Influenza A Virus Assembly Intermediates Fuse in the Cytoplasm

Reassortment of influenza viral RNA (vRNA) segments in co-infected cells can lead to the emergence of viruses with pandemic potential. Replication of influenza vRNA occurs in the nucleus of infected cells, while progeny virions bud from the plasma membrane. However, the intracellular mechanics of vRNA assembly into progeny virions is not well understood. Here we used recent advances in microscopy to explore vRNA assembly and transport during a productive infection. We visualized four distinct vRNA segments within a single cell using fluorescent in situ hybridization (FISH) and observed that foci containing more than one vRNA segment were found at the external nuclear periphery, suggesting that vRNA segments are not exported to the cytoplasm individually. Although many cytoplasmic foci contain multiple vRNA segments, not all vRNA species are present in every focus, indicating that assembly of all eight vRNA segments does not occur prior to export from the nucleus. To extend the observations made in fixed cells, we used a virus that encodes GFP fused to the viral polymerase acidic (PA) protein (WSN PA-GFP) to explore the dynamics of vRNA assembly in live cells during a productive infection. Since WSN PA-GFP colocalizes with viral nucleoprotein and influenza vRNA segments, we used it as a surrogate for visualizing vRNA transport in 3D and at high speed by inverted selective-plane illumination microscopy. We observed cytoplasmic PA-GFP foci colocalizing and traveling together en route to the plasma membrane. Our data strongly support a model in which vRNA segments are exported from the nucleus as complexes that assemble en route to the plasma membrane through dynamic colocalization events in the cytoplasm.

共感染细胞内的流感病毒RNA（vRNA）节段重配，可催生具备大流行潜力的病毒株。流感vRNA的复制发生于感染细胞的细胞核中，而子代病毒颗粒则从细胞质膜出芽释放。然而，vRNA组装为子代病毒颗粒的胞内机制仍未得到充分阐明。
本研究借助近期发展的显微成像技术，探究了增殖性感染过程中vRNA的组装与转运过程。我们通过荧光原位杂交（fluorescent in situ hybridization, FISH）在单个细胞内可视化了四种不同的vRNA节段，观察到包含多种vRNA节段的灶点定位于细胞核外膜附近，这提示vRNA节段并非以单一片段的形式被转运出细胞核。
尽管多数细胞质灶点包含多种vRNA节段，但并非所有vRNA种类都存在于每一个灶点中，这表明八种vRNA节段的完整组装并非发生于细胞核输出之前。
为了拓展固定细胞中的观察结果，我们使用一种编码与病毒聚合酶酸性（PA）蛋白融合的绿色荧光蛋白（GFP）的病毒（WSN PA-GFP），探究了增殖性感染过程中活细胞内vRNA组装的动态过程。由于WSN PA-GFP可与病毒核蛋白及流感vRNA节段共定位，我们将其作为替代标记物，通过倒置选择性平面照明显微镜（inverted selective-plane illumination microscopy）实现了vRNA转运的三维高速可视化。
我们观察到细胞质中的PA-GFP灶点在向细胞质膜转运的途中发生共定位并伴随协同移动。本研究数据强有力地支持了如下模型：vRNA节段以复合物形式从细胞核输出，并在向细胞质膜转运的过程中，通过细胞质内的动态共定位事件完成组装。