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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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3D reconstruction of NL4-3 and NL4-3/ADA gp140 trimers. Density maps of NL4-3 gp140 trimer (grey) and NL4-3/ADA gp140 trimer (cyan) viewed along (A, top view) and perpendicularly to its threefold symmetry axis (B, side view). Both density maps show a trimeric propeller-like Env architecture with an open conformation. The three masses are only connected in the lower part. The bottom row depicts the superposition of the density maps. Note the high overall congruency; the subtle differences in the tilting of the upper masses and the rotation of the triangular lower mass are not significant at the present resolution of 25 Å.
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Figure 5: 3D reconstruction of NL4-3 and NL4-3/ADA gp140 trimers. Density maps of NL4-3 gp140 trimer (grey) and NL4-3/ADA gp140 trimer (cyan) viewed along (A, top view) and perpendicularly to its threefold symmetry axis (B, side view). Both density maps show a trimeric propeller-like Env architecture with an open conformation. The three masses are only connected in the lower part. The bottom row depicts the superposition of the density maps. Note the high overall congruency; the subtle differences in the tilting of the upper masses and the rotation of the triangular lower mass are not significant at the present resolution of 25 Å.

Mentions: Using electron microscopy and single particle analysis, we generated 3D reconstructions from trimeric NL4-3 (EMDB 2657 [43]) and NL4-3/ADA gp140 (EMDB 2659 [44]). Gp140 density maps were generated from negatively stained single particles through an iterative procedure of 3D alignment, classification, and averaging using about 3,000 particles per data set (see random selection in Additional file 7). By these means we obtained structural information from trimeric Env derived from an X4 HIV-1 strain and observed for the first time the effects of exchanging the V3 spanning region on the overall trimer structure at the given resolution of ~25 Å (Figure 5; NL4-3 in grey, NL4-3/ADA in cyan). In side view, the structures display an open upper part with three masses that are connected with one bridge each to a compact mass in the lower part (Figure 5B). The upper masses are directed upwards and are almost parallel to the three-fold symmetry axis. Viewed from the top (Figure 5A), the density maps have a propeller-like appearance. The upper masses are located laterally to the central lower mass with its apices tilting sideways and for NL4-3/ADA also slightly outwards. Superposition of both density maps (Figure 5, bottom row) reveals that they clearly resemble each other in their overall architecture. Whether the small differences between both density maps in tilt and rotation angle of the four masses are significant is not clear at the present resolution.


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)

3D reconstruction of NL4-3 and NL4-3/ADA gp140 trimers. Density maps of NL4-3 gp140 trimer (grey) and NL4-3/ADA gp140 trimer (cyan) viewed along (A, top view) and perpendicularly to its threefold symmetry axis (B, side view). Both density maps show a trimeric propeller-like Env architecture with an open conformation. The three masses are only connected in the lower part. The bottom row depicts the superposition of the density maps. Note the high overall congruency; the subtle differences in the tilting of the upper masses and the rotation of the triangular lower mass are not significant at the present resolution of 25 Å.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: 3D reconstruction of NL4-3 and NL4-3/ADA gp140 trimers. Density maps of NL4-3 gp140 trimer (grey) and NL4-3/ADA gp140 trimer (cyan) viewed along (A, top view) and perpendicularly to its threefold symmetry axis (B, side view). Both density maps show a trimeric propeller-like Env architecture with an open conformation. The three masses are only connected in the lower part. The bottom row depicts the superposition of the density maps. Note the high overall congruency; the subtle differences in the tilting of the upper masses and the rotation of the triangular lower mass are not significant at the present resolution of 25 Å.
Mentions: Using electron microscopy and single particle analysis, we generated 3D reconstructions from trimeric NL4-3 (EMDB 2657 [43]) and NL4-3/ADA gp140 (EMDB 2659 [44]). Gp140 density maps were generated from negatively stained single particles through an iterative procedure of 3D alignment, classification, and averaging using about 3,000 particles per data set (see random selection in Additional file 7). By these means we obtained structural information from trimeric Env derived from an X4 HIV-1 strain and observed for the first time the effects of exchanging the V3 spanning region on the overall trimer structure at the given resolution of ~25 Å (Figure 5; NL4-3 in grey, NL4-3/ADA in cyan). In side view, the structures display an open upper part with three masses that are connected with one bridge each to a compact mass in the lower part (Figure 5B). The upper masses are directed upwards and are almost parallel to the three-fold symmetry axis. Viewed from the top (Figure 5A), the density maps have a propeller-like appearance. The upper masses are located laterally to the central lower mass with its apices tilting sideways and for NL4-3/ADA also slightly outwards. Superposition of both density maps (Figure 5, bottom row) reveals that they clearly resemble each other in their overall architecture. Whether the small differences between both density maps in tilt and rotation angle of the four masses are significant is not clear at the present resolution.

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