JOURNAL OF VIROLOGY, Sept. 2003, p. 10099–10105
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 18
Combinations of Two Capsid Regions Controlling Canine Host Range
Determine Canine Transferrin Receptor Binding by Canine and
Karsten Hueffer,1Lakshman Govindasamy,2Mavis Agbandje-McKenna,2and Colin R. Parrish1*
James A. Baker Institute, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University,
Ithaca, New York 14853,1and Department of Biochemistry and Molecular Biology, Center for Structural Biology, The
Brain Institute, College of Medicine, University of Florida, Gainesville, Florida 32610-02452
Received 10 March 2003/Accepted 23 June 2003
Feline panleukopenia virus (FPV) and its host range variant, canine parvovirus (CPV), can bind the feline
transferrin receptor (TfR), while only CPV binds to the canine TfR. Introducing two CPV-specific changes into
FPV (at VP2 residues 93 and 323) endowed that virus with the canine TfR binding property and allowed canine
cell infection, although neither change alone altered either property. In CPV the reciprocal changes of VP2
residue 93 or 323 to the FPV sequences individually resulted in modest reductions in infectivity for canine cells.
Changing both residues in CPV to the FPV amino acids blocked the canine cell infection, but that virus was
still able to bind the canine TfR at low levels. This shows that both CPV-specific changes control canine TfR
binding but that binding is not always sufficient to mediate infection.
Canine parvovirus (CPV) emerged in the late 1970s as the
cause of a new disease in dogs and is now prevalent in dogs
worldwide. CPV was most likely derived as a host range variant
of the long-known feline panleukopenia virus (FPV), which
infects cats and some other carnivores, but not dogs (11, 18).
FPV became adapted to dogs through a series of steps, the first
being the emergence of the ancestor of the CPV viruses, which
gave rise to a variant (designated CPV type 2) which spread
worldwide during 1978 (30). However, by 1980 the CPV type 2
strain had been replaced worldwide by a variant strain desig-
nated CPV type 2a, which has remained prevalent in dogs with
only a small number of additional changes (19, 30). Each of
these evolutionary steps involved alterations in the capsid,
which changed both its antigenic and host range properties (19,
Parvoviruses have 25-nm-diameter T?1 icosahedral capsids
which are assembled from 60 copies of a combination of the
overlapping VP1 and VP2 proteins (31). The VP1 and VP2
structures contain an eight-stranded antiparallel ?-barrel, with
four large loops inserted between some of the ? strands mak-
ing up much of the viral surface (31). Features of the capsid
surface include cylinders around the fivefold axes, depressions
(the dimples) spanning the twofold axes of symmetry, and
22-A ˚-high raised regions (the threefold spikes) at the threefold
axes of symmetry (1, 31, 33).
Residues in three regions of the capsid surface control the
ability of CPV to infect dog cells. Two of those were seen as
differences between FPV isolates and CPV type 2, where the
changes in FPV of VP2 residue Lys 93 to Asn and Asp 323 to
Asn allowed the virus to infect dog cells (6, 9). In a CPV
background, changing either residue 93 or 323 to the FPV
residue reduced the ability of the virus to infect dog cells (6).
The side chain of residue 93 is exposed on the surface of the
capsid on the top of loop 1 (1, 31), and in FPV Lys 93 forms
two hydrogen bonds with backbone oxygen atoms of residues
225 and 227 of loop 2, while those bonds are not formed by the
Asn in CPV (1, 31, and L. Govindasamy, K. Hueffer, C. R.
Parrish, and M. Agbandje-McKenna, submitted for publica-
tion) (Fig. 1). VP2 residue 323 is separated from residue 93 by
20 A ˚, and the structure near that position is controlled in part
by the binding of up to three divalent ions (25 and L. Govin-
dasamy et al., submitted). A third region controlling infectivity
for dog cells is within a ridge on the side of the threefold spike
(the shoulder), and that contains three differences between
CPV type 2 and FPV (VP2 residues 80, 564, and 568) which
are not exposed on the surface of the capsid (13, 17). The
shoulder region also acquired three additional and surface-
exposed differences during the evolution to the CPV type 2a
strain (19). In addition, some changes in that region can elim-
inate canine cell infection of CPV (13, 17).
