Identification and characterization of a sixth structural protein of Lelystad virus: The glycoprotein GP(2) encoded by ORF2 is incorporated in virus particles

Previously we have shown that Lelystad virus (LV) contains five structural proteins, a nucleocapsid protein N, an integral membrane protein M, and three glycoproteins GP(3), GP(4), and GP(5). 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 be N-glycosylated. It was therefore designated GP(2). The GP(2) protein was also immunoprecipitated by ORF2-specific antipeptide serum from lysates and extracellular virus of CL2621 cells infected with LV. A fraction of the GP(2) protein present in these lysates contained an intrachain disulfide bond. Endoglycosidase H treatment of immunoprecipitates indicated that the endoglycosidase H-sensitive N-glycans of the GP(2) protein become endoglycosidase H-resistant during passage through the Golgi compartment in cells infected with LV. In contrast, the N-glycans of the GP(2) protein expressed individually in a recombinant Semliki Forest virus remained endoglycosidase H-sensitive, indicating that the GP(2) protein 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. (C) 1996 academic Press, Inc.