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 and neoplasia. 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 and 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 structures of the MHC-Iv molecules, m144, m152, and m153. Each of these molecules represents a different mode of immunoevasive action. Previous work from our laboratory examined the structure and function of these MHC-Iv molecules. Recently we have redirected our focus another set of the putative mCMV immunoevasins, the m04 family. This family, that includes m02, m04, and m06 seems to have distinct mechanisms of action. m04 accompanies the MHC-I molecule to the cell surface, and m06 directs MHC-I molecules to an endosomal/lysomal pathway. We have successfully engineered m04 and examined its binding to MHC-I to quantify this interaction. We have previously determined the structure of m04 in solution using NMR in collaboration with Drs.Nik Sgourakis and Ad Bax. NMR structure determination is based on determination of multidimensional spectra that allow assignment of molecular restraints to various interatomic distances within the molecule. Proceeding from these earlier studies, we are now complementing them with cellular imaging experiments designed to examine the precise intermediates of MHC-I folding that proceed within the cell. We have established that m06 routes MHC-I molecules to the endosomal/ysomsomal pathway, thus contributing to the downregulation of cell surface MHC-I, and the resulting functional immunoevasion. Prelinary screening of molecular complexes of the m06 protein bound to the MHC molecule H2-Ld indicate that diffraction quality crystals should be achievable to allow definitive structural determination of the unique mechanism of m06 evasive function.
