Discrimination between sialic acid-containing receptors and pseudoreceptors regulates polyomavirus spread in the mouse.

Abstract

Variations in the polyomavirus major capsid protein VP1 underlie important biological differences between highly pathogenic large-plaque and relatively nonpathogenic small-plaque strains. These polymorphisms constitute major determinants of virus spread in mice and also dictate previously recognized strain differences in sialyloligosaccharide binding. X-ray crystallographic studies have shown that these determinants affect binding to the sialic acids. Here we report results of further experiments designed to test the importance of specific contacts between VP1 and the carbohydrate moieties of the receptor. With minor exceptions, substitutions at positions predicted from crystallography to be important in binding the terminal alpha-2,3-linked sialic acid or the penultimate sugar (galactose) destroyed the ability of the virus to replicate in cell culture. Substitutions that prevented binding to a branched disialyloligosaccharide were found to result in viruses that were both viable in culture and tumorigenic in the mouse. Conversely, substitutions that allowed recognition and binding of the branched carbohydrate chain inhibited spread in the mouse, though the viruses remained viable in culture. Mice of five different inbred strains, all highly susceptible to large-plaque virus, showed resistance to the spread of polyomavirus strains bearing the VP1 type which binds the branched-chain receptor. We suggest that glycoproteins bearing the appropriate O-linked branched sialyloligosaccharide chains are effective pseudoreceptors in the host and that they block the spread of potentially tumorigenic or virulent virus strains.

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JOURNAL OF VIROLOGY,
0022-538X/99/$04.00?0
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
July 1999, p. 5826–5832 Vol. 73, No. 7
Discrimination between Sialic Acid-Containing Receptors and
Pseudoreceptors Regulates Polyomavirus
Spread in the Mouse
PAUL H. BAUER,1CUNQI CUI,1THILO STEHLE,2STEPHEN C. HARRISON,3
JAMES A. DECAPRIO,4AND THOMAS L. BENJAMIN1*
Department of Pathology1and Dana-Farber Cancer Institute,4Harvard Medical School, Boston, Massachusetts 02115;
Laboratory of Developmental Immunology, Massachusetts General Hospital, Boston, Massachusetts 021142;
and Department of Molecular and Cellular Biology, Howard Hughes Medical Institute,
Harvard University, Cambridge, Massachusetts 021383
Received 28 January 1999/Accepted 12 April 1999
Variations in the polyomavirus major capsid protein VP1 underlie important biological differences between
highly pathogenic large-plaque and relatively nonpathogenic small-plaque strains. These polymorphisms
constitute major determinants of virus spread in mice and also dictate previously recognized strain differences
in sialyloligosaccharide binding. X-ray crystallographic studies have shown that these determinants affect
binding to the sialic acids. Here we report results of further experiments designed to test the importance of
specific contacts between VP1 and the carbohydrate moieties of the receptor. With minor exceptions, substi-
tutions at positions predicted from crystallography to be important in binding the terminal ?-2,3-linked sialic
acid or the penultimate sugar (galactose) destroyed the ability of the virus to replicate in cell culture.
Substitutions that prevented binding to a branched disialyloligosaccharide were found to result in viruses that
were both viable in culture and tumorigenic in the mouse. Conversely, substitutions that allowed recognition
and binding of the branched carbohydrate chain inhibited spread in the mouse, though the viruses remained
viable in culture. Mice of five different inbred strains, all highly susceptible to large-plaque virus, showed
resistance to the spread of polyomavirus strains bearing the VP1 type which binds the branched-chain
receptor. We suggest that glycoproteins bearing the appropriate O-linked branched sialyloligosaccharide
chains are effective pseudoreceptors in the host and that they block the spread of potentially tumorigenic or
virulent virus strains.
