8 research outputs found

    An integrated analysis of membrane remodeling during porcine reproductive and respiratory syndrome virus replication and assembly

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    <div><p>Background</p><p>Recently, three-dimensional (3D) imaging techniques have been used to detect viral invasion and the appearance of specialized structures established in virus-infected cells. These methods have had a positive impact in the field of virology and helped to further our knowledge of how viruses invade cells. Nearly all positive-strand RNA viruses propagate their viral genomes in part through intracellular membranes. Porcine reproductive and respiratory syndrome virus (PRRSV), an Arterivirus, accumulates viral RNA that forms replication complexes (RCs) in infected cells. In this study, using immunofluorescence and electron microscopy (EM), we dissected PRRSV-induced membrane structures in infected cells and determined the correlations between PRRSV particles and vesicles stimulated by PRRSV to understand the structural and dynamic aspects of PRRSV infection.</p><p>Methods</p><p>We identified the appropriate time point by determining the 50% tissue culture infectious dose (TCID50) and using qRT-PCR and Western blotting. The co-localization of viruses and organelles was determined by immunofluorescence and immune-electron microscopy (IEM). The ultrastructure of cells infected by PRRSV was observed using EM and electron tomography (ET).</p><p>Results</p><p>In our study, we found that PRRSV dsRNA was located at the endoplasmic reticulum (ER) and autophagosomes; in addition, the N protein was located at the mitochondria, ER and autophagosomes. Vesicles induced by PRRSV appeared at 16 hours post-infection (h.p.i.) and increased in size with time during the infection period. In addition, our findings demonstrated that the virus vesicles originated from the ER, and these two organelle structures connected with each other to form a reticulovesicular network (RVN) that provided a site for virus replication and assembly.</p><p>Conclusion</p><p>Our results revealed that membrane vesicles induced by PRRSV were derived from the ER. The vesicles may provide a location for PRRSV replication and assembly.</p></div

    ET Reveals the RVN of interconnected ER-derived DMVs.

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    <p>Marc-145 cells were infected with PRRSV at an MOI of 1, fixed at 24 h.p.i, and processed for ET as mentioned in the Materials and Methods. (A) Left: slice of a dual-axis tomogram presenting the various membrane changes; right, 3D structure of the integrated tomogram. ER membranes and vesicles interconnected with ER lumens are described in light green; PRRSV-induced vesicles are depicted in light yellow. Scale bars, 100 nm (B) Left: slice of a dual-axis tomogram showing that a DMV is linked to the ER lumen; middle and right: this tomogram and the 3D membrane rendering of the DMVs structure are shown. These tomograms are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200919#pone.0200919.s002" target="_blank">S1 Movie</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200919#pone.0200919.s003" target="_blank">S2 Movie</a>. Scale bars, 50nm.</p

    IEM of PRRSV-infected cells.

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    <p>PRRSV at an MOI of 1 was transfected into Marc-145 cells, and at 24hours post-infection, cells were immobilized, dehydrated, and inserted as described in the Materials and Methods(A’, B’, C’ and D’). Mock infected Marc-145 cells served as controls (A, B, C and D), Ultrathin sections were marked with antibodies against J2 (A, A’, B and B’) and N protein (C, C’ D and D’). All of the gold particles labeled the cytoplasm of infected cells. Anti-dsRNA labeling was mostly associated with ER and MV (A’), and few gold particles were observed in the mitochondria (M) (B’). Anti-N antibodies displayed labeling on the ER and mitochondria in infected cells (C’ and D’).</p

    Progression of alterations in PRRSV-infected cells.

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    <p>Electron micrographs showing fixed PRRSV-infected cells versus mock (A) at 4 h (B), 8 h (C), 12 h (D), 16 h (E), 24 h (F), and 36 h (G). A minimum of 100 cells was detected for morphological alterations that appear during PRRSV invasion. An image of each typical area is represented. Scale bars, 500 nm.</p

    ET manifests the relationship between MVs and virus particles derived from ER membranes and captures virus budding.

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    <p>Marc-145 cells were infected with PRRSV at an MOI of 1, fixed at 24 hpi, and processed for ET as described in the Materials and Methods. (A) Close inspection of an ET ultra-thin slice displays an association between membrane vesicles and ER lumen-containing virus particles, with viral particles assembling at the limiting membrane of the vesicle. (B) The 3D structure model of the virus-induced structures in (A) depicts the relationship between membrane vesicles and the membranes enclosing the virus particles. The membrane vesicle is depicted in yellow and virus particles in red. (C) Final 3D surface-rendered model showing the connection between the viral particle and the ER derived membrane vesicle. (D) A side view of the 3D model represents the connection between virus budding and the membrane vesicle. This tomogram is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200919#pone.0200919.s004" target="_blank">S3 Movie</a>. (E) The particle was connected with the membrane which wrapped the particles, the arrow is the connection.</p

    Timeline of PRRSV replication and assembly.

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    <p>PRRSV was transfected into Marc-145 cells at an MOI of 1and samples were obtained at different time points as mentioned. (A) Total RNA was extracted and quantified by quantitative RT-PCR through a PRRSV-specific TaqMan probe. (B) The cell-related virus titers in infected cell lysates collected at each time point were employed to analyze the extracellular virus titers. (C) Cell extractives were verified by Western blotting using anti-N antibodies; anti-GAPDH antibodies served as an internal reference.</p

    Membranous ultrastructure caused by PRRSV infection in host cells.

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    <p>PRRSV at an MOI of 1 was transfected into Marc-145 cell, fixed at 24 hpi, and used for conventional EM as described in the Materials and Methods. (A) A range of vesicles were detected. DMVs (✱) were labeled with asterisks; autophagosomes are annotated with white arrows. (B) DMVs are often found in clusters by a small network of membranes. Scale bars, 500 nm.</p

    Localization of the viral N protein and dsRNA in Marc-145 cells infected with PRRSV.

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    <p>At the 24 h time point, Marc-145 cells were fixed and performed for immunofluorescence as mentioned in the Materials and Methods after PRRSV infection at an MOI of 1. Images of samples were visualized using a Nikon TE2000-E inverted confocal microscope at 63× magnification. (A)—(H) The cellular nuclei were dyed with DAPI (blue). Cells were co-stained with the J2 monoclonal anti-dsRNA antibody and (A) the anti-calnexin monoclonal antibody, (B) the anti-LC3 monoclonal antibody, (C) LysoTracker Red or (D) MitoTracker Red. Cells were costained with the monoclonal anti-NS1 antibody and (E) the anti-calnexin monoclonal antibody, (F) the anti-LC3 monoclonal antibody, (G) LysoTracker Red or (H) MitoTracker Red. Scale bars, 10 μm. (I) The graph showing Pearson’s correlation coefficients for each markers in (A)~(H), values above 0.5 were considered as colocalization.</p
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