CPV and FPV both bind to the feline transferrin receptor
(TfR) and use that receptor to infect feline cells (16), and the
host range of CPV for canine cells is determined by the specific
ability of that virus to bind the canine TfR (11). Binding to the
canine TfR was lost in a host range mutant of CPV which had
a single substitution within the shoulder region of the threefold
spike (Gly 299 to Glu), but whether the other residues con-
trolling host range also affect canine TfR binding and its use
for infection is not known (11). Here we examined the specific
* Corresponding author. Mailing address: James A. Baker Institute
for Animal Health, Department of Microbiology and Immunology,
College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.
Phone: (607) 256-5649. Fax: (607) 256-5608. E-mail: crp3@cornell
roles of residues 93 and 323 in controlling the binding of the
canine TfR and the effects on infection of canine cells.
To determine the functions of residues 93 and 323, we re-
placed VP2 residue 93 and neighboring residues on an adja-
cent loop with alternative amino acids (Fig. 1 and 2). Residue
323 was also changed alone or in combination with residue 93
so as to explore the functional interactions between those
residues (Fig. 1 and 2). Mutant viruses were prepared either by
site-directed mutagenesis of M13 uracilated single-stranded
DNA (12) or by using the Gene Editor protocol on the intact
infectious plasmid (Promega, Madison, Wis.). Some mutants
had previously been isolated as antibody neutralization escape
When mutant genomes were tested for viability and host
range, it was apparent that considerable variation was allowed
in the structure of the CPV capsid near residue 93 without
affecting feline cell infection. All the viruses retained feline
TfR binding and feline cell infection (Fig. 2). Most CPV mu-
tants with single substitutions also retained the ability to infect
dog cells, although many showed lower titers in those cells than
in feline cells (Fig. 2B). In that assay, NLFK or A72 cells in
96-well plates were inoculated with 10-fold dilutions of virus,
FIG. 1. Structures of CPV and FPV within the areas examined in this study. (A) Atomic model of the structure of the CPV capsid in the vicinity
of VP2 residue 93. Labels indicate the VP2 residues that were substituted in these studies. Loop 1 is shown between VP2 residues 91 and 95, while
loop 2 is shown between residues 222 and 229. (B) Atomic model of FPV around residue 93 in the same orientation as in panel A. Labels indicate
the VP2 residues that were substituted during these studies. Hydrogen bonds between residue 93 and the backbone oxygen atoms of residues 225
and 227 are indicated by green lines. (C) Residues mutated in these studies shown on a roadmap of one asymmetric unit of the CPV capsid (24),
showing surface-exposed residues examined in this study. The number following the VP2 residue indicates the relationship of the VP2 molecule
containing the residue relative to the reference VP2 monomer in the standard orientation (2?, twofold; 3?, threefold; 5?, fivefold) (1). Residues
that were changed in CPV are marked in blue, those changed in FPV are marked in green, and those changed in both viruses are colored in red.
Residues 299 and 300, which were not altered in these studies but which also affect canine host range, are shown in brown. Other surface-exposed
residues are not shown here. The position of residue 323 in a neighboring asymmetric unit is indicated by the dashed lines.
10100 NOTESJ. VIROL.
were incubated for 1 h, and then were incubated for a further
2 days in growth medium, after which the cells were fixed and
stained for viral antigen (17). The ratio between the titers
obtained for each virus in dog and cat cells measured the
efficiency of canine cell infection (Fig. 2B). Wild-type CPV
infected both NLFK and A72 cells to similar titers, but all CPV
mutants with single changes of residue 93 infected dog cells at
10- to 100-fold lower titers than were seen with the feline cells
(Fig. 2B). CPV with a single change of residue 323 to Asp
(CPV-N323D) also showed a 10-fold reduction in canine cell
infection, but the double mutant CPV-N93K/N323D was re-
stricted by 10?4, similar to the case of FPV (Fig. 2B).