Wild-type laboratory strains of polyomavirus are all potent
transforming agents in culture, but only some are able to in-
duce the high frequency and broad array of tumors in mice (12)
typical of early virus isolates (14, 21). Previous studies with
recombinants between a strain producing tumors at a high
frequency and a strain producing tumors at a low frequency
indicated that a single-site polymorphism in VP1 was a major
determinant of tumorigenicity (15), although differences in
noncoding sequences between the two strains also had effects
(16, 18). Similarly, comparisons between a virulent strain that
causes a rapidly lethal infection of newborn mice and its pa-
rental strain, which induces tumors at a high frequency,
showed a single amino acid difference in VP1 to be the major
determinant of virulence (2). Table 1 summarizes the findings
with respect to critical amino acid substitutions in the VP1s of
three prototype wild-type strains of widely different pathoge-
nicities. It is significant that the important biological determi-
nants at positions 91 and 296 map to regions of VP1 that form
parts of the oligosaccharide binding site on the virus surface
(32–34).
Differences in selectivity and avidity of binding to sialyloli-
gosaccharides dictated by VP1 clearly correlate with pathoge-
nicity. The directions of these correlations are surprising and
have interesting implications, in two respects. First, although
small-plaque virus strains have broader binding specificities,
recognizing branched- as well as straight-chain sialyloligosac-
charides (6, 7, 19), they are far less pathogenic than large-
plaque strains, which bind only the straight chain (12, 15, 18).
The operative change is at position 91 (17). The presence of
glutamic acid at this position prevents binding to the branched-
chain oligosaccharide (through electrostatic repulsion with the
?-2,6-linked sialic acid), while glycine passively accommodates
the branched carbohydrate chain (32, 33). These results imply
the existence in the mouse of virus inhibitors or pseudorecep-
tors with branched-chain sialyloligosaccharides. Second, strain
PTA disseminates broadly and induces multiple tumors, while
its derivative strain LID (26, 31) spreads even more rapidly and
kills newborn mice before tumors can develop (2, 4). The
important difference between these two large-plaque strains is
at position 296, where valine in PTA is replaced by alanine in
LID. This seemingly conservative substitution results in the
loss of a hydrophobic contact between VP1 and the terminal
?-2,3-linked sialic acid. These results imply that weaker bind-
ing by the virus to its true receptor translates into a more
virulent phenotype (2).
In this study, we first report unsuccessful attempts to identify
a unique polyomavirus receptor through the isolation of mono-
clonal antibodies and then turn to analyzing the recognition of
the carbohydrate moiety of the receptor by VP1 as the key to
understanding the importance of virus-receptor interactions in
pathogenesis. Site-directed mutagenesis is used to introduce
substitutions into the VP1s of different wild-type strains,
guided by results of X-ray crystallography (32–34). Differences
in noncoding sequences among wild-type strains are common,
and these can have significant effects on the extent and tissue
* Corresponding author. Mailing address: Department of Pathology,
Harvard Medical School, 200 Longwood Ave., Boston, MA 02115.
Phone: (617) 432-1960. Fax: (617) 277-5291. E-mail: thomas_benjamin
@hms.harvard.edu.
5826

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specificity of virus replication (1, 9, 28–30) as well as on tumor
induction (16, 18). By comparing site-directed mutants with
parental strains, the effects of VP1 mutations can be assessed
in the absence of differential contributions from viral enhanc-
ers. Mutations affecting all sites predicted from structural stud-
ies to be involved in oligosaccharide binding have been stud-
ied. The results show that proper recognition of the terminal
sialic acid as well as of the penultimate galactose are critical for
viability of both large- and small-plaque strains. Substitutions
that allow recognition of the branched disialyloligosaccharide
are viable in culture but lead to restricted spread and reduced
pathogenicity in the mouse. These results confirm and extend
earlier findings on the structural basis for selectivity in sialic
acid binding by the virus, the importance of receptor affinity
and selectivity, and the likely role of branched-chain sialyloli-
gosaccharides as pseudoreceptors capable of inhibiting virus
spread in the mouse.
