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. 2000 Oct;74(19):8953-65.
doi: 10.1128/jvi.74.19.8953-8965.2000.

Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles

D A Suhy  1 , T H Giddings Jr, K Kirkegaard
Affiliations

Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles

D A Suhy et al. J Virol. 2000 Oct.

Abstract

All positive-strand RNA viruses of eukaryotes studied assemble RNA replication complexes on the surfaces of cytoplasmic membranes. Infection of mammalian cells with poliovirus and other picornaviruses results in the accumulation of dramatically rearranged and vesiculated membranes. Poliovirus-induced membranes did not cofractionate with endoplasmic reticulum (ER), lysosomes, mitochondria, or the majority of Golgi-derived or endosomal membranes in buoyant density gradients, although changes in ionic strength affected ER and virus-induced vesicles, but not other cellular organelles, similarly. When expressed in isolation, two viral proteins of the poliovirus RNA replication complex, 3A and 2C, cofractionated with ER membranes. However, in cells that expressed 2BC, a proteolytic precursor of the 2B and 2C proteins, membranes identical in buoyant density to those observed during poliovirus infection were formed. When coexpressed with 2BC, viral protein 3A was quantitatively incorporated into these fractions, and the membranes formed were ultrastructurally similar to those in poliovirus-infected cells. These data argue that poliovirus-induced vesicles derive from the ER by the action of viral proteins 2BC and 3A by a mechanism that excludes resident host proteins. The double-membraned morphology, cytosolic content, and apparent ER origin of poliovirus-induced membranes are all consistent with an autophagic origin for these membranes.

