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Structural Biology of Viruses and the Human Immune System
Summary: Unitil his recent death, Don Wiley studied the atomic structure and biochemical functions of membrane glycoproteins on viral and human cellular surfaces that are involved in infectivity and human immune responses. His lab continues his work.
My research group studies how proteins from the surfaces of viruses initiate viral infections and how proteins of the human cellular immune system respond by presenting antigens and mobilizing defensive cells. We use x-ray crystallography to determine the atomic structures of proteins isolated from viruses and human cells. The structures provide us with a framework for determining how the molecules carry out their biological functions. We focus on the function of viral membrane glycoproteins, especially their functions in receptor binding, viral entry by membrane fusion, and evolution to evade the host immune response. We study proteins from influenza, HIV-1, herpes, and human respiratory syncytial viruses. We also study the functions of a number of human membrane glycoproteins that are involved in presenting antigens as part of the human cellular immune response, including T cell receptors (TCRs), major histocompatibility complex (MHC) glycoproteins, antigen peptide transporters (TAPs), and the proteins involved in trafficking and loading of antigenic peptides onto MHC molecules (Ii, tapasin, and DM).
In the past year, our studies of virus proteins have included determining the structure of a complex between a human cytomegalovirus protein (US2) and a human class I MHC molecule (with Hidde Ploegh, Harvard Medical School), showing how the virus subverts antigen presentation to avoid destruction by the immune system, and determining the structure of a complex between the HIV-1 glycoprotein responsible for viral entry into cells (gp41) and a synthetic inhibitor of that membrane fusion event that we had discovered earlier in collaboration with Stuart Schreiber and Stephen Harrison (both HHMI, Harvard University). (HIV-1 studies were supported in part by a grant from the National Institutes of Health). We have also found methods for producing large quantities of the soluble trimer of SIV-1 glycoprotein gp140, closely related to the HIV-1 gp140, which is responsible for initiating AIDS infections (in collaboration with Harrison and Ellis Reinherz, Harvard University). (These studies were partially funded by the National Institutes of Health.)
We also determined the three-dimensional structures of avian H5 and swine H9 influenza hemagglutinin glycoproteins (HAs) from viruses closely related to those that caused outbreaks of human disease in Hong Kong in 1997 (which caused 6 fatalities of 18 known cases) and 1999, and bound them to avian and human cell receptor analogs (in collaboration with John Skehel, National Institute for Medical Research, London, and with support from the National Institutes of Health). Emerging influenza pandemics have been accompanied by the evolution of receptor-binding specificity—from the preference of avian viruses for sialic acid receptors in α2,3 linkage to the preference of human viruses for α2,6 linkages. When compared with our previously reported crystal structures of HA/sialoside complexes of the H3 subtype that caused the 1968 Hong Kong influenza virus pandemic, the new structures make clearer how receptor-binding sites of HAs from avian viruses evolve as the virus adapts to humans.
To understand how viruses enter cells by membrane fusion, we have studied influenza, HIV-1, and Ebola virus in the past and found they share many elements of their atomic mechanism. While continuing to study those mechanisms, we have also begun to study human herpes simplex virus and human respiratory syncytial virus (HRSV), which initiate membrane fusion differently. This year we determined the structure of a complex of the herpes glycoprotein gD with one of its cellular receptors, a member of the tumor necrosis factor receptor family (in collaboration with Roselyn Eisenberg and Gary Cohen, University of Pennsylvania; partial funding from the National Institutes of Health). These studies revealed a conformational change induced upon receptor binding, which we are now studying. Herpes viruses are a major human health problem: HSV-1 infections become latent, emerging sporadically to cause oral lesions in humans; HSV-2 causes genital lesions. Glycoprotein gD of herpes virus is required for infection and the cell-to-cell spread involved in emergence from latency. This is the first of the 11 glycoproteins of any herpes virus to be seen at atomic detail. Our studies on HRSV, in collaboration with John Skehel (London) and Jose Molero (Madrid), used biochemistry and electron microscopy to define two conformations of the membrane fusion glycoprotein F and to show that two proteolytic processing events are required before this glycoprotein is able to be activated for membrane fusion.
In the past few years we have determined five structures of human αβTCRs bound to class I MHC molecules presenting peptides, revealing an atomic picture of the recognition event that occurs between a cytotoxic (killer) T cell and an antigen-presenting cell (in collaboration with William Biddison, National Institutes of Health). In the past year we determined a similar complex but with a TCR recognizing a class II MHC molecule presenting an influenza virus peptide, which showed the intercellular recognition event that a T helper (CD4+) cell would make with an antigen-presenting cell during an influenza virus infection. Despite these structures and a few from other research groups, it remains unclear how the T cell knows that an MHC-peptide complex has bound to an MHC molecule. In one study last year using an array of physical chemical methods, we showed that the soluble TCR-MHC complexes do not dimerize; dimer formation had been favored as the mechanism of signal transduction. Our work, repeating experiments from other laboratories and extending the study by applying other methods, indicates that the results from two previously published studies cannot be generalized to the two different TCR-MHC complexes we studied. It remains possible that the entire (not just αβ) TCR in membranes will form dimers once bound to MHC-peptide complexes between cells. The new result thus serves to focus our efforts on the more complicated membrane-bound forms of the TCR.
Susceptibility to autoimmune diseases such as multiple sclerosis and insulin-dependent diabetes (IDDM) is linked to specific class II MHC molecules. Recently, in collaboration with Kai Wucherpfennig (Dana-Farber Cancer Institute), we determined the structure of the class II MHC molecule most closely associated with IDDM, DQ8, complexed with a peptide from insulin. Comparisons and modeling of peptide-binding pockets suggest DQ8 is similar to other human allelic products and the mouse MHC molecule associated with autoimmune diabetes. These similarities suggest that diabetes is caused by the same antigen-presentation event(s) in humans and the non-obese diabetic (NOD) mice and provide evidence that the NOD mouse is a good model for the human disease. The structure also suggests the locations on the DQ molecule that might be blocked specifically to inhibit antigen presentation of DQ. In a related study we also determined the structure of another class II MHC molecule, DR1, with a hybrid-peptide inhibitor blocking its antigen-binding site (in collaboration with Gary Olson [Provid Research]; David Bolin [Hoffmann-LaRoche]; and Andrew Benowitz, Paul Sprengeler, Amos Smith III, and Ralph Hirschmann [University of Pennsylvania]).
We also recently determined the structure of human MHC class I molecules complexed with modified peptides from antigens presented by melanomas. Such peptides are candidates for antimelanoma vaccines. (This work was a collaboration with Olivier Michielin, Jean-Charles Cerottini, Immanuel Luescher, and Pedro Romero, from Strasbourg and Lausanne, and Martin Karplus, from Harvard University).
Antigenic peptides are transported into the endoplasmic reticulum to be loaded on MHC class I molecules by an ATP-dependent transporter, TAP. To determine how this membrane-bound peptide-loading machinery operates, we recently determined the structure of one of its pieces, the TAP1 ATPase domain. The structure revealed how TAP1 binds ADP and provided a framework for experiments on its activity.
Last updated January 29, 2002
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