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Published Ahead of Print 24 April 2013.
2013, 87(13):7255. DOI: 10.1128/JVI.03518-12. J. Virol.
Prasad and Mary K. Estes
Robert F. Ramig, Jacques Le Pendu, B. V. Venkataram
E. Crawford, Rita Czako, David F. Smith, Gagandeep Kang,
Sasirekha Ramani, Nicolas W. Cortes-Penfield, Liya Hu, Sue
Glycans
Strain G10P[11] Binds to Type II Precursor
The VP8* Domain of Neonatal Rotavirus
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The VP8* Domain of Neonatal Rotavirus Strain G10P[11] Binds to
Type II Precursor Glycans
Sasirekha Ramani,
a
Nicolas W. Cortes-Penfield,
a
Liya Hu,
b
Sue E. Crawford,
a
Rita Czako,
a
David F. Smith,
c
Gagandeep Kang,
d
Robert F. Ramig,
a
Jacques Le Pendu,
e
B. V. Venkataram Prasad,
a,b
Mary K. Estes
a
Departments of Molecular Virology and Microbiology
a
and Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine,
Houston, Texas, USA
b
; Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA
c
; Christian Medical College, Vellore, Tamilnadu, India
d
;
INSERM, U892, and CNRS, UMR 6299, Université de Nantes, Nantes, France
e
Naturally occurring bovine-human reassortant rotaviruses with a P[11] VP4 genotype exhibit a tropism for neonates. In-
teraction of the VP8* domain of the spike protein VP4 with sialic acid was thought to be the key mediator for rotavirus
infectivity. However, recent studies have indicated a role for nonsialylated glycoconjugates, including histo-blood group
antigens (HBGAs), in the infectivity of human rotaviruses. We sought to determine if the bovine rotavirus-derived VP8* of
a reassortant neonatal G10P[11] virus interacts with hitherto uncharacterized glycans. In an array screen of >600 glycans,
VP8* P[11] showed specific binding to glycans with the Gal1-4GlcNAc motif, which forms the core structure of type II
glycans and is the precursor of H type II HBGA. The specificity of glycan binding was confirmed through hemagglutination
assays; GST-VP8* P[11] hemagglutinates type O, A, and B red blood cells as well as pooled umbilical cord blood erythro-
cytes. Further, G10P[11] infectivity was significantly enhanced by the expression of H type II HBGA in CHO cells. The bo-
vine-origin VP4 was confirmed to be essential for this increased infectivity, using laboratory-derived reassortant viruses
generated from sialic acid binding rotavirus SA11-4F and a bovine G10P[11] rotavirus, B223. The binding to a core glycan
unit has not been reported for any rotavirus VP4. Core glycan synthesis is constitutive in most cell types, and modification
of these glycans is thought to be developmentally regulated. These studies provide the first molecular basis for understand-
ing neonatal rotavirus infections, indicating that glycan modification during neonatal development may mediate the age-
restricted infectivity of neonatal viruses.
Interaction with cell surface glycans is a critical step in the initi-
ation of many enteric infections (1). For rotavirus, this is medi-
ated by the outer capsid spike protein VP4 through its glycan
binding domain, VP8*. Sialic acid (Sia) is a key binding partner for
VP8*; previous studies have shown that some animal rotaviruses
bind to terminal Sia residues on cell surfaces and some human
rotaviruses interact with gangliosides, such as GM1, that contain
internal Sia moieties (2–6). Recently, some human rotaviruses
have been shown to interact with nonsialylated glycoconjugates,
including histo-blood group antigens (HBGAs) that are found in
mucosal secretions and on the surface of epithelial cells (7–9).
Structural studies on the VP8* of one such human rotavirus,
HAL1166, showed that HBGA binding occurs in the same pocket
where Sia binds in animal rotaviruses and that binding is mediated
by specific sequence changes in this region. Further, the infectivity
of HAL1166 is enhanced by HBGA expression in vitro (7). These
new data are shifting the paradigm in understanding human ro-
tavirus infectivity, and while they may explain zoonotic transmis-
sion of some animal rotaviruses, their relevance to different hu-
man strains remains unclear.
