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Antigenic and 3D structural characterization of soluble X4 and hybrid X4-R5 HIV-1 Env trimers.

Arnold P, Himmels P, Weiß S, Decker TM, Markl J, Gatterdam V, Tampé R, Bartholomäus P, Dietrich U, Dürr R - Retrovirology (2014)

Bottom Line: V3 mAbs showed significant binding differences to the three constructs, which were refined by SPR analysis.The first 3D reconstruction of an Env construct from an X4 TCLA HIV-1 strain reveals an open conformation, in contrast to recently published more closed structures from R5 Env.Exchanging the X4 V3 spanning region for that of R5 ADA did not alter the open Env architecture as deduced from its very similar 3D reconstruction. 3D EM analysis showed an apparent open trimer configuration of X4 NL4-3 gp140 that is not modified by exchanging the V3 spanning region for R5 ADA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Virology, Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Paul-Ehrlich-Str, 42-44, 60596 Frankfurt, Germany. Ralf.Duerr@nyumc.org.

ABSTRACT

Background: HIV-1 is decorated with trimeric glycoprotein spikes that enable infection by engaging CD4 and a chemokine coreceptor, either CCR5 or CXCR4. The variable loop 3 (V3) of the HIV-1 envelope protein (Env) is the main determinant for coreceptor usage. The predominant CCR5 using (R5) HIV-1 Env has been intensively studied in function and structure, whereas the trimeric architecture of the less frequent, but more cytopathic CXCR4 using (X4) HIV-1 Env is largely unknown, as are the consequences of sequence changes in and near V3 on antigenicity and trimeric Env structure.

Results: Soluble trimeric gp140 Env constructs were used as immunogenic mimics of the native spikes to analyze their antigenic properties in the context of their overall 3D structure. We generated soluble, uncleaved, gp140 trimers from a prototypic T-cell line-adapted (TCLA) X4 HIV-1 strain (NL4-3) and a hybrid (NL4-3/ADA), in which the V3 spanning region was substituted with that from the primary R5 isolate ADA. Compared to an ADA (R5) gp140, the NL4-3 (X4) construct revealed an overall higher antibody accessibility, which was most pronounced for the CD4 binding site (CD4bs), but also observed for mAbs against CD4 induced (CD4i) epitopes and gp41 mAbs. V3 mAbs showed significant binding differences to the three constructs, which were refined by SPR analysis. Of interest, the NL4-3/ADA construct with the hybrid NL4-3/ADA CD4bs showed impaired CD4 and CD4bs mAb reactivity despite the presence of the essential elements of the CD4bs epitope. We obtained 3D reconstructions of the NL4-3 and the NL4-3/ADA gp140 trimers via electron microscopy and single particle analysis, which indicates that both constructs inherit a propeller-like architecture. The first 3D reconstruction of an Env construct from an X4 TCLA HIV-1 strain reveals an open conformation, in contrast to recently published more closed structures from R5 Env. Exchanging the X4 V3 spanning region for that of R5 ADA did not alter the open Env architecture as deduced from its very similar 3D reconstruction.

Conclusions: 3D EM analysis showed an apparent open trimer configuration of X4 NL4-3 gp140 that is not modified by exchanging the V3 spanning region for R5 ADA.

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Kinetics of 447-52D antibody binding to gp140 constructs with SPR measurements. ProteOn XPR36 measurements were performed with immobilized 447-52D antibody (ligand) and rising concentrations of the different gp140 constructs (analyte). Note the remarkable slower dissociation of the NL4-3/ADA construct from the 447-52D mAb in contrast to the NL4-3 and ADA construct, which is responsible for its higher affinity to 447-52D (see also Figure 2 and table of Additional file 6 with listed KD, kon and koff rates).
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Figure 3: Kinetics of 447-52D antibody binding to gp140 constructs with SPR measurements. ProteOn XPR36 measurements were performed with immobilized 447-52D antibody (ligand) and rising concentrations of the different gp140 constructs (analyte). Note the remarkable slower dissociation of the NL4-3/ADA construct from the 447-52D mAb in contrast to the NL4-3 and ADA construct, which is responsible for its higher affinity to 447-52D (see also Figure 2 and table of Additional file 6 with listed KD, kon and koff rates).

