MEMBRANE ASSOCIATION OF THE C-TERMINAL HALF OF THE OPEN READING FRAME1A PROTEIN OF LACTATE DEHYDROGENASE-ELEVATING VIRUS

ORF 1a of lactate dehydrogenase-elevating virus, strain P (LDV-P), enc odes a protein of 2206 amino acids. Eisenberg hydrophobic moment analy sis of the protein predicted the presence of eleven transmembrane segm ents in the C-terminal half of the molecule (amino acids 980-1852) tha t flank the serine protease domain. cDNAs encoding ORF 1a protein segm ents encompassing transmembrane segments 5 to 11 and its amphipathic C -terminal end as well as the N-terminal 80 amino acids of the downstre am ORF Ib protein were transcribed and the transcripts in vitro transl ated in the absence and presence of microsomal membranes. The synthesi s of the protein products with putative transmembrane segments was enh anced by the presence of the microsomal membranes and the proteins bec ame membrane associated. When synthesized in the absence of membranes they were recovered in the supernatant upon ultracentrifugation of the translation reaction mixtures, whereas they were recovered in the mem brane pellet when synthesized in the presence of membranes. Furthermor e, the latter proteins were not released from the membranes by disrupt ion of the membrane vesicles in carbonate buffer, pH 11.5, and large p ortions of the proteins were resistant to digestion by trypsin, chymot rypsin and proteinase K. No N-glycosylation was observed and only litt le, if any, processing of the protein by the putative serine protease. The results indicate that the C-terminal half of the ORF la protein r epresents a non-glycosylated integral membrane protein. Potential mode s of synthesis and function of the protein are discussed. In addition, the results showed that the synthesis of the ORF 1a protein was gener ally terminated at its termination codon, but that read-through into t he ORF 1b gene occurred with low frequency.