The Human Immunodeficiency Virus Type 1 gp120 V2 Domain Mediates gp41-Independent Intersubunit Contacts

Abstract

The envelope protein of human immunodeficiency virus type 1 HIV-1 undergoes proteolytic cleavage in the Golgi complex to produce subunits designated gp120 and gp41, which remain noncovalently associated. While gp41 has a well-characterized oligomeric structure, the maintenance of gp41-independent gp120 intersubunit contacts remains a contentious issue. Using recombinant vaccinia virus to achieve high-level expression of gp120 in mammalian cells combined with gel filtration analysis, we were able to isolate a discrete oligomeric form of gp120. Oligomerization of gp120 occurred intracellularly between 30 and 120 min after synthesis. Analysis by sedimentation equilibrium unequivocally identified the oligomeric species as a dimer. In order to identify the domains involved in the intersubunit contact, we expressed a series of gp120 proteins lacking various domains and assessed the effects of mutation on oligomeric structure. Deletion of the V1 or V3 loops had little effect on the relative amounts of monomer and dimer in comparison to wild-type gp120. In contrast, deletion of either all or part of the V2 loop drastically reduced dimer formation, indicating that this domain is required for intersubunit contact formation. Consistent with this, the V2 loop of the dimer was less accessible than that of the monomer to a specific monoclonal antibody. Previous studies have shown that while the V2 loop is not an absolute requirement for viral entry, the absence of this domain reduces viral resistance to neutralization by monoclonal antibodies or sera. We propose that the quaternary structure of gp120 may contribute to resistance to neutralization by limiting the exposure of conserved epitopes.The envelope (env) proteins of human immunodeficiency virus type 1 (HIV-1) mediate viral entry and are the primary targets of neutralizing antibodies. Following synthesis and posttranslational modifications in the endoplasmic reticulum, including N-linked glycosylation, disulfide bond formation, and oligomerization, the env protein precursor gp160 passes through the Golgi complex where it is cleaved to form subunits designated gp120 and gp41 (11,41). A noncovalently associated oligomeric gp120-gp41 complex is transported to the surface of infected cells, where incorporation into budding virions occurs. The binding of virion-associated or infected-cell-associated gp120 to the CD4 receptor induces conformational changes that promote subsequent interaction with one of a number of chemokine receptors (19,46,49). Movement of the variable (V) loop structure V1/V2 following CD4 binding has been shown to result in increased exposure of an antibody epitope which overlaps with the chemokine receptor binding site (44,52). Receptor binding-induced env conformational changes are believed to culminate in the exposure of the gp41 fusion peptide and its repositioning toward the target cell prior to fusion of the membranes of the infected cell or virion and the target cell (5,14,26,48).Despite numerous investigations, the oligomeric structure of the nativ...

Identifying epitopes of HIV-1 that induce protective antibodies

“…Given that viruses lacking the V1/V2 region or the V2 variable loop are capable of replication 74,75 , this region cannot be essential for infectivity and therefore is relatively unconstrained in terms of mutation. Therefore, despite the fact that this region of the virus envelope carries out important functions -mediating gp41-independent interaction within the gp120 trimer 76 and shielding the region in and around the bridging sheet of gp120 responsible for interaction with chemokine receptors 74,75,77-79 -the V1/V2 loop is an improbable target for vaccines and, in fact, is specifically being deleted in some vaccine constructs to provide better access to neutralizing epitopes in and near the co-receptor binding site 80 .…”

Measuring Protein‐Protein Interactions by Equilibrium Sedimentation

“…Methods for the estimation of average partial specific volumes have been described by Shire (1992), Junghans et al (1996), Fairman et al (1999), and Lewis and Junghans (2000). For glycosylated proteins, the availability of molar mass data from mass spectrometry is extremely helpful, because, together with the molar mass of the protein component as calculated from amino acid composition, the molar mass of the carbohydrate component can be estimated (Fairman et al, 1999;Center et al, 2000). A technique in which two SE experiments are performed simultaneously, one in D 2 O and one in H 2 O (Edelstein and Schachman, 1967), can be useful in the study of glycosylated proteins.…”

Oligomerization of the SARS-CoV S glycoprotein: dimerization of the N-terminus and trimerization of the ectodomain

“…Major findings of this work are that (i) the SU subunit of the SARS-CoV S glycoprotein (S1) forms dimers, (ii) the dimerization domain does not overlap and is upstream of the RBD, (iii) its deletion abolishes fusion, (iv) dimeric S1 binds receptor molecules much more efficiently than monovalent fragments containing the RBD, and (v) the soluble S ectodomain forms trimers under gel filtration conditions. It has been previously reported that some attachment SU subunits of class I fusion proteins that bind receptor molecules can form dimers including gp120 of the retrovirus HIV-1 [17] and S1 of the coronavirus MHV [16]. What is the role of S1 dimerization for mediation of membrane fusion however remains unclear.…”