Subcellular localization and topology of porcine reproductive and respiratory syndrome virus E protein

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) open reading frame (ORF) 2a contains a small internal ORF (2b) capable of encoding a protein of 70 or 73 amino acids (aa), termed E protein. The function and biochemical information of the E protein are currently not clear. In the present investigation, it was shown that the E protein was mainly located in the endoplasmic reticulum (ER) and Golgi complex in MARC-145 cells. Deletion studies identified the N-terminal 15 residues as an ER localization sequence of the E protein, besides two other localization sequences within positions 23-50 and 50-73, and the N-myristoylation site significantly affected the subcellular localization of the N-terminal 15 residues. The membrane association assay demonstrated that the E protein was an integral membrane protein embedded in the phospholipid bilayer. However, neither the N-myristoylation site nor the hydrophilic C-terminal domain was essential to the membrane association of the E protein. The topology analysis revealed that this protein had N-terminus oriented toward the cytoplasm and C-terminus toward the ER lumen. Finally, immunofluorescence assay indicated that the E protein colocalized with GP2, GP3, GP4 and M protein in cotransfected cells, but not N protein.