Human rotaviruses are widely known to be the leading cause of
diarrheal mortality in children worldwide. However, rotavirus in-
fections in neonates are distinct from those in older children. Neo-
natal infections are predominantly asymptomatic and are often
associated with unusual rotavirus strains. These strains appear to
be geographically restricted, show remarkable stability, and can
persist in specific settings for long periods of time (10,11). Rota-
viruses are classified into G and P genotypes using a binary no-
menclature system based on the sequence variability in the genes
encoding the outer capsid glycoprotein VP7 (G type) and pro-
tease-sensitive spike protein VP4 (P type) (12). A group of un-
usual rotaviruses possessing the P[11] VP4 type and a G9 or G10
VP7 genotype has been associated with neonatal infections in In-
dia. Of these, the G10P[11] rotaviruses were initially detected in
diarrheal samples from cattle in many studies and in some asymp-
tomatic infections in neonates (13–15). The understanding of the
epidemiology of these viruses, however, changed when a 4-year
hospital surveillance study involving 1,300 neonates in southern
India showed that nearly 50% of neonates were positive for rota-
virus and 81% of the samples were genotyped as G10P[11]; fur-
thermore, the virus was significantly associated with gastrointes-
tinal symptoms in this study (16). In concomitant hospital-based
surveillance studies for rotavirus diarrhea among 960 infants from
⬎1 month to 36 months of age, only 1 case of G10P[11] was seen
out of 342 rotavirus-positive cases, clearly indicating a predilec-
tion of this strain for neonates (17). A high incidence of neonatal
infections with the G10P[11] strain was also detected in a com-
munity-based birth cohort in this region, confirming the predi-
lection of this strain for neonates (18,19). Through whole-ge-
Received 21 December 2012 Accepted 12 April 2013
Published ahead of print 24 April 2013
Address correspondence to Mary K. Estes, mestes@bcm.edu.
S.R. and N.W.C.-P. contributed equally to this article.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/JVI.03518-12.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JVI.03518-12
July 2013 Volume 87 Number 13 Journal of Virology p. 7255–7264 jvi.asm.org 7255
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nome sequencing of a clinical isolate, the neonatal G10P[11] virus
was identified to be a bovine-human reassortant, with the genes
encoding nonstructural proteins NSP1 to NSP3 being of human
rotavirus origin and the remaining 8 genes, including VP4, being
of bovine rotavirus origin (20). Another P[11] rotavirus strain
associated with neonatal infections is G9P[11]. This strain causes
asymptomatic neonatal infections in northern India and forms
the basis for a new rotavirus vaccine currently in phase III trials
(21,22). G9P[11] is also a bovine-human reassortant virus, with
all genes being of human rotavirus origin except VP4, which is of
bovine rotavirus origin (23,24).
Given the high disease burden of rotavirus gastroenteritis and
the cocirculation of many genotypes, including globally prevalent
strains, the predominance of a single genotype in this age-re-
stricted population sharply contrasts with the strain diversity seen
among older children (16,17,25). With recent evidence for the
interaction of the VP8* of human rotaviruses with novel glycan
partners, we sought to determine if the bovine rotavirus-derived
VP8* of neonatal G10P[11] rotavirus interacts with hitherto un-
characterized glycan partners and whether this interaction is sig-
nificant for infection.
