Intracellular synthesis, processing, and transport of proteins encoded by ORFs 5 to 7 of porcine reproductive and respiratory syndrome virus

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 5. 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 compartmenl. During the first 30 min of chase, 5 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 5 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 5 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. (C) 1996 Academic Press, Inc.