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

BACKGROUND: 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.

METHODS: 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).

RESULTS: 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.

CONCLUSION: 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.