One of the main mechanisms by which viruses gain entry to mammalian cells is through the recognition of specific mammalian cellular receptors by the virus’ “entry spike”. Understanding the structure of these “entry spike” protein complexes is important both for understanding the molecular mechanisms underlying virus-cell interactions and for the design of effective immunogens to combat diseases such as HIV/AIDS, influenza, Ebola, and other viruses. A video describing much of what we know about HIV viral entry is available here.
One highlight of our work on viruses has been the study of the envelope glycoprotein (the “entry spike”) of HIV-1, and its relative SIV, by cryo-electron microscopy. Our early work on the HIV and SIV envelope glycoproteins (Env) revealed that the Env trimer undergoes a major structural reorganization upon binding to the cellular receptor CD4. We have since built upon that initial work, undertaking a series of studies of HIV-1 Env bound to a number of neutralizing antibodies. Using cryo-electron tomography, we determined the structures of native HIV-1 Env bound to broadly neutralizing antibodies b12, VRC01, and VRC03, as well as the 17b co-receptor binding site antibody, the membrane proximal external region specific antibody Z13e1, and the small antibody derivative molecules A12 and m36. These antibody binding studies suggested that some strains of HIV (and SIV) can “sample” multiple conformations when unbound, effectively allowing co-receptor binding site antibodies to engage the spike in the absence of the CD4 receptor, while others are more tightly closed, and require receptor binding to achieve the “open” state. These structures also led to the hypothesis that broadly neutralizing antibodies such as VRC01 and VRC03 work by blocking the Env trimer from transitioning to the open state, and thus interfering with co-receptor binding and subsequent viral entry.
Many of the techniques that we developed in our study of the HIV-1 envelope glycoprotein are directly applicable to the study of envelope proteins on the surface of other viruses, such as influenza and Ebola. We applied our tomographic techniques to studying the mechanism of antibody-mediated neutralization of the 2009 H1N1 strain of influenza, determining the structure of the influenza HA trimer in complex with neutralizing antibodies. We followed this with a structural study of a chimeric HA complex, where the head domain from the H5N1 HA protein was paired with the stalk domain from H1N1. These chimeric proteins are designed as vaccine immunogens that could potentially elicit immune responses towards the conserved stalk domain rather than the highly variable head domain. Our structural studies indicate that these chimeric HA proteins fold correctly into a trimer, but have a different twist than native H1N1 or H5N1 proteins.
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, its envelope glycoprotein, on the surface of mature virions. Our initial tomographic studies of the Ebola envelope glyoprotein visualized the large mucin domain at the apex of the trimeric glycoprotein spike, an area thought to be important in immune recognition. Our more recent studies of three neutralizing antibodies included in the ZMapp anti-Ebola therapeutic have shown that one antibody binds to the Ebola envelope glycoprotein mucin cap, while two others recognize the base of the glycoprotein, suggesting that this antibody cocktail works by a multi-pronged attack to prevent viral entry.