Analysis of Receptor Usage by Ecotropic Murine Retroviruses, Using Green Fluorescent Protein-Tagged Cationic Amino Acid Transporters

Abstract

Entry of ecotropic murine leukemia virus (MuLV) into host cells is initiated by interaction between the receptor-binding domain of the viral SU protein and the third extracellular domain (TED) of the receptor, cationic amino acid transporter 1 (CAT1). To study the molecular basis for the retrovirus-receptor interaction, mouse CAT1 (mCAT1) was expressed in human 293 cells as a fusion protein with jellyfish green fluorescent protein (GFP). Easily detected by fluorescence microscopy and immunoblot analysis with anti-GFP antibodies, the mCAT1-GFP fusion protein was expressed in an N-glycosylated form on the cell surface and in the Golgi apparatus, retaining the ecotropic receptor function. The system was applied to compare Friend MuLV (F-MuLV) and its neuropathogenic variant, PVC-211 MuLV, which exhibits a unique cellular tropism and host range, for the ability to use various CAT family members as a receptor. The results indicated that F-MuLV and PVC-211 MuLV could infect the cells expressing wild-type mCAT1 at comparable efficiencies and that rat CAT3, but not mCAT2, conferred a low but detectable level of susceptibility to F-MuLV and PVC-211 MuLV. The data also suggested that CAT proteins might be expressed in an oligomeric form. Further application of the system developed in this study may provide useful insights into the entry mechanism of ecotropic MuLV.

Expression of solute carrier 7A4 (SLC7A4) in the plasma membrane is not sufficient to mediate amino acid transport activity

“…Similar results have been obtained with other hCAT isoforms (results not shown). These results are also consistent with an earlier report demonstrating that a fusion protein between murine CAT-1 and GFP retained its function as receptor for murine ecotropic leukaemia viruses [18]. In addition, SLC7A4-EGFP exhibited a similar subcellular localization to hCAT-1-EGFP.…”

Neuropathology induced by infection with Friend murine leukemia viral clone A8‐V depends upon the level of viral antigen expression

“…This might imply the importance of infection in the blood vessel wall for neuro-pathogenesis, as was claimed in previous reports. 5,19 However, our recent study comparing the distribution of the viral antigen and lesional tropism suggests that the distribution of glial cells expressing viral antigens is more correlated to lesional tropism than the distribution of blood vessels that are positive for viral antigens, 20 which are often encountered in areas where the tissue remains normal in appearance without any spongiotic degeneration.…”

Visualization of Retroviral Replication in Living Cells Reveals Budding into Multivesicular Bodies

“…Friend MLV Env‐YFP was generated by YFP insertion into the NheI site of the construct pFr‐MuLV273/274 (49) and transfer of the Env gene into pcDNA3. MCAT‐CFP was a variant of MCAT‐GFP (50). Lamp1‐YFP was created by insertion of the Lamp1‐coding region of pLamp1‐GFP (Norma Andrews, Yale University, New Haven, CT, USA) (51) into the EcoRI‐BamHI sites of pEYFP‐N1 (Clontech, Palo Alto, CA, USA).…”