基孔肯雅病毒(CHIKV)是一种蚊子传播的病原体,可导致人类急性肌肉骨骼疾病。病毒RNA基因组的复制发生在专门的膜复制细胞器(RO)或球体中,其中含有病毒复制复合体。最初由RNA合成相关的质膜变形产生,甲病毒RO通常被快速内吞以产生I型细胞病变液泡(CPV-I),从中挤出新生的RNA用于细胞质翻译。相比之下,CHIKVRO内在化程度很低,在感染后期提出了他们的命运和功能问题。这里,使用原位低温电子显微镜方法,我们调查了CHIKVRO的结果和感染的人类细胞中相关的复制机制。我们证明了CHIKVRO在质膜上的后期持续存在,在球颈处有一个冠状的蛋白质复合物,类似于最近解决的复制复合物。这些隔室出乎意料的异质和大直径表明,这些细胞器的动态生长超出了单个RNA基因组的复制。周围细胞质区域的超微结构分析支持长出的CHIKVRO在病毒RNA合成中保持动态活性并输出到细胞胞质溶胶以进行蛋白质翻译。有趣的是,具有均匀直径的稀有RO也在CPV-I中被少量内在化,接近未知功能的蜂窝状排列,在未感染的对照中不存在,从而暗示了这种内在化的时间调节。总之,这项研究揭示了感染细胞中CHIKVRO的动态模式和与细胞膜界面相关的病毒复制。重要意义基孔肯雅病毒(CHIKV)是一种正链RNA病毒,其基因组复制需要专门的膜复制细胞器(RO)。我们对这个病毒周期阶段的了解仍然不完整,特别是关于感染细胞中CHIKVRO的命运和功能动力学。这里,我们表明,CHIKVRO维持在质膜超过第一个病毒周期,继续生长并在病毒RNA复制和向细胞胞质溶胶的输出中动态活跃,其中翻译发生在RO附近。这与细胞质液泡内化过程中RO的均匀直径相反,通常与功能未知的蜂窝状排列有关,提出了一种调节机制。这项研究为人类细胞中CHIKVRO的动力学和命运提供了新的思路,因此,我们对基孔肯雅病毒周期的理解。
Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for an acute musculoskeletal disease in humans. Replication of the viral RNA genome occurs in specialized membranous replication organelles (ROs) or spherules, which contain the viral replication complex. Initially generated by RNA synthesis-associated plasma membrane deformation, alphavirus ROs are generally rapidly endocytosed to produce type I cytopathic vacuoles (CPV-I), from which nascent RNAs are extruded for cytoplasmic translation. By contrast, CHIKV ROs are poorly internalized, raising the question of their fate and functionality at the late stage of infection. Here, using in situ cryogenic-electron microscopy approaches, we investigate the outcome of CHIKV ROs and associated replication machinery in infected human cells. We evidence the late persistence of CHIKV ROs at the plasma membrane with a crowned protein complex at the spherule neck similar to the recently resolved replication complex. The unexpectedly heterogeneous and large diameter of these compartments suggests a continuous, dynamic growth of these organelles beyond the replication of a single RNA genome. Ultrastructural analysis of surrounding cytoplasmic regions supports that outgrown CHIKV ROs remain dynamically active in viral RNA synthesis and export to the cell cytosol for protein translation. Interestingly, rare ROs with a homogeneous diameter are also marginally internalized in CPV-I near honeycomb-like arrangements of unknown function, which are absent in uninfected controls, thereby suggesting a temporal regulation of this internalization. Altogether, this study sheds new light on the dynamic pattern of CHIKV ROs and associated viral replication at the interface with cell membranes in infected cells.IMPORTANCEThe Chikungunya virus (CHIKV) is a positive-stranded RNA virus that requires specialized membranous replication organelles (ROs) for its genome replication. Our knowledge of this viral cycle stage is still incomplete, notably regarding the fate and functional dynamics of CHIKV ROs in infected cells. Here, we show that CHIKV ROs are maintained at the plasma membrane beyond the first viral cycle, continuing to grow and be dynamically active both in viral RNA replication and in its export to the cell cytosol, where translation occurs in proximity to ROs. This contrasts with the homogeneous diameter of ROs during internalization in cytoplasmic vacuoles, which are often associated with honeycomb-like arrangements of unknown function, suggesting a regulated mechanism. This study sheds new light on the dynamics and fate of CHIKV ROs in human cells and, consequently, on our understanding of the Chikungunya viral cycle.