MATERIALS AND METHODS
Screening for monoclonal antibodies to polyomavirus receptors. Hamster
anti-mouse cell hybridomas were prepared through Monoclonal Core Facility of
Program Project grant PO1-CA50661 (D. Livingston, principal investigator; J.
DeCaprio, core director). Golden Armenian hamsters were immunized by three
intraperitoneal inoculations of 2 ? 106to 5 ? 106NIH 3T3 or primary baby
mouse kidney epithelial cells 1 month apart. Animals were sacrificed, and spleen
cells were harvested and fused with NS-1 cells 1 week after the third inoculation.
Hybridoma supernatants were screened for their ability to block infection of NIH
3T3 cells by a cytopathic effect (CPE)-protection assay. NIH 3T3 cells were
grown in 96-well plates to ?50% confluence; cells were preincubated at 37°C for
30 min in medium containing 50 ?l of supernatant. Wild-type (PTA) virus was
then added at a multiplicity of 2 to 5 PFU/cell, and the cultures were monitored
for development of CPE over a 5- to 6-day period.
Virus strains. Genomic clones of viral DNAs of the large-plaque strains PTA
(15, 18) and LID (2) and the small-plaque strain RA (15, 18) were used to
generate VP1 mutants by oligonucleotide mutagenesis. The EcoRI-BamHI large
(3.2-kb) fragments encoding all of VP1 were subcloned into pUC19 (Gibco BRL,
Gaithersburg, Md.), and the desired mutations were introduced with a Trans-
former site-directed mutagenesis kit (Clontech, Palo Alto, Calif.). In most cases,
silent mutations were introduced along with the desired mutation to allow dis-
crimination between possible reversion and contamination. Whole virus genomes
were reconstituted by ligation of the large fragment confirmed to carry the
desired mutations with the PTA wild-type EcoRI-BamHI small (2.1-kb) frag-
ment. The ligation mixtures were transfected into NIH 3T3 cells by electropo-
ration (2 ? 105to 5 ? 105cells in 400 ?l; 260 V, 1,050 ?F). Transfected cells
were plated in Dulbecco’s modified Eagle’s medium with 10% calf serum, and
the cultures were monitored as described in Results. Viable mutants were further
propagated on primary baby mouse kidney epithelial cells or NIH 3T3 cells.
Plaque assays were performed on NIH 3T3 cells with PTA and RA controls to
discern the large- versus the small-plaque phenotype as described previously
(17).
Mouse strains and animal experiments. For most experiments, mice of the
C3H/BiDa strain were used (obtained from Clarence Reeder, National Cancer
Institute, Frederick, Md.). AKR/J, CBA/J, PERA/Ei, and Czech II/Ei mice were
obtained from the Jackson Laboratory (Bar Harbor, Maine). Newborn mice
(?18 h old) were inoculated intraperitoneally with 50 ?l of crude virus of known
titer as indicated in Results. Animals were sacrificed at the times indicated below
for whole-mouse section hybridization as described previously (13) or main-
tained for survival studies (2) or tumor development (12). Viral genotypes were
confirmed by sequencing viral DNAs amplified from kidneys of infected mice.
RESULTS
The likelihood of occurrence of multiple sialo-glycoprotein
receptors. Cell surface glycoproteins with specific sialic acid
linkages are critical determinants for hemagglutination (6, 7)
and infection of host cells (19) by polyomavirus. Receptors on
NIH 3T3 cells are predominantly N-linked glycoproteins (10),
but it is not known whether a single or multiple receptor
species exist. We have attempted to answer this question
through the isolation and screening of hamster anti-mouse
cell monoclonal antibodies. Hybridoma supernatants were
screened by preincubation of NIH 3T3 cells followed by viral
infection and monitoring for inhibition of development of
CPE, a procedure similar to that used successfully in other
systems to identify virus receptors (3, 11, 20). Roughly 2,000
hybridomas were screened. Some clones gave preliminary in-
dications of causing a delayed CPE and showed cell surface
staining on NIH 3T3 cells by indirect immunofluorescence;
however, upon subcloning and further characterization, none
proved to be clearly protective. While these negative results do
not rule out a single receptor, they suggest the likelihood of
multiple glycoprotein receptor species, with each bearing a
terminal ?-2,3-linked sialic acid(s). Such linkages are com-
monly found on glycoproteins with N-linked sugars expressed
in a variety of cells. Four different ?-galactoside ?-2,3 sialyl-
transferases have been cloned from the mouse, with overlap-
ping but distinguishable acceptor specificities, tissue distribu-
tions, and developmental patterns of expression (24). These
observations are also consistent with the existence of multiple
polyomavirus receptor species and with the broad range of cell
types (?30) that polyomavirus is known to infect (12).