FPV was highly restricted in dog cells, infecting only to 10?5
the titer seen in cat cells. However, FPV-K93N/D323N in-
fected canine cells to levels only 10-fold lower than levels for
CPV, but changing either residue 93 or 323 alone to Asn
resulted in no increase in FPV infectivity for canine cells (Fig.
2B). Replacing residue 93 in FPV with Arg instead of Lys
along with the 323 change to Asn (FPV-K93R/D323N) re-
sulted in a 250-fold increase in canine cell infectivity compared
to that for wild-type FPV (Fig. 2B).
Infectivities for NLFK and A72 cells were also examined by
testing for replicative-form (RF) DNA production after inoc-
ulation with CPV and FPV mutants containing reciprocal sub-
stitutions of residues 93 and 323 or with alternative changes of
residue 93 (CPV-N93D, CPV-N93R, FPV K93R, and FPV-
K93R/D323N). A72 or NLFK cells seeded at 104cells per cm2
in 9.6-cm2cultures were inoculated with a 50% tissue culture
infective dose (TCID50) of each virus of 0.5 per cell (as deter-
mined in NLFK cells), and then low-molecular-weight DNA
was recovered 2 days later according to a modification of the
method of Hirt (8, 17). After electrophoresis in a 1% agarose
gel containing 1 ?g of ethidium bromide/ml, the DNA was
blotted to a membrane and was probed with a32P-labeled DNA
sequence from the CPV genome. All mutants were infected at
similar levels in NLFK cells, while in A72 cells CPV-N93K,
CPV-N93D, CPV-N93R, and CPV-N323D showed reduced
levels of both infection and RF DNA production compared to
those of wild-type CPV, but CPV-N93K/N323D did not infect
to a detectable level (Fig. 2C). FPV-K93N/D323N infected
more efficiently than FPV-K93R/D323N but did so at lower
levels than CPV, and little or no infection was observed for
wild-type FPV, FPV-K93N, FPV-D323N, or FPV-K93R (Fig.
2C). Although the results of the TCID50and Southern blots
were closely correlated, differences in the assays occurred for
CPV-N93D, which showed more DNA in the canine cell cul-
tures than might be predicted from the TCID50assay. This is
likely explained by the fact that the TCID50detects only the
initial cell infection, while the RF DNA would measure mul-
tiple rounds of replication for the viruses that can infect the
In earlier studies using plaque titrations, a greater decrease
in canine cell infection was observed for single-point mutants
in a CPV background, although the results of DNA analysis
were similar to those observed here (6). The TCID50assay
used here is considerably more sensitive than the plaque assay
for detecting infections by viruses in cells that are relatively
resistant to the virus, as multiple rounds of infection of cells
would be required to form a detectable plaque (6).
The antigenic properties of the mutant viruses were tested
FIG. 2. Mutants prepared in these studies and their relative titers
in feline and canine cells. (A) The genetic background (CPV or FPV)
of each virus is indicated, as well as the changes(s) introduced into the
VP2 sequence. The single-letter code for amino acids was used, with
the letter before the number indicating the amino acid of the wild-type
virus and that following indicating the change(s) introduced. (B) Rel-
ative infectivity of each virus for canine cells shown as the ratio of the
TCID50titer of the virus stock in A72 cells to that in NLFK cells. Error
bars show one standard deviation of the mean for at least three inde-
pendent experiments. For the CPV-P229A mutant, data from one
(ratio, 2.8 log10) of seven independent experiments was excluded from
the analysis. (C) Infection and replication in NLFK and A72 cells of
selected viruses examined by the production of viral RF and single-
stranded DNA (ss). Cells seeded in six-well plates were inoculated with
0.5 TCID50per cell and were incubated for 2 days, and then the
low-molecular-weight DNA was recovered, electrophoresed in an aga-
rose gel, transferred to a membrane, and detected with a32P-labeled
DNA probe. The samples from NLFK cells were exposed for half the
exposure of the A72 cell-derived samples.
