The focus of this work has been to understand the molecular details that control initial steps in the recognition of cells infected with pathogens such as viruses by cells of the innate and adaptive immune systems. Understanding the function, mechanism, structure, and evolution of the interaction of virus-encoded molecules recognized by the immune system can lead not only to a deeper understanding of molecular interactions in general and of cell-cell interactions in the immune system, but also may lead to rational approaches to intervention in virus infection. In particular, we study representative members of the large family of major histocompatibility complex (MHC)-encoded molecules from a biophysical and structural perspective. We are interested in how MHC-I molecules interact with receptors on natural killer (NK) cells or on T lymphocytes through their NK and T cell receptors, respectively. Large DNA viruses of the herpesvirus family produce proteins that mimic host MHC-I molecules as part of their immunoevasive strategy, and we have directed our efforts to understand the function, cellular expression, and structure of a set of these MHC-I (referred to as MHC-Iv) molecules encoded by the mouse cytomegalovirus (mCMV). We have analyzed the expression of several of these genes after transfection in different cell types, and have established that, unlike the classical MHC-I molecules, the viral MHC-I molecules do not require either the light chain component of the classical MHC-I molecule, beta-2 microglobulin, or self-peptide for expression. Although several of these MHC-Iv molecules are expressed at the surface of virus-infected cells early after infection, several others, including m152 and m155 are not expressed well at the cell surface, suggesting that their functions result from intracellular activities. In earlier studies, we determined the structure of the MHC-Iv molecule, m144, and showed that it preserved an MHC-I like fold, though it was devoid of bound peptide. In the past year we have extended our studies to include expression, binding and structural studies of m152, m153, and m157. Each of these molecules represents a different mode of action. m152 down-regulates host molecules crucial for recognition by either T cells or NK cells, specifically it down regulates host MHC-I molecules to elude CD8 T cell recognition. In addition, m152 down-regulates ligands of the NKG2D NK cell activating receptor, in particular the RAE-1 family of stress induced molecules. m153 has an unknown function, but is highly conserved in sequence among a number of mCMV strains that derive from wild mice, suggesting a conserved function. and m157 has been shown to be a primary ligand for NK cell receptors, Ly49I, an NK cell inhibitory receptor expressed in BALB/c (virus-susceptible) mice, and Ly49H, an NK cell activation receptor expressed in resistant C57BL/6 mice. Our studies of m152 demonstrated the direct interaction of this mCMV encoded MHC-Iv protein both with host MHC-I and the stress-induced RAE-1 molecules, and we have determined the X-ray crystallographic structure of m152 in complex with its RAE-1 ligand. This structure reveals a novel adaptation of the MHC-I protein fold for binding to RAE-1, another member of the MHC-I family. Since the NK activating receptor, NKG2D also interacts with RAE-1, it was important to compare the interaction of m152 with RAE-1 with the interaction of NKG2D. Surprisingly, the sites of interaction are the same, and competition experiments confirm this. The details of the interaction of m152 with RAE-1 have been confirmed by examination of the binding of some 18 site directed mutants of RAE-1. Thus m152 provides a novel example of an mCMV-encoded MHC-I-like protein that binds two different classes of MHC-I-like proteins. The mCMV protein m153 provides another unique example of the varied functions of mCMV MHC-Iv molecules. Although the precise function of m153 is not known, we have explored this extensively using a reporter cell system, in which m153 expressing cells become fluorescent on ligation of their surface expressed m153 by cells bearing an m153 ligand. A number of murine cell lines from various origins were screened with these reporter cells, but none stimulated the production of GFP. Freshly isolated spleen cells and in particular CD11c+ dendritic cells (DC) are particularly potent in activation the indicator cells. Further fractionation of the spleen cell populations indicate that CD11c+ dendritic cells (DC) are the most potent in activating the indicator cells. Several complementary approaches are now underway to identify the ligand(s) of m153 expressed on DC: staining and competition for staining of known DC markers with an m153 tetramer both by flow cytometry and by confocal microscopy;antibody blocking of the reporter cell assay;subfractionation of the DC population that carries the stimulatory ligand;mass spectrometric identification of molecules that pull down from DC lysates with the m153 tetramer;and screening of a cDNA expression library generated from DC. The consistent finding, that m153, an early mCMV encoded cell surface molecule, engages a molecule expressed at the surface of DCs, is itself a provocative finding. mCMV is known to replicate in epithelial cells as well as DC, and we hypothesize that one of the functions of m153 is to promote the infectious spreading of mCMV virus particles to DCs as a further site for secondary replication. Our studies of the function of m153 have been complemented by the determination of the crystallographic structure of this molecule, which has been solved and refined to 2.4 Angstrom resolution. The most striking feature of this new MHC-like structure is that the molecule forms a stable head to tail homodimer. To confirm the dimerization interface observed in the crystal structure of m153 we have analyzed a number of interface mutants, confirming the site of dimerization. The biological function of m153 is unknown. In addition to the m145 family of mouse CMV molecules, we have directed considerable attention to another novel family of putative immunoevasins of the MCMV, the m04 family. These molecules, that include m02, m04, and m06 seem to have distinct functions. m04 accompanies the MHC-I molecule to the cell surface, and m06 directs MHC-I molecules to an endosomal/lysomal pathway. This is a distinct protein family, and efforts to explore the structure of any of its members crystallographically have been difficult. We have successfully engineered m04 and examined its binding to MHC-I to quantify this interaction. Because of the difficulties we encountered in crystallization of the m04 protein, we have collaborated with Drs. Nik Sgourakis and Ad Bax to determine the structure of m04 by nuclear magnetic resonance techniques. NMR structure determination is based on determination of multidimensional spectra that allow assignment of molecular restraints to various interatomic distances within the molecule. Because we expressed the recombinant molecule in bacteria, we were able to label it with a variety of isotopic precursors (13C, 15N, 2D, in various combinations) permitting gathering of spectra allowing interpretation of different interatomic distances. To reduce the amount of data needed, allowing us to employ sparse NMR data, Drs. Sgourakis and Bax exploited a new technology using Rosetta modeling to determine the solution structure of the m04 protein. This structure is a unique beta-fold, based distantly on the Ig-fold, that is representative of the full m02-m06 family of viral proteins. Thus, we not only determined a completely new structure, indicative of a previously elusive protein family, but have applied a novel methodology was recently published in Structure. Further NMR studies are now underway to map the MHC binding site on m04.
