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Topogenic analysis of the human immunodeficiency virus type 1 envelope glycoprotein, gp160, in microsomal membranes
O K Haffar1, D J Dowbenko, P W Berman
1Department of Molecular Biology, Genentech, Inc., South San Francisco, California 94080.
Abstract:
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.
Insights
Researchers determined the membrane orientation of the human immunodeficiency virus type 1 envelope glycoprotein (gp160). The external domain spans amino acids 1-665, with a transmembrane domain and a single cytoplasmic domain identified.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein precursor (gp160) is crucial for viral entry.
- Understanding gp160's membrane orientation is key to elucidating its function and developing therapeutics.
Purpose of the Study:
- To investigate the in vitro membrane orientation of the HIV-1 gp160.
- To identify specific domains within gp160, including transmembrane and cytoplasmic regions.
Main Methods:
- In vitro transcription and translation of env gene variants.
- Translocation into canine pancreatic microsomal membranes.
- Immunoprecipitation, protease protection, and membrane extraction studies.
Main Results:
- The external domain of gp160 extends from the mature amino terminus through amino acid 665.
- A stop-transfer sequence (transmembrane domain) was located between amino acids 665 and 732.
- A single cytoplasmic domain was identified C-terminal to residue 707; a hydrophobic domain (512-541) did not function as a stop-transfer sequence.
Conclusions:
- The study delineates the precise membrane topology of HIV-1 gp160.
- A secondary association of the gp160 cytoplasmic tail with microsomal membranes is proposed.