Use of a transient assay for studying the genetic determinants of Fv1 restriction

To probe the genetic determinants controlling the interaction between the r etroviral restriction gene Fv1 and its murine leukemia virus target, we set out to develop rapid, transient assays for Fv1 function. Cells were transf ected or transduced with Fv1 expression plasmids which can produce green fl uorescent protein via an internal ribosome entry site positioned between th e Fv1 and green fluorescent protein coding sequences. Fv1 function was then assessed by comparing virus replication in green fluorescent protein-posit ive and -negative cells, using retroviral vectors encoding a second fluores cent marker, yellow fluorescent protein, or beta-galactosidase, Using this assay, we could show that Fv1 specificities were not as absolute as previou sly thought, since the Fv1(b) allele was capable of interacting with "nonre stricted"' B- and NB-tropic viruses and by shuffling the n- and b-alleles o f Fv1, it was possible to generate a Fv1 molecule capable of restricting N- , B-, and NB-tropic viruses equally efficiently. Further, we could show tha t the presence of nonrestricting Fv1 in the same cell as restrictive Fv1 ab rogates restriction, implying competition for binding to the retroviral tar get.