VOL. 77, 2003NOTES10101
using monoclonal antibodies (MAb) in a hemagglutination
inhibition (HI) assay (20, 21, 28). Most mutated viruses showed
reduced binding to several MAb that recognize antigenic site
A, which includes the regions around residue 93 (28) (Fig. 3).
An Asn at residue 93 creates an epitope recognized by all the
CPV-specific MAb tested (MAb 14, 7, 13, and 2A9), and sub-
stitution of residue 93 with any of several other amino acids
reduced or eliminated that epitope (Fig. 3). Antibodies recog-
nizing antigenic site A were clearly subdivided into two groups:
the CPV-specific antibodies that were affected by changes of
residue 93 from Asn to most other amino acids and those that
were not affected by changes of residue 93 but which were
affected by changes of residues 222 and 224 (Fig. 3). These
mutants show that the wild-type CPV structure was not re-
quired for the virus to infect canine cells, as many mutant
viruses showing reduced or no reactivity with CPV-specific
MAb still infected canine cells, albeit with reduced efficiency
compared to that of the wild type (Fig. 2 and 3). Changing
residue 323 alone did not alter the reactivity with any of the
MAb tested, and none of the mutants showed altered reactivity
with MAb 8, which recognizes antigenic site B (Fig. 3).
Infection of canine cells partially correlated with the degree
of virus binding and uptake into those cells (Fig. 4). For the
binding assays, cells seeded at 5 ? 104cells per cm2in 10-cm2
dishes were incubated for 1 h at 37°C with a 10-?g/ml concen-
tration of purified empty capsids of the indicated viruses (Fig.
4) (11). After being washed twice in cold Hanks buffered saline
solution without Mg2?or Ca2?(HBSS), cells were detached
with 1 mM EDTA in HBSS for 10 min at 4°C and then were
fixed for 10 min at 22°C with 4% paraformaldehyde. Cell-
associated virus was detected with MAb 8 conjugated to Cy2
(Amersham Biosciences, Piscataway, N.J.) in phosphate-buff-
ered saline with 0.5% (wt/vol) bovine serum albumin and 0.5%
(vol/vol) Triton X-100. Cells were analyzed by using a FACS-
calibur flow cytometer and Cell Quest software (Becton Dick-
inson, San Jose, Calif.). All viruses tested bound efficiently to
the feline NLFK cells, although FPV, FPV mutants, and CPV-
N93D and CPV-N93R all showed two- to threefold reduced
binding levels compared to those for CPV (Fig. 4A).
Levels of virus binding to canine Cf2Th cells were more
variable than those for binding to feline cells (Fig. 4B), and
similar results were obtained for A72 cell binding of the same
capsids (data not shown). CPV showed the strongest canine
cell binding, while mutants of CPV with residue 93 or 323
replaced bound at lower levels, some at close to background
levels (Fig. 4). The correlation between binding and infectivity
of dog cells was most clearly seen for FPV and its mutants,
where FPV-K93N/D323N bound and infected while wild-type
FPV and the mutants FPV-K93N and FPV-K93R/D323N did
not bind or infect those cells (Fig. 2 and 4B).
In previous studies viruses labeled with35S or3H were in-
cubated with cells at 4°C, and no significant differences in
binding to canine cells were seen between the closely related
mink enteritis virus and CPV or between a host range mutant
of CPV and wild-type CPV (10, 17). We examined the binding
of the viruses to feline and canine cells at 37 and 4°C and found
that at 4°C the CPV capsids bound to canine cells at levels
close to background, similar to the binding seen for FPV cap-
sids or for no capsids (Fig. 4C). At 4°C the binding to NLFK
cells showed moderate decreases of three- to fourfold for CPV
and 1.5- to 3.5-fold for FPV (Fig. 4C). This shows that binding
is temperature dependent and that temperature directly influ-
ences the association between the capsids and the cell surface
or the receptor(s).
