。Southern印迹法鉴定出VP9改变了着丝粒周围的染色质结构，使其更容易被微球菌核酸酶消化。RNA微阵列显示，VP9表达还导致细胞基因表达发生显著变化。我们的研究结果提供了证据，证明VP9改变了细胞的高阶染色质结构，揭示了WSSV为促进其复制而采用的一种潜在策略。
