Single Mutations in the VP2 300 Loop Region of the Three-Fold Spike of the Carnivore Parvovirus Capsid Can Determine Host Range

Abstract

Sylvatic carnivores, such as raccoons, have recently been recognized as important hosts in the evolution of canine parvovirus (CPV), a pandemic pathogen of domestic dogs. Although viruses from raccoons do not efficiently bind the dog transferrin receptor (TfR) or infect dog cells, a single mutation changing an aspartic acid to a glycine at capsid (VP2) position 300 in the prototype raccoon CPV allows dog cell infection. Because VP2 position 300 exhibits extensive amino acid variation among the carnivore parvoviruses, we further investigated its role in determining host range by analyzing its diversity and evolution in nature and by creating a comprehensive set of VP2 position 300 mutants in infectious clones. Notably, some position 300 residues rendered CPV noninfectious for dog, but not cat or fox, cells. Changes of adjacent residues (residues 299 and 301) were also observed often after cell culture passage in different hosts, and some of the mutations mimicked changes seen in viruses recovered from natural infections of alternative hosts, suggesting that compensatory mutations were selected to accommodate the new residue at position 300. Analysis of the TfRs of carnivore hosts used in the experimental evolution studies demonstrated that their glycosylation patterns varied, including a glycan present only on the domestic dog TfR that dictates susceptibility to parvoviruses. Overall, there were significant differences in the abilities of viruses with alternative position 300 residues to bind TfRs and infect different carnivore hosts, demonstrating that the process of infection is highly host dependent and that VP2 position 300 is a key determinant of host range.

Importance: Although the emergence and pandemic spread of canine parvovirus (CPV) are well documented, the carnivore hosts and evolutionary pathways involved in its emergence remain enigmatic. We recently demonstrated that a region in the capsid structure of CPV, centered around VP2 position 300, varies after transfer to alternative carnivore hosts and may allow infection of previously nonsusceptible hosts in vitro. Here we show that VP2 position 300 is the most variable residue in the parvovirus capsid in nature, suggesting that it is a critical determinant in the cross-species transfer of viruses between different carnivores due to its interactions with the transferrin receptor to mediate infection. To this end, we demonstrated that there are substantial differences in receptor binding and infectivity of various VP2 position 300 mutants for different carnivore species and that single mutations in this region can influence whether a host is susceptible or refractory to virus infection.

FIG 1

Structure of the parvovirus capsid and the 3-fold-spike region. (A) The four major morphological regions of the CPV capsid are shown. The terminal residue at the distal tip of each of the three VP2 monomers (position 224) that constitute the 3-fold spike is highlighted in red for orientation between panels A and B. (B) Magnification of the 3-fold-spike region, viewed from the side. VP2 residues 299 (teal), 300 (white), and 301 (royal blue) are found beneath the apex of the spike, on a region known as the shoulder. Note that each individual position 300 loop region in one VP2 monomer (e.g., dark gray) is in close proximity to two other VP2 monomers, one from the same 3-fold spike (e.g., green) and one from a neighboring subunit (e.g., dark blue).

FIG 2

Host ranges of VP2 position 300 mutants of CPV in cells of different carnivore species. The 11 different amino acids that were created at VP2 position 300 in a CPV-2a infectious clone are shown in the far left column and highlighted in green in the second column from the left, with the flanking positions, residues 299 and 301, also indicated. Viruses were passaged in domestic cat (NLFK), domestic dog (A72), and gray fox (FoLu) cells, and mutations occurring in VP2 (either specifically in residues 299, 300, and 301 or outside this region) were monitored at passages 2, 5, 10, and 20. Viruses that could no longer be detected from culture by PCR during the passage experiments are indicated with a boxed “x” that is shaded gray. Mutations that arose in position 299 or 301 during host cell passage are shown in blue, with the VP2 position 300 mutant in which it occurred highlighted in green. If the mutated position remained polymorphic during the passage series, all amino acid residues that were detected are shown and separated by a slash(es). For mutations occurring outside residues 299, 300, and 301, the VP2 amino acid position and associated change are shown (also see Fig. 7). Viruses in which no mutations were observed outside residues 299, 300, and 301 during cell culture passage are indicated with an asterisk.

FIG 3

Single mutations of VP2 position 300 can dictate the host range of CPV in vitro. Relative infectivities of two VP2 position 300 mutants of CPV (300-Gly and 300-Trp) in domestic cat, gray fox, and domestic dog cells are shown at day 3 postinfection. Immunofluorescence indicates viral antigen detected using a rabbit anti-CPV VP1/VP2 antibody and an Alexa Fluor 488–goat anti-rabbit IgG. Note that the 300-Trp mutation rendered CPV noninfectious to dog cells, while no such restriction was observed during infection of cat or fox cells. Also note the lower relative infectivity for dog cells than for cat or fox cells for the prototype CPV (300-Gly), the virus normally found in dogs.

