Effects of mutations in glycosylation sites and disulphide bonds on processing, CD4-binding and fusion activity of human immunodeficiency virus envelope glycoproteins

Abstract

Site-directed mutagenesis was used to study the biological significance of a disulphide bridge and two N-linked oligosaccharides in the CD4-binding region of the envelope glycoproteins of human immunodeficiency virus type 1. Mutagenesis was performed in a phage M13 system at sites corresponding to the cysteine residue (amino acid 402) and the asparagine residues (390 and 447) of the env gene. The mutated env gene was inserted into a recombinant vaccinia virus under the control of the vaccinia virus 7.5K promoter and the expression of mutated env proteins was analysed by SDS-PAGE, a conventional indirect immunofluorescence assay and by a fluorescenceactivated cell sorter. Cysteine 402 was found to be essential for the specific cleavage of gp 160 into gp 120 and gp41, and for intracellular transport of the protein to the cell surface. CD4-binding and syncytium formation assays demonstrated that the disulphide bridge of cysteine 402 stabilized a conformation essential for receptor binding as well as syncytium formation by CD4 + cells. No altered biological activity compared to that of the wild-type proteins could be detected for the mutant proteins lacking the Nglycosylation sites. These data show that the two conserved glycans attached to asparagine residues 390 and 447 do not play any active role in the formation of the disulphide bridge involving cysteine 402 or in the maintenance of an active conformation of the protein, despite their location within the functionally important CD4-binding region.

Nonrandom distribution of gp120 N-linked glycosylation sites important for infectivity of human immunodeficiency virus type 1.

“…Bolmstedt et al (27) reported that the removal of N-linked glycosylation sites represented by our mutants 406 and 463 from the envelope recombinant proteins expressed by a vaccinia expression vector did not affect CD4 receptor binding or syncytium formation (27). Their results are compatible with our finding that CD4-positive SupT1 cells were readily infected by our mutants 406 and 463, and support our hypothesis that the N-linked glycosylation sites located in the carboxyl-terminal half ofgp120 are more dispensable for viral infectivity than those located in the amino-terminal half.…”

Neutralizing Antibody Response During Human Immunodeficiency Virus Type 1 Infection: Type and Group Specificity and Viral Escape

“…First, patient sera were assayed for their ability to block binding between gp 120 and cell-associated CD4 in a kinetic assay (B. Svennerholm, unpublished results), gpl20, purified from green monkey kidney cells infected with a gpl20-encoding recombinant virus (Bolmstedt et al, 1991), was allowed to bind to CD4 ÷ HeLa cells with or without preincubation with dilutions of serum for 15 rain at 37 °C. The amount of gpl20 adsorbed to the cell surface was measured directly in an ELISA system using the anti-V3 (IIIB) MAb F58/H3 as detecting antibody (negative control CD4-HeLa cells).…”

Removal of N-Linked Glycosylation Sites in the V1 Region of Simian Immunodeficiency Virus gp120 Results in Redirection of B-Cell Responses to V3

“…However, once fully glycosylated proteins have been produced, these carbohydrate moieties do not appear to be required to maintain native protein structure, since enzymatically deglycosylated core envelope proteins retain their ability to bind CD4 and many conformationally dependent antibodies (28)(29)(30). In addition, despite the general requirement for carbohydrates in the production of envelope proteins, it is possible to remove some individual sites without impairing the ability of these glycosylation mutant envelope proteins to bind CD4 or yield replication-competent viruses (3,4,26). For years, it has been suggested that these carbohydrates serve as a barrier to shield the virus from effective immune recognition and control.…”