title: CD4 molecules with a diversity of mutations encompassing the CDR3 region efficiently support human immunodeficiency virus type 1 envelope glycoprotein-mediated cell fusion.
abstract: The third complementarity-determining region (CDR3) within domain 1 of the human CD4 molecule has been suggested to play a critical role in membrane fusion mediated by the interaction of CD4 with the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein. To analyze in detail the role of CDR3 and adjacent regions in the fusion process, we used cassette mutagenesis to construct a panel of 30 site-directed mutations between residues 79 and 96 of the full-length CD4 molecule. The mutant proteins were transiently expressed by using recombinant vaccinia virus vectors and were analyzed for cell surface expression, recombinant gp120-binding activity, and overall structural integrity as assessed by reactivity with a battery of anti-CD4 monoclonal antibodies. Cells expressing the CD4 mutants were assayed for their ability to form syncytia when mixed with cells expressing the HIV-1 envelope glycoprotein. Surprisingly in view of published data from others, most of the mutations had little effect on syncytium-forming activity. Normal fusion was observed in 21 mutants, including substitution of human residues 85 to 95 with the corresponding sequences from either chimpanzee, rhesus, or mouse CD4; a panel of Ser-Arg double insertions after each residue from 86 to 91; and a number of other charge, hydrophobic, and proline substitutions and insertions within this region. The nine mutants that showed impaired fusion all displayed defective gp120 binding and disruption of overall structural integrity. In further contrast with results of other workers, we observed that transformant human cell lines expressing native chimpanzee or rhesus CD4 efficiently formed syncytia when mixed with cells expressing the HIV-1 envelope glycoprotein. These data refute the conclusion that certain mutations in the CDR3 region of CD4 abolish cell fusion activity, and they suggest that a wide variety of sequences can be functionally tolerated in this region, including those from highly divergent mammalian species. Syncytium formation mediated by several of the CDR3 mutants was partially or completely resistant to inhibition by the CDR3-directed monoclonal antibody L71, suggesting that the corresponding epitope is not directly involved in the fusion process.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
authors.forename: C C
authors.initials: CC
authors.lastname: Broder
authors.forename: E A
authors.initials: EA
authors.lastname: Berger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015704:CD4 Antigens / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D002459:Cell Fusion / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D004252:DNA Mutational Analysis; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology; D011323:Primates / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014170:Transformation, Genetic; D014616:Vaccinia virus / Q000235:genetics; D014760:Viral Fusion Proteins / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014760:Viral Fusion Proteins