Our research demonstrated that the transformation of cellular membranes into poliovirus replication complexes in infected cells is a well-coordinated multistage process. Previous reports of poliovirus replication structures described conflicting results and generated alternative models of their development. Nevertheless, these structures were generally visualized as “vesicles” whether surrounded by a single-membrane (11
) or a double-membrane (48
) bilayer. It is indeed tempting to interpret the round membranous contours on 2-dimensional electron microscopy images as representing cross-sections of “vesicles,” but our data demonstrate that this perception is misleading. Electron tomography reconstruction shows that, until the final stages of infection, the membranous matrix associated with viral replication is represented by irregularly shaped branching convoluted single-membrane tubular structures that look more like a porous sponge rather than like clusters of spherical individual vesicles.
Other data supporting a vesicular organization of poliovirus replication complexes were obtained from transmission EM images of viral replication-associated membranes isolated from infected cells. They appeared as clusters of elongated vesicles with protruding interwoven narrow necks. It was suggested that the inflated and the narrow portions, called “compact membranes,” of the vesicles may be associated with different forms of viral replication complexes (10
). In our experiments, we were able to investigate samples that were 200 nm in thickness. We did not see the “rosette-like” arrangements of replication membranes previously described for isolated replication complexes (10
); neither was a segregation of areas corresponding to “compact membranes” obvious. It is possible that the shapes of the structures isolated from infected cells disrupted by homogenization in buffered aqueous solution resulted from fragmentation of the continuous structures that we had directly observed in infected cells.
Many positive-strand RNA viruses form their replication complexes on specific cellular organelles. For example, the outer mitochondrial membrane (40
), ER (38
), chloroplasts (16
), and plasma membrane (50
) were previously shown to be sites of assembly of replication complexes for different viruses. The data available for poliovirus suggest that it utilizes membranes from multiple cellular sources (48
). In agreement with some previous reports (10
), the first virus-induced membrane structures that we observed were associated with a cis
-Golgi protein GM130, suggesting that Golgi membranes may be the initial site of poliovirus replication complex formation. The redistribution of this protein into multiple small foci in infected cells supports this idea. Other biochemical data have also pointed to the Golgi membrane as an important target of poliovirus replication. It has been shown that poliovirus proteins specifically engage components of the cellular secretory pathway, such as the small GTPase Arf and the Arf guanine nucleotide exchange factors GBF1, BIG1, and BIG2 (5
), that are known to play a role in Golgi homeostasis. However, pre-existing Golgi membranes likely could not provide sufficient material for the masses of membranous replication complexes that can occupy most of the cytoplasmic space in infected cells later in infection. The ER is the most likely source of membranes for growth and expansion of viral replication complexes. Previous studies showed virus-induced structures and tubules of the ER in close proximity (9
). However, we believe it is unlikely that the ER is directly transformed into viral replication structures. The resident ER protein calnexin was excluded from the poliovirus replication structures, and the tomographic reconstruction did not reveal any apparent direct connections between the ER and the clusters of virus-induced membranous chambers. These data suggest that ER membranes must be modified somehow before they are used for virus replication.
The role of double-membrane structures in the infectious cycle of poliovirus remains uncertain. The exponential stage of viral RNA replication is essentially finished by 4 h.p.i., when these structures begin to form. It is possible that the wrapping of single-membrane early structures that generates the double-membrane tubules or vesicles is the result of the cell's response to virus infection and that they represent a dead-end development of the replication complexes. On the other hand, the amount of RNA synthesized after 4 h constitutes the major portion of the total viral RNA yield; thus, either double-membrane structures significantly contribute to the overall RNA production or single-membrane replication-active membranous complexes are being constantly generated throughout the course of infection. Our estimates of the intracellular constituents based on thin-section EM images are not quantitative, and there might be a sufficient source of membranes in infected cells that could contribute to the continuous formation of replication structures. Indeed, we saw regions of typical single-membrane early-looking membranous clusters in cells even at 7 h.p.i (). Given the high level of RNA synthesis associated with the single-membrane chambers, their contribution to RNA production may be higher than that of more copious double-membrane structures. Unfortunately, analysis of thin-section EM images does not allow reliable statistical evaluation of the true distribution of different structures in cells. It is possible that the double-membrane structures are responsible not for viral RNA replication but for other stages in the infectious cycle such as RNA encapsidation or, as has been recently proposed, for nonlytic exit of virus from infected cells (52
). The latter phenomenon may be important for virus spread in an animal host, although the amount of virus released in the absence of cell lysis represents only a tiny fraction of the total amount of virus synthesized.