We have shown that the specificity of capsid binding to the
feline and canine TfRs correlates with the host range of the
viruses and the susceptibility of the host cells (11). The binding
of mutant capsids to the canine TfR was therefore tested on
TRVb cells expressing the canine TfR from a plasmid after
transfection, and the cells were simultaneously incubated with
iron-loaded canine transferrin conjugated to Cy5 (11). All vi-
ruses tested efficiently bound the feline TfR expressed on
TRVb cells (results not shown) (11, 16). All of the viruses that
infected canine cells showed efficient binding to the expressed
canine TfR at 37°C (Fig. 5). Mutants CPV-N93K, CPV-N93D,
and CPV-N93R all bound the canine TfR, but they did so at
levels lower than were seen for the wild-type virus, as higher
expression levels of the TfR on the cells (as seen by transferrin
binding) were necessary before virus binding was detected
(Fig. 5A). Although CPV-N93K/N323D was not able to infect
the canine cells, it still bound the canine TfR, although at levels
that were lower than those for the single mutants (Fig. 5A).
Of the FPV mutants, FPV-K93N and FPV-D323N did not
show detectable binding to the canine TfR, while FPV-K93N/
D323N, which was able to infect canine cells, bound at levels
that were similar to those of CPV-N93K/N323D (Fig. 5B). It
FIG. 3. Antigenic analysis of the viruses examined by using MAb in
an HI assay. Eight hemagglutinin units of virus were incubated for 1 h
with twofold dilutions of monoclonal antibodies in the assay, and then
erythrocytes were added. The results are shown as the titer of the
antibodies for each virus relative to that of wild-type CPV. Filled
boxes, mutant HI at 10 to 100% wild-type titer; cross-hatched boxes,
less than 10% but greater than 1% of the wild-type titer; open boxes,
less than 1% of the wild-type titer.
10102NOTES J. VIROL.
therefore appears that canine TfR binding is required for in-
fection of canine cells, that additional host-specific steps are
required for successful infection, and that infection is influ-
enced by residues that act in combination with VP2 residues 93
and 323. In previous studies of CPV binding to the canine TfR,
binding to the canine TfR expressed on TRVb cells led to only
low levels of infection by the CPV type 2 strain, while those
cells were more efficiently infected by the CPV type 2b strain
VP2 residues 93 and 323 are separated by 20 A ˚in the capsid
structure and there is no direct association between them, but
both sites are required for successful capsid interactions with
the canine TfR. Other sites in the CPV capsid are also involved
in controlling canine TfR binding and canine cell infection.
This was seen for CPV-N93K/N323D, which bound the canine
TfR despite the FPV-derived Lys and Asp at residues 93 and
323, indicating that other structural differences in that virus
must allow binding to the canine TfR in the presence of those
changes. However, that virus-receptor interaction was not suf-
ficient for infection. Mutant CPV-G299E does not infect ca-
nine cells or bind the canine TfR (11, 17), and residue 299 is
?30 A ˚from both residues 93 and 323 (Fig. 1C), indicating that
FIG. 4. Binding of CPV, FPV, or mutant virus capsids to feline or canine cells. Cells were incubated with 10 ?g of virus/ml for 1 h at 37°C, and
then after cell detachment, fixation, and permeabilization the capsid antigen was detected by antibody staining and flow cytometry. (A) Binding
to feline cells of CPV capsids and mutants in a CPV background (upper panel) or FPV capsids and mutants in an FPV background (lower panel).
(B) Binding to canine cells of CPV capsid and mutants in a CPV background (upper panel) or FPV capsids and mutants in an FPV background
(lower panel). (C) Effect of temperature on binding of CPV or FPV capsids to feline or canine cells. Cells were incubated with 10 ?g of virus/ml
for 1 h at 4°C or at 37°C, and then after detachment, fixation, and permeabilization viral antigen was detected by antibody staining and flow cytometry.
VOL. 77, 2003 NOTES10103
there is a third capsid region that also influences the canine
TfR-virus interaction (Fig. 1C). From the structures of CPV
and FPV, and also from new structures of additional mutant
viruses CPV-N93D and CPV-N93R (L. Govindasamy et al.,
submitted), it is clear that the changes within each host-range-
controlling region do not affect the structure at the other site.
