Many viruses take advantage of cellular trafficking machineries to assemble and release new infectious particles. Using RNA interference (RNAi), we demonstrate that the Golgi/autophagosome-associated Rab33B is required for hepatitis B virus (HBV) propagation in hepatoma cell lines. While Rab33B is dispensable for the secretion of HBV subviral envelope particles, its knockdown reduced the virus yield to 20% and inhibited nucleocapsid (NC) formation and/or NC trafficking. The overexpression of a GDP-restricted Rab33B mutant phenocopied the effect of deficit Rab33B, indicating that Rab33B-specific effector proteins may be involved. Moreover, we found that HBV replication enhanced Rab33B expression. By analyzing HBV infection cycle steps, we identified a hitherto unknown membrane targeting module in the highly basic C-terminal domain of the NC-forming core protein. Rab33B inactivation reduced core membrane association, suggesting that membrane platforms participate in HBV assembly reactions. Biochemical and immunofluorescence analyses provided further hints that the viral core, rather than the envelope, is the main target for Rab33B intervention. Rab33B-deficiency reduced core protein levels without affecting viral transcription and hampered core/NC sorting to envelope-positive, intracellular compartments. Together, these results indicate that Rab33B is an important player in intracellular HBV trafficking events, guiding core transport to NC assembly sites and/or NC transport to budding sites.

译文

:许多病毒利用细胞运输设备来组装和释放新的传染性颗粒。使用RNA干扰(RNAi),我们证明了高尔基体/自噬体相关的Rab33B是乙型肝炎病毒(HBV)在肝癌细胞系中繁殖所必需的。尽管Rab33B对于HBV亚病毒包膜颗粒的分泌是必不可少的,但其敲除将病毒的产量降低到20%,并抑制了核衣壳(NC)的形成和/或NC的运输。 GDP受限的Rab33B突变体的过表达表型化了缺乏的Rab33B的作用,表明可能参与了Rab33B特异的效应蛋白。此外,我们发现HBV复制增强了Rab33B的表达。通过分析HBV感染的循环步骤,我们在形成NC的核心蛋白的高碱性C末端结构域中确定了迄今未知的膜靶向模块。 Rab33B失活减少了核心膜的缔合,表明膜平台参与了HBV组装反应。生化和免疫荧光分析提供了进一步的提示,即病毒核心而不是包膜是Rab33B干预的主要目标。 Rab33B缺乏症降低了核心蛋白水平,而没有影响病毒的转录,也没有阻碍核心/ NC对包膜阳性细胞内区室的分选。总之,这些结果表明,Rab33B是细胞内HBV转运事件的重要参与者,指导核心转运至NC装配位和/或NC转运至萌芽位。

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