title: Intracellular synthesis, processing, and transport of proteins encoded by ORFs 5 to 7 of porcine reproductive and respiratory syndrome virus.
abstract: Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a small enveloped virus containing a positive-strand RNA genome, possesses at least three major structural proteins designated N, M, and E. The N protein is considered as the major component of the nucleocapsid, whereas M and E are membrane-associated. Previous studies using peptide-specific antibodies assigned these proteins to ORFs 7, 6, and 5, respectively. In the present report, monospecific antisera raised against Escherichia coli-expressed ORFs 5, 6, and 7 products were used to study the synthesis and processing of PRRSV structural proteins in the highly permissive MARC-145 cell line. Treatment of viral proteins with various glycosidases showed that only E was modified by N-linked glycans. Pulse-chase experiments revealed that intracellular transport of the major envelope glycoprotein was delayed in the premedial Golgi compartment. During the first 30 min of chase, E undergoes a gradual downward shift of its apparent molecular weight, thought to result from trimming of the mannose-rich glycan structures. Once E is transported to the medial Golgi or proximal elements, some molecules undergo complete processing of all their high-mannose N-linked oligosaccharides to complex type, while in other molecules only a fraction of N-linked glycans are terminally glycosylated. These two differentially glycosylated forms of E were found to be incorporated into extracellular virions. In cells and virions, both M and E were shown to occur in heterodimeric complexes linked by disulfide bonds. The oligomerization process, as analyzed from pulse-chase experiments, showed that M and E are incorporated into M-E complexes with different kinetics and efficiencies, in a fashion similar to their counterparts in equine arteritis virus. Apparently, all steps of E protein N-glycans processing proceed after its association with M which occurs in the endoplasmic reticulum (ER). In the infected cells, E and M appear highly membrane-associated, while N is predominantly cytosolic.
authors.affiliation: Centre de recherche en virologie, Institut Armand-Frappier, Université du Québec, Laval, Canada.
authors.forename: H
authors.initials: H
authors.lastname: Mardassi
authors.forename: B
authors.initials: B
authors.lastname: Massie
authors.forename: S
authors.initials: S
authors.lastname: Dea
mesh_terms: D000818:Animals; D018018:Arterivirus / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D001483:Base Sequence; D001692:Biological Transport; D002460:Cell Line; D004279:DNA, Viral; D006031:Glycosylation; D007700:Kinetics; D008358:Mannose / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D016366:Open Reading Frames; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D014759:Viral Envelope Proteins; D014764:Viral Proteins / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000378:metabolism; D019065:Virus Assembly
chemical_list: D004279:DNA, Viral; D009844:Oligosaccharides; D014759:Viral Envelope Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C114789:glycoprotein 5, PRRSV; D008358:Mannose