We have taken several steps towards these goals. 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. 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.
| 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 |
