Feline aminopeptidase N is not a functional receptor for avian infectious bronchitis virus

BACKGROUND: Coronaviruses are an important cause of infectious diseases in humans, including severe acute respiratory syndrome (SARS), and have the continued potential for emergence from animal species. A major factor in the host range of a coronavirus is its receptor utilization on host cells. In many cases, coronavirus-receptor interactions are well understood. However, a notable exception is the receptor utilization by group 3 coronaviruses, including avian infectious bronchitis virus (IBV). Feline aminopeptidase N (fAPN) serves as a functional receptor for most group 1 coronaviruses including feline infectious peritonitis virus (FIPV), canine coronavirus, transmissible gastroenteritis virus (TGEV), and human coronavirus 229E (HCoV-229E). A recent report has also suggested a role for fAPN during IBV entry (Miguel B, Pharr GT, Wang C: The role of feline aminopeptidase N as a receptor for infectious bronchitis virus. Brief review. Arch Virol 2002, 147:2047–2056. RESULTS: Here we show that, whereas both transient transfection and constitutive expression of fAPN on BHK-21 cells can rescue FIPV and TGEV infection in non-permissive BHK cells, fAPN expression does not rescue infection by the prototype IBV strain Mass41. To account for the previous suggestion that fAPN could serve as an IBV receptor, we show that feline cells can be infected with the prototype strain of IBV (Mass 41), but with low susceptibility compared to primary chick kidney cells. We also show that BHK-21 cells are slightly susceptible to certain IBV strains, including Ark99, Ark_DPI, CA99, and Iowa97 (<0.01% efficiency), but this level of infection is not increased by fAPN expression. CONCLUSION: We conclude that fAPN is not a functional receptor for IBV, the identity of which is currently under investigation

Evolutionary Reconstructions of the Transferrin Receptor of Caniforms Supports Canine Parvovirus Being a Re-emerged and Not a Novel Pathogen in Dogs

Parvoviruses exploit transferrin receptor type-1 (TfR) for cellular entry in carnivores, and specific interactions are key to control of host range. We show that several key mutations acquired by TfR during the evolution of Caniforms (dogs and related species) modified the interactions with parvovirus capsids by reducing the level of binding. These data, along with signatures of positive selection in the TFRC gene, are consistent with an evolutionary arms race between the TfR of the Caniform clade and parvoviruses. As well as the modifications of amino acid sequence which modify binding, we found that a glycosylation site mutation in the TfR of dogs which provided resistance to the carnivore parvoviruses which were in circulation prior to about 1975 predates the speciation of coyotes and dogs. Because the closely-related black-backed jackal has a TfR similar to their common ancestor and lacks the glycosylation site, reconstructing this mutation into the jackal TfR shows the potency of that site in blocking binding and infection and explains the resistance of dogs until recent times. This alters our understanding of this well-known example of viral emergence by indicating that canine parvovirus emergence likely resulted from the re-adaptation of a parvovirus to the resistant receptor of a former host

Early Steps in Cell Infection by Parvoviruses: Host-Specific Differences in Cell Receptor Binding but Similar Endosomal Trafficking ▿ †

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) are closely related parvoviruses that differ in their host ranges for cats and dogs. Both viruses bind their host transferrin receptor (TfR), enter cells by clathrin-mediated endocytosis, and traffic with that receptor through endosomal pathways. Infection by these viruses appears to be inefficient and slow, with low numbers of virions infecting the cell after a number of hours. Species-specific binding to TfR controls viral host range, and in this study FPV and strains of CPV differed in the levels of cell attachment, uptake, and infection in canine and feline cells. During infection, CPV particles initially bound and trafficked passively on the filopodia of canine cells while they bound to the cell body of feline cells. That binding was associated with the TfR as it was disrupted by anti-TfR antibodies. Capsids were taken up from the cell surface with different kinetics in canine and feline cells but, unlike transferrin, most did not recycle. Capsids labeled with fluorescent markers were seen in Rab5-, Rab7-, or Rab11-positive endosomal compartments within minutes of uptake, but reached the nucleus. Constitutively active or dominant negative Rab mutants changed the intracellular distribution of capsids and affected the infectivity of virus in cells

Effect of Lys or Asn at position 384 in the black-backed jackal TfR on FPV and CPV binding and infection, compared to feline or canine TfRs under the same conditions.