FIG 4

Compensatory mutations at residues that flank VP2 position 300 can significantly alter the fitness of CPV in domestic dog cells. (A) Single growth curves for CPV 300-Val containing either the wild-type Thr or mutant Pro residue at position 301 in A72 cells. Note that the 301-Pro mutation resulted in a 118-fold increase in titer by day 4 postinfection. Data shown are from experiments performed in triplicate, with error bars indicating standard deviations. (B) A72 cells from the experiment for panel A at day 6 postinfection, showing significant cytopathic effects resulting from the increase in replication conferred by the 301-Pro mutation, while cellular morphology in the cells infected with the 301-Thr virus is similar to that for the uninfected control. Bars = 200 μm. Structural models of the position 301 mutations in the CPV-2a infectious clone are shown on the right.

FIG 5

Host ranges of VP2 position 300 mutants of FPV in cells of different carnivore species. The five different amino acids that were created at VP2 position 300 in an FPV infectious clone are shown in the far left column and highlighted in green in the second column from the left, with the flanking positions, residues 299 and 301, also indicated. Note that position 299 was also mutated (from a Gly to a Glu) in the prototype FPV (300-Ala), based on the occurrence of this 299-Gly-to-Glu change observed during passage of CPV-2a in domestic cat cells (see Fig. 2). Viruses were passaged in domestic cat (NLFK), domestic dog (A72), and gray fox (FoLu) cells, and mutations occurring in VP2 (either specifically in residues 299, 300, and 301 or outside this region) were monitored at passages 2, 5, 10, and 20. Viruses that could no longer be detected from culture by PCR during the passage experiments are indicated with a boxed “x” that is shaded gray. Note that FPV mutants cannot be maintained in dog cells. Mutations that arose in residues 299, 300, and 301 during host cell passage are shown in blue. If the mutated position remained polymorphic during the passage series, all amino acid residues that were detected are shown and are separated by a slash(es). For mutations occurring outside residues 299, 300, and 301, the VP2 amino acid position and associated change are shown (also see Fig. 7).

FIG 6

Effects of mutations at VP2 position 300 on receptor binding and infectivity of FPV in domestic cat cells. (A) Passage of FPV containing 300-Asp results in the selection of a His residue through a single mutation at the first base in the codon (GAT to CAT). Note that the position 300 loop region is shown in the same configuration as the capsid surface rendition in Fig. 1. (B) Single growth curves demonstrating that the Asp-to-His mutation at position 300 results in a 152-fold increase in peak viral titer in NLFK cells. Data shown are from experiments performed in triplicate, with error bars indicating standard deviations. (C) Passage of FPV containing 300-Gly results in the selection of a Val residue through a single mutation at the second base in the codon (GGT to GTT). (D) Single growth curves showing that the Gly-to-Val mutation at position 300 results in an 18-fold increase in peak viral titer in NLFK cells. Data shown are from experiments performed in triplicate, with error bars indicating standard deviations. (E) Coomassie blue-stained SDS-PAGE gel with the purified FPV 300-Asp and 300-His mutants used for biolayer interferometry, showing the constituent protein composition (VP1 and VP2) of the capsid. (F) Biolayer interferometry analysis of FPV 300-Asp and 300-His mutants. His-tagged recombinant domestic cat TfR was immobilized on a Ni-NTA biosensor and loaded with either FPV 300-Asp or 300-His. Note the increase in virus binding of the 300-His virus in comparison to that of the 300-Asp virus, corroborating the infectivity data shown in panel B.

FIG 7

Capsid locations of additional amino acid changes that were observed in areas outside the region of residues 299, 300, and 301 during passage of VP2 position 300 mutants of CPV-2a and FPV in domestic dog (A72), domestic cat (NLFK), and gray fox (FoLu) cells. Mutations that occurred in various VP2 positions during passage in dog, cat, or fox cells were mapped onto the surface renditions of the crystal structures of their cognate viruses (CPV-2a or FPV). Note that VP2 position 300 is highlighted in white in all panels for orientation and that positions 299 and 301 are highlighted only if they were also mutated during passage in a particular host (see Fig. 2 and 5). (A to E) Structural location of each of the changes observed during passage of the CPV and FPV VP2 position 300 mutants in the three cell lines (note that FPV does not grow in dog cells and that data for this combination are thus not included). Amino acids are color coded as shown in the lower panel, and the virus and host of passage are indicated. Note that most of the additional mutations are in close structural proximity to the position 300 loop region, suggesting that such changes may be coordinated. The bottom panel gives additional information on mutations occurring outside the region of residues 299, 300, and 301, including the VP2 position 300 mutants in which they were observed, hosts of passage, amino acid changes, locations on capsid, and known or suspected functional roles in host range and associated references.

