Molecular architecture of the uncleaved HIV-1 envelope glycoprotein trimer

Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02215.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 06/2013; 110(30). DOI: 10.1073/pnas.1307382110
Source: PubMed


The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer, a membrane-fusing machine, mediates virus entry into host cells and is the sole virus-specific target for neutralizing antibodies. Binding the receptors, CD4 and CCR5/CXCR4, triggers Env conformational changes from the metastable unliganded state to the fusion-active state. We used cryo-electron microscopy to obtain a 6-Å structure of the membrane-bound, heavily glycosylated HIV-1 Env trimer in its uncleaved and unliganded state. The spatial organization of secondary structure elements reveals that the unliganded conformations of both glycoprotein (gp)120 and gp41 subunits differ from those induced by receptor binding. The gp120 trimer association domains, which contribute to interprotomer contacts in the unliganded Env trimer, undergo rearrangement upon CD4 binding. In the unliganded Env, intersubunit interactions maintain the gp41 ectodomain helical bundles in a "spring-loaded" conformation distinct from the extended helical coils of the fusion-active state. Quaternary structure regulates the virus-neutralizing potency of antibodies targeting the conserved CD4-binding site on gp120. The Env trimer architecture provides mechanistic insights into the metastability of the unliganded state, receptor-induced conformational changes, and quaternary structure-based strategies for immune evasion.

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    • "The gp140 construct from the TCLA X4 strain NL4-3 shows a marked open configuration, which resembles the open architecture of CD4 independent SIV Env [51,52] but differs considerably from published R5 trimer structures derived from HIV-1 subtype B [20,21,24,53,54] (see Additional files 11 and 12). These subtype B R5 structures share a closed Env configuration with a tight assembly of the three gp120 protomers at the top. "
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    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.
    Retrovirology 05/2014; 11(1):42. DOI:10.1186/1742-4690-11-42 · 4.19 Impact Factor
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    • "The results with the I559P mutant described a possible hairpin structure for HR1 (Sanders et al., 2002), and mutations within the N-terminal half of HR2 in our study support the hypothesis that a flexible or even unstructured loop conformation could exist within HR2. Based on these observations and recent high resolution cryo-EM data (Mao et al., 2012, 2013; Tran et al., 2012), we could propose a pre-fusion model for gp41 in which both HR1 and HR2 are broken in the middle (or more sites) and a coordinated loop-to-helix transition is necessary for viral fusion (Fig. 5). HIV gp41 could go through the following steps to drive membrane fusion. "
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    ABSTRACT: HIV CRF07 B'/C is a strain circulating mainly in northwest region of China. The gp41 region of CRF07 is derived from a clade C virus. In order to compare the difference of CRF07 gp41 with that of typical clade B virus, we solved the crystal structure of the core region of CRF07 gp41. Compared with clade B gp41, CRF07 gp41 evolved more basic and hydrophilic residues on its helix bundle surface. Based on sequence alignment, a hyper-mutant cluster located in the middle of HR2 heptads repeat was identified. The mutational study of these residues revealed that this site is important in HIV mediated cell-cell fusion and plays critical roles in conformational changes during viral invasion.
    Virology 11/2013; 446(1-2):86-94. DOI:10.1016/j.virol.2013.07.024 · 3.32 Impact Factor
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    ABSTRACT: The envelope glycoprotein (Env) is the sole antigenic feature on the surface of HIV and the target for the humoral immune system. Soluble, uncleaved gp140 Env constructs truncated at the transmembrane domain are being investigated intensively as potential vaccine immunogens by many groups, and understanding their structural properties is essential. Here we use Hydrogen/Deuterium-exchange mass spectrometry and small-angle X-ray scattering to probe structural order in a panel of commonly used gp140 constructs and matched gp120 monomers. We observe that oligomeric forms of uncleaved gp140, generally presumed to be trimeric, contain a protease-resistant form of gp41 akin to the post-fusion, helical bundle conformation and appear to lack specific interactions between gp120 and gp41. In contrast, the monomeric form of gp140 shows significant stabilization of the gp120 inner domain imparted by the gp41 region, demonstrating excellent agreement with past mutagenesis studies. Moreover, the gp140 monomers respond to CD4 binding in manner that is consistent with the initial stages of Env activation: CD4 binding induces structural ordering throughout gp120 while loosening its association with gp41. The results indicate that uncleaved gp140 oligomers do not represent an authentic pre-fusion, soluble form Env, while gp140 monomers isolated from the same glycoprotein preparations in many ways exhibit function and internal structural order that are consistent with expectations for certain aspects of native Env. gp140 monomers may thus be a useful reagent for advancing structural and functional studies.
    Journal of Virology 08/2013; 87(21). DOI:10.1128/JVI.01681-13 · 4.44 Impact Factor
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