Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China.
Research Unit, Shanghai Public Health Clinical Center, Fudan University, Shanghai, China.
PLoS Pathog. 2021 Aug 9;17(8):e1009838. doi: 10.1371/journal.ppat.1009838. eCollection 2021 Aug.
Through evolution, Hepatitis B Virus (HBV) developed highly intricate mechanisms exploiting host resources for its multiplication within a constrained genetic coding capacity. Yet a clear picture of viral hitchhiking of cellular processes with spatial resolution is still largely unsolved. Here, by leveraging bDNA-based fluorescence in situ hybridization (FISH) combined with immunofluorescence, we developed a microscopic approach for multiplex detection of viral nucleic acids and proteins, which enabled us to probe some of the key aspects of HBV life cycle. We confirmed the slow kinetics and revealed the high variability of viral replication at single-cell level. We directly visualized HBV minichromosome in contact with acetylated histone 3 and RNA polymerase II and observed HBV-induced degradation of Smc5/6 complex only in primary hepatocytes. We quantified the frequency of HBV pregenomic RNAs occupied by translating ribosome or capsids. Statistics at molecular level suggested a rapid translation phase followed by a slow encapsidation and maturation phase. Finally, the roles of microtubules (MTs) on nucleocapsid assembly and virion morphogenesis were analyzed. Disruption of MTs resulted in the perinuclear retention of nucleocapsid. Meanwhile, large multivesicular body (MVB) formation was significantly disturbed as evidenced by the increase in number and decrease in volume of CD63+ vesicles, thus inhibiting mature virion secretion. In conclusion, these data provided spatially resolved molecular snapshots in the context of specific subcellular activities. The heterogeneity observed at single-cell level afforded valuable molecular insights which are otherwise unavailable from bulk measurements.
通过进化，乙型肝炎病毒（HBV）发展出了高度复杂的机制，利用宿主资源在有限的遗传编码能力内进行复制。然而，HBV 劫持细胞过程的详细情况，其分辨率仍不明确。在这里，我们利用基于 bDNA 的荧光原位杂交（FISH）结合免疫荧光技术，开发了一种用于检测病毒核酸和蛋白质的多重检测的显微镜方法，使我们能够探究 HBV 生命周期的一些关键方面。我们证实了病毒复制的缓慢动力学，并在单细胞水平上揭示了其高度的变异性。我们直接观察到 HBV 微小染色体与乙酰化组蛋白 3 和 RNA 聚合酶 II 接触，并仅在原代肝细胞中观察到 HBV 诱导的 Smc5/6 复合物降解。我们量化了翻译核糖体或衣壳占据 HBV 前基因组 RNA 的频率。分子水平的统计数据表明存在快速翻译阶段，随后是缓慢的包装和成熟阶段。最后，我们分析了微管（MTs）在核衣壳组装和病毒粒子形态发生中的作用。MT 的破坏导致核衣壳在核周滞留。同时，大的多泡体（MVB）形成受到显著干扰，证据是 CD63+囊泡的数量增加和体积减小，从而抑制成熟病毒粒子的分泌。总之，这些数据提供了特定亚细胞活动背景下的空间分辨分子快照。在单细胞水平上观察到的异质性提供了有价值的分子见解，而这些见解是从批量测量中无法获得的。
