Cryoelectron Tomographic Analysis of an HIV-neutralizing Protein and Its Complex with Native Viral gp120

Abstract

Identifying structural determinants of human immunodeficiency virus (HIV) neutralization is an important component of rational drug and vaccine design. We used cryoelectron tomography and atomic force microscopy to characterize the structure of an extremely potent HIV-neutralizing protein, D1D2-Ig␣tp (abbreviated as D1D2-IgP), a polyvalent antibody construct that presents dodecameric CD4 in place of the Fab regions. We show that D1D2-IgP has a novel structure, displaying greater flexibility of its antibody arms than the closely related IgM. Using simian immunodeficiency virus in complex with D1D2-IgP, we present unequivocal evidence that D1D2-IgP can cross-link surface spikes on the same virus and on neighboring viruses. The observed binding to the viral envelope spikes is the result of specific CD4-gp120 interaction, because binding was not observed with MICA-IgP, a construct that is identical to D1D2-IgP except that major histocompatibility complex Class I-related Chain A (MICA) replaces the CD4 moiety. CD4-mediated binding was also associated with a significantly elevated proportion of ruptured viruses. The ratio of inactivated to CD4-liganded gp120-gp41 spikes can be much greater than 1:1, because all gp120-gp41 spikes on the closely apposed surfaces of cross-linked viruses should be incapable of accessing the target cell surface and mediating entry, as a result of inter-virus spike cross-linking. These results implicate flexibility rather than steric bulk or polyvalence per se as a structural explanation for the extreme potency of D1D2-IgP and thus suggest polyvalence presented on a flexible scaffold as a key design criterion for small molecule HIV entry inhibitors. Human immunodeficiency virus type 1 (HIV-1)3 infection of target cells is initiated by binding of the viral envelope glycoprotein gp120 to the cell surface receptor CD4 (1-3). The first step in the infection process, gp120-CD4 binding is an attractive drug target because the CD4 binding site of gp120 is well conserved and because de novo infection of target cells would be blocked, preventing all subsequent stages of the viral life cycle. No therapeutic agents that target gp120-CD4 binding are currently available, although certain small molecules are in clinical trials (reviewed in Ref. 4).Soluble monomeric CD4 (sCD4) neutralizes primary isolates poorly at pharmacologically realizable concentrations (5) and is therefore not useful as a therapeutic agent against HIV-1. Minimally passaged clinical primary isolates, which best model the in vivo virus, are unaffected by physiologically sustainable concentrations of sCD4 in infectivity assays (6). Recent reports suggest that the sCD4-induced conformational fixation in gp120 (7-9) makes sCD4 binding energetically unfavorable (6).In contrast to the soluble monomeric form, CD4 on the cell surface is thought to be clustered (10, 11). The clustered CD4 may mediate an avidity effect, so that soluble CD4 competes poorly with target cell CD4 for viral gp120 (6). To mimic the clustering of cell surface CD4, Ar...

Exceptionally Potent and Broadly Cross-Reactive, Bispecific Multivalent HIV-1 Inhibitors Based on Single Human CD4 and Antibody Domains

“…Each D1D2Ig-␣tp molecule carries Ն12 D1D2 units and has molecular sizes ranging from 600 to 1,200 kDa, with a 12-nm average hydrodynamic radius. Cryo-electron tomographic analysis showed that D1D2Ig-␣tp cross-links the viral spikes on the same virus particle and on neighboring viruses, resulting in inactivation of not only the bound spikes but also those on the closely apposed surfaces of cross-linked viruses (65). The novel mode of action leads to the very potent neutralizing activity (IC 90 s, Ͻ3 nM) of D1D2Ig-␣tp (66).…”

Intraprotomer masking of third variable loop (V3) epitopes by the first and second variable loops (V1V2) within the native HIV-1 envelope glycoprotein trimer

“…1, 8, and 37). Although the Env trimer has not yet proven amenable to X-ray crystallography, recent cryoelectron tomography studies have yielded ultrastructural images of the native Env trimers in unliganded, CD4-bound, and antibody-bound forms (29,(38)(39)(40)(41)(42)(43)(44). Differences in the reconstructed images and in the fitting of X-ray crystallographic structures of monomeric forms of gp120 into trimer models have led to evolving interpretations.…”

Electron tomography of viruses

“…The examples presented are segmented renderings of the 3-D structures of (a) herpes simplex virus (HSV) capsid [6], (b) simian immunodeficiency virus [7], (c) influenza virus [20], (d-f) views of the herpesvirus capsid portal vertex from three different groups: a cross-sectional rendering (d) highlighting the unique portal vertex in HSV-1 by Chang et al [24], rendering of surface views (e) of portal (top) and non-portal vertices (bottom), also of HSV-1 by Cardone et al [23] and views of the Kaposi's sarcoma-associated herpesvirus (f) with non-portal penton vertex (left) and portal (right) vertex shown by Deng et al [25], (g) averaged structure of the surface spike from moloney murine leukemia virus [5] and (h,i) divergent, averaged 3D structures of the surface spike on SIV derived from the work of Zhu et al [10] and Zanetti et al [9]. Cryo-electron tomography of SIV complexed with the potent neutralizing protein D1D2-IgP [7,33]. Electron tomographic analysis of the contact between HIV and T-cells.…”

“…One example where tomography has proved to be useful has been reported in a recent study of the binding of a highly potent neutralizing antibody construct to native gp120 on immunodeficiency viruses. Bennett et al [33] have shown that the potent HIV neutralizing antibody construct D1D2-IgP works by bridging neighboring spikes on the same virus and spikes on neighboring viruses by virtue of its flexible, polyvalent structure, suggesting that spike cross-linking is likely to be central to the mechanism of neutralization by compounds with these characteristics (fig. 3).…”