Topogenic analysis of the human immunodeficiency virus type 1 envelope glycoprotein, gp160, in microsomal membranes.
The orientation in cellular membranes of the 856 amino acid envelope glycoprotein precursor, gp160, of human immunodeficiency virus type 1 was investigated in vitro. Variants of the env gene were transcribed using the bacteriophage SP6 promoter, translated using a rabbit reticulocyte lysate, and translocated into canine pancreatic microsomal membranes. Immunoprecipitation studies of gp160 variants using antibodies specific for various gp160-derived polypeptides provided evidence that the external (cell surface) domain of gp160 begins at the mature amino terminus of the protein and continues through amino acid 665. A stop-transfer sequence (transmembrane domain) was identified in a hydrophobic region COOH-terminal to amino acid 665 and NH2-terminal to amino acid 732. Protease protection experiments demonstrated that gp160 possesses a single cytoplasmic domain COOH-terminal to residue 707. Membrane extraction studies using carbonate buffer provided evidence that the 29 amino acid hydrophobic domain (residues 512-541) of gp160 was unable to serve as a stop-transfer sequence. Finally, we propose that the cytoplasmic tail of gp160 forms a secondary association with the microsomal membranes.
D000818:Animals; D000918:Antibody Specificity; D001692:Biological Transport; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000494:pharmacology; D006678:HIV / Q000235:genetics / Q000502:physiology*; D015483:HIV Antibodies / Q000276:immunology; D019302:HIV Envelope Protein gp160; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000032:analysis*; D008861:Microsomes / Q000032:analysis*; D008954:Models, Biological; D010957:Plasmids; D011233:Precipitin Tests; D011487:Protein Conformation; D011498:Protein Precursors / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D012191:Retroviridae Proteins / Q000032:analysis* / Q000235:genetics; D013462:Sulfur Radioisotopes; D009626:Terminology as Topic; D014158:Transcription, Genetic; D014759:Viral Envelope Proteins / Q000032:analysis* / Q000235:genetics
D015483:HIV Antibodies; D019302:HIV Envelope Protein gp160; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D012191:Retroviridae Proteins; D013462:Sulfur Radioisotopes; D014759:Viral Envelope Proteins; D010450:Endopeptidases