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 continued to make significant progress towards these goals over the last year. HIV-1 infection begins with the binding of trimeric viral envelope glycoproteins (Env) to CD4 and a co-receptor on target T-cells. Understanding how these ligands influence the structure of Env is of fundamental interest for HIV vaccine development. Using cryo-electron microscopy, we first described the dramatically different structural outcomes of trimeric Env binding to soluble CD4, to the broadly neutralizing, CD4-binding site antibodies VRC01, VRC03 and b12, or to the monoclonal antibody 17b, a co-receptor mimic. Binding of trimeric HIV-1 BaL Env to either soluble CD4 or, surprisingly, 17b alone, is sufficient to trigger formation of the open quaternary conformation of Env. In contrast, VRC01 locks Env in the closed state, while b12 binding requires a partial opening in the quaternary structure of trimeric Env. Our results have shown that, despite general similarities in regions of the HIV-1 gp120 polypeptide that contact CD4, VRC01, VRC03 and b12, there are important differences in quaternary structures of the complexes these ligands form on native trimeric Env, and potentially explain differences in the neutralizing breadth and potency of antibodies with similar specificities. Using the knowledge that VRC03 and 17b binding stabilize the closed and open Env conformations, we carried out cryo-electron microscopic analysis of a cleaved, soluble version of trimeric Env in both the closed, pre-fusion and open, activated conformations to attain higher resolution. These structures of the closed and open Env states, at resolutions of 6 Angstrom and 9 Angstrom respectively, establish that a principal structural signature of Env trimer structure is a three-helix motif composed of alpha-helical segments derived from highly conserved, non-glycosylated N-terminal regions of the gp41 trimer. The three N-terminal helices in this novel, activated Env conformation are much less compactly packed than in the post-fusion, six-helix bundle state. We show that three gp41 helices at the core of the trimer serve as an anchor around which the rest of Env is reorganized upon activation to the open quaternary conformation. The architecture of trimeric HIV-1 Env in pre-fusion and activated intermediate states resembles the corresponding states of influenza hemagglutinin trimers, providing direct evidence for the similarity in entry mechanisms employed by HIV-1, influenza and related enveloped viruses. These findings suggest a new structural template for designing immunogens that can elicit antibodies targeting HIV at a vulnerable, pre-entry stage. The extensive carbohydrate coat and protein structural features on HIV-1 envelope glycoproteins (Env), and the steric constraints of the virus-cell interface during infection, present challenges to the elicitation of effective full-length ( 150 kDa), neutralizing antibodies against HIV. This has motivated the engineering of smaller antibody derivatives that can bind Env and neutralize the virus. To further understand the mechanisms by which these proteins neutralize HIV-1, we carried out cryo-electron tomography of native HIV-1 BaL virions complexed separately to two small ( 15 kDa) HIV-neutralizing proteins: A12, which binds the CD4-binding site on Env, and m36, whose binding to Env is enhanced by CD4 binding. We show that despite their small size, the presence of these proteins and their effects on the quaternary conformation of trimeric Env can be visualized in molecular structures derived by cryo-electron tomography combined with sub-volume averaging. Binding of Env to A12 results in a conformational change that is comparable to changes observed upon its binding to the CD4-binding site antibody, b12. In contrast, binding of Env to m36 results in an """"""""open"""""""" quaternary conformation similar to that seen with binding of soluble CD4 or the CD4i antibody, 17b. Because these small neutralizing proteins are less sterically hindered than full-length antibodies at zones of virus-cell contact, the finding that their binding has the same structural consequences as that of other broadly neutralizing antibodies highlights their potential for use in therapeutic applications. In a related study, we reported cryo-electron microscopic analysis of the interaction between the ectodomain of trimeric HIV-1 envelope glycoprotein (Env) and Z13e1, a broadly neutralizing antibody that targets the membrane proximal external region (MPER) of the gp41 subunit. We showed that Z13e1-bound Env displays an open quaternary conformation similar to the CD4-bound conformation, supporting the idea that MPER directed antibodies, such as Z13e1, block viral entry by interacting with Env at a step after CD4 activation. In addition to studies of the structural aspects of the interaction of HIV with cellular receptors, we have also investigated the mechanisms underlying cell-cell spread of the virus. HIV transmission efficiency is greatly increased when viruses are transmitted at virological synapses formed between infected and uninfected cells. We have previously shown that virological synapses formed between HIV-pulsed mature dendritic cells (DCs) and uninfected T cells contain interdigitated membrane surfaces, with T cell filopodia extending toward virions sequestered deep inside invaginations formed on the DC membrane. To explore membrane structural changes relevant to HIV transmission across other types of intercellular conjugates, we used a combination of light and focused ion beam scanning electron microscopy (FIB-SEM) to determine the 3D architectures of contact regions between HIV-1 infected CD4+ T cells and either uninfected human CD4+ T cells or human fetal astrocytes. We present evidence that in each case, membrane extensions that originate from the uninfected cells, either as membrane sheets or filopodial bridges, are present and may be involved in HIV transmission from infected to uninfected cells. We show that individual virions are distributed along the length of astrocyte filopodia, suggesting that virus transfer to the astrocytes is mediated, at least in part, by processes originating from the astrocyte itself. Mechanisms that selectively disrupt the polarization and formation of such membrane extensions could thus represent a possible target for reducing viral spread.
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