Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein

van der Schaar, H. M.; Melia, C. E.; van Bruggen, J. A. C.; Strating, J. R. P. M.; van Geenen, M. E. D.; Koster, A. J.; Bárcena, M.; van Kuppeveld, F. J. M.

The morphology of the enterovirus-induced membrane structures (often termed replication organelles [ROs]) has been a subject of intense investigation. Twodimensional electron microscopy (EM) studies have shown both single-membrane and double-membrane structures, depending on the cell type, time point, and experimental procedure (9) (10) (11) (12) (13) (14) (15) (16) . Recently, two studies with poliovirus and coxsackievirus B3 (CVB3) were conducted to reveal the three-dimensional (3D) structure of the ROs in the course of infection using electron tomography (17, 18) . Both studies showed that the first structures detected upon infection are single-membrane tubular structures. These tubules appear to be formed at the expense of Golgi membranes, since in most cell sections, the Golgi apparatus is no longer detected when the tubules are present. The tubules emerge during the exponential phase of viral RNA replication, suggesting that they are the preeminent structures supporting viral genome synthesis. Later in infection, the tubular ROs morph into double-membrane vesicles (DMVs) and multilamellar structures, a phenomenon that is reminiscent of autophagy. It was shown for poliovirus that newly synthesized viral RNA localizes not only to the tubular structures but also to the DMVs, implying that the DMVs may also facilitate genome replication (17) . In addition, DMVs have been proposed to mediate nonlytic release of progeny virions (19) (20) (21) .