Residues in the apical domain of the feline and canine transferrin receptors control host-specific binding and cell infection of canine and feline parvoviruses

Abstract

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) capsids bind to the transferrin receptors (TfRs) of their hosts and use these receptors to infect cells. The binding is partially host specific, as FPV binds only to the feline TfR, while CPV binds to both the canine and feline TfRs. The host-specific binding is controlled by a combination of residues within a raised region of the capsid. To define the TfR structures that interact with the virus, we altered the apical domain of the feline or canine TfR or prepared chimeras of these receptors and tested the altered receptors for binding to FPV or CPV capsids. Most changes in the apical domain of the feline TfR did not affect binding, but replacing Leu221 with Ser or Asp prevented receptor binding to either FPV or CPV capsids, while replacing Leu221 with Lys resulted in a receptor that bound only to CPV but not to FPV. Analysis of recombinants of the feline and canine TfRs showed that sequences controlling CPV-specific binding were within the apical domain and that more than one difference between these receptors determined the CPV-specific binding of the canine TfR. Single changes within the canine TfR which removed a single amino acid insertion or which eliminated a glycosylation site gave that receptor the expanded ability to bind to FPV and CPV. In some cases, binding of capsids to mutant receptors did not result in infection, suggesting a structural role for the receptor in cell infection by the viruses.

FIG. 1.

(A) The aligned sequences of the human, feline, and canine TfRs containing residues between positions 190 and 441 in the feline TfR sequence which transferred the CPV-specific binding to the feline TfR. Residues that are identical to those in the human TfR sequence are indicated by periods, while residues that differ between any of the three receptors are indicated. Residues that were mutated in the feline or canine TfR in these studies are marked with asterisks. The sequence corresponding to the apical domain of the human TfR is indicated in black, while protease-like domain sequences are shown in blue. Potential N-linked glycosylation sites in the sequences are boxed in red. (B) (Right) A ribbon diagram of the human TfR dimer structure, with the protease-like domain (red), the apical domain (green), and the helical domain (yellow) shown on one copy of the dimer. (Left) An enlarged view of the apical domain shows the positions of sequences analyzed by mutation in the feline or canine TfRs. Substitutions of positions indicated in blue had no clear effect on virus binding. Changes of residue 221 shown in red prevented CPV and FPV binding (feline TfR-L221S or feline TfR-L221D) or specifically prevented FPV binding (feline TfR-L221K). Other residues shown in red altered the specificity of virus binding of the canine TfR either when an Asn was deleted from position 205 of the canine TfR (delN205) or when Asn383 was changed to Lys, which altered a unique glycosylation site in the canine TfR. Positions marked in black affected receptor transport to the cell surface when mutated.

FIG. 2.

Chimeras of the feline and canine TfRs that were prepared and their binding to CPV or FPV capsids. (A) Feline-canine TfR chimeras tested. Clones of the feline (blue) and canine (red) TfRs were joined using either existing common restriction enzyme sites or sites engineered into the two sequences. Numbers indicate the residue positions (from the feline TfR sequence) at the recombination points. Can, canine; Fel, feline. (B) The binding of FPV or CPV capsids to the canine or feline TfRs or to chimeras of those receptors. The receptors were expressed in TRVb cells, and the virus capsid and Tf binding were measured by flow cytometry. Binding results were not obtained for the feline/canine chimera at position 350, as that receptor was not expressed on the cell surface. Symbols: +, binding; −, no binding. (C) A cartoon showing the TfR sequence, indicating the locations of the different domains of the proteins defined for the human TfR structure. Numbers indicate the residue positions in the feline TfR sequence at the boundaries of the structural domains.

FIG. 3.

Western blot showing the size and level of expression of various TfRs. Wild-type TfR and examples of mutant feline TfRs recovered from transfected TRVb cells compared to proteins from feline CRFK cells or from nontransfected TRVb cells are shown. TRVb cells were transfected with plasmids expressing either the wild-type feline TfR or mutants of that receptor. Cells were lysed in SDS sample buffer, electrophoresed on a 10% acrylamide gel, and transferred to a membrane. The TfR was detected with an antibody against the cytoplasmic tail. The positions of molecular size markers (in kilodaltons) are indicated to the right of the gel. del, deletion.

FIG. 4.

Binding of canine Tf (y axis) or CPV or FPV capsids (x axis) to wild-type or mutant canine or feline TfRs expressed in TRVb cells. Receptors were expressed in TRVb cells by transfection, and the cells were then incubated with Cy5-labeled canine Tf and CPV or FPV capsids for 1 h at 37°C. Capsids were detected by antibody staining, and the cell-associated capsids and Tf were quantified by flow cytometry.

FIG. 5.

Binding of canine Tf and CPV or FPV capsids to TfRs that were chimeras of the feline and canine receptors (Fig. 2). Feline225canine (Fel225/Can) and canine225feline (Can225/Fel) were reciprocal chimeras recombined around residue 225 in the feline TfR sequence, while feline191canine439feline (Fel191/Can439/Fel) was a double recombinant with residues 192 to 439 of the feline TfR replaced by the equivalent sequence of the canine TfR. Capsid and Tf binding to the wild-type canine and feline TfRs are depicted as shown in Fig. 4. Receptors were expressed in TRVb cells by transfection, and the cells were then incubated with canine Tf and CPV or FPV capsids and examined as described in the legend to Fig. 4.

FIG. 6.

Expression and N-linked glycosylation of TfRs from feline (NLFK and CRFK) and canine (A72, Cf2Th, and Walter Reed) cells or of wild-type or mutant TfRs expressed from plasmids by transfection of TRVb cells. Proteins were collected by lysis of the cells and then were either not treated or incubated with PNGase before electrophoresis on 10% acrylamide gels and detection of the TfR by Western blotting. (A) The feline or canine TfR recovered from cell lines of feline or canine origin or after expression from cDNA clones in hamster TRVb cells. Samples were either not treated or were treated with PNGase before electrophoresis. (B) The wild-type feline and canine TfRs and mutants containing an insertion (ins) or deletion (del) of Asn at position 205 and with an Asn or Lys at position 383 to alter those positions to the sequence of the alternative host receptor. All plasmids were expressed in TRVb cells.

FIG. 7.

Microscopic analysis of the feline TfR expressed in TRVb cells, showing the cellular localization of wild-type receptor (Feline TfR) compared to feline TfRs containing mutations which prevented virus binding (Y220T/L221S) or which affected cell surface transport (V209E/I211G/V212D and N381D/V382T/K383D). Cells were fixed with PFA. The cells were then either permeabilized with 0.5% Triton X-100 and stained with an antibody against the cytoplasmic domain of the receptor or were not permeabilized and stained with a rabbit anti-TfR antipeptide antibody that recognizes the ectodomain of the receptor.

FIG. 8.

Virus susceptibility of TRVb cells expressing the wild-type feline or canine TfR or mutant TfRs that were altered within the apical domain. The cells were transfected with the plasmids expressing the receptors, incubated for 2 days at 37°C, and then inoculated with 10 TCID₅₀ per cell of CPV type 2 or 2b or FPV. The cells were fixed 2 days later and stained for the presence of infected cells using an antibody against the NS1 protein. The data are the mean percentages of infected cells among the cells that became transfected in the experiment ± 1 standard deviation (error bars) of the data from at least three separate infection experiments for all the receptors tested. Fel, feline; Can, canine; ins, insertion; del, deletion.