Parvovirus infection of cells by using variants of the feline transferrin receptor altering clathrin-mediated endocytosis, membrane domain localization, and capsid-binding domains

Abstract

The feline and canine transferrin receptors (TfRs) bind canine parvovirus to host cells and mediate rapid capsid uptake and infection. The TfR and its ligand transferrin have well-described pathways of endocytosis and recycling. Here we tested several receptor-dependent steps in infection for their role in virus infection of cells. Deletions of cytoplasmic sequences or mutations of the Tyr-Thr-Arg-Phe internalization motif reduced the rate of receptor uptake from the cell surface, while polar residues introduced into the transmembrane sequence resulted in increased degradation of transferrin. However, the mutant receptors still mediated efficient virus infection. In contrast, replacing the cytoplasmic and transmembrane sequences of the feline TfR with those of the influenza virus neuraminidase (NA) resulted in a receptor that bound and endocytosed the capsid but did not mediate viral infection. This chimeric receptor became localized to detergent-insoluble membrane domains. To test the effect of structural virus receptor interaction on infection, two chimeric receptors were prepared which contained antibody-variable domains that bound the capsid in place of the TfR ectodomain. These chimeric receptors bound CPV capsids and mediated uptake but did not result in cell infection. Adding soluble feline TfR ectodomain to the virus during that uptake did not allow infection.

FIG. 1.

Mutant or chimeric receptors analyzed in this study and comparison of the TfR sequences mutated. (A) Aligned sequences of the cytoplasmic, transmembrane, and stalk domains of the human, feline, and canine TfRs. The YTRF internalization motif is shaded in red. Differences from the human TfR are shaded in blue for the feline and canine receptors. Red underlining, cytoplasmic sequence; green underlining, transmembrane sequences; blue underlining, stalk sequences. The arrows indicate residues mutated in TfR-75-81-83, and the black line above the cytoplasmic sequences indicates the deleted residues in TfR-Δ3-32. (B) A model of the human TfR ectodomain structure is shown, along with diagrams of the altered regions of the other receptors. In the TfR-Δ3-32 mutant, residues 3 to 32 of the cytoplasmic domain of the feline TfR were deleted. Three residues of the YTRF internalization motif were changed to Ala in TfR-ATAA. In the TfR-75-81-83 mutant, three nonpolar amino acids in the transmembrane domain were replaced by polar residues. Chimeric receptors prepared included an NA-TfR chimera, which had residues 1 to 35 of the influenza virus NA fused to residues 94 to 769 of the TfR, and TfR-scFv8 and TfR-scFv14, which contained residues 1 to 122 of the feline TfR fused to the variable domains derived from MAb 8 and MAb 14, respectively. In the previously described mutant TfR-L221K, a Leu at position 221 was replaced with a lycine; this mutant receptor binds capsids but does not mediate efficient infection (52). The colors in the TfR model indicate the different domains defined for the human receptor as follows: blue, protease-like domain; red, apical domain; and green, helical domain. For the scFv model, the red ovals represent the light and heavy variable domains of the antibodies, and the line connecting those domains indicates the linker peptide added during the production of the single chains.

FIG. 2.

Flow cytometric analysis of the binding of CPV capsids and canine Tf to TRVb cells transiently expressing the feline TfR or receptors with altered transmembrane or cytoplasmic sequences. Cells were incubated with Cy5-labeled Tf and CPV together for 1 h at 37°C, and then after detachment from the tissue culture dishes and fixation, the cell-associated CPV was detected with Cy2-labeled MAb 8. y axis, Tf binding; x axis, capsid binding.

FIG. 3.

Distribution and uptake of Tf bound to TRVb cells expressing the wild-type or mutant feline TfRs. (A) Rate of ¹²⁵I-labeled canine Tf uptake into TRVb cells expressing various mutant receptors. Tf was incubated with the cells on ice, and then after warming to 37°C for various times, the extracellular Tf was removed by acid wash. The data are shown for three independent experiments ±1 standard deviation. (B) Degradation of Tf in TRVb cells expressing TfR-75-81-83 or wild-type TfR. Cells were incubated with ¹²⁵I-labeled Tf at 37°C for 1 h, washed on ice, and then incubated for a further 2 h at 37°C. The protein released into the supernatant was precipitated by using 10% TCA, and the amounts of TCA-soluble and insoluble radioactivity were counted. Data are for three independent experiments ±1 standard deviation. wt, wild type.

FIG. 4.

Association of wild-type (wt) TfR and NA-TfR with detergent-insoluble lipid rafts. TRVb cells expressing either feline TfR or NA-TfR were incubated with ¹²⁵I-labeled Tf for 30 min on ice and then extracted with PBS containing 1% Triton X-100. The radioactivity in the extracted supernatant and in the cells was measured. To disrupt lipid rafts the cells were pretreated with 5 mM methyl-β-cyclodextrin prior to incubation with the labeled Tf. The data are shown for three independent experiments. Error bar, ±1 standard deviation.

FIG. 5.

Cell surface and intracellular distribution of capsids in TRVb cell lines stably expressing the wild-type (wt) or mutant feline TfR. The cells were incubated for 1 h at 37°C with CPV capsids and then fixed. Cell-associated virus was detected with Cy2-labeled MAb 8 in Triton X-100-permeabilized or nonpermeabilized cells. The ratio of total virus and surface-accessible virus was determined by flow cytometry. Data are for three independent experiments ±1 standard deviation.

FIG. 6.

Localization of CPV capsids in TRVb cells expressing the feline TfR or altered receptors and Rab11-GFP (green). Cells were incubated with Cy3.5-labeled capsids (red) for 30 min at 37°C and then fixed and examined by confocal microscopy. One section through the center of the cell body is shown. Scale bar, 10 μm. The inset boxes show threefold enlargements of a portion of each image.

FIG. 7.

Infection of TRVb cells expressing altered receptors containing the feline TfR ectodomain. Cells were inoculated with between 0.001 and 1 TCID₅₀ of CPV per cell for 1 h at 37°C and then incubated in growth medium for 24 h at 37°C. The proportion of cells expressing each TfR was determined by flow cytometry in parallel cultures. The data show the percentages of cells expressing the mutant TfR that became infected. Error bars represent data for at least five independent experiments ± standard deviations. wt, wild type.

FIG. 8.

Flow cytometric analysis of the binding of virus to cells expressing receptors with altered ectodomains. (A) CPV binding to TRVb cells expressing TfR-scFv8 or TfR-scFv14 or to the control feline TfR-L221K which has a mutation in the apical domain that affects capsid binding and infection. Cells were incubated with Cy5-labeled CPV for 1 h at 37°C and then detached; TfR expression was detected with a MAb against the cytoplasmic tail of the TfR. y axis, receptor expression; x axis, CPV binding; wt, wild type. (B) Specificity of capsid binding to TfR-scFv chimeras. Cloned cell lines expressing TfR-scFv8 or TfR-scFv14 constructs were incubated with 10 μg of CPV or FPV capsids per ml for 1 h at 37°C; after detachment the cells were fixed and permeabilized, and the cell-associated virus was detected with Cy2-labeled MAb 8. Mock, mock-transfected cells.

FIG. 9.

Infection of TRVb cells expressing TfR-scFv8, TfR-scFv14, or TfR-L221K after inoculation with 1 TCID₅₀ per cell of CPV, as described in the legend of Fig. 8. The data are shown for three independent experiments ±1 standard deviation.