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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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Scheme of the NL4-3 and NL4-3/ADA constructs and protein analysis by SDS-PAGE. (A) Scheme of gp140 expression constructs indicating cloning of the HIV-1 NL4-3 Env ectodomain with tPA leader sequence and deleted cleavage site. The hybrid construct NL4-3/ADA was generated by exchanging the V3 domain in conjunction with adjacent constant regions of the X4 tropic NL4-3 construct with the respective sequences of the R5 tropic HIV-1 ADA strain. The exchange of the extended V3 region contains two short linear elements of the discontinuous CD4 binding site (see also Additional file 1 and 2). (B, C) SDS-PAGE analysis of purified NL4-3 and NL4-3/ADA gp140 trimers under non-reducing (B) and reducing conditions (+50 mM DTT) (C).
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Figure 1: Scheme of the NL4-3 and NL4-3/ADA constructs and protein analysis by SDS-PAGE. (A) Scheme of gp140 expression constructs indicating cloning of the HIV-1 NL4-3 Env ectodomain with tPA leader sequence and deleted cleavage site. The hybrid construct NL4-3/ADA was generated by exchanging the V3 domain in conjunction with adjacent constant regions of the X4 tropic NL4-3 construct with the respective sequences of the R5 tropic HIV-1 ADA strain. The exchange of the extended V3 region contains two short linear elements of the discontinuous CD4 binding site (see also Additional file 1 and 2). (B, C) SDS-PAGE analysis of purified NL4-3 and NL4-3/ADA gp140 trimers under non-reducing (B) and reducing conditions (+50 mM DTT) (C).

Mentions: Two uncleaved gp140 constructs were generated, NL4-3 gp140 derived from the X4 prototypic HIV-1 subtype B strain NL4-3 and a hybrid mutant (NL4-3/ADA) with a V3 spanning region exchanged for that of the R5 subtype B strain ADA (Figure 1A). The exchanged region comprises the complete V3 loop as well as adjacent elements of C2 (45 aa) and C3 (14 aa). This strategy enables V3 presentation in its parental context, preserves all N-glycosylation sites as well as adjacent CD4bs elements, whereas the exchange of threonine for asparagine at position 277 marginally affects the epitopes of some CD4bs mAbs (see sequence alignment and epitopes in Additional files 1 and 2). The recombinantly expressed gp140 proteins were purified with at least two sequential purification steps to obtain pure gp140 trimers devoid of monomers and dimers (Figure 1B, Additional file 3A,B). Gp140 trimers are composed of gp140 monomers that migrate around 180 kDa upon DTT treatment in SDS-PAGE (Figure 1C). The uncleaved R5 ADA gp140 construct was biochemically analyzed in detail [17], and was also highly purified in its trimeric form for comparison (see Additional file 3C).


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)

Scheme of the NL4-3 and NL4-3/ADA constructs and protein analysis by SDS-PAGE. (A) Scheme of gp140 expression constructs indicating cloning of the HIV-1 NL4-3 Env ectodomain with tPA leader sequence and deleted cleavage site. The hybrid construct NL4-3/ADA was generated by exchanging the V3 domain in conjunction with adjacent constant regions of the X4 tropic NL4-3 construct with the respective sequences of the R5 tropic HIV-1 ADA strain. The exchange of the extended V3 region contains two short linear elements of the discontinuous CD4 binding site (see also Additional file 1 and 2). (B, C) SDS-PAGE analysis of purified NL4-3 and NL4-3/ADA gp140 trimers under non-reducing (B) and reducing conditions (+50 mM DTT) (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Scheme of the NL4-3 and NL4-3/ADA constructs and protein analysis by SDS-PAGE. (A) Scheme of gp140 expression constructs indicating cloning of the HIV-1 NL4-3 Env ectodomain with tPA leader sequence and deleted cleavage site. The hybrid construct NL4-3/ADA was generated by exchanging the V3 domain in conjunction with adjacent constant regions of the X4 tropic NL4-3 construct with the respective sequences of the R5 tropic HIV-1 ADA strain. The exchange of the extended V3 region contains two short linear elements of the discontinuous CD4 binding site (see also Additional file 1 and 2). (B, C) SDS-PAGE analysis of purified NL4-3 and NL4-3/ADA gp140 trimers under non-reducing (B) and reducing conditions (+50 mM DTT) (C).
Mentions: Two uncleaved gp140 constructs were generated, NL4-3 gp140 derived from the X4 prototypic HIV-1 subtype B strain NL4-3 and a hybrid mutant (NL4-3/ADA) with a V3 spanning region exchanged for that of the R5 subtype B strain ADA (Figure 1A). The exchanged region comprises the complete V3 loop as well as adjacent elements of C2 (45 aa) and C3 (14 aa). This strategy enables V3 presentation in its parental context, preserves all N-glycosylation sites as well as adjacent CD4bs elements, whereas the exchange of threonine for asparagine at position 277 marginally affects the epitopes of some CD4bs mAbs (see sequence alignment and epitopes in Additional files 1 and 2). The recombinantly expressed gp140 proteins were purified with at least two sequential purification steps to obtain pure gp140 trimers devoid of monomers and dimers (Figure 1B, Additional file 3A,B). Gp140 trimers are composed of gp140 monomers that migrate around 180 kDa upon DTT treatment in SDS-PAGE (Figure 1C). The uncleaved R5 ADA gp140 construct was biochemically analyzed in detail [17], and was also highly purified in its trimeric form for comparison (see Additional file 3C).

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