Human complement receptor type 2, CR2, is the B lymphocyte receptor for the d region of the third component of complement. CR2 also serves as the receptor for the Epstein-Barr virus, which is associated with several human cancers and which immortalizes human B lymphocytes in vitro. Although CR2 is thought to mediate growth or differentiation signals during B cell responses, the mechanism of its action is unknown. The long term goals of this applicant are to define the role of CR2 in the normal immune response by identifying its functional domains and sites of interaction with other cellular components and to define the genomic elements controlling its expression under normal and abnormal conditions. Additionally, the function of this receptor in the uptake and infection of the Epstein-Barr virus will be investigated. Analysis of a B lymphocyte specific complement receptor will enhance our understanding of the role of complement in the B cell response to antigen. Furthermore, the study of the interaction of the Epstein-Barr virus with its receptor could serve as a model for viral-host cell interactions with implications for vaccine development. Molecular biology will be utilized to answer these questions. A cDNA encoding CR2 has recently been identified, allowing analysis of the primary structure of this protein and the identification of genomic sequences associated with it. CR2 cDNA will be inserted into eucaryotic expression vectors for transfection into murine and human lymphocyte cell lines, and expression on the surface of these cells. Deletion mutants will be generated from the cDNA in order to map the ligand binding sites, and site directed mutagenesis will be employed to identify the actual residues involved. The transmembrane and cytoplasmic regions from other proteins will be exchanged for the CR2 sequences, allowing identification of the regions of the protein required for the internalization and capping properties of CR2. The generation of cell lines which are expressing transfected CR2 will also be used to study the uptake and early events of infection by the Epstein-Barr virus. The genomic sequences which direct the tissue-specific expression of CR2, and function to promote and enhance transcription of the CR2 gene will be identified within genomic phage spanning this gene. Regions 5' o the CR2 coding sequences will be subcloned into vectors containing the chloramphenicol acetyltransferase gene, and the effect of these CR2 sequences on the expression of the CAT gene in various cell types will be determined.
