Use of a Transient Assay for Studying the Genetic Determinants of Fv1 Restriction

To probe the genetic determinants controlling the interaction between the retroviral restriction geneFv1 and its murine leukemia virus target, we set out to develop rapid, transient assays forFv1 function. Cells were transfected or transduced withFv1 expression plasmids which can produce green fluorescent protein via an internal ribosome entry site positioned between theFv1 and green fluorescent protein coding sequences.Fv1 function was then assessed by comparing virus replication in green fluorescent protein-positive and -negative cells, using retroviral vectors encoding a second fluorescent marker, yellow fluorescent protein, or β-galactosidase. Using this assay, we could show thatFv1 specificities were not as absolute as previously thought, since theFv1b allele was capable of interacting with “nonrestricted” B- and NB-tropic viruses and by shuffling the n- and b-alleles ofFv1 , it was possible to generate a Fv1 molecule capable of restricting N-, B-, and NB-tropic viruses equally efficiently. Further, we could show that the presence of nonrestricting Fv1 in the same cell as restrictive Fv1 abrogates restriction, implying competition for binding to the retroviral target.