Studies with crosslinking reagents on the oligomeric structure of the env glycoprotein of HIV

Schawaller, Manfred; Smith, Gale; Skehel, J.J.; Wiley, Don C.

doi:10.1016/0042-6822(89)90142-6

Cited by 159 publications

(86 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Evidence from cross-linking studies and gradient centrifngation suggests that HIV-1 gp160 exists as a tetramer consisting of two non-covalently associated dimers Schawaller et al, 1989). This structure is similar to that observed previously for paramyxovirus envelope proteins F and HN.…”

supporting

confidence: 67%

Expression, characterization and purification of simian immunodeficiency virus soluble, oligomerized gp160 from mammalian cells

Rhodes

Spitali²,

Hutchinson³

et al. 1994

Journal of General Virology

View full text Add to dashboard Cite

The envelope glycoprotein, gp160, of human (HIV) and simian (SIV) immunodeficiency viruses mediates virushost cell binding followed by fusion of the viral and plasma membranes. The envelope proteins are known to exist as non-covalently associated oligomers on the virus surface. The production of permanent mammalian cell lines that constitutively secrete relatively high levels of soluble forms of SIV gpl60 is described and we show that these proteins are secreted predominantly as tetramers with lower levels of dimer forms. Oligomeric forms were purified to greater than 90 % purity using a simple gel filtration method. The purified proteins bind CD4 suggesting that they remain in their native conformation. The purified oligomeric proteins provide the basis for more relevant structural, functional and immunological studies than recombinant gpl20 as they more closely resemble the envelope protein oligomer.

show abstract

supporting

confidence: 67%

Expression, characterization and purification of simian immunodeficiency virus soluble, oligomerized gp160 from mammalian cells

Rhodes

Spitali²,

Hutchinson³

et al. 1994

Journal of General Virology

View full text Add to dashboard Cite

show abstract

“…Further confirmation of the functional configuration of gp120 and its mutants was demonstrated by binding of conformation-demanding MAbs, such as b12 (50,51) and b6 (52,53), as well as the glycomoiety-specific MAb 2G12. SDS-PAGE analyses of the gp120 preparations indicated the presence of both monomeric and oligomeric forms, as previously reported (54)(55)(56)(57)(58)(59)(60)(61)(62)(63). The aberrant disulfide-linked oligomers continued to bind CD4 as expected.…”

Section: Methodsmentioning

confidence: 70%

Range of CD4-Bound Conformations of HIV-1 gp120, as Defined Using Conditional CD4-Induced Antibodies

Kaplan

Roitburd-Berman

Lewis

et al. 2016

J Virol

View full text Add to dashboard Cite

The HIV envelope binds cellular CD4 and undergoes a range of conformational changes that lead to membrane fusion and delivery of the viral nucleocapsid into the cellular cytoplasm. This binding to CD4 reveals cryptic and highly conserved epitopes, the molecular nature of which is still not fully understood. The atomic structures of CD4 complexed with gp120 core molecules (a form of gp120 in which the V1, V2, and V3 loops and N and C termini have been truncated) have indicated that a hallmark feature of the CD4-bound conformation is the bridging sheet minidomain. Variations in the orientation of the bridging sheet hairpins have been revealed when CD4-liganded gp120 was compared to CD4-unliganded trimeric envelope structures. Hence, there appears to be a number of conformational transitions possible in HIV-1 monomeric gp120 that are affected by CD4 binding. The spectrum of CD4-bound conformations has been interrogated in this study by using a well-characterized panel of conditional, CD4-induced (CD4i) monoclonal antibodies (MAbs) that bind HIV-1 gp120 and its mutations under various conditions. Two distinct CD4i epitopes of the outer domain were studied: the first comprises the bridging sheet, while the second contains elements of the V2 loop. Furthermore, we show that the unliganded extended monomeric core of gp120 (core e ) assumes an intermediate CD4i conformation in solution that further undergoes detectable rearrangements upon association with CD4. These discoveries impact both accepted paradigms concerning gp120 structure and the field of HIV immunogen design. IMPORTANCE Elucidation of the conformational transitions that the HIV-1 envelope protein undergoes during the course of entry into CD4؉ cells is fundamental to our understanding of HIV biology. The binding of CD4 triggers a range of gp120 structural rearrangements that could present targets for future drug design and development of preventive vaccines. Here we have systematically interrogated and scrutinized these conformational transitions using a panel of antibody probes that share a specific preference for the CD4i conformations. These have been employed to study a collection of gp120 mutations and truncations. Through these analyses, we propose 4 distinct sequential steps in CD4i transitions of gp120 conformations, each defined by antibody specificities and structural requirements of the HIV envelope monomer. As a result, we not only provide new insights into this dynamic process but also define probes to further investigate HIV infection. V iral tropism is mediated by the specific binding of the viral spike protein to its corresponding cell surface receptor. Evolution has driven human immunodeficiency virus (HIV) to elaborate on this canonical paradigm, introducing a series of orchestrated sequential events involving two receptors: CD4 as a primary receptor (1-3) and a chemokine receptor (CXCR4 or CCR5) as a subsequent coreceptor (4-10). However, many critical details of the molecular mechanisms by which CD4 triggers a number of conformati...

