Cystein 402 of HIV gp 120 is essential for CD4-binding and resistance of gp 120 to intracellular degradation.
A DNA fragment encoding the CD4-binding region of human immunodeficiency virus type 1 (HIV) gp 120 was excised from an SV40-based expression vector containing gp 160, 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 gp 120/gp 160, and subsequent SDS-polyacrylamide gel electrophoresis demonstrated presence of gp 160, whereas gp 120 was replaced by [3H]-GlcN-labelled material, migrating as a diffuse band corresponding to 80-105k, suggesting increased sensitivity of mutant env gene products to proteolysis after cleavage to gp 120. Wild type gp 120 and gp 160 bound to CD4, whereas neither gp 160 nor gp 120 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 of env gene products and for (ii) the physical stability of gp 120.
Hemming
A
A
Department of Clinical Virology, University of Göteborg, Sweden.
Bolmstedt
A
A
Flodby
P
P
Lundberg
L
L
Gidlund
M
M
Wigzell
H
H
Olofsson
S
S
D000595:Amino Acid Sequence; D015704:CD4 Antigens / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003545:Cysteine / Q000502:physiology*; D004279:DNA, Viral / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015686:Gene Products, env / Q000378:metabolism; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000378:metabolism*; D019302:HIV Envelope Protein gp160; D008969:Molecular Sequence Data; D009154:Mutation; D011233:Precipitin Tests; D011498:Protein Precursors / Q000378:metabolism; D015728:Receptors, HIV / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000502:physiology
D015704:CD4 Antigens; D004279:DNA, Viral; D015686:Gene Products, env; D015699:HIV Envelope Protein gp120; D019302:HIV Envelope Protein gp160; D011498:Protein Precursors; D015728:Receptors, HIV; D012694:Serine; D003545:Cysteine