Knowledge of the molecular structure of trimeric Env on intact viruses and delineating the mechanisms of transmission are central to the design of effective immunogens and therapeutic agents to combat HIV/AIDS. In addition, related enveloped viruses such as influenza and Ebola may share similar mechanisms for viral entry, and as such structural studies of these viruses may offer insight towards vaccine design for all three of these viruses. We have continued to make significant progress towards these goals over the last year. In addition to the work on HIV, we have extended the tomographic methods we have developed to study structures of glycoproteins displayed on enveloped viruses such as influenza and Ebola. Our studies highlight the basic similarities in the envelope glycoproteins structures of these viruses, which also potentially share similar mechanisms for cellular entry. The Ebola virus is an emerging pathogen that has become a critical target for vaccine and therapeutic development. Ebola displays many copies of a single complex, the envelope glycoprotein, on the surface of mature virions. Broadly neutralizing antibodies directed at the envelope glycoprotein have proven effective in preventing viral fusion; however, the precise binding and mechanism of action of these antibodies has been poorly understood. Using cryo-electron tomography, we undertook a structural study of the Zmapp antibody cocktail, which is composed of the c2G4, c4G7 and c13C6 antibodies, bound to native, full-length Ebola envelope glycoprotein from the West African 2014 isolate embedded in filamentous viral-like particles. Our tomographic studies revealed that while c13C6 binds to the top of the envelope glycoprotein spike, c2G4 and c4G7 bind to the base, and can potentially bridge multiple spikes. These latter antibodies don't appear to impede receptor binding, but they do appear to inhibit viral entry by preventing the required conformational changes of the envelope glycoprotein spike. Several years ago, we reported the first determination of the structure of the native influenza HA trimer on the 2009 pandemic strain bound to the neutralizing antibody C179 using cryo-electron tomography. This year, we have expanded upon our work on the influenza HA trimer, exploring the structural differences between the native trimer and chimeric HA species that have been posited as potential candidates as immunogens for a universal influenza vaccine. While most neutralizing antibodies directed towards influenza bind the HA head domain, this region is highly variable, leading to poor cross-reactivity between strains. In contrast, the stalk domain of the HA trimer is relatively highly conserved, making this region an attractive target for vaccine development. In order to select for antibodies directed against the stalk region, it has been proposed that one could create chimeric structures of HA, pairing the stalk region of one strain with the head domain of another. We undertook a comparative structural study of native HA from H1N1 with a chimeric HA composed of the head domain from H5N1 and the stalk from H1N1 (cH5/1N1), each expressed on the surface of viral-like particles. Interestingly, we found that cH5/1N1 HA timers were structural different than native H1N1 HA trimers, with a 60 degree rotation of the head domain. This leads to a more open conformation, suggesting that interactions between the head and stalk domains conserve trimer structure. Our tomographic studies show that despite the change in conformation, chimeric HA molecules retain the ability to bind neutralizing antibodies, suggesting that the variability in spike structure we observed does not prevent neutralization.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010825-10
Application #
9343714
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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
Earl, Lesley A; Subramaniam, Sriram (2016) Cryo-EM of viruses and vaccine design. Proc Natl Acad Sci U S A 113:8903-5
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
Frank, Gabriel A; Narayan, Kedar; Bess Jr, Julian W et al. (2015) Maturation of the HIV-1 core by a non-diffusional phase transition. Nat Commun 6:5854
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
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
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
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
Earl, Lesley A; Lifson, Jeffrey D; Subramaniam, Sriram (2013) Catching HIV 'in the act' with 3D electron microscopy. Trends Microbiol 21:397-404

Showing the most recent 10 out of 33 publications