Selective elimination of high constitutive activity or chemokine binding in the human herpesvirus 8 encoded seven transmembrane oncogene ORF74

Open reading frame 74 (ORF74) encoded by human herpesvirus 8 is a highly co nstitutively active seven transmembrane (7TM) receptor stimulated by angiog enic chemokines, e.g, growth-related oncogene-cv, and inhibited by angiosta tic chemokines e.g, interferon-gamma-inducible protein. Transgenic mice exp ressing ORF74 under control of the CD2 promoter develop highly vascularized Kaposi's sarcoma-like tumors. Through targeted mutagenesis we here create three distinct phenotypes of ORF74: a receptor with normal, high constituti ve signaling through the phospholipase C pathway but deprived of binding an d action of chemokines obtained through deletion of 22 amino acids from the N-terminal extension; an ORF74 with high constitutive activity but with se lective elimination of stimulatory regulation by angiogenic chemokines obta ined through substitution of basic residues at the extracellular ends of TR I-V or TM-VI; and an ORF74 lacking constitutive activity but with preserved ability to be stimulated by agonist chemokines obtained through introducti on of an Asp residue on the hydrophobic, presumed membrane-exposed face of TM-II, It is concluded that careful molecular dissection can selectively el iminate either agonist or inverse agonist modulation as well as high consti tutive activity of the virally encoded oncogene ORF74 and that these mutant forms presumably can be used in transgenic animals to identify the molecul ar mechanism of its transforming activity.