Multimerization Potential of the Cytoplasmic Domain of the Human Immunodeficiency Virus Type 1 Transmembrane Glycoprotein gp41

• Published in: The Journal of biological chemistry Vol. 275; no. 21; pp. 15809 - 15819
• Main Authors: Lee, Sheau-Fen, Wang, Chin-Tien, Liang, Judy Y.-P., Hong, Shi-Lan, Huang, Chin-Cheng, Chen, Steve S.-L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 26.05.2000; American Society for Biochemistry and Molecular Biology
• Subjects: AIDS/HIV; ATP-Binding Cassette Transporters; Carrier Proteins - genetics; Centrifugation, Density Gradient; Chromatography, Gel; Cross-Linking Reagents - chemistry; Escherichia coli; Escherichia coli Proteins; Glutaral - chemistry; glycoprotein gp41; HIV Envelope Protein gp41 - chemistry; HIV Envelope Protein gp41 - genetics; HIV-1 - chemistry; HIV-1 - genetics; Human immunodeficiency virus 1; Humans; Maltose-Binding Proteins; Monosaccharide Transport Proteins; Periplasmic Binding Proteins; Protein Conformation; Recombinant Fusion Proteins - chemistry; Viral Envelope Proteins - chemistry; Viral Envelope Proteins - genetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M000601200

We previously demonstrated that an envelope mutant of human immunodeficiency virus type 1 lacking the entire cytoplasmic domain interferes in trans with the production of infectious virus by inclusion of the mutant envelope into the wild-type envelope complex. We also showed that the envelope incorporation into virions is not affected when the wild-type envelope is coexpressed with the mutant envelope. These results suggest that an oligomeric structure of the cytoplasmic domain is functionally required for viral infectivity. To understand whether the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein gp41 has the potential to self-assemble as an oligomer, in the present study we fused the coding sequence of the entire cytoplasmic domain at 3′ to theEscherichia coli malE gene, which encodes a monomeric maltose-binding protein. The expressed fusion protein was examined by chemical cross-linking, sucrose gradient centrifugation, and gel filtration. The results showed that the cytoplasmic domain of gp41 assembles into a high-ordered structural complex. The intersubunit interaction of the cytoplasmic domain was also confirmed by a mammalian two-hybrid system that detects protein-protein interactions in eucaryotic cells. A cytoplasmic domain fragment expressed in eucaryotic cells was pulled down by glutathione-Sepharose 4B beads via its association with another cytoplasmic domain fragment fused to the C terminus of the glutathione S-transferase moiety. We also found that sequences encompassing the lentiviral lytic peptide-1 and lentiviral lytic peptide-2, which are located within residues 828–856 and 770–795, respectively, play a critical role in cytoplasmic domain self-assembly. Taken together, the results from the present study indicate that the cytoplasmic domain of gp41 by itself is sufficient to assemble into a multimeric structure. This finding supports the hypothesis that a multimeric form of the gp41 cytoplasmic domain plays a crucial role in virus infectivity.