THE ENVELOPE PROTEINS OF LACTATE DEHYDROGENASE-ELEVATING VIRUS AND THEIR MEMBRANE TOPOGRAPHY

We have studied the membrane topography and N-glycosylation of the env elope proteins of lactate dehydrogenase-elevating virus (LDV, strain P ), Transcripts of open reading frames (ORFs) 2, 5, and 6 were in vitro translated in the absence and presence of microsomal membranes, and t he products analyzed for molecular weight, sensitivity to endoglycosid ase F/N-glycosidase F and proteinases, and reaction with anti-LDV anti bodies. The ORF 6 mRNA translation was enhanced in the presence of mic rosomal membranes. ORF 6 encodes a polytopic class III membrane protei n identified as the nonglycosylated virion envelope protein (M/VP-2; s imilar to 18 kDa). The protein has a very short (about 11 amino acids) ectodomain, a longer (about 79 amino acids) C-terminal endodomain, an d crosses the membrane three times between these domains. ORF 5 encode s the primary virion envelope glycoprotein (VP-3P) (25-42 kDa). Our re sults suggest that it is a polytopic class I glycoprotein. After remov al of a signal peptide, the processed protein of about 171 amino acids consists of a short (approximately 30 amino acids) N-terminal ectodom ain with three asparagine residues that appear to be N-glycosylated, a segment that crosses the membrane three times, and an about 74 amino acid long C-terminal endodomain. Neutralizing anti-LDV antibodies are probably directed to an epitope(s) in the N-terminal ectodomain. The O RF 2 protein is a standard class I glycoprotein with a single C-termin al membrane anchor segment and its signal peptide is removed during me mbrane-associated synthesis. The remaining ectodomain (about 165 amino acids) contains three asparagine residues which appear to be N-glycos ylated. Our results suggest that the ORF 2 protein may be present in a low concentration in LDV virions (VP-3M). (C) 1995 Academic Press, In c.