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

Abstract

BackgroundRecently, 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.MethodsWe 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).ResultsIn 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.ConclusionOur results revealed that membrane vesicles induced by PRRSV were derived from the ER. The vesicles may provide a location for PRRSV replication and assembly.

Coronavirus infection induces progressive restructuring of the endoplasmic reticulum involving the formation and degradation of double membrane vesicles

“…The morphologically earliest stage of DMV we observed were the “budding” DMVs, which were characterized by a fully formed inner vesicle, an outer membrane continuous with the ER, and extensive pairing of the inner and outer membrane aside from the connected region. Budding DMVs have similar structure to those observed in the distantly-related Arterivirus porcine reproductive and respiratory syndrome virus ( Zhang et al, 2018 ). The formation mechanism for Arterivirus DMVs is still being explored, and tomographic evidence of potential intermediates exists for both the wrapping and double budding model ( van der Hoeven et al, 2016 ).…”

The SARS-Coronavirus Infection Cycle: A Survey of Viral Membrane Proteins, Their Functional Interactions and Pathogenesis

“…There can be up to 1000 vesiculations in a single SARS-infected cell [ 90 ], and separate VPs fuse together to form GVPs which may be interconnected with LVCVs and Golgi membranes containing both vesicles and significant numbers of budding complete virions [ 90 , 95 ]. In all CoVs, at every recorded stage, no DMVs exhibit openings to the cytosol [ 64 , 90 , 91 , 92 , 93 , 94 , 95 , 96 ]. DMVs close to the ER exhibit tightly apposed inner and outer membranes, but perinuclear residing DMVs have looser organization between the two membranes [ 90 ].…”

A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis

“…In general, the morphological and functional characterization of ROs of the second, vesiculotubular type is lagging behind. Such structures, which always include double-membrane vesicles (DMVs), commonly derive from membranes of the secretory pathway and have been found in cells infected with, e.g., picornaviruses [10][11], noroviruses [12], hepatitis C virus (HCV) [13], and different nidoviruses, including the arterivirus and coronavirus (CoV) families [14][15][16][17][18].…”