show abstract

“…Efforts to evaluate HIV-1 binding/fusion-inhibition antibodies were made here using a method more representative of natural conditions, where an assay was developed to detect antibodies capable ofblocking whole virus particles from binding or fusing to viable cells. This assay differed from the radiolabeled gpl20 assay in that gpl20 was in its native, multimeric form attached to gp4l (52,53), and was capable of mediating fusion owing to the presence of gp41 and an intact viral membrane (14)(15)(16)(17). It was determined that boosting elicited HIV-l binding/fusion-inhibition antibodies in all 11 volunteers and that much of this activity could be removed by a synthetic peptide corresponding to the V3 loop, indicating that these antibodies were V3 loop-specific (Table I).…”

Section: Discussionmentioning

confidence: 99%

V3-specific neutralizing antibodies in sera from HIV-1 gp160-immunized volunteers block virus fusion and act synergistically with human monoclonal antibody to the conformation-dependent CD4 binding site of gp120. NIH-NIAID AIDS Vaccine Clinical Trials Network.

Montefiori

Graham²,

Zhou³

et al. 1993

J. Clin. Invest.

View full text Add to dashboard Cite

Sera from 11 volunteers immunized with a recombinant HIV-1 gp160-expressing vaccinia virus (HIVAC-le; Oncogen/Bristol-Myers Squibb, Seattle, WA) and boosted with baculovirusderived rgpl60 (VaxSyn; MicroGeneSys, Inc., Meriden, CT) were evaluated for functional serum antibodies and their epitopes. Sera obtained prior to boosting had undetectable HIV-1-specific IgG and neutralizing activity, and did not block HIV-1 from binding or fusing to CD4+ MT-2 cells. 14 d after boosting, sera from each volunteer contained HIV-1-specific IgG titers of 1:40 to 1:1,280. Five of these sera also contained neutralizing antibodies, where most or all neutralizing activity was blocked by a synthetic peptide corresponding to amino acids 307-330 ofthe V3 loop ofgpl 20, indicating that neutralizing antibodies were mostly V3 loop-specific. All sera obtained after boosting contained HIV-1 binding/fusion-inhibition antibodies, and a significant portion of their activity was blocked by the V3 loop peptide, a result consistent with the presence of antibodies against the region of the V3 loop that participates in fusion. Three sera with V3 loop-specific neutralizing and fusion-inhibition antibodies were studied further. In competitive antibody binding experiments, antibodies reactive with the conformation-dependent, CD4 binding site ofgpl20 were undetectable in each serum. When evaluated in combination with a monoclonal antibody to the CD4 binding site of gpl 20, two sera demonstrated synergism in neutralizing assays, and all three sera demonstrated synergism in binding/fusion-inhibition assays, further indicating that the functional antibodies were primarily V3 loop-specific. The synergism also suggests that a vaccine that elicits strong serum antibody responses to both regions of gpl20 may improve the potential for inducing protective immunity. (J. Clin. Invest. 1993. 92:840-847.)

show abstract

Studies with crosslinking reagents on the oligomeric structure of the env glycoprotein of HIV

Cited by 159 publications

References 8 publications

Expression, characterization and purification of simian immunodeficiency virus soluble, oligomerized gp160 from mammalian cells

Expression, characterization and purification of simian immunodeficiency virus soluble, oligomerized gp160 from mammalian cells

Range of CD4-Bound Conformations of HIV-1 gp120, as Defined Using Conditional CD4-Induced Antibodies

V3-specific neutralizing antibodies in sera from HIV-1 gp160-immunized volunteers block virus fusion and act synergistically with human monoclonal antibody to the conformation-dependent CD4 binding site of gp120. NIH-NIAID AIDS Vaccine Clinical Trials Network.

Contact Info

Product

Resources

About