Knowledge of the molecular structure of trimeric Env on intact viruses and delineating the mechanisms of cell-cell transmission are central to the design of effective immunogens and therapeutic agents to combat HIV/AIDS. We have taken several important steps over the last year towards these goals. Cell-cell interactions that play a critical role in normal immune system function are exploited by HIV to facilitate its transmission from antigen-presenting cells such as dendritic cells to susceptible target CD4+ T-cells via a specialized structure designated a virological synapse. Using ion abrasion scanning electron microscopy, electron tomography, and super-resolution light microscopy, we analyzed the spatial architecture of cell-cell contacts and distribution of HIV virions at virological synapses formed between T-cells and both mature and immature dendritic cells. We demonstrated the striking envelopment of T-cells by sheet-like membrane extensions derived from mature dendritic cells, resulting in a shielded region for formation of virological synapses. Within the synapse, filopodial extensions emanating from CD4+ T-cells make contact with HIV virions sequestered deep within a 3D network of surface-accessible compartments in the dendritic cell. We show that HIV-1 induces membrane extensions in immature dendritic cells through activation of Cdc42. We demonstrated that these extensions are induced following engagement of DC-SIGN by HIV-1 Env via a cascade that involves Src kinases, Cdc42, Pak1 and Wasp. Silencing of Cdc42 or treatment with a specific Cdc42 inhibitor, Secramine A, dramatically reduced the number of membrane protrusions visualized on the cell surface and decreased HIV-1 transfer via infectious synapses. Ion abrasion scanning electron microscopy of cell-cell contact regions showed that cellular extensions from immature dendritic cells that have the appearance of thin filopodia in thin section images are in fact extended membranous sheets with a narrow cross-section. Our results demonstrate that HIV-1 binding on immature DC enhances the formation of membrane extensions that facilitate HIV-1 transfer to CD4+ T lymphocytes. While there are striking differences in the synapses formed by immature dendritic cells, the common theme of the burial of the site of HIV transfer and the receptor-dependent initiation of virion transfer by T-cells are new discoveries that highlight novel aspects of cell-cell HIV transmission. An immunogen that can stimulate the production of broadly neutralizing antibodies to the HIV-1 envelope glycoproteins (Env) gp120 and gp41 is a major goal in the quest for a HIV/AIDS vaccine. Using cryo-electron tomography combined with subvolume averaging, we have analyzed the structure of SOSIP gp140 trimers, which are cleaved, solubilized versions of the ectodomain of trimeric HIV-1 Env. We show that unliganded gp140 trimers adopt a quaternary arrangement similar to that displayed by native unliganded trimers on the surface of intact HIV-1 virions. When complexed with soluble CD4 (sCD4), Fab 17b, which binds to gp120 at its chemokine co-receptor binding site or both sCD4 and 17b Fab, gp140 trimers display an open conformation in which there is an outward rotation and displacement of each gp120 protomer. We demonstrate that the molecular arrangements of gp120 trimers in the closed and open conformations of the soluble trimer are the same as those observed for the closed and open states, respectively of trimeric gp120 on intact HIV-1 BaL virions, establishing that soluble gp140 trimers can be designed to mimic the quaternary structural transitions displayed by native trimeric Env. The discovery that trimeric gp140 immunogens can mimic functionally relevant Env quaternary structural changes on infectious virions is an important step towards development of an Env-based HIV-1 vaccine, and also represents an important technical advance in the use of tomographic methods to determine the structures of protein complexes. Our previous studies on cryo-electron tomography of HIV-1 established several basic features of the function of trimeric Env and the conformational changes associated with CD4 binding. We have extended these investigations to a variety of SIV and HIV-1 strains, including some that were part of earlier controversies in the field about spike structure. Our results showed unequivocally that the molecular architectures of trimeric Env from SIVmneE11S and SIVmac239 strains are closely comparable to that previously determined for HIV-1 BaL, with the V1 and V2 variable loops located at the apex of the spike, close to the contact zone between virus and cell. The location of the V1/V2 loops was definitively confirmed by structural analysis of trimeric Env in an HIV-1 strain with deletions of these loops. Strikingly, in SIV CP-MAC, a CD4-independent strain, trimeric Env is in a constitutively open conformation with gp120 trimers splayed out in a conformation similar to that seen for HIV-1 BaL Env when it is complexed with sCD4 and the CD4i antibody 17b. Our findings suggest for the first time a structural explanation for the molecular mechanism of CD4-independent viral entry and further establish that cryo-electron tomography can be used to discover distinct, functionally relevant quaternary structures of Env displayed on intact viruses.
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