Specificity in receptor usage by T-cell-tropic feline leukemia viruses: implications for the in vivo tropism of immunodeficiency-inducing variants

Abstract

Cytopathic, T-cell-tropic feline leukemia viruses (FeLV-T) evolve from FeLV-A in infected animals and demonstrate host cell specificities that are distinct from those of their parent viruses. We recently identified two cellular proteins, FeLIX and Pit1, required for productive infection by these immunodeficiency-inducing FeLV-T variants (M. M. Anderson, A. S. Lauring, C. C. Burns, and J. Overbaugh, Science 287:1828-1830, 2000). FeLV-T is the first example of a naturally occurring type C retrovirus that requires two proteins to gain entry into target cells. FeLIX is an endogenous protein that is highly related to the N-terminal portion of the FeLV envelope protein, which includes the receptor-binding domain. Pit1 is a multiple-transmembrane phosphate transport protein that also functions as a receptor for FeLV-B. The FeLV-B envelope gene is derived by recombination with endogenous FeLV-like sequences, and its product can functionally substitute for FeLIX in facilitating entry through the Pit1 receptor. In the present study, we tested other retrovirus envelope surface units (SUs) with their cognate receptors to determine whether they also could mediate infection by FeLV-T. Cells were engineered to coexpress the transmembrane form of the envelope proteins and their cognate receptors, or SU protein was added as a soluble protein to cells expressing the receptor. Of the FeLV, murine leukemia virus, and gibbon ape leukemia virus envelopes tested, we found that only those with receptor-binding domains derived from endogenous FeLV could render cells permissive for FeLV-T. We also found that there is a strong preference for Pit1 as the transmembrane receptor. Specifically, FeLV-B SUs could efficiently mediate infection of cells expressing the Pit1 receptor but could only inefficiently mediate infection of cells expressing the Pit2 receptor, even though these SUs are able to bind to Pit2. Expression analysis of feline Pit1 and FeLIX suggests that FeLIX is likely the primary determinant of FeLV-T tropism. These results are discussed in terms of current models for retrovirus entry and the interrelationship among FeLV variants that evolve in vivo.

FIG. 1

Summary of the receptor specificities of feline, murine, and primate type C retrovirus envelopes used in this study. The SU for each virus is shown in schematic form. A-MLV, SU from A-MLV; GALV, SU from GALV. White boxes, FeLV-A-derived sequences; vertical lines, appropriate positions where FeLV-A-61E and FeLV-T-61C differ. The six-amino-acid deletion (X) and six-amino-acid insertion (inverted triangle) in FeLV-T-61C are also shown. FeLV-B-90Z, SU of the 90Z molecular clone. 90Z_RBD is a chimeric envelope containing amino acids 1 to 244 from 90Z and the rest from 61E. The codons for FeLIX are 95% identical to those for 90Z within the portion of the envelope gene coding for the mature SU (2). The approximate location of the RBD, as defined for MLVs, is indicated at the top (6). The receptor specificity of each of the viral envelopes is indicated to the right (27, 28, 31, 45, 48, 50). Receptor usage by FeLV-Bs is as described previously (; Anderson et al., unpublished data).

FIG. 2

Expression and infection analyses of cell lines stably expressing retrovirus envelopes and Pit proteins. (A) Histograms from flow-cytometric analyses of cells stained with monoclonal antibody C11D8, which recognizes an epitope in the SU common to FeLV subgroups A, B, and T (15). In all cases, the x axis is fluorescence intensity (log scale) and the y axis is cell number. Open profiles, parental MDTF-Pit cell lines stained with C11D8 and the secondary antibody; filled profiles, MDTFs expressing envelopes and Pit proteins (upper right corner) stained with C11D8 and the secondary antibody. (B) Interference in stable cell lines expressing Pit-envelope combinations to challenge with homologous virus. Titers were measured as β-galactosidase focus-forming units per milliliter as in Table 1. Percent interference was calculated as (1 − [titer of virus on Pit cell line/titer of virus on Pit-envelope cell line]) × 100. For example, the percent interference by the 90Z envelope in HuPit1–90Zenv cells would be (1 − [FeLV-B-90Z titer on HuPit1 cells/FeLV-B-90Z titer on HuPit1–90Zenv cells]) × 100. N.D., no data, because HuPit2 cells and derivatives are not susceptible to infection by FeLV-B-90Z and GALV. (C) FeLV-T-61C titer in focus-forming units (ffu) per milliliter on stable cell lines expressing Pit-envelope combinations. The data in both panels are representative of at least two independent experiments.