FIG 8

Glycosylation patterns in the TfRs of different carnivore hosts. (A) N-linked glycosylation sites identified in the domestic dog and cat TfRs by mass spectrometry. A total of five potential glycosylation sites—TfR positions 261, 327, 384, 732, and 737 (amino acid residues and numbering are based on the dog TfR)—were present, of which only four were glycosylated. A surface rendition of the crystal structure of the human TfR ectodomain (35) is shown, with the subdomains color coded as follows: white, apical subdomain; gray, protease-like subdomain; and aqua, helical subdomain. N-acetylglucosamine (GlcNAc)-linked asparagine (Asn) residues in the human TfR are shown as green stick representations (which are equivalent to the blue boxes shown in panel B) and are present at positions 261, 327, and 737 (the human TfR does not contain an Asn at position 384 or 732) (35). Note that the TfR position 384 glycosylation site (shown in red) is in close proximity to Leu-221/222 (shown in purple), a residue that has previously been shown to be critical in parvovirus binding (13). (B) Identification of the glycan present at position 384 in the domestic dog TfR but absent in the domestic cat or gray fox receptor. The MS/MS spectrum shows the N-linked glycopeptide ³⁸²NVNLTVNNVLK³⁹² with an attached 1,710.6-Da glycan [(Hex)₂(HexNAc)₁(NeuAc)₁ + (Man)₃(GlcNAc)₂]. Note that the glycan contains oligomannose and complex antennae (i.e., is a hybrid glycan [see the upper right corner]). A series of y ions (y3 to y11) were observed with the complete knockout of the glycan molecule, leading to the identification of the glycopeptide.

FIG 9

Effects of glycanase treatment of domestic cat and dog TfRs and cells on CPV and FPV infectivity. (A) Cleavage site (arrow) of endoglycosidase H (endo H), located between the chitobiose core of GlcNAc₂ residues in high-mannose and hybrid N-glycans. The glycan identified by mass spectrometry at position 384 in the domestic dog TfR is shown as an example. (B) Coomassie blue-stained SDS-PAGE gel with the purified recombinant domestic dog TfR ectodomain treated with endo H, showing an increase in electrophoretic mobility indicative of glycan cleavage. Lane 1, dog TfR without endo H treatment; lane 2, dog TfR treated with endo H; lane 3, endo H without TfR. (C) Treatment of domestic cat (NLFK) cells with endo H does not result in an increase in infection with either CPV or FPV over control (non-endo H treated) levels, suggesting that the cat TfR high-mannose and hybrid N-glycans do not influence parvovirus infection. (D) Treatment of domestic dog (A72) cells with endo H results in an increase in infection with both CPV and FPV. (Left) Although A72 cells incubated with the prototype dog-specific CPV strain (300-Gly) became infected (control), endo H treatment led to a substantial increase in the number of infected cells detected by immunofluorescence, suggesting that the unique glycan found at TfR position 384 may reduce CPV infection. (Right) A72 cells are refractory to infection with FPV (control). However, endo H treatment allows the host range barrier of dog cells to FPV to be overcome, most likely through cleavage of the TfR position 384 glycan.

FIG 10

Virus capsid-host receptor interactions and the pandemic emergence of CPV. (A) VP2 position 300 is highly variable among parvovirus isolates recovered from different carnivore species, suggesting that it is a key site in dictating cross-species transfers by binding to the host TfR. Parvovirus infections in a new host resulting in a change at VP2 position 300 may also result in additional compensatory mutations of surface-exposed capsid residues (such as those at VP2 position 299 or 301) that increase infectivity. For example, mutation of prototype CPV-2a 300-Gly to a 300-Ser mutant, followed by its passage in domestic cat cells, selects for a 301-Ala residue identical to the 301-Ala residue seen in 300-Ser CPV-2a isolates recovered in nature from masked civets (27), another feliform species. (B) Glycan mapping of the domestic dog and cat TfRs demonstrated that the domestic dog TfR contains an N-linked glycan at position 384 that is not found in the cat receptor, and this glycan is a key determinant in blocking FPV infection in dogs and other species with identical TfRs (coyote and gray wolf). As shown here, endo H treatment of dog cells allows for FPV infection, reiterating the role that the glycan plays in dictating the host range of FPV. Note that endo H does not cleave complex glycans, such as the one shown at position 261. (C) The pandemic emergence of CPV-2a was facilitated by the ability of the virus to overcome the host range barrier induced by the position 384 glycan on the dog TfR by mutations in its capsid, including VP2 position 300. The role of the TfR position 384 glycan in determining host susceptibility was further demonstrated because its removal allowed for an increase in CPV infection in dog cells. For purposes of clarity, note that the viruses and receptors/glycans are not shown to scale.