We have taken several steps last year towards our goals of understanding structural aspects of HIV entry and neutralization. One important advance we have made is towards understanding viral entry better with our discovery of the HIV entry claw. We have recently discovered that the initial contact of SIV or HIV with T-lymphocytes occurs via formation of a viral entry claw that is composed of about 5-7 anchors ( 100 wide and 100 long) spanning the space between the membranes, with an average center-to-center spike spacing of about 150 . The entry claw formed between wild-type HIV-1 viruses and T-cells display the same architectural features observed for SIV viruses interacting with T-cells, implying that the similar structural and biochemical mechanisms are likely to underlie entry of these viruses into human and simian T-cells, respectively. Our working hypothesis is that each of the anchors is derived from a single viral spike, although we cannot yet be sure about its molecular composition. The observed average length ( 100 ) of the rods of density connecting the viral and cell membranes is consistent with the expected dimensions of a potentially fully extended state of TM, representing the pre-hairpin intermediate proposed almost a decade ago. (see Sougrat et al (2007) for more details). A second advance we have made concerns identification of structural determinants of HIV neutralization, as relevant to rational drug and vaccine design. We used cryo electron 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 (SIV) in complex with D1D2-IgP, we present unequivocal evidence that D1D2-IgP can crosslink 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, since binding was not observed with MICA-IgP, a construct that is identical to D1D2-IgP, except that MHC 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 crosslinked viruses should be incapable of accessing the target cell surface and mediating entry, as a result of inter-virus spike crosslinking. 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. (see Bennett et al (2007) for more details). A third advance we have made concerns description of the structure of trimeric gp120 on the surface of the viral membrane. The envelope glycoproteins (Env) of human and simian immunodeficiency viruses (HIV and SIV, respectively) mediate virus binding to the cell surface receptor CD4 on target cells to initiate infection. Env is a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120), and forms trimers on the surface of the viral membrane. Using cryo-electron tomography combined with 3D image classification and averaging, we have reported the three-dimensional structures, at resolutions of 20 , of trimeric Env displayed on native HIV-1 in the unliganded state, in complex with the broadly neutralizing antibody b12 and in a ternary complex with CD4 and the 17b antibody. By fitting the known crystal structures of the monomeric gp120 core in the b12- and CD4/17b-bound conformations into the density maps derived by electron tomography, we derive molecular models for the native HIV-1 gp120 trimer in unliganded and CD4-bound states. We demonstrate that CD4 binding results in a major reorganization of the Env trimer, causing an outward rotation and displacement of each gp120 monomer. This appears to be coupled with a rearrangement of the gp41 region along the central axis of the trimer, leading to closer contact between the viral and target cell membranes. Our findings elucidate the structure and conformational changes of trimeric HIV-1 gp120 relevant to antibody neutralization and attachment to target cells. (see Liu et al (2008) and Subramaniam et al (2007) for more details).
| Bennett, Adam; Liu, Jun; Van Ryk, Donald et al. (2007) Cryoelectron tomographic analysis of an HIV-neutralizing protein and its complex with native viral gp120. J Biol Chem 282:27754-9 |
