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      Allosteric Modulation of the HIV-1 gp120-gp41 Association Site by Adjacent gp120 Variable Region 1 (V1) N-Glycans Linked to Neutralization Sensitivity

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          Abstract

          The HIV-1 gp120-gp41 complex, which mediates viral fusion and cellular entry, undergoes rapid evolution within its external glycan shield to enable escape from neutralizing antibody (NAb). Understanding how conserved protein determinants retain functionality in the context of such evolution is important for their evaluation and exploitation as potential drug and/or vaccine targets. In this study, we examined how the conserved gp120-gp41 association site, formed by the N- and C-terminal segments of gp120 and the disulfide-bonded region (DSR) of gp41, adapts to glycan changes that are linked to neutralization sensitivity. To this end, a DSR mutant virus (K601D) with defective gp120-association was sequentially passaged in peripheral blood mononuclear cells to select suppressor mutations. We reasoned that the locations of suppressors point to structural elements that are functionally linked to the gp120-gp41 association site. In culture 1, gp120 association and viral replication was restored by loss of the conserved glycan at Asn 136 in V1 (T138N mutation) in conjunction with the L494I substitution in C5 within the association site. In culture 2, replication was restored with deletion of the N 139INN sequence, which ablates the overlapping Asn 141-Asn 142-Ser-Ser potential N-linked glycosylation sequons in V1, in conjunction with D601N in the DSR. The 136 and 142 glycan mutations appeared to exert their suppressive effects by altering the dependence of gp120-gp41 interactions on the DSR residues, Leu 593, Trp 596 and Lys 601. The 136 and/or 142 glycan mutations increased the sensitivity of HIV-1 pseudovirions to the glycan-dependent NAbs 2G12 and PG16, and also pooled IgG obtained from HIV-1-infected individuals. Thus adjacent V1 glycans allosterically modulate the distal gp120-gp41 association site. We propose that this represents a mechanism for functional adaptation of the gp120-gp41 association site to an evolving glycan shield in a setting of NAb selection.

          Author Summary

          The envelope glycoprotein gp120-gp41 complex of HIV-1 mediates receptor attachment and virus-cell membrane fusion, leading to cellular entry. A shield of asparagine-linked oligosaccharides occludes the gp120-gp41 protein surface and evolution of this glycan shield provides a means for evading circulating neutralizing antibody. Here we examined how conserved structural elements of the glycoprotein complex, in particular the gp120-gp41 association site, retain functionality in the context of glycan shield evolution. This information is important for the evaluation and exploitation of such conserved functional determinants as potential drug and/or vaccine targets. Our data indicate that the loss of either of 2 adjacent glycans in variable region 1 of gp120 leads to changes in local and remote glycan-dependent epitopes and that this is linked to a remodelling of gp120-gp41 interactions in order to maintain a functional gp120-gp41 complex. We propose that this represents a mechanism for the functional adaptation of the gp120-gp41 association site to an evolving glycan shield in a setting of neutralizing antibody selection.

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          Most cited references95

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          Core structure of gp41 from the HIV envelope glycoprotein.

          The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) consists of a complex of gp120 and gp41. gp120 determines viral tropism by binding to target-cell receptors, while gp41 mediates fusion between viral and cellular membranes. Previous studies identified an alpha-helical domain within gp41 composed of a trimer of two interacting peptides. The crystal structure of this complex, composed of the peptides N36 and C34, is a six-helical bundle. Three N36 helices form an interior, parallel coiled-coil trimer, while three C34 helices pack in an oblique, antiparallel manner into highly conserved, hydrophobic grooves on the surface of this trimer. This structure shows striking similarity to the low-pH-induced conformation of influenza hemagglutinin and likely represents the core of fusion-active gp41. Avenues for the design/discovery of small-molecule inhibitors of HIV infection are directly suggested by this structure.
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            Atomic structure of the ectodomain from HIV-1 gp41.

            Fusion of viral and cellular membranes by the envelope glycoprotein gp120/gp41 effects entry of HIV-1 into the cell. The precursor, gp160, is cleaved post-translationally into gp120 and gp41 which remain non-covalently associated. Binding to both CD4 and a co-receptor leads to the conformational changes in gp120/gp41 needed for membrane fusion. We used X-ray crystallography to determine the structure of the protease-resistant part of a gp41 ectodomain solubilized with a trimeric GCN4 coiled coil in place of the amino-terminal fusion peptide. The core of the molecule is found to be an extended, triple-stranded alpha-helical coiled coil with the amino terminus at its tip. A carboxy-terminal alpha-helix packs in the reverse direction against the outside of the coiled coil, placing the amino and carboxy termini near each other at one end of the long rod. These features, and the existence of a similar reversal of chain direction in the fusion pH-induced conformation of influenza virus HA2 and in the transmembrane subunit of Moloney murine leukaemia virus (Fig. 1a-d), suggest a common mechanism for initiating fusion.
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              Mechanisms of viral membrane fusion and its inhibition.

              Viral envelope glycoproteins promote viral infection by mediating the fusion of the viral membrane with the host-cell membrane. Structural and biochemical studies of two viral glycoproteins, influenza hemagglutinin and HIV-1 envelope protein, have led to a common model for viral entry. The fusion mechanism involves a transient conformational species that can be targeted by therapeutic strategies. This mechanism of infectivity is likely utilized by a wide variety of enveloped viruses for which similar therapeutic interventions should be possible.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                April 2013
                April 2013
                4 April 2013
                : 9
                : 4
                : e1003218
                Affiliations
                [1 ]Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Victoria, Australia
                [2 ]Department of Microbiology, Monash University, Clayton, Victoria, Australia
                [3 ]Department of Microbiology and Immunology, The University of Melbourne, Melbourne, Victoria, Australia
                [4 ]Department of Immunology, Monash University, Alfred Medical Research and Education Precinct, Melbourne, Victoria, Australia
                [5 ]Department of Surgery, Austin Health, The University of Melbourne, Heidelberg, Victoria, Australia
                [6 ]Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
                [7 ]Deakin University School of Medicine, Geelong, Victoria, Australia
                Harvard University, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Performed the molecular modelling: PAR PP. Conceived and designed the experiments: HED PP. Performed the experiments: HED MKH ALM SW JM PP. Analyzed the data: HED MKH ALM SW PAR JM PP. Contributed reagents/materials/analysis tools: HED MKH ALM SW PAR JM PP. Wrote the paper: PP HED.

                Article
                PPATHOGENS-D-12-01453
                10.1371/journal.ppat.1003218
                3616969
                23592978
                3dc615fa-2aad-4ed9-80e4-3b2915c34272
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 18 June 2012
                : 15 January 2013
                Page count
                Pages: 18
                Funding
                This work was supported by The National Health and Medical Research Council of Australia (Grants 1009809 and 603735) and its Infrastructure Support Scheme, and the Australian Centre for HIV and Hepatitis Virology Research. HED is a recipient of an R. D. Wright Award (433929). JM is an Australian Research Council Future Fellow. The authors gratefully acknowledge the contribution to this work of the Victorian Operational Infrastructure Support Program received by the Burnet Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Microbiology
                Host-Pathogen Interaction
                Medical Microbiology
                Microbial Pathogens
                Virology
                Medicine
                Infectious Diseases
                Viral Diseases
                HIV
                Retrovirology and HIV immunopathogenesis
                Sexually Transmitted Diseases

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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