Effects of mutations in the receptor binding pockets of VP1.
The virus surface shows three distinct pockets, which bind
different moieties of sialic acid-containing oligosaccharide re-
ceptors (32, 34). Pocket 1 accommodates the terminal sialic
acid, and pocket 2 accommodates the penultimate ?-2,3-linked
galactose. Pocket 3 accommodates the branched sialic acid,
which is bound through an ?-2,6 linkage to the third sugar,
typically N-acetylglucosamine or N-acetylgalactosamine. A
fourth pocket can also be discerned but has not been impli-
cated thus far in binding any carbohydrate moiety. Figure 1
shows a view of the pockets on the virus surface along with a
schematic representation of interactions between VP1 side
chains and the sugars. The VP1 contacts are based on cocrystal
structures with purified virus (resolution, 3.65 Å) or recombi-
nant VP1 pentamers (resolution, 1.9 Å) and various sialyloli-
gosaccharides (32–34).
Amino acid substitutions were introduced at each position in
VP1 involved in carbohydrate binding. In most instances, con-
servative substitutions were chosen to alter specific contacts
with the sugar and to minimize the possibility of long-range
effects that might interfere with VP1 folding or virus assembly.
The latter possibilities seem unlikely based on the structure.
All of the mutations reside in surface loops of VP1, which are
exposed to solvent, and involve sites that are far from either
interpentamer contacts or contacts between neighboring VP1s
within a pentamer (32, 33). Complete mutant viral genome
constructs were made on either an RA or a PTA background
and transfected by electroporation into NIH 3T3 cells. Trans-
fection efficiencies were between 1 and 5% based on immuno-
fluorescent staining for T antigen. Transfections with compa-
rable amounts of wild-type viral DNAs were carried out in
parallel as positive controls. Transfected cultures were moni-
TABLE 1. Properties of prototype wild-type strains of polyomavirus
Virus strain
(plaque
type)
Pathogenicity
Sialic acid
recognitiona
VP1
sequence
at
position:
Straight
chain
Branched
chain
91 296
RA (small)
PTA (large)
LID (large)
Induces few or no tumors
Highly tumorigenic
Virulent
?
?
?
?
?
?
Gly
Glu
Glu
Val
Val
Ala
aStraight chain, NeuNAc-(?-2,3)-Gal-(?-1,3)-GalNAc or -GlcNAc-; branched
chain, NeuNAc-(?-2,3)-Gal-(?-1,3 or ?-1,4)-[(NeuAc-(?-2,6)]-GlcNAc-.
VOL. 73, 1999POLYOMAVIRUS RECEPTORS AND PSEUDORECEPTORS5827

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tored for development of CPE as an indication of virus growth.
CPE typically developed in wild-type-transfected cultures
within the first week and was complete after 8 to 10 days. In
most instances, the VP1 mutants showed little or no CPE even
after 3 weeks of incubation. Cultures with or without overt
CPE were harvested, and virus yields on NIH 3T3 cells were
determined by plaque assay. From any mutant lysate showing
plaques, several plaques were picked, the virus was amplified
on NIH 3T3 or primary baby mouse kidney cells, and the viral
DNA was sequenced to confirm the presence of the mutation
and to rule out the possibilities of reversion and wild-type
contamination. Experiments on animals were carried out with
selected mutants. Table 2 summarizes the results with all the
mutants tested.
