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. 2018 Aug 7;9(4):e01253-18.
doi: 10.1128/mBio.01253-18.

Reovirus σNS and μNS Proteins Remodel the Endoplasmic Reticulum to Build Replication Neo-Organelles

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Free PMC article

Reovirus σNS and μNS Proteins Remodel the Endoplasmic Reticulum to Build Replication Neo-Organelles

Raquel Tenorio et al. mBio. .
Free PMC article

Abstract

Like most viruses that replicate in the cytoplasm, mammalian reoviruses assemble membranous neo-organelles called inclusions that serve as sites of viral genome replication and particle morphogenesis. Viral inclusion formation is essential for viral infection, but how these organelles form is not well understood. We investigated the biogenesis of reovirus inclusions. Correlative light and electron microscopy showed that endoplasmic reticulum (ER) membranes are in contact with nascent inclusions, which form by collections of membranous tubules and vesicles as revealed by electron tomography. ER markers and newly synthesized viral RNA are detected in inclusion internal membranes. Live-cell imaging showed that early in infection, the ER is transformed into thin cisternae that fragment into small tubules and vesicles. We discovered that ER tubulation and vesiculation are mediated by the reovirus σNS and μNS proteins, respectively. Our results enhance an understanding of how viruses remodel cellular compartments to build functional replication organelles.IMPORTANCE Viruses modify cellular structures to build replication organelles. These organelles serve as sites of viral genome replication and particle morphogenesis and are essential for viral infection. However, how these organelles are constructed is not well understood. We found that the replication organelles of mammalian reoviruses are formed by collections of membranous tubules and vesicles derived from extensive remodeling of the peripheral endoplasmic reticulum (ER). We also observed that ER tubulation and vesiculation are triggered by the reovirus σNS and μNS proteins, respectively. Our results enhance an understanding of how viruses remodel cellular compartments to build functional replication organelles and provide functions for two enigmatic reovirus replication proteins. Most importantly, this research uncovers a new mechanism by which viruses form factories for particle assembly.

Keywords: endoplasmic reticulum; membrane remodeling; reovirus; virus factory biogenesis.

