| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2003: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2002: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
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.
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