ORF-3 OF LACTATE DEHYDROGENASE-ELEVATING VIRUS ENCODES A SOLUBLE, NONSTRUCTURAL, HIGHLY GLUCOSYLATED, AND ANTIGENIC PROTEIN

Open reading frame (ORF) 3 of the genome of lactate dehydrogenase-elev ating virus (LDV), strain P, was cloned into the plasmid pcDNA/Amp and in vitro transcribed and translated. Translation of ORF 3 yielded a s oluble protein of the expected size (about 21 kDa). When synthesized i n the presence of endoplasmic reticulum (ER) membranes the resulting g lycoprotein of about 36 kDa became associated with the membranes. Howe ver, disruption of the ER Vesicles by incubation in carbonate buffer, pH 11.5, resulted in the release of the protein from the membranes. Hy drophobic moment analysis of the ORF 3 protein indicated the absence o r any potential transmembrane segments, except for a N-terminal signal peptide, but no cleavage or the signal peptide was observed during me mbrane-associated in vitro synthesis. The ORF 3 protein elicited a str ong antibody response in infected mice. The antibodies from infected m ice as well as a monoclonal antibody specifically precipitated the in vitro-synthesized ORF 3 protein, but no protein from LDV virions. The overall results suggest that the ORF 3 protein is a nonstructural, hig hly glycosylated, and antigenic glycoprotein that is probably soluble and secreted or at most only weakly associated with membranes via the signal peptide. (C) 1997 Academic Press