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, two of which are summarized below. 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 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 show 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. From cryo-electron microscopic analysis at 9 Angstrom resolution of a cleaved, soluble version of trimeric Env, we show that a structural signature of the open Env conformation 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. 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 showed 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.

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Tran, Erin E H; Podolsky, Kira A; Bartesaghi, Alberto et al. (2016) Cryo-electron Microscopy Structures of Chimeric Hemagglutinin Displayed on a Universal Influenza Vaccine Candidate. MBio 7:e00257
Tran, Erin E H; Nelson, Elizabeth A; Bonagiri, Pranay et al. (2016) Mapping of Ebolavirus Neutralization by Monoclonal Antibodies in the ZMapp Cocktail Using Cryo-Electron Tomography and Studies of Cellular Entry. J Virol 90:7618-27
Earl, Lesley A; Subramaniam, Sriram (2016) Cryo-EM of viruses and vaccine design. Proc Natl Acad Sci U S A 113:8903-5
Moulaei, Tinoush; Alexandre, Kabamba B; Shenoy, Shilpa R et al. (2015) Griffithsin tandemers: flexible and potent lectin inhibitors of the human immunodeficiency virus. Retrovirology 12:6
Tran, Erin E H; Simmons, James A; Bartesaghi, Alberto et al. (2014) Spatial localization of the Ebola virus glycoprotein mucin-like domain determined by cryo-electron tomography. J Virol 88:10958-62
Do, Thao; Murphy, Gavin; Earl, Lesley A et al. (2014) Three-dimensional imaging of HIV-1 virological synapses reveals membrane architectures involved in virus transmission. J Virol 88:10327-39
Narayan, Kedar; Danielson, Cindy M; Lagarec, Ken et al. (2014) Multi-resolution correlative focused ion beam scanning electron microscopy: applications to cell biology. J Struct Biol 185:278-84
Merk, Alan; Subramaniam, Sriram (2013) HIV-1 envelope glycoprotein structure. Curr Opin Struct Biol 23:268-76
Harris, Audray K; Bartesaghi, Alberto; Milne, Jacqueline L S et al. (2013) HIV-1 envelope glycoprotein trimers display open quaternary conformation when bound to the gp41 membrane-proximal external-region-directed broadly neutralizing antibody Z13e1. J Virol 87:7191-6
Meyerson, Joel R; Tran, Erin E H; Kuybeda, Oleg et al. (2013) Molecular structures of trimeric HIV-1 Env in complex with small antibody derivatives. Proc Natl Acad Sci U S A 110:513-8

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