MATERIALS AND METHODS
Comparison of VP8* sequences. To determine if the VP8* amino acid
sequence of P[11] rotaviruses was different from that of other strains with
known glycan partners, multiple alignments were performed using the
Clustal W algorithm on BioEdit software (version 7.0.5.3). The G10P[11]
sequences included those from (i) a human neonatal isolate (N155) whose
whole genome has previously been sequenced (20), (ii) a human neonatal
isolate (N1509) that has been adapted to culture through multiple pas-
sages on African green monkey kidney epithelial (MA104) cells, and (iii) a
bovine G10P[11] isolate (strain B223). Both N155 and N1509 were iso-
lated from symptomatic neonates with feed intolerance. The VP8* amino
acid sequences of P[11] rotaviruses were compared to the VP8* amino
acid sequences of other animal and human rotaviruses with known glycan
partners (Fig. 1). These included sialidase-sensitive animal rotavirus
strains that bind to glycans with a terminal Sia moiety (rhesus rotaviruses
RRV and SA11, porcine rotavirus CRW-8) and human rotaviruses that
bind to glycans with internal Sia residues (Wa and DS-1). Wa and DS-1
represent the two most common global genotypes, G1P[8] and G2P[4],
respectively. In addition, the VP8* amino acid sequence of a P[14] siali-
dase-insensitive human rotavirus, HAL1166, that binds to A-type HBGA
at the same location as Sia in the VP8* of animal rotaviruses was also
included in the analysis.
GST-VP8* protein expression and purification. The gene sequences
of VP8* corresponding to amino acids (aa) 64 to 224 of human neonatal
G10P[11] rotaviruses N155 and N1506 as well as the bovine G10P[11]
rotavirus B223 were synthesized (Epoch Life Science) and cloned with an
N-terminal glutathione S-transferase (GST) tag into a pGEX-2T expres-
sion vector (GE Healthcare) (7). The recombinant proteins were ex-
pressed in Escherichia coli BL21(DE3) cells (Novagen) and purified using
glutathione Sepharose 4 Fast Flow chromatography medium (GE Health-
care). The VP8* segments of SA11 variant 4F (SA11-4F) and human ro-
tavirus Wa were also expressed and purified for use as controls. The pu-
rified GST-VP8* proteins were used in glycan array and hemagglutination
assays.
Glycan array screen for GST-VP8* proteins. The carbohydrate bind-
ing specificity of GST-VP8* P[11] was determined using a glycan array
comprised of ⬎600 glycans (v5.0; Consortium for Functional Glycomics,
Protein-Glycan Interaction Core-H, Emory University School of Medi-
cine [http://www.functionalglycomics]) as previously described (7).
Briefly, the recombinant protein from neonatal G10P[11] N155 was used
in decreasing concentrations (200, 20, 2.0, and 0.2 g/ml) in individual
arrays and detected using a fluorescently labeled anti-GST monoclonal
antibody (Sigma). The strength of binding to a glycan was expressed in
terms of relative fluorescence units (RFU). To normalize the results be-
tween the different arrays, a rank was assigned to each glycan using the
formula 100 ⫻(test glycan RFU/highest number of RFU in that assay). An
average of the ranks determined the final rank for each glycan (26). Sim-
ilar glycan array studies were carried out with GST-VP8* proteins from
SA11-4F, Wa, N1509, and B223. These proteins were tested in a single
concentration (200 g/ml) on the array.
Hemagglutination assay. Adult human red blood cells (RBCs) corre-
sponding to blood types O, A1, A2, and B were obtained from Immucor
Inc. Pooled umbilical cord blood erythrocytes were obtained from My-
biosource. The cells were centrifuged for 10 min at 500 ⫻g, and 0.5%
suspensions of each RBC type were prepared in 0.85% saline (pH 6.2). The
GST-VP8* proteins were serially diluted on 96-well V-bottom plates
(Nunc) and mixed with an equal volume of each RBC suspension. The
suspension was incubated for about1hat4°Candassessed for hemag-
glutination. The hemagglutination titer was recorded as the highest dilu-
tion of sample that resulted in complete hemagglutination. Recombinant
Norwalk virus virus-like particles (VLPs) known to hemagglutinate type
A and type O but not type B RBCs and GST-VP8* P[14] from rotavirus
strain HAL1166, which hemagglutinates type A RBCs, were included as
positive controls. The assay was also performed with GST to rule out false
positivity due to the GST tag.
