Stabilizing Exposure of Conserved Epitopes by Structure Guided Insertion of Disulfide Bond in HIV-1 Envelope Glycoprotein

Kassa, Aemro; Dey, Antu; Sarkar, Pampi; LaBranche, Celia C.; Go, Eden P.; Clark, Daniel F.; Sun, Yuliang; Nandi, Avishek; Hartog, Karin; Desaire, Heather; Montefiori, David C.; Carfı́, Andrea; Srivastava, Indresh K.; Barnett, Susan W.

doi:10.1371/journal.pone.0076139

Cited by 17 publications

(29 citation statements)

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“…The canonical disulfide-bonded peptides were detected in each of the three CZA97.012 gp140 populations, which is consistent with several prior reports on various gp120 and gp140 proteins (53)(54)(55)74). However, aberrant forms were also detected ( Table 3).…”

Section: Resultssupporting

confidence: 91%

“…The gp120 proteins from most HIV-1 isolates contain nine intermolecular disulfide bonds that must form for the protein to fold and remain in a native conformation. However, it was reported several years ago that gp120 monomers and uncleaved gp140 proteins contain inappropriate intramolecular disulfide bonds (53)(54)(55)74). Thus, in at least some fraction of the total population of Env proteins, some Cys residues can pair incorrectly and create nonnative proteins, with the V1V2 loop structure being particularly vulnerable to disulfide bond scrambling.…”

Section: Resultsmentioning

confidence: 99%

“…Finally, the uncleaved CZA97.012 gp140 proteins contain a substantial proportion of gp120 subunits with scrambled disulfide bonds, particularly in the V1V2 region; the aberrant subunits further contribute to the nonnative antigenicity and associated aberrant structural and aggregation properties of these proteins. Based on prior reports and our unpublished work, this problem is generic to the standard design of uncleaved gp140s (e.g., CZA97.012 and 92UG037.8 gp140 UNC -Fd-His), but it also affects gp120 monomers of multiple genotypes (53)(54)(55)74;Go et al,unpublished). Moreover, the scrambling of disulfide bonds explains why the uncleaved gp140s are prone to forming aggregated, higher-MW forms; internally unpaired Cys residues are available to bond with counterparts on a neighboring gp140 protein.…”

Section: Discussionmentioning

confidence: 99%

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Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

Ringe

Yasmeen

Ozorowski

et al. 2015

J Virol

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166

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We have investigated factors that influence the production of native-like soluble, recombinant trimers based on the env genes of two isolates of human immunodeficiency virus type 1 (HIV-1), specifically 92UG037.8 (clade A) and CZA97.012 (clade C). When the recombinant trimers based on the env genes of isolates 92UG037.8 and CZA97.012 were made according to the SOSIP.664 design and purified by affinity chromatography using broadly neutralizing antibodies (bNAbs) against quaternary epitopes (PGT145 and PGT151, respectively), the resulting trimers are highly stable and they are fully native-like when visualized by negative-stain electron microscopy. They also have a native-like (i.e., abundant) oligomannose glycan composition and display multiple bNAb epitopes while occluding those for nonneutralizing antibodies. In contrast, uncleaved, histidine-tagged Foldon (Fd) domain-containing gp140 proteins (gp140 UNC -Fd-His), based on the same env genes, very rarely form native-like trimers, a finding that is consistent with their antigenic and biophysical properties and glycan composition. The addition of a 20-residue flexible linker (FL20) between the gp120 and gp41 ectodomain (gp41 ECTO ) subunits to make the uncleaved 92UG037.8 gp140-FL20 construct is not sufficient to create a native-like trimer, but a small percentage of native-like trimers were produced when an I559P substitution in gp41 ECTO was also present. The further addition of a disulfide bond (SOS) to link the gp120 and gp41 subunits in the uncleaved gp140-FL20-SOSIP protein increases native-like trimer formation to ϳ20 to 30%. Analysis of the disulfide bond content shows that misfolded gp120 subunits are abundant in uncleaved CZA97.012 gp140 UNC -Fd-His proteins but very rare in native-like trimer populations. The design and stabilization method and the purification strategy are, therefore, all important influences on the quality of trimeric Env proteins and hence their suitability as vaccine components. IMPORTANCESoluble, recombinant multimeric proteins based on the HIV-1 env gene are current candidate immunogens for vaccine trials in humans. These proteins are generally designed to mimic the native trimeric envelope glycoprotein (Env) that is the target of virus-neutralizing antibodies on the surfaces of virions. The underlying hypothesis is that an Env-mimetic protein may be able to induce antibodies that can neutralize the virus broadly and potently enough for a vaccine to be protective. Multiple different designs for Env-mimetic trimers have been put forth. Here, we used the CZA97.012 and 92UG037.8 env genes to compare some of these designs and determine which ones best mimic virus-associated Env trimers. We conclude that the most widely used versions of CZA97.012 and 92UG037.8 oligomeric Env proteins do not resemble the trimeric Env glycoprotein on HIV-1 viruses, which has implications for the design and interpretation of ongoing or proposed clinical trials of these proteins. R ecombinant, soluble envelope glycoprotein (Env) gp140 trimers from...

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Section: Resultssupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

Ringe

Yasmeen

Ozorowski

et al. 2015

J Virol

Self Cite

166

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“…The resistance of most primary HIV-1 Envs to sCD4 engagement may explain why sCD4 exhibited only minimal in vivo potential as an antiviral agent (44). CD4mc have been shown to sensitize HIV-1 virions to neutralization by otherwise nonneutralizing CD4i Abs that can easily be generated through vaccination with CD4-bound stabilized gp120s (25,45,46). Importantly, these types of Abs were isolated from RV144 vaccinees and possessed ADCC potential (10,12).…”

Section: Discussionmentioning

confidence: 99%

CD4 mimetics sensitize HIV-1-infected cells to ADCC

Richard

Veillette

Brassard

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

128

291

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HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 has evolved a sophisticated mechanism to avoid exposure of ADCC-mediating Env epitopes by down-regulating CD4 and by limiting the overall amount of Env at the cell surface. Here we report that small-molecule CD4-mimetic compounds induce the CD4-bound conformation of Env, and thereby sensitize cells infected with primary HIV-1 isolates to ADCC mediated by antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we identified one CD4 mimetic with the capacity to sensitize endogenously infected ex vivo-amplified primary CD4 T cells to ADCC killing mediated by autologous sera and effector cells. Thus, CD4 mimetics hold the promise of therapeutic utility in preventing and controlling HIV-1 infection.

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“…The formation of oligomeric forms of gp120 in transfected HEK 293T cells due to aberrant disulfide bridges is well documented (55,58,(79)(80)(81). Oligomerization can result specifically from rearranged disulfides in the V2 loop region.…”

Section: Figmentioning

confidence: 99%

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

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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...

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Stabilizing Exposure of Conserved Epitopes by Structure Guided Insertion of Disulfide Bond in HIV-1 Envelope Glycoprotein

Cited by 17 publications

References 50 publications

Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

CD4 mimetics sensitize HIV-1-infected cells to ADCC

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

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