Changes in a murine leukemia virus (MLV) receptor encoded by an alphavirusvector during passage in cells expressing the MLV envelope

We constructed alphavirus vectors encoding the ecotropic murine leukemia vi rus (MLV) receptor mCAT1. Cells electroporated with these Vector RNAs expre ssed mCAT1 protein and fused with cells that expressed a fusogenic form of the MW envelope on the cell surface. Electroporated cells also released sub micron particles that were infectious in envelope-expressing cells. infecti on resulted in giant syncytia that could be enumerated by plaque assay. Cel l-free supernatants could be serially passaged and contained up to 10(4) in fectious units/ml. To determine whether repeated passage would select for f unctional variants of the receptor, we analyzed mCAT1 amplified by RT-PCR a fter 12 serial passages. Several amino acid substitutions were identified t hat encoded functional receptor variants. In independent experiments, varia nts containing an isoleucine or a leucine instead of a phenylalanine at pos ition 224 in the third extracellular domain of the receptor arose spontaneo usly and outgrew the parental vector; indicating that mutations at this sit e are highly selected in this system. This region of the receptor has previ ously been implicated in the envelope-receptor interaction. This alphavirus vector system provides a novel method for generating and selecting functio nal variants of viral receptors (C) 2000 Academic Press.