To determine the effect of neuraminidase treatment of RBCs on hem-
agglutination, O-type RBCs were treated with neuraminidase from Vibrio
cholerae (Sigma). Briefly, a 10% suspension was prepared using 10 lof
packed RBCs in 250 mM sodium acetate buffer (pH 5.8) containing bo-
vine serum albumin (1.25 mg/ml), NaCl (5 mM), and CaCl
2
(4 mM). The
RBCs were treated with or without 25 mU neuraminidase for2hat37°C
(modified from reference 27). Following incubation, the cells were
washed and hemagglutination assays were carried out as described above.
The neuraminidase treatment studies were carried out with a subset of the
GST-VP8* proteins. These included VP8* from human and bovine
G10P[11] rotaviruses (N155 and B223, respectively), SA11-4F, and
HAL1166.
Cell lines and virus strains for binding and infectivity assays. Bind-
ing and infectivity assays were performed on Chinese hamster ovary
(CHO) cells that differ in the production of HBGAs (7). The parental CHO
cells do not express HBGAs (H⫺/A⫺/B⫺). Single-transfectant CHO cells
that stably express the enzyme fucosyltransferase (Fut2) express the H antigen
(H⫹/A⫺/B⫺). Double-transfectant CHO cells expressing Fut2 and either
A-type glycosyltransferase or B-type glycosyltransferase express the A antigen
(H⫹/A⫹/B⫺) or B antigen (H⫹/A⫺/B⫹), respectively (28). Polylac-
tosamines were assayed on CHO cells by flow cytometry using the biotin-
ylated lectins STL from Solanum tuberculosum (potato; Biovalley), LEL
from Lycospersum (tomato; Biovalley), and streptavidin-peridinin chlo-
rophyll protein conjugate (BD Biosciences) at 1 g/ml.
The binding studies were carried out using VP8* P[11] from N155 and
VP8* P[14]. Infectivity on CHO cells was assessed using a panel of virus
strains. These included the human neonatal G10P[11] rotavirus isolate
(N1509) that was adapted to culture through multiple passages on MA104
cells, well-characterized laboratory strains (simian rotavirus SA11 variant
4F [SA11-4F], human rotaviruses Wa and HAL1166, and bovine
G10P[11] rotavirus B223), as well as 6 reassortant viruses (R-179, R-144,
R-141, R-491, R-004, and R-198) generated using SA11-4F and B223 as
parental strains. The reassortant viruses differed in their overall genetic
background (see Fig. 6), and their characterization has been described
previously (29,30).
Binding and infectivity assays on CHO cells. Binding of VP8* to the
different CHO cells was assessed by flow cytometry using fluorescein iso-
thiocyanate (FITC)-labeled GST-VP8* proteins. The binding studies were
carried out on parental CHO cells or CHO cells stably expressing the
H-type or the A-type glycosyltransferase with FITC-GST-VP8* P[11]
from human neonatal G10P[11] N155. FITC-GST and FITC-GST-V8*
Ramani et al.
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P[14] from HAL1166 were included as controls. Briefly, 2 mg/ml of pu-
rified recombinant GST, GST-VP8* P[11], or P[14] in phosphate-buff-
ered saline (pH 8.0) was labeled using an FITC antibody-labeling kit
(Thermo Scientific). The proteins were incubated with one vial of FITC
reagent each at room temperature for 1 h with protection from light. The
labeled proteins were then purified using the purification resin provided
in the kit and stored at 4°C before examining their binding to CHO cells.
The binding studies were carried out at 4°C for 30 min using 25 gof
protein for 1 ⫻10
6
to 2 ⫻10
6
cells. In addition, binding studies were also
carried out following treatment of CHO cells with neuraminidase at 37°C
for1hataconcentration of 50 mU per 10
6
cells.
