FUNCTIONAL AND DNA ALALYSIS OF THE NOVEL PROTEIN (R21 PROTEIN) INVOLVED IN MEMBRANE FUSION

By immunizing rats with cocultured HTLV-I-positive ILT8M2 and HTLV-I-negative MOLT-4cells，we isolated one monoclonal antibody(MAb)，designated as mAb R21，which enhances the syncytium formation induced by coculturing ILT8M2 cells with MOLT-4cells.Since the enhancing activity by mAb R21 of syncytium formation was observed only in the presence of a factor contained in fetal calf serum(FCS)which seems to bind to mAb R21，we purified this serum factor from FCS using a mAb R21-coupled Sepharose 4B column.The partial amino acid sequence of the purified protein indicates that R21 protein is a novel bovine serum protein which has approximately 90%amino acid homology with bovine platelet factor4，a member of CXC chemokine family.Next，to study the mechanism of viral entry and cell fusion mediated by HTLV-I env.Glycoproteins，and to identify the HTLV-I cellular receptor(S)，we constructed a cell fusion-dependent reporter gene activation assay and a vesicular stomatitis virus(VSV)pse…More udotype assay.The cell fusion-dependent reporter gene activation assay，in which T7RNA polymerase in donor cells co-expressing env glycoproteins activates a luciferase gene in recipient cells upon cell fusion，revealed that a broad range of cells are susceptible to HTLV-I env-mediated cell fusion。This indicated that cell-to-cell transmission of HTLV-I can occur in a wider range of cells than previously reported.The VSV pseudotype assay，which consists of recombinant VSV containing the green fluorescent protein gene instead of the receptor-binding G protein gene(VSV G I i D1*i D 1)and complemented with HTLV-I env glycoproteins(VSV G I i D 1*I e D 1-Env)，revealed that VSV G I i D 1*E D 1-Env infects efficiently only HepG 2 and 293T cells，indicating that cell-free G I D 1*E D 1-Env infects efficiently only HepG 2 and 293T cells，The characterization of the HTLV-I receptor(S)by various chemical modifications of HepG2 and 293T cells indicated that some glycosaminoglycans and phospholipids but not proteins on the cell surface may play a major role in HTLV-I env-mediated viral entry。The expression cloning of HTLV-I receptor is now underway using cDNA library from HepG2 cells.Less：Less

By immunizing rats with cocultured HTLV-I-positive ILT8M2 and HTLV-I-negative MOLT-4 cells, we isolated one monoclonal antibody (mAb), designated as mAb R21, which enhances the syncytium formation induced by coculturing ILT8M2 cells with MOLT-4 cells. Since the enhancing activity by mAb R21 of syncytium formation was observed only in the presence of a factor contained in fetal calf serum(FCS) which seems to bind to mAb R21, we purified this serum factor from FCS using a mAb R21- coupled Sepharose 4B column. The partial amino acid sequence of the purified protein indicates that R21 protein is a novel bovine serum protein which has approximately 90% amino acid homology with bovine platelet factor 4, a member of CXC chemokine family.Next, to study the mechanism of viral entry and cell fusion mediated by HTLV-I env. glycoproteins, and to identify the HTLV-I cellular receptor(s), we constructed a cell fusion-dependent reporter gene activation assay and a vesicular stomatitis virus (VSV) pse … More udotype assay. The cell fusion-dependent reporter gene activation assay, in which T7 RNA polymerase in donor cells co-expressing env glycoproteins activates a luciferase gene in recipient cells upon cell fusion, revealed that a broad range of cells are susceptible to HTLV-I env-mediated cell fusion. This indicated that cell-to-cell transmission of HTLV-I can occur in a wider range of cells than previously reported. The VSV pseudotype assay, which consists of recombinant VSV containing the green fluorescent protein gene instead of the receptor-binding G protein gene(VSV△GィイD1*ィエD1) and complemented with HTLV-I env glycoproteins (VSV△GィイD1*ィエD1-Env), revealed that VSV△GィイD1*ィエD1-Env infects efficiently only HepG2 and 293T cells, indicating that cell-free HTLV-I transmission is less efficient than cell-to-cell transmission. The characterization of the HTLV-I receptor(s) by various chemical modifications of HepG2 and 293T cells indicated that some glycosaminoglycans and phospholipids but not proteins on the cell surface may play a major role in HTLV-I env-mediated viral entry. The expression cloning of HTLV-I receptor is now underway using cDNA library from HepG2 cells. Less