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

通过用协同HTLV-I-阳性ILT 8 M2和HTLV-I-阴性MOLT-4细胞免疫大鼠，我们分离了一个单克隆抗体（mAb），设计为mAb R21，通过协同化ILT 8 M2细胞增强了合成形成与MOLT-4细胞相关的ILT 8 M2细胞。自从mAb R21合成的mAb R21的增强活动仅在胎儿钙血清（FCS）中发现要绑定到mAb R21的时候，我们使用mAb R21偶联的Sepharose 4 B列的FCS净化了这个血清因子。R21蛋白是一种新颖的骨胶原血清蛋白，其含有骨胶原因子4的近90%的骨胶原同源性，是CXC化学家族的成员。下一个，研究由HTLV-I env介导的病毒入口和细胞融合的机制。糖蛋白质，并识别HTLV-I细胞受体（s），我们构建了一个细胞融合依赖性报告器基因激活试验和一个血管性胶原蛋白病毒（VSV） ... More udotype assay。细胞融合-依赖性报告员基因激活试验中，在供体细胞中的T7 RNA聚合酶协同表达env糖蛋白质激活受体细胞中的荧光酶基因，揭示了细胞的一个宽范围可追溯到HTLV-I env介导细胞融合。这表明HTLV的细胞对细胞传输--我可以在细胞的宽范围内使用比以前报道过的更多。VSV伪型分析，关于重组的VSV含有绿色荧光蛋白基因的受体结合G蛋白基因的观点（VSV△ Gyy D1* yye D1） and complemented with HTLV-I env glycoproteins（VSV△G D1* D1-Env）， revealed that VSV△G D1* D1-Env infects efficiently only HepG2 and 293T cells，指示无细胞HTLV的传输比细胞到细胞传输的有效性要小。HTLV-I受体的特征（s）是由HepG 2和293 T细胞的各种化学修饰所指示的，某些糖胺聚糖和磷脂，但在细胞表面上没有蛋白质可能在HTLV-I介导的病毒条目中扮演主要角色。HTLV-I受体的表达克隆现在正在利用HepG 2细胞的cDNA库。Less（低）

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