Figures

FIG 1
FIG 1
ER remodeling in reovirus-infected cells as visualized by confocal microscopy, 3D TEM, and CLEM. HeLa cells were adsorbed with reovirus T1L M1-P208S. At 14 h postadsorption, cells were immunolabeled with a rabbit anti-calreticulin (CLT) polyclonal antiserum, a mouse anti-σNS monoclonal antibody, and the corresponding secondary antibodies conjugated with Alexa 488 (green) and Alexa 594 (red). Nuclei were stained with DAPI (blue). (A) A mock-infected cell with normal ER cisternae. (B) Reovirus-infected cell with altered ER. White arrows indicate thin, fragmented ER membranes around and inside VIs. The arrowhead indicates thin, undulated ER attached to a VI. (C) TEM of serial sections and 3D reconstruction. VIs (yellow) containing viral particles (light blue) are surrounded by a network of abnormally thin, undulated ER cisternae (brown) that contact the VI (arrowheads). Mitochondria are colored in red, and the nuclear envelope is in dark blue. (D to F) CLEM of reovirus inclusions. HeLa cells engineered to express mCherry-μNS-MT were adsorbed with reovirus, incubated for 14 h, and imaged using bright-field and fluorescence microscopy. Cell nuclei are labeled with asterisks. Selected fluorescent cells (dashed squares) were imaged using TEM (F). An early VI is surrounded by rough ER (RER) and mitochondria (mi). Membranes distribute inside (black arrows) and at the periphery (arrowheads) of the inclusion. Bars, 10 µm (A and B), 500 nm (C), and 200 nm (F).
FIG 2
FIG 2
Immunogold labeling of ER proteins in Tokuyasu cryosections of reovirus inclusions. HeLa cells were adsorbed with reovirus and incubated for 24 (A, B, and E) or 14 (C, D, and F to H) h, frozen in liquid nitrogen, and sectioned at −120°C. (A to D) Thawed cryosections were processed for immunogold labeling using a rabbit polyclonal anti-PDI antiserum and a secondary antibody bound to 10-nm colloidal gold particles (A and B) or a rabbit polyclonal anti-calreticulin (CLT) antiserum and a secondary antibody bound to 10-nm colloidal gold particles (C and D). (E) Cryosections were double labeled with a rabbit polyclonal anti-PDI antiserum and a secondary antibody bound to 5-nm colloidal gold particles (arrows) and a mouse monoclonal anti-dsRNA antibody and a secondary antibody bound to 15-nm colloidal gold particles (arrowheads). Anti-PDI antibody labels RER cisternae in the cytosol and membranes inside inclusions. Anti-dsRNA antibody labels membranes and viral particles inside VIs. (F) Cryosection labeled with a mouse monoclonal anti-BrU antibody and a secondary antibody bound to 10-nm colloidal gold particles. The anti-BrU antibody labels membrane fragments (white arrows) and viral particles (black arrows). (G and H) Cryosections were double labeled with a mouse monoclonal anti-BrU antibody and a secondary antibody bound to 15-nm colloidal gold particles (arrowheads) and an antiserum specific for the λ3 viral RNA-dependent RNA polymerase and a secondary antibody bound to 5-nm colloidal gold particles (arrows). Low- and high-magnification views of VIs show that both antibodies label viral particles and membrane fragments (inset in panel G). Bars, 200 nm (A, B, and E), 100 nm (C, D, F, G, and inset in G), and 50 nm (H).
FIG 2
FIG 2
Immunogold labeling of ER proteins in Tokuyasu cryosections of reovirus inclusions. HeLa cells were adsorbed with reovirus and incubated for 24 (A, B, and E) or 14 (C, D, and F to H) h, frozen in liquid nitrogen, and sectioned at −120°C. (A to D) Thawed cryosections were processed for immunogold labeling using a rabbit polyclonal anti-PDI antiserum and a secondary antibody bound to 10-nm colloidal gold particles (A and B) or a rabbit polyclonal anti-calreticulin (CLT) antiserum and a secondary antibody bound to 10-nm colloidal gold particles (C and D). (E) Cryosections were double labeled with a rabbit polyclonal anti-PDI antiserum and a secondary antibody bound to 5-nm colloidal gold particles (arrows) and a mouse monoclonal anti-dsRNA antibody and a secondary antibody bound to 15-nm colloidal gold particles (arrowheads). Anti-PDI antibody labels RER cisternae in the cytosol and membranes inside inclusions. Anti-dsRNA antibody labels membranes and viral particles inside VIs. (F) Cryosection labeled with a mouse monoclonal anti-BrU antibody and a secondary antibody bound to 10-nm colloidal gold particles. The anti-BrU antibody labels membrane fragments (white arrows) and viral particles (black arrows). (G and H) Cryosections were double labeled with a mouse monoclonal anti-BrU antibody and a secondary antibody bound to 15-nm colloidal gold particles (arrowheads) and an antiserum specific for the λ3 viral RNA-dependent RNA polymerase and a secondary antibody bound to 5-nm colloidal gold particles (arrows). Low- and high-magnification views of VIs show that both antibodies label viral particles and membrane fragments (inset in panel G). Bars, 200 nm (A, B, and E), 100 nm (C, D, F, G, and inset in G), and 50 nm (H).
FIG 3
FIG 3
