Probing sequence variation in the receptor-targeting domain of feline leukemia virus envelope proteins with peptide display libraries

Abstract

Determinants of cellular tropism and receptor targeting lie within a short peptide in the Vr1 region of the envelope (Env) proteins of feline leukemia virus (FeLV) subgroups A and C. Libraries of FeLV Env proteins with random amino acid substitutions in the peptide were screened for their ability to deliver a marker gene to D17 and AH927 cells. Screening on D17 canine cells yielded D17-specific Env proteins that used the FeLV-C receptor. Screening on AH927 cells yielded Env proteins with a broader host range, with maximal titers on AH927 cells and similar or lower titers on other cells. These Env proteins used an unidentified non-FeLV receptor for entry. The A5 isolate obtained from the AH927 screen was readily concentrated to yield titers of 10(5) on human PC-3 prostate tumor cells. The sequence divergence observed among targeting peptides of library-selected Env proteins was greater than that found in parental FeLV isolates. Substitution analyses of a conserved R in the middle of the targeting peptide held constant during screening indicated that maximal titers were obtained only when R was present in both a D17 selected isolate and an AH927 selected isolate. The ability to isolate Env proteins with unique tropisms dependent on the cells on which the library is screened has direct implications for targeting gene delivery vectors.

FIG. 1.

Peptide library screening strategy for targeting retroviruses. (A) The Ψ⁺ retroviral vector expressing the library of peptides in the receptor-targeting region of the Env protein is bicistronic. Black boxes are long terminal repeat (LTR) sequences. The sequence surrounding the 10 randomized amino acids denoted by N is shown. W is amino acid 52 of the mature 61E FeLV-A Env protein (6). The neo resistance gene is expressed from the simian virus 40 (SV40) promoter. (B) Retroviruses carrying the peptide library env gene cassettes and expressing corresponding peptide-substituted Env proteins on their surfaces were tested for cell targeting. Successful targeting leads to drug resistance gene transfer, followed by characterization of the linked env gene.

FIG. 2.

Tropism of library-derived Env proteins is influenced by the cell line on which the library is screened. Library-derived Env proteins were expressed on retroviral particles produced from the TELCeB6 lacZ packaging cell line (5) and titered on feline AH927, canine D17, and human 293T cells. Env proteins from the library screen on AH927 cells are indicated by the AH prefix, those obtained from the screen on D17 cells are indicated by the D prefix. Results with wild-type FeLV-A and -C Env proteins are shown for comparison. Titers are TELCeB6 lacZ CFU per ml.

FIG. 3.

Library-derived A5 and A9a proteins do not use FeLV-A or FeLV-C receptors. Interference assays were used to examine receptor usage. lacZ transduction mediated by A5 or A9a on either AH927 (AH) or 293T cells infected with FeLV-A (A) and/or FeLV-C (C) was examined and compared with titers obtained on uninfected cells. The titer on infected cells as a percentage of that on uninfected is given in parentheses. Titers are TELCeB6 lacZ CFU per ml. Assays with 293T cells and AH927 cells were performed on different days and are each representative of two separate experiments.

FIG. 4.

(A) Library-derived A5 and A9a proteins do not use Pit-1 or Pit-2 receptors. Interference assays were performed on AH927 (AH) and 293T cells infected with either FeLV-B (B) or amphotropic murine leukemia virus 4070A (4070). AHCeB/A5 cells express the A5 Env protein. The titer on infected cells as a percentage of that on uninfected is given in parentheses. Titers are TELCeB6 lacZ CFU per ml. Assays with 293T cells and AH927 cells were performed on different days and are each representative of two separate experiments. (B) A5 Env expression does not interfere with FeLV-A or FeLV-C entry. Experimental details are as described for panel A.

FIG. 5.

Library-derived targeting peptide sequences show little if any homology to each other or to wild-type FeLV-A and FeLV-C isolates. Targeting peptide sequences of wild-type isolates are shown at the top of the figure. Library-derived peptides are at the bottom of the figure, below the sequence of the peptide region randomized for library screening. Randomized residues are denoted by X and are numbered from 1 to 10. Dots indicate gaps in the sequence. AH927 indicates selection on AH927 cells. D17 indicates selection on D17 cells except for the previously reported EF isolate, which was selected on AH927 cells but specifically infects D17 cells (2).