In pocket 1, substitutions for R77 that removed the salt
bridge to the negatively charged sialic acid were lethal, as
expected. These results stress the critical role of this salt bridge
in sugar binding. Removal of the hydrogen bond between Y72
and the N-acetyl, however, was not necessarily lethal. The
Y72F mutant was viable, though it grew less well than wild-
type virus. Evaluation of the structure indicates that a water
molecule can substitute for the tyrosine hydroxyl group in the
binding pocket. The Y72A mutant, on the other hand, was not
viable. Since the Y72 side chain stacks against the R77 guani-
dinium group in the wild-type structure and is important for
proper orientation of the R77 side chain, removal of the phenyl
group is likely to affect the conformation of the crucial R77
side chain and therefore also carbohydrate binding. Although
extension of the N-acetyl side chain through metabolic incor-
poration of synthetic analogues partially blocks infection by
polyomavirus (22), the shorter and naturally occurring N-gly-
colyl neuraminic acid (23) was expected to interact normally
with the virus. The H298Q mutant was nonviable, indicating
that the hydrogen bond to the O-4 of the terminal sugar is an
important interaction for binding. Although Q298 can in prin-
ciple hydrogen bond to O-4, such an interaction is ruled out
because to do so Q298 would have to assume a conformation
that would result in unfavorable contacts with other residues.
Substitutions for V296 are viable but with different biologi-
cal consequences. The V296I PTA mutant (PTA-V296I) is
expected to preserve the important van der Waals contact with
the sugar ring (2). This mutant induced multiple tumors in
FIG. 1. Interactions between polyomavirus VP1 and sialyloligosaccharide receptors, as seen in their crystal structures (32–34). The oligosaccharide receptor is shown
as a ball-and-stick model, with the straight-chain receptor being represented with light shading and the branching ?-2,6-linked sialic acid being represented with darker
shading. Hydrogen bonds between VP1 residues and the receptor are shown as broken lines; hydrophobic interactions are represented with arrows. The molecular
surface of a polyomavirus VP1 pentamer complexed with the branched oligosaccharide is shown in the inset. The figure was prepared with RIBBONS (M. Carson,
University of Alabama at Birmingham) and GRASP (B. Honig and A. Nicholls, Columbia University).
5828BAUER ET AL.J. VIROL.

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each of six mice inoculated and is thus essentially similar to
PTA encoding V296. Computer modeling indicated that the
I296 side chain can assume a conformation similar to that of
the V296 side chain and in which the additional methyl group
does not contact the sugar. PTA-V296G remained viable but
grew more slowly and to lower titer than PTA in culture.
However, in animals, this mutant resembles PTA-V296A and
LID (encoding A296) in acquiring a virulent phenotype. The
V296G substitution, introducing what is likely to be an even
looser fit of the sugar with complete loss of the van der Waals
contact, appears to be even more virulent than LID. None of
32 mice receiving 104PFU of PTA-V296G survived 32 days,
while in a previous study four of five mice receiving a similar
dose of LID survived (2).
Two substitutions in pocket 2 (G78V and N93A) appeared
to be nonviable. These results are consistent with the cocrystal
structures which show a hydrogen bond between N93 and O-6
of the galactose and also indicate that any side chain at posi-
tion 78 would collide with this sugar (32, 34). In pocket 3, the
substitution E91L on a PTA background preserved normal
viability in culture and also the essential tumorigenic behavior
of PTA, with four of four mice developing multiple tumors
with an average latency period of less than 70 days. Electro-
static repulsion between E91 and the carboxyl of the branched
sialic acid is the likely basis for the failure of large-plaque
strains to bind the branched chain (32, 34). Although the L91
substitution does not result in unfavorable contacts with the
branched sugar, the presence of the hydrophobic and com-
pletely solvent-exposed leucine might trigger a more extended
structural rearrangement that partially buries this side chain,
thereby altering the pocket shape so that it can no longer
accommodate the branched sugar.
