Functional contribution of cysteine residues to the human immunodeficiency virus type 1 envelope.
Although the envelope gene of human immunodeficiency virus type 1 shows considerable strain variability, cysteine residues of the envelope protein are strongly conserved, suggesting that they are important to the envelope structure. We constructed and analyzed mutants of a biologically active molecular clone of human immunodeficiency virus type 1 in which different cysteines were replaced by other amino acids in order to determine their functional importance. Substitution of cysteines 296 and 331, on either side of a region recognized by type-specific neutralizing antibodies, or on either side (residues 418 and 445) of a region important for CD4 binding, resulted in noninfectious mutants. These mutants were blocked early in the viral life cycle. Their gp160 envelope precursor polypeptides were poorly cleaved, and CD4 binding was also strongly impaired. Similar substitutions in the first variable region (residue 131) or between the first and second variable regions (residue 196) also gave noninfectious mutant virus, but here the block was late in the virus life cycle; these mutants were defective for syncytium formation. Substitution of cys386, between the neutralization and CD4 binding regions, resulted in a virus which retained infectivity but which spread much more slowly than the wild type. As with the cys131 and cys196 mutants, the cys386 mutant appeared to be defective in syncytium formation. These results show that all seven of the tested cysteines are vital for envelope function and suggest that this is likely true for all envelope cysteines. The results further show that regions important for CD4 binding, proteolytic cleavage recognition, and syncytium formation are all multiple and distributed over a relatively large part of the gp120 and therefore are likely dependent on protein tertiary structure.
Tschachler
E
E
Laboratory of Tumor Cell Biology, National Cancer Institute, Bethesda, Maryland 20892.
Buchow
H
H
Gallo
R C
RC
Reitz
M S
MS
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000032:analysis / Q000235:genetics; D000954:Antigens, Surface / Q000032:analysis; D001483:Base Sequence; D015704:CD4 Antigens / Q000032:analysis / Q000235:genetics; D002460:Cell Line; D003545:Cysteine; D005455:Fluorescent Antibody Technique; D005814:Genes, Viral; D015726:Giant Cells / Q000166:cytology / Q000276:immunology; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D014162:Transfection; D014759:Viral Envelope Proteins / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics*
D015703:Antigens, CD; D000954:Antigens, Surface; D015704:CD4 Antigens; D015345:Oligonucleotide Probes; D014759:Viral Envelope Proteins; D015678:Viral Structural Proteins; D003545:Cysteine