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

Abstract

To probe the genetic determinants controlling the interaction between the retroviral restriction gene Fv1 and its murine leukemia virus target, we set out to develop rapid, transient assays for Fv1 function. Cells were transfected or transduced with Fv1 expression plasmids which can produce green fluorescent protein via an internal ribosome entry site positioned between the Fv1 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 beta-galactosidase. Using this assay, we could show that Fv1 specificities were not as absolute as previously thought, since the Fv1(b) allele was capable of interacting with "nonrestricted" B- and NB-tropic viruses and by shuffling the n- and b-alleles of Fv1, 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.

FIG. 1

Schematic drawings of the plasmid constructs used in the assays. (A) pIRES2-EGFP/Fv1n and pIRES2-EGFP/Fv1b are Fv1 expression vectors used for the transfection assay. Driven by the CMV immediate-early promoter, the Fv1 alleles and EGFP are expressed from a bicistronic mRNA. (B) Constructs used for transient production of delivery viruses after transfection into 293T cells. pLFv1nIEG, pLFv1bIEG, and pLFv1MMxIEG are retroviral packaging vectors for Fv1 and EGFP expression in the target cell; pHIT60 is a gag-pol expression plasmid; and pczVSV-G is a VSV G expression plasmid. (C) Constructs for tester viruses. pCI G3 N, pCI G3 B, and pHIT60 are N-, B-, or NB-tropic gag-pol expression plasmids (Bsr is the blastocidin resistance gene); pMFG-NLSLacZ is a retroviral vector containing lacZ with a nuclear localization signal (NLS); pCFG2fEYFP is a retroviral vector for EYFP. SV40, simian virus 40; LTR, long terminal repeat.

FIG. 2

Transfection assay for Fv1 restriction. (A) Typical example of cells stained for β-galactosidase activity following transfection with Fv1ⁿ and sorting into EGFP-positive and -negative populations. (B) Blue cell counts for determination of Fv1ⁿ restriction. (C) Blue cell counts for determination of Fv1^b restriction.

FIG. 3

Transduction assay for Fv1 restriction. Typical example of a FACS analysis to examine Fv1ⁿ and Fv1^b restriction on M. dunni cells. EGFP expression is shown on the x axis, and EYFP expression is shown on the y axis. Percentages given indicate the proportion of EYFP-positive cells in the EGFP⁻ and EGFP⁺ subpopulations. The delivery virus used in the experiments in the left column was for Fv1ⁿ expression, and that in the right column was for Fv1^b. Top row, B-tropic tester virus; middle row, N-tropic tester virus; bottom row, NB-tropic tester virus.

FIG. 4

Restriction of Fv1-transduced single-cell clones after infection with lacZ tester viruses. (A) A total of 17 single-cell clones for each Fv1 allele and 9 Fv1-negative controls were tested. The mean values and standard error are given. (B) The mean values for the controls were arbitrarily set to 100, and the corresponding values for the single-cell clones were calculated relative to the controls.

FIG. 5

Transduction assay on B-3T3 and N-3T3 cells. (A) Transduction of B-3T3 cells with Fv1^b (left) and transduction of N-3T3 cells with Fv1ⁿ (right). Top row, B-tropic tester virus; middle row, N-tropic tester virus; bottom row, NB-tropic tester virus. (B) Transduction of B-3T3 cells with Fv1ⁿ (left) and transduction of N-3T3 cells with Fv1^b (right). Top row, B-tropic tester virus; middle row, N-tropic tester virus; bottom row, NB-tropic tester virus.

FIG. 5

Transduction assay on B-3T3 and N-3T3 cells. (A) Transduction of B-3T3 cells with Fv1^b (left) and transduction of N-3T3 cells with Fv1ⁿ (right). Top row, B-tropic tester virus; middle row, N-tropic tester virus; bottom row, NB-tropic tester virus. (B) Transduction of B-3T3 cells with Fv1ⁿ (left) and transduction of N-3T3 cells with Fv1^b (right). Top row, B-tropic tester virus; middle row, N-tropic tester virus; bottom row, NB-tropic tester virus.

FIG. 6

Restriction of Fv1 mix-and-match mutants in the transduction assay. The mix-and match mutants are named with three letters; the first and second letters stand for the allelic origin of the amino acids at positions 358 and 399, respectively, and the last letter indicates the allelic origin of the C terminus. The extent of restriction is as follows: +, restriction; (+), slightly less restriction (example, Fv1^b on NB-tropic viruses [Fig. 3]); (−), slight inhibition (example, Fv1^b on B-tropic viruses); −, no restriction. Essentially identical results were obtained on both M. dunni and CCL-64 cells.