<p><b>A</b>) Fluorescently labeled CPV or FPV and Tf were bound to cells expressing empty vector, feline TfR, canine TfR, wild-type jackal TfR, or Lys384Asn mutant jackal TfR at 37°C. The binding of FPV to the 384Asn black-backed jackal TfR exhibits the profile of CPV binding for canine TfR on multiple occasions <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002666#ppat.1002666-Palermo1" target="_blank">[15]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002666#ppat.1002666-Goodman1" target="_blank">[16]</a>. <b>B</b>) Fluorescently labeled FPV or CPV capsids were incubated with cells expressing feline TfR, canine TfR, wild type or mutant black-backed jackal TfR at 37°C. The binding was compared to that of fluorescently labeled Tf. <b>C</b>) Cell expressing these receptors were inoculated with FPV or CPV, and then infection measured by staining for the parvoviral NS1 expression, and the expression of TfR determined by staining for the cytoplasmic tail of the receptor. Error bars = mean ±1SD of three replicates. The wild-type and mutant black-backed jackal receptors were compared by fitting a generalized linear mixed model to the binomial data and considering replication as a random effect; * indicates statistically significant difference in frequency of binding or infection.</p

Analysis of dN/dS along each branch of the carnivore <i>TFRC</i> phylogeny.

<p><b>A</b>) dN/dS was calculated along each branch of the carnivore phylogeny. In this analysis, only the apical domain was analyzed. The number of estimated non-synonymous and synonymous DNA mutations that have occurred along each branch are shown in parentheses (N∶S) after the dN/dS value. <b>B</b>) A secondary analysis was performed with additional canid sequences, and the relevant clade is shown. The lineage leading to dog is highlighted with red branches. Below the phylogeny, key mutations predicted to have occurred along the branches leading to dog (branches 1–3) are shown.</p

Host species examined in this analysis.

<p><i>TFRC</i> sequences were determined from cDNA prepared from mRNA isolated from the samples indicated.</p

Determining the effects of varying residues in the feline TfR apical domain on parvovirus binding.

<p>CPV (<b>A</b>) or FPV (<b>B</b>) and Tf were incubated with TRVb cells expressing receptors with different combinations of the residues 378, 379, and 380 (feline TfR numbering). The name of one host species which contains the combination of residues shown is also given. Ligands were incubated with the cells at 37°C (white) or 4°C (black). Fluorescence of the labeled capsid was divided by fluorescence of the bound Tf to account for differential receptor expression. The mean of this ratio among all receptor-expressing cells was evaluated for each of three trials. The mean and standard deviation of the three trials is shown. Brackets connect groups of receptors that were statistically different in pairwise comparisons by Tukey's HSD at α = 0.05; i.e. samples not covered by a bracket did not differ at the α = 0.05 level in any comparisons. <b>C</b>) Effects of variant residues in the feline TfR on FPV infection. Cells expressing exogenous TfR with different three-amino acid combinations at residues 378–380 were inoculated and the ratio of infected cells (expressing NS1) and those expressing TfR is shown. The expressed receptors were compared for infection percentage by fitting a generalized linear mixed model to the binomial data and considering replication as a random effect, and those differed at the p = 0.034 level. Only one pairwise comparison was statistically significant after controlling for multiple testing by the Tukey-Kramer method: 34% of cells expressing TfR containing QNR (as seen in the mink TfR) were infected by FPV, while 26% of cells containing RNS (as Pallas' cat) were infected.</p

The positions of the positively selected residues in the TfR structure.

<p><b>A</b>) A listing of the TfR residues in the apical domain under positive selection as revealed by PAML analysis, showing the positions in the different TfRs, and the alternative residues found. <b>B</b>) Residues found to be under positive selection mapped in red onto the crystal structure of the human TfR ectodomain, and those in the apical domain were labeled with the corresponding feline TfR coordinates <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002666#ppat.1002666-Abraham1" target="_blank">[28]</a>. The 3 domains of the ectodomain are shown in green (apical domain), blue (protease-like domain), and yellow (helical domain). Strands of the apical domain β-sheet which influence virus binding are labeled. Some residues under positive selection are close to the host-range determinant (feline 383/canine 384) (shown in black) and the leucine at residue 221 (shown in orange) which can be mutated to block capsid attachment, and to discriminate between FPV and CPV <i>in vitro </i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002666#ppat.1002666-Palermo1" target="_blank">[15]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002666#ppat.1002666-Goodman1" target="_blank">[16]</a>.</p