Mentions: We studied the antigenic properties of our gp140 constructs by analyzing the reactivity with monoclonal antibodies (mAbs)/antibody constructs directed against gp120 V3 (447-52D, D19), CD4bs (CD4-Fc, VRC01, VRC03, b12, b13, F105), the coreceptor binding site (CG10, 17b) and gp41 (Md-1, 2F5, 246-D) (Figures 2, 3, 4, Additional file 4 and tables in Additional files 5 and 6). The V3 mAbs D19 and 447-52D reacted with all gp140 recombinant proteins proving the exposure of V3 in all constructs. MAb D19 preferentially recognizing V3 from X4 strains [36], showed enhanced reactivity with the X4 NL4-3 gp140 compared to the R5 ADA gp140, as expected. Consequently, the exchange of the X4 V3 region for the R5 ADA V3 reduced mAb D19 binding to levels of the R5 ADA control, as seen by comparable Amax (Figure 2) and affinity (see KD values in Additional file 5). In contrast, mAb 447-52D, directed against the tip of V3, showed similar binding curves for NL4-3 and ADA gp140, however Amax and affinity for the hybrid NL4-3/ADA construct were remarkably increased. We confirmed this finding by SPR measurements with an inversed binding setup compared to the ELISA experiments, i.e. immobilization of mAb 447-52D and administration of the gp140 constructs as analytes (Figure 3 and table in Additional file 6). The kon rates of the different gp140 constructs for mAb 447-52D were comparable, however we observed a much slower dissociation of the hybrid NL4-3/ADA from mAb 447-52D with koff values 5 times lower compared to the other constructs. This resulted in lower KD values and enhanced binding signals in end point analyses. Gp41 antibodies Md-1, 2F5 and 246-D were reactive with all gp140 constructs (Figure 2 and Additional files 4 and 5). The reactivity with the trimer specific antibody Md-1 confirmed the trimeric state of our gp140 constructs (Figure 2). Despite the presence of several antibody epitopes in all gp140 constructs, we detected quantitative differences: in most cases the mAbs showed best binding to NL4-3 gp140, reduced binding to ADA gp140 and strongly reduced binding to NL4-3/ADA. Exceptions are mAbs D19, Md-1 and b13 with comparable binding levels to ADA and NL4-3/ADA and the V3 mAb 447-52D, which binds by far best to the hybrid NL4-3/ADA.


Antigenic and 3D structural characterization of soluble X4 and hybrid X4-R5 HIV-1 Env trimers.

Arnold P, Himmels P, Weiß S, Decker TM, Markl J, Gatterdam V, Tampé R, Bartholomäus P, Dietrich U, Dürr R - Retrovirology (2014)

Kinetics of 447-52D antibody binding to gp140 constructs with SPR measurements. ProteOn XPR36 measurements were performed with immobilized 447-52D antibody (ligand) and rising concentrations of the different gp140 constructs (analyte). Note the remarkable slower dissociation of the NL4-3/ADA construct from the 447-52D mAb in contrast to the NL4-3 and ADA construct, which is responsible for its higher affinity to 447-52D (see also Figure 2 and table of Additional file 6 with listed KD, kon and koff rates).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4048260&req=5

