Identification and Characterization of a Sixth Structural Protein of Lelystad Virus: The Glycoprotein GP2Encoded by ORF2 Is Incorporated in Virus Particles

Abstract

Previously we have shown that Lelystad virus (LV) contains five structural proteins, a nucleocapsid protein N, an integral membrane protein M, and three glycoproteins GP3, GP4, and GP5. In this study we identified a sixth structural protein of Lelystad virus. The protein has an apparent molecular weight of 29 to 30 kDa, was recognized by an ORF2-specific antipeptide serum in Western immunoblotting using sucrose gradient purified LV virions, and was shown to beN-glycosylated. It was therefore designated GP2. The GP2protein was also immunoprecipitated by ORF2-specific antipeptide serum from lysates and extracellular virus of CL2621 cells infected with LV. A fraction of the GP2protein present in these lysates contained an intrachain disulfide bond. Endoglycosidase H treatment of immunoprecipitates indicated that the endoglycosidase H-sensitiveN-glycans of the GP2protein become endoglycosidase H-resistant during passage through the Golgi compartment in cells infected with LV. In contrast, theN-glycans of the GP2protein expressed individually in a recombinant Semliki Forest virus remained endoglycosidase H-sensitive, indicating that the GP2protein was retained in the endoplasmic reticulum. LV is the first arterivirus which has been demonstrated to contain six virion-associated proteins, one nucleocapsid protein, one integral membrane protein, and four glycoproteins.