Substitution of glycine for glutamic acid-91 in large-plaque
strains reduces tumorigenicity and virulence in C3H/BiDa
mice. The expected consequence of the E3G substitution at
position 91 in pathogenic large-plaque strains was to allow
binding of oligosaccharide chains bearing branched-chain sialic
acids of the type NeuNAc-(?-2,3)-Gal-(?-1,3)-[NeuNAc-(?-
2,6)]-GlcNAc. Introduction of this mutation into the highly
tumorigenic strain PTA resulted in decreased tumorigenicity
(Table 3). At the highest dose tested, PTA-E91G was able to
induce tumors in 100% of the animals but with a latency period
twice as long as that of PTA at an equivalent dose. Moreover,
when the doses of virus were reduced, the frequency of tumor
induction by PTA-E91G fell to 0 while that of PTA was still
100%. Similarly, when the E91G substitution was introduced
into the virulent LID strain, the virus became avirulent. Twelve
of 12 newborn mice inoculated with ?106PFU of LID-E91G
survived, in contrast to mice inoculated with LID itself, which
kills 100% of recipients at the same dose or even 10-fold lower
doses of virus (2, 8).
The E91G substitution reduced the degrees of virus spread
of large-plaque strains in newborn C3H/BiDa mice. Figure 2
shows results of whole-mouse-section hybridization in which
the standard wild-type strains were compared with their sub-
stitution mutants. By 7 days, the spread of PTA was extensive
and became even more extensive with the addition of the
virulence determinant (PTA-V296A), similar to results with
TABLE 2. Substitutions in receptor binding pockets of polyomavirus VP1
Pocket ResidueInteraction with oligosaccharidea
StrainMutationViabilityb
1 R77Salt bridge between R77 and carboxyl of
NeuNAc-1; key interaction in
attracting and orienting NeuNAc-1
PTA or RA
PTA or RA
R77E
R77Q
Nonviable
Nonviable
Y72Hydrogen bond linking the phenolic
oxygen of Y72 with amide nitrogen of
NeuNAc-1
PTA or RA
PTA or RA
Y72F
Y72A
Viable (?)
Nonviable
H298
V296
Hydrogen bond to 0-4 of NeuNAc-1
van der Waals contact with C-3 and 0-4
of NeuNAc-1
PTA or RA
PTA
PTA
H298Q
V296G
V296I
Nonviable
Viable (?), virulent
Viable (??), tumorigenic
2 G78Absence of a side chain important in
accommodating the galactose
Hydrogen bond with 0-4 of galactose
PTA or RAG78VNonviable
N93PTA or RAN93ANonviable
3 E91Electrostatic repulsion of NeuNAc-2 PTAE91L Viable, tumorigenic
aInteractions are those seen in the crystal structures (see the work of Stehle et al. [32–34]).
bViability is scored based on kinetics and the extent of development of CPE and on the titers of virus recovered from transfected cultures. Nonviable indicates no
observable CPE and no virus recovered (?20 PFU/ml). (??) indicates viability equivalent to that of wild-type virus; (?) indicates a delayed and reduced CPE and
virus titers at least 1 order of magnitude less than that of the wild type. See Fig. 1 and the text.
TABLE 3. Tumor development in mice inoculated with PTA or PTA-E91Ga
Virus dose
(PFU/animal)
PTAPTA-E91G
No. of mice with
tumor(s)/total no. of mice
Avg latency (days)
No. of mice with
tumor(s)/total no. of mice
Avg latency (days)
2 ? 105to 5 ? 105
1 ? 102to 3 ? 102
1 ? 101to 3 ? 101
9/9
22/22
20/20
71
92
16/16
2/29
0/39
153
135
143
?180
aNewborn C3H/BiDi mice were inoculated within 24 h of birth intraperitoneally with the virus doses indicated. The fractions of mice developing at least one tumor
are given, along with the average times at which mice came to necropsy as an indication of latency. The experiment with PTA-91EG was terminated at 6 months.
