Structure and Function of Viral Immunoevasins

National Institute Of Allergy And Infectious Diseases

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Abstract

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 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. 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 in solution, indicative of a previously elusive protein family, but have applied a novel methodology. We have extended these findings on m04 to the related MCMV immunoevasin, m06. This molecule has been engineered for expression in E. coli, and binding studies indicate that it interacts with MHC-I molecules with low but detectable affinity. Using a recombinant MHC-I molecule that gives an outstanding multidimensional NMR spectrum, we have recently been able to map the MHC-I binding site of m06. Proceeding from these earlier studies, we are now complementing them with cellular experiments designed to examine the precise intermediates of MHC-I folding that proceed within the cell and that are susceptible to m06 dependent routing to the endosomal/ysomsomal pathway.