This suggests that the TfR apical domain, which mediates
interaction with the capsid (15), engages all three host-range-
determining regions to give a binding that can lead to infection.
These results have parallels with those for other parvovi-
ruses. In the minute virus of mice (MVM), two strains can be
distinguished on the basis of their tropisms for lymphoid and
fibroblastic cells. MVM(p)can infect fibroblasts, while MVM(i)
infects erythropoetic progenitor cells and lymphoid cells, in-
cluding T cells (3, 7, 29). The fibrotropic phenotype of MVM(p)
was controlled by a combination of residues 317 and 321 in
VP2, which determine the host cell specificity in a coordinated
manner (4). Those residues are located on the surface of the
capsid on the side of the threefold spike and correspond to
residues 318 and 323 of CPV (2). When MVM(i)containing
single-point mutations of either residue were grown on fibro-
blasts to select for variants, viruses were isolated with muta-
tions of residues 317 or 321 or of residues in nearby regions of
the capsids. In MVM VP2 residue 95, which corresponds to
CPV residue 93, is a Lys and forms a hydrogen bond to the
backbone oxygen of residue 230, similar to FPV (2). In contrast
to our data for FPV, the restricted infection of MVM strains
was reported to be blocked after cell binding and uptake of the
capsids (23, 26). In Aleutian mink disease virus (ADV) the
ADV Utah strain does not replicate in cell culture but infects
mink, while the ADV-G strain replicates in cell culture but not
in mink (5). The differences controlling these tropisms were
mapped to a region of the capsid gene corresponding to the tip
of loop 1 in CPV (14, 27). One of these changes (Gln 94 to Lys)
aligns with residue 93 in CPV. These similarities of changes
controlling host range and tropism between CPV, MVM, and
ADV suggest that the regions examined in this study play a
central role in the general virus-cell interactions of parvovi-
Most viruses are well-adapted parasites and are restricted to
a defined range of hosts or cell types. Rare transfers of mutant
viruses with new host range properties into new hosts can lead
to widespread distribution. From CPV and from other exam-
ples of host range adaptation, such as that of the influenza
viruses (32), it is clear that emerging viruses of this type can
become very successful in their new host. The finding that in
FIG. 5. Binding of transferrin and capsids of CPV, FPV, or mutant virus to TRVb cells expressing the canine TfR from plasmids. Transiently
transfected TRVb cells expressing the canine TfR were incubated with both capsids and Cy5-labeled canine transferrin for 1 h at 37°C, and then
after detachment, fixation, and permeabilization the viral antigen was detected by antibody staining and flow cytometry. Virus signal is shown on
the X axis, while transferrin is shown on the Y axis. The numbers in the upper quadrants of the graphs represent the percentage of events that fell
within those quadrants, and the numbers in parentheses show the percentage of positive cells (as shown by transferrin binding in the upper two
quadrants) which bound virus in each case. (A) Binding to the canine TfR expressed on TRVb cells of CPV or mutants derived from CPV.
(B) Binding to the canine TfR expressed on TRVb cells as well as on mock-inoculated cells of FPV or mutants derived from FPV.
10104 NOTESJ. VIROL.
CPV specific combinations of residues act in concert to control
the new receptor-virus interactions gives a clearer understand-
ing of the molecular mechanisms that permitted CPV emer-
gence and evolution to occur. Several changes were required
for FPV to become a new successful pathogen of dogs, and
those most likely needed to arise in a particular order (11, 30).
This suggests that the probability of this type of host shift is
extremely low and likely occurs through multiple adaptive
events. Early identification of new viruses may therefore allow
control of the new virus before it becomes fully adapted to its
Wendy Weichert and Gail Sullivan provided expert technical assis-
tance. Hollis Erb provided help with data analysis.
This work was supported by grants AI 28385 and AI 33468 from the
National Institutes of Health to C.R.P.
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