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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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Fitting of gp120 X-ray structures into NL4-3 and NL4-3/ADA gp140 density maps. Automated fitting of a gp120 X-ray structure (pdb 2NY7) into NL4-3 (grey) and NL4-3/ADA gp140 density maps (cyan), viewed from top (A) and side view (B). In the bottom row, the 3D model of NL4-3 gp140 is shown with the locations of the V3 loop and the CD4 binding site indicated with green and yellow balls, respectively. Magenta balls represent the N- and C-terminal stumps of gp120. The bridging sheets are colored in blue.
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Figure 6: Fitting of gp120 X-ray structures into NL4-3 and NL4-3/ADA gp140 density maps. Automated fitting of a gp120 X-ray structure (pdb 2NY7) into NL4-3 (grey) and NL4-3/ADA gp140 density maps (cyan), viewed from top (A) and side view (B). In the bottom row, the 3D model of NL4-3 gp140 is shown with the locations of the V3 loop and the CD4 binding site indicated with green and yellow balls, respectively. Magenta balls represent the N- and C-terminal stumps of gp120. The bridging sheets are colored in blue.

Mentions: At the mass-corrected threshold, the three protruding masses of both 3D reconstructions resembled an unliganded gp120 molecule in size and overall shape (see for example pdb-ID 2NY7 [45]). Copies of the latter were docked as follows (Figure 6): First, the gp120 structures were oriented manually with their N- and C-terminus (magenta spheres in Figure 6 bottom row) directing towards the single larger central mass, which is supposed to represent the ectodomain of gp41. After this pre-orientation, automated rigid-body fitting yielded a stable result, with the N-glycosylation sites (spheres in Additional file 8) and the CD4 binding sites (yellow spheres in Figure 6 bottom row) pointing outwards. Significantly, with these sites pointing inwards, stable automated docking was not achieved. This result was obtained with both 3D reconstructions. In both cases, the only stable alternative was with the N- and C-termini pointing 180° away, i.e. opposite to the supposed ectodomain, which can be excluded for structural reasons. The allocation of the V3 loops and the bridging sheet are indicated with green spheres and blue beta sheets, respectively (Figure 6 bottom row and its animated version in Additional file 9). Automated fitting of both 3D reconstructions revealed that their docked X-ray structures were almost superimposed (Additional file 10). This underlines their similarity, as the slight differences are within the resolution limit.


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)

Fitting of gp120 X-ray structures into NL4-3 and NL4-3/ADA gp140 density maps. Automated fitting of a gp120 X-ray structure (pdb 2NY7) into NL4-3 (grey) and NL4-3/ADA gp140 density maps (cyan), viewed from top (A) and side view (B). In the bottom row, the 3D model of NL4-3 gp140 is shown with the locations of the V3 loop and the CD4 binding site indicated with green and yellow balls, respectively. Magenta balls represent the N- and C-terminal stumps of gp120. The bridging sheets are colored in blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Fitting of gp120 X-ray structures into NL4-3 and NL4-3/ADA gp140 density maps. Automated fitting of a gp120 X-ray structure (pdb 2NY7) into NL4-3 (grey) and NL4-3/ADA gp140 density maps (cyan), viewed from top (A) and side view (B). In the bottom row, the 3D model of NL4-3 gp140 is shown with the locations of the V3 loop and the CD4 binding site indicated with green and yellow balls, respectively. Magenta balls represent the N- and C-terminal stumps of gp120. The bridging sheets are colored in blue.
Mentions: At the mass-corrected threshold, the three protruding masses of both 3D reconstructions resembled an unliganded gp120 molecule in size and overall shape (see for example pdb-ID 2NY7 [45]). Copies of the latter were docked as follows (Figure 6): First, the gp120 structures were oriented manually with their N- and C-terminus (magenta spheres in Figure 6 bottom row) directing towards the single larger central mass, which is supposed to represent the ectodomain of gp41. After this pre-orientation, automated rigid-body fitting yielded a stable result, with the N-glycosylation sites (spheres in Additional file 8) and the CD4 binding sites (yellow spheres in Figure 6 bottom row) pointing outwards. Significantly, with these sites pointing inwards, stable automated docking was not achieved. This result was obtained with both 3D reconstructions. In both cases, the only stable alternative was with the N- and C-termini pointing 180° away, i.e. opposite to the supposed ectodomain, which can be excluded for structural reasons. The allocation of the V3 loops and the bridging sheet are indicated with green spheres and blue beta sheets, respectively (Figure 6 bottom row and its animated version in Additional file 9). Automated fitting of both 3D reconstructions revealed that their docked X-ray structures were almost superimposed (Additional file 10). This underlines their similarity, as the slight differences are within the resolution limit.

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