Influenza A and B viruses cause important diseases in humans and animals, with tremendous socioeconomic consequences, for influenza continues to occur in regular epidemics and occasional pandemics and is a leading cause of morbidity and mortality. Paramyxoviruses cause many biologically and economically important diseases of humans and lower animals. These viruses include parainfluenza virus (PIV) types 1–5, mumps virus, measles virus, respiratory syncytial virus, canine distemper virus, Newcastle disease virus of chickens, and rinderpest of cattle.
Virus-Mediated Cell-to-Cell Fusion
Paramyxoviruses enter cells by fusion of the viral envelope with the cellular plasma membrane, and the viral fusion (F) protein mediates this process. The initial folding of the F protein causes it to be trapped energetically in a metastable form. Activation of the F protein for fusion activity requires coexpression of the viral receptor-binding protein, hemagglutinin-neuraminidase (HN). When HN binds its receptor, sialic acid, HN undergoes a conformational change, which in turn causes a further conformational change in the F protein. Finally, refolding of the F protein and the formation of a six-helix bundle (6HB; core trimer) cause the merger of the cell target membrane with the viral membrane. We are using a combination of cellular assays, biophysical measurements, and microscopic assays, as well as protein structural determination at the atomic level, to approach this problem.
We have hypothesized that the F protein is a biological nanomachine that undergoes a series of conformational changes and is controlled such that fusion only occurs at the right time and in the right place.
Recently we determined the crystal structure of both the prefusion and postfusion forms of the paramyxovirus F protein (in collaboration with Theodore Jardetzky, now at Stanford University). We solved the prefusion F structure for PIV5 and the postfusion F structure for human (h) PIV3. In large part the structures are thought to be representative of the F structures of all paramyxoviruses. The prefusion F structure contains a globular head attached to a trimeric coiled-coil stalk formed by the C-terminal heptad repeat B (HRB) region. The globular head contains three domains (DI–DIII). The fusion peptides at the N terminus of the heptad repeat A (HRA) region are sequestered between adjacent subunits, with a cleavage/activation site exposed at the protein surface.
The postfusion F forms a trimer, which reveals a globular, predominantly β-sheet-containing head domain, a neck region formed by both β sheet and α helices, and a stalk region that is predominantly α helical. The structure contains the 6HB expected of the postfusion conformation of the protein.
The PIV5 prefusion F and hPIV3 postfusion F structures are in strikingly different conformations, consistent with a transition from pre- to postfusion forms. We have observed related forms of the F protein in electron micrographs of F. None of the intersubunit contacts are conserved in the pre- and postfusion forms. The two F structures are related by flipping the stalk and transmembrane domains relative to the F head.
The conformational change requires the opening and translocation of the HRB stalk. In the prefusion form, HRB is located at the base of the head region. During the conversion to the postfusion conformation, HRB segments must separate and swing around the base of the head, to pack against the HRA coiled coil. In the prefusion conformation, HRA is broken up into four helices, two β strands, and five loop, kink, or turn segments. Thus, the conformational changes in HRA involve the refolding of 11 distinct segments into a single, extended α-helical conformation.
The prefusion F structure provides a model for the stepwise induction of membrane fusion by paramyxoviruses and reveals how multiple sequence elements play distinct structural roles in the pre- and postfusion conformations.
We also solved (in collaboration with the Jardetzky lab) the atomic structure of the tetrameric paramyxovirus HN protein to a resolution of 2.8 Å. We propose a model based on this structure for HN involvement in membrane fusion that is consistent with the available data and that involves ligand-dependent changes in the HN oligomer that are driven by surface:surface interactions. In this model, the HN dimer/tetramer forms in the absence of ligands and can interact with the F protein, potentially through lateral interactions on two sides of the tetramer. Engagement of cell surface receptors could trigger the partial disassembly of the HN tetramer, assuming that the energy of binding of the individual HN sites to distinct sialic receptors is sufficient to perturb the weak neuraminidase domain interactions. Opening of the tetrameric head, driven by the energy of receptor engagement, could lead to changes in both the HN stalk region (the HN stalk domain is thought to interact with F) and the interaction with F, thus activating F for membrane fusion.
Structure-Function Analysis of the Influenza Virus Ion Channel
Influenza virus protein M2 is a small (96-residue) integral membrane protein that spans the cell membrane once and is a disulfide-linked homotetramer. The M2 protein acts as an ion channel during the virus-uncoating process in endosomes, permitting a flow of protons into the interior of virus particles to disrupt protein-protein interactions. The M2 ion channel is specifically inhibited by the antiviral drug amantadine, and the M2 protein channel activity is activated by low pH, suggesting that the channel is only switched on in endosomes and the trans-Golgi network—intracellular compartments with lowered pH. In collaboration with Lawrence Pinto (Northwestern University), we showed that the M2 protein has ion channel activity in both mammalian cells and Xenopus laevis oocytes. Recently, we have found that the influenza B virus BM2 protein is a proton-selective ion channel.
Because the M2 protein ion channel is distinct in structure from almost all other ion channels and because of its simplicity of size, it provides a marvelous opportunity to understand how an ion channel functions. We are performing a detailed structure-function analysis of this channel.
Enveloped Virus Assembly
We are investigating the nature of the molecular interactions that are necessary to form a virus particle. The application of reverse genetics—i.e., the ability to rescue infectious influenza virus and paramyxoviruses from cloned DNA—facilitates these studies. We are using an in vitro budding assay that enables us to determine the molecular requirements for virus assembly. We are also determining the possible role of monoubiquitination of viral proteins in correct assembly of cellular protein complexes that may mediate viral budding. Recently, we have identified domains know as "late domains" in the paramyxovirus PIV5 matrix protein that control the budding process (in collaboration with Wesley Sundquist, University of Utah). We have also shown that influenza viruses bud from specialized regions of the plasma membrane that are enriched in cholesterol and sphingomyelin. These patches of membrane (related to rafts) were visualized by immunogold labeling and three-dimensional electron microscopy reconstructions.