Figure 3: Kinetics of 447-52D antibody binding to gp140 constructs with SPR measurements. ProteOn XPR36 measurements were performed with immobilized 447-52D antibody (ligand) and rising concentrations of the different gp140 constructs (analyte). Note the remarkable slower dissociation of the NL4-3/ADA construct from the 447-52D mAb in contrast to the NL4-3 and ADA construct, which is responsible for its higher affinity to 447-52D (see also Figure 2 and table of Additional file 6 with listed KD, kon and koff rates).
Mentions: We studied the antigenic properties of our gp140 constructs by analyzing the reactivity with monoclonal antibodies (mAbs)/antibody constructs directed against gp120 V3 (447-52D, D19), CD4bs (CD4-Fc, VRC01, VRC03, b12, b13, F105), the coreceptor binding site (CG10, 17b) and gp41 (Md-1, 2F5, 246-D) (Figures 2, 3, 4, Additional file 4 and tables in Additional files 5 and 6). The V3 mAbs D19 and 447-52D reacted with all gp140 recombinant proteins proving the exposure of V3 in all constructs. MAb D19 preferentially recognizing V3 from X4 strains [36], showed enhanced reactivity with the X4 NL4-3 gp140 compared to the R5 ADA gp140, as expected. Consequently, the exchange of the X4 V3 region for the R5 ADA V3 reduced mAb D19 binding to levels of the R5 ADA control, as seen by comparable Amax (Figure 2) and affinity (see KD values in Additional file 5). In contrast, mAb 447-52D, directed against the tip of V3, showed similar binding curves for NL4-3 and ADA gp140, however Amax and affinity for the hybrid NL4-3/ADA construct were remarkably increased. We confirmed this finding by SPR measurements with an inversed binding setup compared to the ELISA experiments, i.e. immobilization of mAb 447-52D and administration of the gp140 constructs as analytes (Figure 3 and table in Additional file 6). The kon rates of the different gp140 constructs for mAb 447-52D were comparable, however we observed a much slower dissociation of the hybrid NL4-3/ADA from mAb 447-52D with koff values 5 times lower compared to the other constructs. This resulted in lower KD values and enhanced binding signals in end point analyses. Gp41 antibodies Md-1, 2F5 and 246-D were reactive with all gp140 constructs (Figure 2 and Additional files 4 and 5). The reactivity with the trimer specific antibody Md-1 confirmed the trimeric state of our gp140 constructs (Figure 2). Despite the presence of several antibody epitopes in all gp140 constructs, we detected quantitative differences: in most cases the mAbs showed best binding to NL4-3 gp140, reduced binding to ADA gp140 and strongly reduced binding to NL4-3/ADA. Exceptions are mAbs D19, Md-1 and b13 with comparable binding levels to ADA and NL4-3/ADA and the V3 mAb 447-52D, which binds by far best to the hybrid NL4-3/ADA.

Bottom Line: V3 mAbs showed significant binding differences to the three constructs, which were refined by SPR analysis.The first 3D reconstruction of an Env construct from an X4 TCLA HIV-1 strain reveals an open conformation, in contrast to recently published more closed structures from R5 Env.Exchanging the X4 V3 spanning region for that of R5 ADA did not alter the open Env architecture as deduced from its very similar 3D reconstruction. 3D EM analysis showed an apparent open trimer configuration of X4 NL4-3 gp140 that is not modified by exchanging the V3 spanning region for R5 ADA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Virology, Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Paul-Ehrlich-Str, 42-44, 60596 Frankfurt, Germany. Ralf.Duerr@nyumc.org.

ABSTRACT

Background: HIV-1 is decorated with trimeric glycoprotein spikes that enable infection by engaging CD4 and a chemokine coreceptor, either CCR5 or CXCR4. The variable loop 3 (V3) of the HIV-1 envelope protein (Env) is the main determinant for coreceptor usage. The predominant CCR5 using (R5) HIV-1 Env has been intensively studied in function and structure, whereas the trimeric architecture of the less frequent, but more cytopathic CXCR4 using (X4) HIV-1 Env is largely unknown, as are the consequences of sequence changes in and near V3 on antigenicity and trimeric Env structure.

Results: Soluble trimeric gp140 Env constructs were used as immunogenic mimics of the native spikes to analyze their antigenic properties in the context of their overall 3D structure. We generated soluble, uncleaved, gp140 trimers from a prototypic T-cell line-adapted (TCLA) X4 HIV-1 strain (NL4-3) and a hybrid (NL4-3/ADA), in which the V3 spanning region was substituted with that from the primary R5 isolate ADA. Compared to an ADA (R5) gp140, the NL4-3 (X4) construct revealed an overall higher antibody accessibility, which was most pronounced for the CD4 binding site (CD4bs), but also observed for mAbs against CD4 induced (CD4i) epitopes and gp41 mAbs. V3 mAbs showed significant binding differences to the three constructs, which were refined by SPR analysis. Of interest, the NL4-3/ADA construct with the hybrid NL4-3/ADA CD4bs showed impaired CD4 and CD4bs mAb reactivity despite the presence of the essential elements of the CD4bs epitope. We obtained 3D reconstructions of the NL4-3 and the NL4-3/ADA gp140 trimers via electron microscopy and single particle analysis, which indicates that both constructs inherit a propeller-like architecture. The first 3D reconstruction of an Env construct from an X4 TCLA HIV-1 strain reveals an open conformation, in contrast to recently published more closed structures from R5 Env. Exchanging the X4 V3 spanning region for that of R5 ADA did not alter the open Env architecture as deduced from its very similar 3D reconstruction.

Conclusions: 3D EM analysis showed an apparent open trimer configuration of X4 NL4-3 gp140 that is not modified by exchanging the V3 spanning region for R5 ADA.

Show MeSH
Related in: MedlinePlus