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Figures

FIG. 1
FIG. 1
Electron micrographs of COS-1 cells preserved by high-pressure freezing show the ultrastructure of uninfected cells and cells infected with poliovirus for 4 h at 37°C. (A) Uninfected cell. N, nucleus; G, Golgi. Bar = 1 μm. (B) Infected cell; bar = 1 μm. (C) Infected cell; bar = 0.2 μm. (D) Immunostaining of infected cell to identify poliovirus 2C epitopes using 15-nm gold particles conjugated to secondary antibodies. Arrows indicate double-membraned vesicles. Bar = 0.3 μm.
FIG. 2
FIG. 2
Density gradient analyses of the distributions of subcellular membranes of uninfected and poliovirus-infected cells. Following infection with poliovirus for 4 h, the plasma membranes of COS-1 cells were disrupted and the cytoplasmic organelles were separated on Percoll density gradients. Individual fractions (lightest fractions are at the left) were collected and either tested for enzymatic activity or analyzed by immunoblot assays to identify poliovirus 2C protein (A, B, and C), ER marker p63 (A), Golgi marker p115 (B), and mtHSP70 [HSP70m(mito)] (C). β-Hexosaminidase activity assays were used to identify fractions derived from lysosomes [β-Hex (Lyso)] (C). Stock isotonic Percoll concentrations were 20% (A) and 9.5% (B and C).
FIG. 3
FIG. 3
Density gradient and ultrastructural analysis of COS-1 cells that expressed poliovirus 3A protein. (A) Cytoplasmic membranes from cells expressing 3A protein were separated on a 12% stock isotonic Percoll; proteins in each fraction were detected by immunoblotting using antibodies directed against the poliovirus 3A protein, calnexin (ER), and mtHSP70 [HSP70m(mito); mitochondria]; lightest fractions are at the left. The distribution profiles of Golgi and lysosomal proteins did not vary significantly from the patterns displayed in Fig. 2 and were not included for simplicity. (B) Ultrastructure of COS-1 cell expressing poliovirus 3A protein. N, nucleus. Bar = 1 μm.
FIG. 4
FIG. 4
Density gradient and ultrastructural analysis of COS-1 cells that expressed poliovirus proteins 2C and 3A. (A) Cytoplasmic membranes from cells expressing 2C and 3A proteins were separated on a 12% stock isotonic Percoll gradient; proteins in each fraction were detected by immunoblotting using antibodies directed against poliovirus 2C, poliovirus 3A, calnexin (ER), and mtHSP70 [HSP70m(mito) mitochondria]; lightest fractions are at the left. (B) Electron microscopy reveals the effects of 2C and 3A expression on the ultrastructure of COS-1 cells. N, nucleus. Bar = 1 μm. (C) Section labeled with 15-nm gold particles coupled to secondary antibodies. The primary antibody was directed against poliovirus protein 2C. Bar = 0.5 μm.
FIG. 5
FIG. 5
Density gradient and ultrastructural analysis of COS-1 cells that expressed poliovirus 2BC protein. (A) Cytoplasmic membranes from cells expressing 2BC protein were separated on a 12% stock isotonic Percoll gradient; proteins in each fraction were detected by immunoblotting using antibodies directed against poliovirus 2C, calnexin (ER), and mtHSP70 [HSP70m(mito) mitochondria]; lightest fractions are at the left. (B) Ultrastructure of COS-1 cells transfected with a 2BC-expressing plasmid. G, Golgi. Bar = 2 μm. (C) An immunostained section using anti-2C antibodies and secondary antibodies coupled to 15-nm gold particles. Bar = 0.5 μm.
FIG. 6
FIG. 6
Density gradient and ultrastructural analysis of COS-1 cells cotransfected with plasmids that encode the poliovirus 2BC and 3A proteins. (A) Cytoplasmic membranes from cells expressing 2BC and 3A proteins were separated on a 12% stock isotonic Percoll gradient; proteins in each fraction were detected by immunoblotting using antibodies directed against poliovirus 2C, poliovirus 3A, calnexin (ER), and mtHSP70 [HSP70m(mito) mitochondria]; lightest fractions are at the left. (B) Ultrastructure of COS-1 cells transfected with 2BC- and 3A-expressing plasmids. N, nucleus. Bar = 1 μm. (C) Higher-resolution image of a section of a 2BC- and 3A-transfected cell. Bar = 0.2 μm. (D) A 2BC- and 3A-transfected cell that was immunostained with antibodies directed against the 2C protein. Secondary antibodies were coupled to 15-nm gold particles. Arrows indicate double-membraned vesicles. Bar = 0.5 μm.
FIG. 7
FIG. 7
Effect of increased ionic strength on cellular membranes and poliovirus-induced vesicles. Following infection with wild-type poliovirus for 4 h, the plasma membranes of COS-1 cells were disrupted in homogenization buffer and the cellular organelles were separated on a 12% stock isotonic Percoll density gradient that did not (A and C) or did (B and D) contain 60 mM KCl; lightest fractions are at the left. (A and B) Distributions of ER membranes and poliovirus-induced vesicles identified by immunoblotting as indicated. (C and D) Distributions of Golgi, mitochondrial, and lysosomal (Lyso) membranes identified by immunoblotting against p115, immunoblotting against mtHSP70 [HSP70m(mito), and β-hexosaminidase (β-Hex) assays as indicated.
FIG. 8
FIG. 8
Effect of increased ionic strength on buoyant density distribution of endosomes and poliovirus-induced vesicles. Following infection with poliovirus for 4 h, the plasma membranes of COS-1 cells were disrupted in homogenization buffer in the presence and absence of 60 mM KCl, and the cellular organelles were separated on 12% stock isotonic Percoll density gradient that did or did not contain 60 mM KCl; lightest fractions are at the left. (A) Distribution of Rab9-containing membranes in the presence and absence of 60 mM KCl; (B) distribution of poliovirus 2C-containing membranes in the presence and absence of 60 mM KCl.
FIG. 9
FIG. 9
Model for the formation of poliovirus-induced vesicles. Proteins of the poliovirus replication complex, especially 2BC and 3A (open squares and circles), accumulate in patches on the ER. Double-membraned vesicles derive from these ER membranes by a mechanism that excludes cellular membrane proteins (closed squares and circles) but includes cytoplasmic material (black) in the lumen.
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