The process of forming double-membrane structures in poliovirus-infected cells has been suggested to involve at least the initial steps of the normal cellular autophagy pathway. This hypothesis was based on the morphological similarity of virus-induced double-membrane vesicles to early autophagosomes as well as on the stimulation of cleavage and modification of cellular LC3 protein in human 293T and MCF7 cells and its colocalization with the LAMP1 protein, an indicator of autophagosome development (27
). The data supporting the contribution of the autophagy pathway to enterovirus replication are mixed. It was demonstrated that inhibition or stimulation of autophagy results in modest inhibition or stimulation of poliovirus and coxsackie B3 virus yield, and data were also presented suggesting involvement of autophagy in replication of rhinoviruses 2 and 14 (27
). However, Brabec-Zaruba et al. reported that replication of rhinovirus 2 was insensitive to manipulation of autophagy with pharmacological agents and did not induce detectable modification of LC3 (13
). This may indicate that different cells or cell types incubated under different conditions respond to poliovirus infection with different levels of activation of the autophagy program. It is also possible that the double-membrane structures in infected cells bear an only superficial morphological resemblance to those of autophagosomes. Our data indicate that the poliovirus double-membrane structures are formed by collapsing and/or wrapping of the early single-membrane structures, whereas recent advanced imaging studies of autophagosome formation demonstrate that they are formed by de novo
synthesis of the isolation membrane and not by the simple wrapping of pre-existing organelle membranes (22
). It is possible that changes in the concentration of intraluminal solutes result in luminal collapse due to change of osmotic pressure. Enterovirus 2B proteins are known viroporins (2
), and accumulation of this protein during the time course of infection could increase the permeability of these structures, contributing to their collapse at later times. Recently, the 3-D architecture of membranous replication complexes of several positive-strand RNA viruses was investigated by electron tomographic methods similar to those used in the studies reported here. Together with the previously accumulated data on membrane modifications induced by positive-strand RNA viruses, the images reveal that viruses from different families infecting different hosts demonstrate significant similarities in the overall organization of their replication complexes. Flock House virus of the Nodaviridae
), dengue virus of the Flaviviridae
), and Semliki Forest virus of the Togaviridae
) remodel membranes of mitochondria, ER, and plasma, respectively, to form invaginations harboring the viral replication machinery. Similar invaginated spherules were previously described as replication sites for other plant and animal viruses (21
). In those studies, invaginations were formed by inducing negative curvature in the pre-existing membrane bilayer. On the other hand, our study and previous data on membrane remodeling by picornaviruses and coronaviruses showed that these viruses shape membranes into tubular and/or spherical vesicle-like structures with a positive curvature (3
). The total spectrum of positive-strand RNA viruses can be classified into three superfamilies of alpha-like, flavi-like, and picorna-like viruses on the basis of their genome sequences and organization (20
). These higher-order taxonomic units encompass diverse viruses infecting different hosts from almost all kingdoms of life. It appears that negative membrane curvature structures initiated by membrane invaginations are a predominant characteristic of the replication complexes of alpha- and flavi-like viruses, while picorna-like viruses almost exclusively develop complexes with positive membrane curvature. This division implies that two different subsets of supporting cellular membrane remodeling pathways are recruited by those groups of viruses and may suggest that there are two basic strategies evolved by these pathogens to develop their replication complexes.