Studies on the regulatory mechanisms for expression of ecotropic murine leukemia virus receptor

生态型小鼠白血病病毒(EMuLV)以阳离子氨基酸转运蛋白1(CAT1)作为进入受体。然而，CAT1蛋白表达的调控机制以及eMuLV感染对CAT1蛋白表达的影响在很大程度上仍不清楚。为了阐明这些问题，将绿色荧光蛋白(GFP)标记的表达小鼠CAT1的载体导入水貂来源的CCL64细胞(Masuda等人，1999)，并获得转化子。这些细胞表面表达GFP标记的mCAT1(mCAT1-GFP)，对eMuLV感染敏感，感染后细胞表面mCAT1-GFP的表达显著降低。激光共聚焦显微镜(LCM)分析表明，mCAT1-GFP从跨高尔基网络到细胞表面的细胞内转运受到负面影响。为了验证这种可能性，将mCAT1-GFP载体导入犬肾上皮细胞来源的MDCK细胞，建立了稳定的转化子。LCM分析表明，mCAT1-GFP被转运到极化上皮细胞的基底外侧表面，并与之共定位。接头复合体AP1和AP2参与蛋白质转运。免疫沉淀和免疫印迹分析也表明mCAT1-GFP与AP1和AP2相关。当表达mCAT1-GFP的MDCK细胞感染eMuLV后，细胞表面niCAT1-GFP的表达显著降低。有趣的是，在eMuLV感染的细胞中，mCAT1-GFP与API的关联性也降低，而与AP3的关联性增加。结果表明，eMuLV感染可影响CAT1与各种接头复合体之间的相互作用。本研究可能为研究受体干扰eMuLV感染的机制提供有益的启示。

Ecotropic murine leukemia virus (eMuLV) uses cationic amino acid transporter 1 (CAT1) as the entry receptor. However, regulatory mechanisms for CAT1 protein expression and the effects of eMuLV infection on CAT1 expression are still largely unknown. To elucidate these issues, mink-derived CCL64 cells were transfected with a vector expressing mouse CAT1 tagged with the green fluorescent protein (GFP) (Masuda et al., 1999), and the transformants were obtained. Expressing GFP-tagged mCAT1 (mCAT1-GFP) on the surface membrane, these cells became susceptible to eMuLV infection, and upon infection, cell surface expression of mCAT1-GFP was dramatically decreased. Analysis by laser confocal microscopy (LCM) suggested that intracellular trafficking of mCAT1-GFP from trans-Golgi network to the cell surface was negatively affected. To examine this possibility, MDCK cells derived from canine renal epithelial cells were transfected with the mCAT1-GFP vector, and stable transformants were established. LCM analysis revealed that mCAT1-GFP was transported to the basolateral surface of polarized epithelial cells and co-localized with. adaptor complexes AP1 and AP2 involved in protein trafficking. Immunoprecipitation and western blot analysis also indicated that mCAT1-GFP was associated with AP1 and AP2. When mCAT1-GFP-expressing MDCK cells were infected with eMuLV, cell surface expression of niCAT1-GFP was dramatically decreased. Interestingly, association between mCAT1-GFP and API was also decreased in the eMuLV-infected cells, whereas association with AP3 was increased. The results demonstrate that eMuLV infection could affect interaction between CAT1 and various adaptor complexes. This study may provide useful insights into the mechanism of the receptor interference with eMuLV infection.