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.

Differential permissivity of human cerebrovascular endothelial cells to enterovirus infection and specificities of serotype EV-A71 in crossing an in vitro model of the human blood–brain barrier

“…In the echovirus infections, we found evidence of single-and double-membrane vesicles organized in compact arrangements near the nucleus and of structures that displayed positive membrane invagination. Both features were reported previously in Vero and HeLa cells infected by CV-B3 and PV-1, respectively (Limpens et al, 2011;Belov et al, 2012). The vesicular structure in the EV-A71-infected hCMEC/D3 cells was characterized by less condensed vesicles and an increased proportion of multilamellar and large vesicles.…”

Influenza A virus ribonucleoproteins modulate host recycling by competing with Rab11 effectors

“…Induction of vesicular clustering and concentration of viral material for facilitated viral production is a well-known mechanism that operates in many positive-sense RNA viruses and poxviruses (Limpens et al, 2011;Monaghan et al, 2004;Welsch et al, 2009;Laliberte and Moss, 2010). Nevertheless, this hypothesis, as appealing as it might be, awaits compelling experimental support because, at the moment, the relevance of the clustered Rab11 vesicles is still uncertain.…”

BPIFB6 Regulates Secretory Pathway Trafficking and Enterovirus Replication

“…Unlike autophagy, which may facilitate unique aspects of enterovirus replication in a virus-specific manner, such as in virus reliance on flux through the autophagic pathway (36,37), all enteroviruses rely on intact secretory pathway trafficking to facilitate their replicative life cycles. Enteroviruses utilize ER exit sites to form tubular structures that extend into the cytoplasm to anchor the viral replication machinery on the cytoplasmic membrane of the ER (34,38). The formation of enterovirus replication organelles also specifically requires COPI (39)(40)(41), which is recruited to replication complexes via the inhibition of the activity of the Arf1 GTPase by the virally encoded 3A protein (42,43).…”