Virus titers on the parental and the singly and doubly transfected CHO
cell lines were determined using fluorescent focus assays (3). Briefly, the
cells were grown to confluence on 96-well plates (Costar; Corning) and
inoculated with 2-fold dilutions of trypsin-activated virus. After 12 to 14 h
of infection, the cells were fixed with ice-cold methanol and stained with a
rabbit polyclonal antirotavirus antibody and a fluorescently labeled don-
key anti-rabbit IgG secondary antibody (Invitrogen). Virus titers were
calculated from dilutions that gave countable numbers of foci (20 to 200)
per well. Differences in virus titers between cells lines were compared
using a two-tailed Student ttest.
Nucleotide sequence accession number. The VP8* sequence of neo-
natal G10P[11] N1509 was deposited in GenBank and assigned the acces-
sion number KC807203.
RESULTS
The VP8* sequence of P[11] rotaviruses is distinct in the glycan
binding domain. Many animal rotaviruses are known to bind
glycans with a terminal Sia residue on cell surface glycans. The
crystal structures of the VP8* of animal rotaviruses RRV and
CRW-8 with bound Sia have been determined, and the residues
FIG 1 The VP8* P[11] sequence is distinct from the sequence in rotavirus strains with known glycan partners. Alignment of the VP8* amino acid sequences from
neonatal G10P[11] rotavirus isolates (N155 [GI:164632843] and culture-adapted neonatal isolate N1509) with the VP8* amino acid sequences from a bovine
(Bo) G10P[11] isolate (B223; GI:408959), simian (Si) rotaviruses RRV (GI:61869) and SA11 (GI:61892), SA11 variant 4F (GI:61946), porcine (Po) rotavirus
CRW-8 (GI:226699783), and human (Hu) rotavirus strains Wa (GI:333781), DS-1 (GI:28268531), and HAL1166 (GI: 452131) is shown. The species of origin,
the names of the isolates, and their VP4 types are indicated. *, residues known to interact with Sia for the animal viruses RRV, SA11, and CRW-8. The VP8* amino
acid sequence of cell culture-adapted human neonatal rotavirus strain N1509 G10P[11] was ⬎98% identical to that of clinical isolate N155. N155 and N1509
showed the same amino acid changes in the Sia or HBGA binding pocket as RRV, CRW-8, and HAL1166.
Neonatal Rotavirus VP8* Binds Type II Precursor
July 2013 Volume 87 Number 13 jvi.asm.org 7257
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that interact with Sia have been identified (2,4). Studies on a
human rotavirus (HAL1166) have shown that VP8* binds A-type
HBGA in the same location as Sia in the VP8* of the animal viruses
(7). An insertion in amino acid position 187 of HAL1166 VP8*
results in a change in the side chain orientation of the conserved
residues R101 and Y188, which sterically hinders Sia binding in
this pocket but allows A-type HBGA binding (7). The VP8* do-
mains of neonatal P[11] strains (N155 and N1509) show signifi-
cant alterations in the residues that interact with either Sia in the
VP8* domain of animal rotaviruses or the A-type HBGA in the
VP8* domain of HAL1166 (Fig. 1). In P[11] rotaviruses, R101 is
replaced by a phenylalanine, while the conserved Y188 and Y189
residues are not seen. The VP8* crystal structures of two other
human rotavirus strains representing the globally prevalent P[8]
(Wa) and P[4] (DS-1) genotypes have also been reported previ-
ously (2,5,6). Using nuclear magnetic resonance, the Wa VP8*
was shown to bind gangliosides, such as GM1, with internal Sia
residues. Although the structural determinants of binding to the
internal Sia residues are not characterized for these human vi-
ruses, the VP8* sequence of P[11] strains varied from the VP8*
sequences of these strains (Fig. 1). These sequence comparisons
suggest that the amino acid sequence of the VP8* glycan binding
domain of P[11] rotavirus strains is distinct from that of the other
rotavirus strains whose glycan partners have been characterized
and that VP8* P[11] could potentially bind previously uncharac-
terized glycan partners.