Electron tomography of a reovirus inclusion. HeLa cells were adsorbed with reovirus, incubated for 14 h, frozen in liquid nitrogen, and sectioned at −120°C. Thawed cryosections were processed by electron tomography. A single-axis tilt series was obtained between −63° and +60° with an angular interval of 1.5°. Images were recorded using an Eagle 4k-by-4k slow-scan charge-coupled device (FEI, Eindhoven, The Netherlands) with FEI software and a Tecnai G2 microscope (FEI) operating at 200 kV. Images were aligned, and the tomogram was reconstructed using the IMOD software package. The tomogram was subjected to noise filtering and automated segmentation to visualize membranes. The 3D model was constructed using Amira. RER, yellow; viral particles, light blue; mitochondria, red; tubules and membrane fragments inside the VI, brown; vesicles inside the VI, orange. The VI is a collection of vesicles and tubules with viral particles attached to membranes (arrows).
FIG 4
FIG 4
Live-cell microscopy, confocal microscopy, and STED of ER remodeling during reovirus infection. (A) HeLa cells were transfected with mCherry-KDEL, adsorbed with reovirus, and incubated for 24 h. Images were collected every 15 min. A cell is shown at 6 h 45 min, 7 h, 7 h 15 min, and 7 h 30 min postadsorption. Normal ER elements are progressively fragmented, collapsed, and aggregated (arrows). (B) HeLa cells were transfected with mCherry-KDEL and, at 24 h posttransfection, adsorbed with reovirus, incubated for 24 h, and imaged using confocal microscopy. The ER in infected cells is progressively thinned (1), fragmented (2), and collapsed and aggregated (3). (C) STED microscopy of reovirus-infected cells. HeLa cells were adsorbed with reovirus and fixed at 26 h. Cells were immunolabeled with σNS-specific antibody and calreticulin (CLT)-specific antibody followed by secondary antibodies conjugated with Alexa 488 (green) and Alexa 546 (red). σNS (red) associates with remodeled ER (green). Some VIs are marked with white asterisks. σNS and ER marker CLT colocalize (arrows) over the network of remodeled ER with VIs. Bars, 10 µm (A), 7.5 µm (B), and 2.5 µm (C).
FIG 5
FIG 5
Effect of T3D σNS and μNS expression on ER morphology. (A) HeLa cells were transfected with mCherry-KDEL, σNS, and μNS and, at 24 h posttransfection, imaged using immunofluorescence and confocal microscopy. ER alterations are similar following cotransfection of σNS and μNS and reovirus infection: (1) linear thinning, (2) fragmentation, and (3) collapse and aggregation. (B and C) HeLa cells were transfected with mCherry-KDEL in combination with σNS (B) or μNS (C) and imaged using confocal microscopy at 24 h posttransfection. (B) Cells expressing σNS showed an altered ER with long, separated, branched thin tubules (arrows). σNS concentrates in the gaps between the tubules, producing a ring-like pattern (arrowheads). (C) Cells expressing μNS showed an altered ER with μNS associated with ER fragments (arrows). (D) HeLa cells engineered to express mCherry-μNS-MT were adsorbed with reovirus, incubated for 8 h, fixed, incubated with gold and silver, and imaged using fluorescence microscopy and TEM. mCherry-μNS-MT gold-silver distributes with vesicles (arrows) near the nucleus (N) where the Cherry red fluorescent signal concentrates (inset on the right; nucleus in blue). Insets on the left show vesicles with mCherry-μNS-MT gold-silver from different cells. The dashed line marks the periphery of the nucleus. (E) mCherry-μNS-MT gold-silver also localizes to strangled ER cisternae (arrows). Asterisks mark normal ER cisternae, and arrowheads indicate ribosomes adjacent to normal or thin, strangled ER cisternae. Bars, 5 µm (A), 3 µm (B), 2.5 µm (C), and 200 nm (D and E and insets in D).
FIG 6
FIG 6
Quantitative confocal microscopy data. (A) Summary showing the number and percentage of cells with various ER morphologies and alterations (normal ER, branched thin tubules, unbranched thin tubules, fragmented ER, and collapsed ER) under different experimental conditions: mock infected, virus infected, cotransfected with σNS and μNS, transfected with σNS alone, and transfected with μNS alone. Large ER-free zones (*) are areas of the cell with a surface of ≥15 µm2 that contain few or no ER elements, like those in Fig. 5A. (B) Comparative analysis of ER alterations under different experimental conditions. Cells at 14 h postinfection and 24 h posttransfection were included in the quantification. This quantitative analysis confirms that σNS causes a thinning of the tubular ER, whereas μNS disrupts the branches of the thin tubules and fragments those structures into small pieces. At later stages of infection, the ER collapses and aggregates, leaving large gaps in the cytosol.
FIG 7
FIG 7
Model of ER remodeling induced by reovirus infection and the specific action of σNS and μNS. Normal ER is composed of ER sheets and tubules. Reovirus targets ER tubules, leaving the sheets untouched. (A) Early in infection, σNS binds to ER cisternae and transforms these structures into thin tubules. (B) μNS binds to thin tubules, eliminates their branches, and severs them into small membranous pieces that aggregate, attach to the remodeled ER, and form VIs. Inside inclusions, replicating viral cores and newly synthesized vRNPs bind to membranes that most likely serve as assembly sites for new viral particles. Schematics at the bottom show how σNS and μNS might remodel ER tubules.

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