FIG. 3

Detection of HA-tagged retrovirus SUs in conditioned media. (A) Human embryonic kidney 293T cells were transfected with constructs expressing the indicated SU proteins, and cell-free supernatants were harvested 48 h posttransfection. SUs were immunoprecipitated from 1 ml of each supernatant using a monoclonal antibody directed against the HA epitope. One-half of each immunoprecipitate was resolved by SDS-PAGE and analyzed by Western blotting using a polyclonal antibody directed against the same epitope. Mock, sample of cell-free supernatant from cells transfected with an untagged version of FeLIX. Molecular mass markers (in kilodaltons) are indicated to the left. (B) GALV-SU_1–262 encodes amino acids 1 to 262 of the GALV SU with a C-terminal HA tag. Immunoprecipitation and Western blot analysis were performed as for panel A except that only one-fifth of each immunoprecipitate was loaded on the gel.

FIG. 4

Receptor-binding properties of HA-tagged SUs. MDTF or MDTF-Pit cells were incubated with 500 μl of conditioned medium containing the SUs indicated (upper right corner) as described in Materials and Methods. Bound SUs were detected by staining with a monoclonal antibody directed against the HA epitope. In all cases the x axis is fluorescence intensity (log scale) and the y axis is cell number. Mock samples (open profiles), cells incubated in standard media and stained with the same antibody. Pit receptor and SU abbreviations (upper right corner) are as described in the legends to Fig. 1 and 3.

FIG. 5

Receptor binding and FeLV-T cofactor activity of SU fragments at different concentrations. (A to D) MDTF-FePit1 and MDTF-FePit2 cells were incubated with 1 to 500 μl of SU-conditioned media in 1-ml total volumes. Fluorescence-activated cell sorter profiles obtained using the indicated amounts of supernatant are shown. Flow-cytometric analyses of SU fragment binding were performed as described in the legend to Fig. 4 and Materials and Methods. Mock, samples of cells incubated in standard media and stained with the same antibody. In all cases the x axis is fluorescence intensity (log scale) and the y axis is cell number. Abbreviations are as described in the legends to Fig. 1 and 3. (E) Data for a single-cycle infection assay using FeLV-T particles that packaged the gene for β-galactosidase. The total volume of medium in each infection was 1 ml. Variable amounts (1 to 500 μl) of SU-conditioned media were added to the infection for each cofactor. x axis, receptors and cofactor pairs tested. A negative result (arrows) indicates that no blue foci were observed with as much as 100 μl of the FeLV-T virus pseudotype, which corresponds to about 10⁵ particles that can infect cells using FeLIX-Pit1.

FIG. 6

Infection of feline fibroblasts in the presence of conditioned media containing retrovirus SUs. (A) Analysis of FeLV-A-61E SU binding to AH927 feline fibroblasts as described in the legend to Fig. 4. (B) FeLV-T-61C titer on AH927 cells in focus-forming units (ffu) per milliliter. Titers are based on challenge with vectors packaging a genome containing the gene for β-galactosidase. HA-tagged SUs were added at the time of infection as a 1:1 dilution of conditioned media harvested from transiently transfected 293T cells.

FIG. 7

Northern blot analysis of FeLIX and FePit1 expression in feline tissues. Total cellular RNA was isolated from the indicated tissues. (Top) RNA (10 μg) was loaded in each lane. The filter was probed with a portion of the FeLIX cDNA. Bands corresponding to the predicted unspliced and spliced FeLIX mRNAs are indicated on the right. (Bottom) The same filter was stripped and reprobed with a portion of the FePit1 cDNA. Molecular mass markers (in kilobases) are indicated at the left.