Puffer B A, Sharron M, Coughlan C M, Baribaud F, McManus C M, Lee B, David J, Price K, Horuk R, Tsang M, Doms R W
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, 19104, USA.
Virology. 2000 Oct 25;276(2):435-44. doi: 10.1006/viro.2000.0557.
APJ is a seven transmembrane domain G-protein-coupled receptor that functions as a coreceptor for some primate immunodeficiency virus strains. The in vivo significance of APJ coreceptor function remains to be elucidated, however, due to the lack of an antibody that can be used to assess APJ expression, and because of the absence of an antibody or ligand that can block APJ coreceptor activity. Therefore, we produced a specific monoclonal antibody (MAb 856) to APJ and found that it detected this receptor in FACS, immunofluorescence, and immunohistochemistry studies. MAb 856 also recognized APJ by Western blot, enabling us to determine that APJ is N-glycosylated. Using this antibody, we correlated APJ expression with coreceptor activity and found that APJ had coreceptor function even at low levels of expression. However, we found that APJ could not be detected by FACS analysis on cell lines commonly used to propagate primate lentiviruses, nor was it expressed on human PBMC cultured under a variety of conditions. We also found that some viral envelope proteins could mediate fusion with APJ-positive, CD4-negative cells, provided that CD4 was added in trans. These findings indicate that in some situations APJ use could render primary cell types susceptible to virus infection, although we have not found any evidence that this occurs. Finally, the peptide ligand for APJ, apelin-13, efficiently blocked APJ coreceptor activity.
APJ是一种具有七个跨膜结构域的G蛋白偶联受体，它作为某些灵长类免疫缺陷病毒株的共受体发挥作用。然而，由于缺乏可用于评估APJ表达的抗体，以及缺乏能够阻断APJ共受体活性的抗体或配体，APJ共受体功能在体内的意义仍有待阐明。因此，我们制备了一种针对APJ的特异性单克隆抗体（单克隆抗体856），并发现它在流式细胞术、免疫荧光和免疫组织化学研究中能检测到这种受体。单克隆抗体856在蛋白质印迹法中也能识别APJ，这使我们能够确定APJ是N-糖基化的。使用这种抗体，我们将APJ表达与共受体活性相关联，发现即使在低表达水平下APJ也具有共受体功能。然而，我们发现在常用于繁殖灵长类慢病毒的细胞系上通过流式细胞术分析无法检测到APJ，在各种条件下培养的人外周血单核细胞上也未表达。我们还发现，只要反式添加CD4，一些病毒包膜蛋白就能介导与APJ阳性、CD4阴性细胞的融合。这些发现表明，在某些情况下，APJ的使用可能使原代细胞类型易受病毒感染，尽管我们尚未找到任何证据证明这种情况会发生。最后，APJ的肽配体apelin-13能有效阻断APJ共受体活性。