VOL. 73, 1999POLYOMAVIRUS RECEPTORS AND PSEUDORECEPTORS5829

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LID. The E91G substitution in each of the large-plaque viruses
led to attenuation of spread to a degree similar to that of the
small-plaque strain RA-encoding 91G. Virus titers in kidney
homogenates were approximately 100-fold higher in 7-day-old
PTA-infected mice than in PTA-E91G-infected or RA-in-
fected mice. The E91G substitution in all three strains altered
the plaque size phenotype from large to small, as expected.
Newborn mice of different strains specifically block the
spread of PTA-E91G. Our experiments on mice were carried
out primarily with the susceptible C3H/BiDa strain. Suscepti-
bility to tumor induction in this strain is based on its major
histocompatability type and the expression of an endogenous
mouse mammary tumor virus superantigen, which together
prevent the development of effective T-cell responses to poly-
omavirus tumors (25). Three other unrelated but highly sus-
ceptible standard inbred strains (AKR/J, CBA/J, and RF/J)
also carry these host determinants. Two recently wild-derived
inbred strains (PERA/Ei and Czech II/Ei) are also highly sus-
ceptible to tumor induction by PTA but have genetic bases for
susceptibility distinctly different from that of the standard in-
bred strains (unpublished data). To investigate how general
the resistance to the spread of small-plaque viruses is among
different mouse strains, newborn mice of four of these strains
were inoculated with the large-plaque virus PTA or A2 (both
91E) or PTA-E91G and examined by whole-mouse-section
hybridization (Fig. 3). The results for each strain were similar
to those with C3H/BiDa mice (Fig. 2). Particularly striking is
the effect of the E91G substitution in preventing high-level
virus replication in the kidneys of all mouse strains tested.
These observations indicate that similar or identical mecha-
nisms operate in different host strains to discern the difference
in VP1 types and to selectively block the spread of the 91G
type of virus.
Substitution of glutamic acid for glycine-91 in the small-
plaque RA strain increases virus spread and pathogenicity.
The predicted effect of replacing glycine-91 with glutamic acid
in the relatively nonpathogenic small-plaque strain RA is to
block the ability of the virus to bind the branched disialyloli-
gosaccharide. If glycoproteins bearing the branched oligosac-
charide act in the intact host as pseudoreceptors, the effect of
this substitution would be to increase virus spread and patho-
genicity. This prediction was tested by whole-mouse-section
hybridization of neonatally infected C3H/BiDa mice (Fig. 4,
top row). The G91E substitution clearly led to greater virus
spread, particularly in the kidney, which is the major site of
amplification and also the critical target in causing death (2, 4).
Titers of virus recovered from kidneys were roughly 50-fold
higher in mice infected with RA-G91E than in those infected
with RA, and the plaque type went from small to large in the
substitution mutant.
Dominance of pseudoreceptor recognition over virulence.
When the virulence determinant at position 296 was intro-
duced into RA, there was no discernable increase in virus
spread (Fig. 4, bottom row); animals inoculated with RA or
RA-V296A were sacrificed at 12 rather than 7 days, and blots
were exposed for longer than usual to look for signs of in-
creased spread. The same substitution on the RA-G91E back-
ground produced a rapidly spreading virus. This RA-G91E
FIG. 2. Whole-mouse-section hybridization. Newborn C3H/BiDa mice were inoculated with 2 ? 105to 5 ? 105PFU of viruses of the indicated strains and sacrificed
at 7 days. See Materials and Methods.
FIG. 3. Whole-mouse-section hybridization with different mouse strains. Newborn mice of the indicated strains were inoculated with 2 ? 105to 5 ? 105PFU of
wild-type PTA or A2, each encoding E91 (top in each pair), or PTA-E91G (bottom in each pair) and sacrificed at 10 to 12 days. See Materials and Methods.
5830BAUER ET AL.J. VIROL.