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

Philipp Arnold,Svenja Weiß,Ralf Dürr,Volker Gatterdam,Tim-Michael Decker,Patricia Himmels,Ursula Dietrich,Robert Tampé,Patrick Bartholomäus,Jürgen Markl

doi:10.1186/1742-4690-11-42

Abstract

BackgroundHIV-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.ResultsSoluble 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.Conclusions3D 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.

Highlights

HIV-1 is decorated with trimeric glycoprotein spikes that enable infection by engaging CD4 and a chemokine coreceptor, either CCR5 or CXCR4
Recombinant expression and characterization of NL4-3 and NL4-3/ADA gp140 trimers 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 variable loop 3 (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 CD4 binding site (CD4bs) elements, whereas the exchange of threonine for asparagine at position 277 marginally affects the epitopes of some CD4bs monoclonal antibodies (mAbs)

Summary

Introduction

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 envelope glycoproteins (Env) of HIV are the key elements mediating viral entry and represent the major target for HIV-neutralizing antibodies [1,2]. Env derives from gp160 precursors that trimerize in the endoplasmic According to their coreceptor usage, HIV-1 strains can be subdivided into CCR5- (R5) and CXCR4-tropic (X4) variants. CXCR4 usage is mainly mediated by mutations in the variable loop 3 (V3) of gp120, especially at the V3 stem, providing an increased positive net charge [6,7,8]. Besides coreceptor selection and interaction, V3′s conserved structural constraints render it into one of four Env regions able to induce cross-clade neutralizing antibodies [11,12,13,14]

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Results

Discussion

Conclusion