The Role of Electron Microscopy in Studying the Continuum of Changes in Membranous Structures during Poliovirus Infection

Abstract

Replication of the poliovirus genome is localized to cytoplasmic replication factories that are fashioned out of a mixture of viral proteins, scavenged cellular components, and new components that are synthesized within the cell due to viral manipulation/up-regulation of protein and phospholipid synthesis. These membranous replication factories are quite complex, and include markers from multiple cytoplasmic cellular organelles. This review focuses on the role of electron microscopy in advancing our understanding of poliovirus RNA replication factories. Structural data from the literature provide the basis for interpreting a wide range of biochemical studies that have been published on virus-induced lipid biosynthesis. In combination, structural and biochemical experiments elucidate the dramatic membrane remodeling that is a hallmark of poliovirus infection. Temporal and spatial membrane modifications throughout the infection cycle are discussed. Early electron microscopy studies of morphological changes following viral infection are re-considered in light of more recent data on viral manipulation of lipid and protein biosynthesis. These data suggest the existence of distinct subcellular vesicle populations, each of which serves specialized roles in poliovirus replication processes.

Keywords: RNA replication; RNA virus; cell morphology; electron microscopy; membrane; membrane remodeling; poliovirus; replication organelle.

Figure 1

Changes in cellular morphology after poliovirus infection. Schematized cells from electron micrographs in Schlegel et al., (A,D) [54], Dales et al., (B,E) [53], and Mattern and Daniels (C) [57]. Representative structures are marked: nuclear membrane (blue), mitochondria (red), vacuoles (orange). Lines denote cytoplasm, and dots represent “viroplasm”. Endoplasmic reticulum (yellow) becomes enlarged and sometimes called “nuclear extrusions” (as in panel C). Early in infection single membrane vesicles (purple) are visible, and later the appearance of U bodies/horseshoe-shaped vesicles (green) and double membrane vesicles (teal). In panel C, an image of a fractionated rosette (from Figure 3) is shown to scale, highlighting its similarity to structures seen within infected cells. Scale bar 1 μm.

Figure 2

Subcellular fractionation of functionally distinct vesicles associated with replication processes. In work by Caliguiri and Tamm, Dounce homogenates of HeLa cells were layered in a discontinuous sucrose gradient, centrifuged at 86,000 x g. Upon fractionation of the gradient, membrane bands with the indicated densities were collected. Properties of fractions 2, 3, and 5 are shown. Vesicle content was examined by electron microscopy [58]; RNA polymerase activity of the isolated fraction was measured by tritiated ATP incorporation; the abundance of RNA species (RI, RF, SS) was determined by gel electrophoresis; and viral titer was measured by plaque assay [58,66]. Note: data from Caliguiri and Tamm [58,66,67].

Figure 3

Morphology of rosette isolated from poliovirus-infected cells. (A) U body-like densities surround a dense central region of smaller vesicles. Cell fractionated material was prepared and imaged by Evan Rossignol using methods from Schlegel et al., 1996 [54]. Sample from 4 hpi is negatively stained and imaged by electron microscopy; (B) Cartoon highlights the horseshoe-shaped (U body-like) configurations of individual vesicles of the cluster. Magnification bar, 100 nm.

Figure 4

Virus-induced membrane remodeling and expansion for poliovirus replication. (A) As early as 2.5 hpi, lipid buds off the ER [52], producing structures comprised of mostly virally induced lipids [11]; (B) New and pre-existing cellular vesicles are dynamically remodeled [55] and invaginated from tubules into vesicles, and U-bodies, using a unique lipid composition and specific virus proteins (listed in Table 1 and Table 2). Only a fraction of the membranous structures are involved in active RNA replication within lumenal spaces. Throughout infection (+) RNA exits the replication factory, destined for translation or packaging. For virus assembly, (+) RNA interacts with membrane-associated capsid protein pentamers on membranes that are distinct from replication factory membranes [70]; (C) Possible mechanism by which closed DMVs transitions to U-body with gated lumen. These structures are visible at timepoints beginning 4 hpi and are believed to continue active replication; (D) Autophagosome-like vesicle formation engulfs immature virus particles. These vesicles have twice the diameter of replication vesicles [71], and we posit that these membranes are distinct from those in RNA replication factories, which are still present in the cell; (E) Acidification of the autophagosome-like vesicles produces mature virus particles [72].