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

We constructed alphavirus vectors encoding the ecotropic murine leukemia virus (MLV) receptor mCAT1. Cells electroporated with these vector RNAs expressed mCAT1 protein and fused with cells that expressed a fusogenic form of the MLV envelope on the cell surface. Electroporated cells also released submicron particles that were infectious in envelope-expressing cells. Infection resulted in giant syncytia that could be enumerated by plaque assay. Cell-free supernatants could be serially passaged and contained up to 10⁴ infectious units/ml. To determine whether repeated passage would select for functional variants of the receptor, we analyzed mCAT1 amplified by RT- PCR after 12 serial passages. Several amino acid substitutions were identified that encoded functional receptor variants. In independent experiments, variants containing an isoleucine or a leucine instead of a phenylalanine at position 224 in the third extracellular domain of the receptor arose spontaneously and outgrew the parental vector, indicating that mutations at this site are highly selected in this system. This region of the receptor has previously been implicated in the envelope-receptor interaction. This alphavirus vector system provides a novel method for generating and selecting functional variants of viral receptors. (C) 2000 Academic Press.