The transformation of enterovirus replication structures: a three-dimensional study of single- and double-membrane compartments

Abstract

All positive-strand RNA viruses induce membrane structures in their host cells which are thought to serve as suitable microenvironments for viral RNA synthesis. The structures induced by enteroviruses, which are members of the family Picornaviridae, have so far been described as either single- or double-membrane vesicles (DMVs). Aside from the number of delimiting membranes, their exact architecture has also remained elusive due to the limitations of conventional electron microscopy. In this study, we used electron tomography (ET) to solve the three-dimensional (3-D) ultrastructure of these compartments. At different time points postinfection, coxsackievirus B3-infected cells were high-pressure frozen and freeze-substituted for ET analysis. The tomograms showed that during the exponential phase of viral RNA synthesis, closed smooth single-membrane tubules constituted the predominant virus-induced membrane structure, with a minor proportion of DMVs that were either closed or connected to the cytosol in a vase-like configuration. As infection progressed, the DMV number steadily increased, while the tubular single-membrane structures gradually disappeared. Late in infection, complex multilamellar structures, previously unreported, became apparent in the cytoplasm. Serial tomography disclosed that their basic unit is a DMV, which is enwrapped by one or multiple cisternae. ET also revealed striking intermediate structures that strongly support the conversion of single-membrane tubules into double-membrane and multilamellar structures by a process of membrane apposition, enwrapping, and fusion. Collectively, our work unravels the sequential appearance of distinct enterovirus-induced replication structures, elucidates their detailed 3-D architecture, and provides the basis for a model for their transformation during the course of infection.

Importance: Positive-strand RNA viruses hijack specific intracellular membranes and remodel them into special structures that support viral RNA synthesis. The ultrastructural characterization of these "replication structures" is key to understanding their precise role. Here, we resolved the three-dimensional architecture of enterovirus-induced membranous compartments and their transformation in time by applying electron tomography to cells infected with coxsackievirus B3 (CVB3). Our results show that closed single-membrane tubules are the predominant initial virus-induced structure, whereas double-membrane vesicles (DMVs) become increasingly abundant at the expense of these tubules as infection progresses. Additionally, more complex multilamellar structures appear late in infection. Based on compelling intermediate structures in our tomograms, we propose a model for transformation from the tubules to DMVs and multilamellar structures via enwrapping events. Our work provides an in-depth analysis of the development of an unsuspected variety of distinct replication structures during the course of CVB3 infection.

FIG 1

Time course analysis of CVB3 infection in Vero E6 cells. (A and B) Fluorescent images of CVB3-infected cells stained for dsRNA (green) and for viral proteins 3A and 3D (red). Nuclei are stained with Hoechst (blue). The asterisks in panel A indicate cells with a clear cytopathic effect that are rounding up. Enlarged images of infected cells at 5 h p.i. are shown in B. (C) Lysates of CVB3-infected cells were prepared in parallel and analyzed for expression of viral proteins on Western blot using antibodies directed against viral proteins 3A and 3D and β-actin, which served as a loading control. (D) In the same experiment, the amount of intracellular viral RNA was determined by quantitative PCR, and the total number of infectious particles was measured by endpoint titration analysis. For clarity, the results are expressed as fold induction relative to the quantities at 1 h p.i.

FIG 2

Electron micrographs of CVB3-infected Vero E6 cells, high-pressure frozen at 5 h p.i. (A and A′), 6 h p.i. (B and B′), and 7 h p.i. (C and C′). (A) At 5 h p.i., large clusters of virus-induced modifications are observed in the perinuclear region. (A′) The clusters consist predominantly of single-membrane elongated structures, which appear to be tubules sometimes visualized in cross section (inset). Some DMVs (white arrows) can also be detected. (B) As infection progresses (6 h p.i.), the perinuclear clusters change. (B′) Single-membrane structures (black arrowheads) are sparser, whereas DMVs (inset) become more common. Compartments surrounded by more than two membranes (white arrowhead) start to be frequently observed. (C) At 7 h p.i., severe cytopathic effects, such as dilation of the ER and nuclear envelope, become apparent (asterisks). (C′) At this stage, CVB3-induced modifications consist of DMVs and more complex multilamellar structures (inset). (D and D′) Micrographs of a mock-infected Vero E6 cell are shown for comparison. N, nucleus; M, mitochondrion, G, Golgi complex. Panels A′, B′, C′, and D′ show higher-resolution images of the boxed areas in panels A, B, C, and D, respectively. Scale bars, 2 µm (A, B, and C), 500 nm (A′, B′, and C′), 5 µm (D), and 1 µm (D′).

FIG 3

ET of the early CVB3-induced membrane modifications (5 h p.i.) (see also Movie S1 in the supplemental material). (A) Tomographic slice through the serial tomogram, with clusters of single-membrane structures and sparsely embedded DMVs. (B) Top and side views of a surface-rendered model of the region boxed in panel A showing single-membrane tubules (green), open (orange) and closed (yellow) DMVs, and ER (blue). All DMVs appear open at the top and bottom in the rendered model due to the intrinsic limitations of ET, as a result of which membrane planes perpendicular to the third dimension are not resolved (24). (C) Example of laterally connected tubules. These connections (white arrowhead) encompass short stretches (boxed area in the model, ~30 nm in length) and are relatively rare. (D) In contrast, membrane-membrane interactions between tubules (white arrowheads) are common. (E) Example of an open DMV with a single opening towards the cytosol, here large enough to partially engulf a tubular structure. Panels A, C, D, and E show 5-nm-thick tomographic slices. Scale bars, 500 nm (A) and 100 nm (C, D, and E).

FIG 4

ET of the CVB3-induced modifications typical of late infection (7 h p.i.) (see also Movie S2 in the supplemental material). (A) The multiple membranes in the vesicles found at this stage are clearly resolved in the virtual sections of the serial tomographic reconstructions. (B) Surface-rendered model of the CVB3-induced compartments, colored according to their 3-D architecture: orange, DMVs; red, multilamellar structures. Parts of the neighboring ER are rendered in blue. In addition to the isolated DMVs (C and C′), the multilamellar structures were also found to be essentially DMVs, being enwrapped by one cisterna (D and D′) or several layered cisternae (E and E′). In panels C′, D′, and E′, the different membranes have been delineated for clarity. All the tomographic slices (A and C to E′) are 5 nm thick. Scale bars, 500 nm (A) and 100 nm (C, D, and E).

FIG 5

Tomographic xy slices through different structures possibly involved in the transition from a tubule to a closed DMV (see also Movie S3 in the supplemental material). (A) A longitudinally oriented tubule reaches its maximum width in the middle panel and fades in the first and last slices. (B) The walls of the tubule have become closer, flattening the compartment and extending it in the z direction, thereby forming a cisterna. Closer membrane pairing can be observed in long stretches of the cisternae shown in panels C and D. (E) Slices through a rounded-up cisterna, essentially an open DMV in a vase-like configuration with an opening to the cytosol (black arrowhead). Fusion of the ends of an open DMV could then give rise to a closed DMV (F). All the panels are 5-nm-thick slices separated by an interval of 15 nm in their respective tomograms; thereby, the encompassed z height is identical for all rows. Scale bars, 100 nm.

FIG 6

ET-based transition model for CVB3-induced replication structures. The single membrane tubules present early in infection transform into double- and multiple-membrane structures as CVB3 infection proceeds. The membranes of the tubules become more tightly apposed, resulting in flattened cisternae. Via an enwrapping mechanism, the cisterna curves into a vase-like configuration, and its membrane ends fuse, producing a closed, spherical DMV. The DMVs then become enwrapped by other cisternae.