application abstract): This submission by Dr. Erich Mackow proposes to investigate the potential significance of a conserved immunoglobulin tyrosine activation motif (ITAM) found within the cytoplasmic tail of the hantavirus G1 protein. Only those hantaviruses that cause hantavirus pulmonary syndrome (HPS) appear to carry this motif which consists of two tyrosine and leucine/isoleucine (YxxL/I) sequences separated by 7-12 amino acids. This motif is not found in non pathogenic hantavirus strains or those that cause hemorrhagic fever with renal syndrome although these viruses do conserve a single YxxL/I sequence. Cellular ITAMs are sequence-specific and have two tyrosine phosphorylation sites which are required for receptor activation of a variety of mononuclear hematopoietic cells. These receptors include Fc (FcR), T-cell and B cell receptors which, when triggered, cause ITAM aggregation and phosphorylation and the initiation of a signaling cascade that eventuates in receptor activation, activated FcR mediate phagocytosis, antibody dependent cytotoxicity, cytokine induction and immune complex clearance. The applicant speculates that the G1 ITAM provides a potential mechanism for the induction of HPS through abnormal or dysfunctional activation of ITAM-containing cellular receptors. Thus, HPS viral ITAMs might inhibit FcR activation, thereby reducing phagocytosis of immune complexes which, in turn, could damage the pulmonary endothelium. Alternatively, HPS viral ITAMs might activate signaling pathways that result in abnormal cytokine responses which are cytotoxic. They might also interfere normal receptor activation by competing for cellular receptor signaling proteins. There are two specific aims: 1) To investigate the function of HPS hantavirus G1 ITAM in Fc receptor signaling, and 2) To identify the cellular signaling elements that interact with the G1 ITAM. The key experimental approach in these studies is to replace the cytoplasmic tail of FcR with one from an HPS hantavirus G1 protein, express the chimeric FcR in COS cells and evaluate FcR activation (phagocytosis) following receptor crosslinking. The latter would be achieved using performed complexes of sheep erythrocytes and rabbit anti-sheep antibodies. Requirements for HPS ITAM function will be defined by mutagenesis and by comparison to equivalent regions from non-pathogenic strains. The ability of G1 ITAMs to inhibit FcR-mediated phagocytosis will be tested by transfecting COS cells to express FcR and either infecting them with HPS hantavirus or co-transfecting them with G1.
