Ecotropic murine leukemia viruses (MLVs) recognize the third extracellular loop of the receptor, cationic amino acid transporter type 1 (CAT1). The CAT1 protein contains two conserved N-linked glycosylation sites in the third extracellular loops of the mouse, rat, and hamster receptors (mCAT1, rCAT1, and hCAT1, respectively). Glycosylation of the rCAT1 and hCAT1 receptors inhibits ecotropic MLV infection of CAT1-expressing cells, but that of the mCAT1 does not afford the cells this protection. As compared to the mCAT1 protein, the rCAT1 and hCAT1 proteins possess three and six amino acid insertions, respectively, in the third extracellular loop. To determine whether these inserted amino acids are associated with ecotropic MLV infection inhibition by glycosylation, several mutants of mCAT1 and rCAT1 receptors were constructed. Of all the mutants generated in the present study, only rCAT1 mutant 1 exhibited detectable protein expression levels. The rCAT1 mutant 1-expressing human NP2 cells were more susceptible to transduction by ecotropic MLV vectors than the wild-type rCAT1-expressing cells. Tunicamycin, an N-glycosylation inhibitor, increased transduction titer in the wild-type rCAT1-expressing cells, but did not do so in the cells expressing either the mCAT1 or rCAT1 mutation 1. An amino acid substitution in the glycosylation site of the wild-type rCAT1 conferred higher infection susceptibility, but that of the rCAT1 mutant 1 did not. As with the wild-type mCAT1 and rCAT1 proteins, the rCAT1 mutants were detected on the cell surface by immunofluorescence microscopy. Tunicamycin treatment did not affect cellular distribution of the rCAT1 mutant 1, wild-type mCAT1 or rCAT1 proteins. These results indicate that the extra amino acids in the rCAT1 (as compared to the mCAT1) are associated with inhibition of ecotropic MLV infection by the rCAT1 glycosylation. © 2004 Elsevier Inc. All rights reserved.
Kubo, Y., Ishimoto, A., Ono, T., Yoshii, H., Tominaga, C., Mitani, C., … Yamamoto, N. (2004). Determinant for the inhibition of ecotropic murine leukemia virus infection by N-linked glycosylation of the rat receptor. Virology, 330(1), 82–91. https://doi.org/10.1016/j.virol.2004.09.011