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.

Immune response in pigs vaccinated with plasmid DNA encoding ORF5 of porcine reproductive and respiratory syndrome virus.

“…For indirect immunofluorescence (IIF), cells were incubated at 37 mC and fixed with 80 % cold acetone for 20 min at 4 mC at various times (18-72 h) post-transfection. The monolayers were then reacted for 30 min with anti-ORF5 rabbit monospecific hyperimmune serum (Mardassi et al, 1996) and the immune reaction was determined following incubation with fluorescein-conjugated goat anti-rabbit Ig (Boehringer Mannheim), as previously described (Loemba et al, 1996). † ELISA : reciprocal of highest serum dilution reacting with the recombinant ORF5-pH expressed in E. coli.…”

“…Six putative structural proteins have been identified and assigned to distinct smaller ORFs, ORFs 2-7, located at the 3h end of the genome (Mardassi et al, 1995(Mardassi et al, , 1996Meulenberg et al, 1993). The virion contains three major structural proteins, a 25 kDa envelope glycoprotein (GP & ), an 18-19 kDa unglycosylated membrane protein (M), and a 15 kDa nucleocapsid (N) protein, encoded by ORFs 5, 6 and 7, respectively.…”

Alternative codon usage of PRRS virus ORF5 gene increases eucaryotic expression of GP5 glycoprotein and improves immune response in challenged pigs

“…Rabbit monospecific hyperimmune serum (α5) to E. coli-expressed ORF5 product of the IAF-Klop PRRSV reference strain was obtained from previous studies [5].…”

“…Sucrose gradient purified-PRRSV was prepared in LB-2 lysis buffer [5], denatured by boiling in the presence of 5% (V/V) β-mercaptoethanol, subjected to 12% SDS-PAGE and electrotransferred onto nitrocellulose membranes as previously described [5,20]. Reactivity of pig sera (dilution 1/50) was tested and the α5 serum was used as a positive control.…”

“…The viral genome consists of a positive single-stranded RNA molecule of approximately 15 kb in length, composed of nine open reading frames [2][3][4]. The ORFs 5 to 7 encode the three major structural proteins of virions which are the envelope glycoprotein GP5 (25)(26), the non-glycosylated membrane protein M (18-19 kDa) and the nucleocapsid protein N (14-15 kDa) [5]. These PRRSV structural proteins are closely associated, GP5 and M proteins being associated in the form of heterodimers [5].…”