Structural Characterization of HIV gp41 with the Membrane-proximal External Region

Abstract

Human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein (gp120/gp41) plays a critical role in virus infection and pathogenesis. Three of the six monoclonal antibodies considered to have broadly neutralizing activities (2F5, 4E10, and Z13e1) bind to the membrane-proximal external region (MPER) of gp41. This makes the MPER a desirable template for developing immunogens that can elicit antibodies with properties similar to these monoclonal antibodies, with a long term goal of developing antigens that could serve as novel HIV vaccines. In order to provide a structural basis for rational antigen design, an MPER construct, HR1-54Q, was generated for x-ray crystallographic and x-ray footprinting studies to provide both high resolution atomic coordinates and verification of the solution state of the antigen, respectively. The crystal structure of HR1-54Q reveals a trimeric, coiled-coil six-helical bundle, which probably represents a postfusion form of gp41. The MPER portion extends from HR2 in continuation of a slightly bent long helix and is relatively flexible. The structures observed for the 2F5 and 4E10 epitopes agree well with existing structural data, and enzyme-linked immunosorbent assays indicate that the antigen binds well to antibodies that recognize the above epitopes. Hydroxyl radical-mediated protein footprinting of the antigen in solution reveals specifically protected and accessible regions consistent with the predictions based on the trimeric structure from the crystallographic data. Overall, the HR1-54Q antigen, as characterized by crystallography and footprinting, represents a postfusion, trimeric form of HIV gp41, and its structure provides a rational basis for gp41 antigen design suitable for HIV vaccine development.The HIV 2 glycoprotein is initially expressed as the precursor protein gp160, which is post-translationally cleaved into two non-covalently associated proteins: gp120, the receptor-binding protein that targets the CD4 receptor, and gp41, which mediates the fusion of the viral and host cellular membranes (1, 2). The native, prefusion form of the gp120-gp41 complex is thought to be a trimer comprising three gp120 subunits and three membrane-anchored gp41 subunits and is in a metastable conformation with the heavily glycosylated gp120 shielding gp41 (3, 4). Upon binding of gp120 to CD4 and a co-receptor (CCR5 or CXCR4), gp41 undergoes a large conformational change to form the prehairpin fusion intermediate in which the N-terminal and C-terminal helices separate from each other and the fusion peptide extends toward the host membrane (1, 5). This ultimately leads to the fusion of the viral and host membranes and leaves a postfusion form of gp41 with a structure of hairpins in a trimer.Several crystal structures of HIV-1 and simian immunodeficiency virus gp41 have been reported (6 -9). All structures contain the gp41 core, which is composed of the two heptad repeat regions, the N-terminal helical heptad repeat (HR1) and the C-terminal helical heptad repeat (HR2). The largest structur...

Targeting HIV-1 gp41 in Close Proximity to the Membrane Using Antibody and Other Molecules

“…Shi and colleagues [49] determined a high-resolution crystal structure of a trimeric form of gp41 consisting of the HR1, HR2 and the MPER region (HR1-54Q), which binds strongly to 2F5, 4E10 and Z13e1 as well as to an HR2-specific antibody 98-6. In a preliminary animal immunization study, HR1-54Q elicited binding antibodies, but neutralization titers were at best moderate [49]. Nevertheless, these more complete gp41 structures provide a basis to test hypotheses about how MPER structure transitions from the untriggered, through to the receptor-activated and post-fusion forms of gp41.…”

“…helix bundle containing: N-terminal MPER residues (E 659 LLELDK 665 )[185]; extended MPER[49]; extended MPER plus fusion peptide[48] Putative prefusion trimeric state of the MPER; 2-stranded antiparallel alpha-helices in a putative fusion intermediate state…”

Carbohydrate-functionalized nanovaccines preserve HIV-1 antigen stability and activate antigen presenting cells

“…Presently, one of the main foci in the development of efficacious HIV-1 vaccines is the use of recombinant proteins as antigens [16,17,26]. Even though these antigens are safe, they are poorly immunogenic, and therefore need an adjuvant to elicit a robust immune response [26].…”

“…Presently, one of the main foci in the development of efficacious HIV-1 vaccines is the use of recombinant proteins as antigens [16,17,26]. Even though these antigens are safe, they are poorly immunogenic, and therefore need an adjuvant to elicit a robust immune response [26]. In this regard, biodegradable polyanhydride nanoparticles have shown promise as adjuvants and/or delivery vehicles with beneficial properties, including antigen stabilization, sustained antigen release, APC activation, and immune modulation, that can be exploited to formulate a successful HIV-1 vaccine [27–34].…”

“…In this regard, biodegradable polyanhydride nanoparticles have shown promise as adjuvants and/or delivery vehicles with beneficial properties, including antigen stabilization, sustained antigen release, APC activation, and immune modulation, that can be exploited to formulate a successful HIV-1 vaccine [27–34]. This study focuses on the use of gp41-54Q–GHC as the immunogen (to be described in more detail elsewhere), which contains the Heptad Repeat Region 2 (HR2) and the membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 [26]. Polyanhydride nanoparticles based on sebacic acid (SA) 1,6-bis(p-carboxyphenoxy) hexane (CPH), and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and CPH were used to encapsulate and release stable gp41-54Q–GHC immunogen.…”