Cystein 402 of HIV gp120 is essential for CD4-binding and resistance of gp120 to intracellular degradation

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Abstract
Summary A DNA fragment encoding the CD4-binding region of human immunodeficiency virus type 1 (HIV) gp120 was excised from an SV40-based expression vector containing gp160, and subcloned into phage M13 for site-directed mutagenesis. Mutant vectors were constructed and CV-1 cells were transfected with constructs, where Cys402 was substituted for a serine, and metabolically labelled with [3H]-N-acetylglucosamine (GlcN). Radioimmunoprecipitation with an hyperimmunserum, specific for gp120/gp160, and subsequent SDS-polyacrylamide gel electrophoresis demonstrated presence of gp160, whereas gp120 was replaced by [3H]-GlcN-labelled material, migrating as a diffuse band corresponding to 80–105k, suggesting increased sensitivity of mutantenv gene products to proteolysis after cleavage to gp120. Wild type gp120 and gp160 bound to CD4, whereas neither gp160 nor gp120 from mutant-transfected cell lysates did bind to CD4. Altogether the results indicated that Cys402, probably by participating in a disulfide bridge, is essential for (i) the CD4-binding ability ofenv gene products and for (ii) the physical stability of gp120.