VP8* P[11] binds glycans with Gal1-4GlcNAc. Potential
glycan partners for VP8* P[11] were screened using a glycan array
comprising ⬎600 glycans. GST-VP8* P[11] showed distinct bind-
ing to a number of glycans, including structures typically found as
both N- and O-linked glycans. Overall, the highest rank was seen
for glycans containing the Gal1-4GlcNAc motif (N-acetyllac-
tosamine; also called LacNAc), in particular, for glycans with mul-
tiple units of LacNAc (polylactosamine). Binding to polylac-
tosamine glycans was seen for GST-VP8* from all P[11] viruses
tested, including N155, N1509, and B223 (Table 1; see Table S1
and Fig. S1 in the supplemental material). Previously known ro-
tavirus binding partners, such as GM1 and GD1a-like glycans,
showed much lower binding (Table 1). The GST-VP8* P[11]
bound with high affinity to the polylactosamine of N-glycans with
no preference for the number of branches (Table 1). The strength
of binding was, however, directly related to the length of the
polylactosamine and less dependent on the terminal monosaccha-
ride. As seen in Table 1, GST-VP8* P[11] bound with less affinity
to the shorter, linear glycans on the array. The addition of other
monosaccharides to polylactosamine had marginal effects on
binding. As can be seen from the glycans listed in Table 1, the
presence of an ␣-1,2-linked fucose (H type II glycan) resulted in
an increase in the strength of binding to glycans with shorter
polylactosamine chains, whereas the presence of Sia (Neu5Ac,
␣-2,3, or ␣-2,6 linkage) resulted in a reduction in binding
strength. In addition to N-glycans, strong binding to polylac-
tosamine was also seen, which is typical of O-glycans. Thus, the
glycan array results suggest that VP8*P[11] can bind to extended
polylactosamine glycans, which are the precursor for H type II
HBGAs (1). H type II HBGAs are formed by the addition of a
terminal fucose to LacNAc through the activity of ␣-1,2-fucosyl-
transferase. The glycan array results also indicate that VP8*P[11]
can bind H type II derivatives, while binding is clearly reduced by
the addition of Sia to LacNAc. Binding to polylactosamine glycans
was not seen for GST-VP8* from SA11-4F or Wa (see Table S1 and
Fig. S1 in the supplemental material). GST by itself does not bind
to the glycans on the array (data not shown).
GST-VP8* P[11] hemagglutinates adult and cord blood
erythrocytes. H type II glycans are present on the surface of O-
type RBCs and serve as the precursor for the synthesis of A- and
B-type blood group antigens through the activity of glycosyltrans-
ferases (1). Thus, hemagglutination assays serve as a useful tool to
assess the specificity and biological significance of the glycan array
data. In hemagglutination assays with RBCs from adults, GST-
VP8* from the P[11] strains as well as SA11-4F hemagglutinated
type O, type A, and type B RBCs. The Norwalk virus VLP control
TABLE 1 GST-VP8* P[11] shows the highest binding to glycans containing Gal1-4GlcNAc
a
Glycan
Chart
no.
No. of LacNAc
residues
Average
rank
b
Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-
2Man␣1-6(Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-
3Gal1-4GlcNAc1-2Man␣1-3)Man1-4GlcNAc1-4(Fuc␣1-6)GlcNAc-Sp19
582 ⬎6 83.18
Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-
3Gal1-4GlcNAc1-2Man␣1-6(Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-
3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-2Man␣1-3)Man1-4GlcNAc1-
4GlcNAc-Sp25
569 ⬎6 78.09
Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-
2Man␣1-6(Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-
3Gal1-4GlcNAc1-2Man␣1-3)Man1-4GlcNAc1-4GlcNAc-Sp25
566 ⬎6 77.88
Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc-Sp0 163 3 9.03
Fuc␣1-2Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc-Sp0 (H type II) 75 3 (terminal fucose) 25.61
Neu5Ac␣2- 3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc-Sp0 259 3 (terminal Sia) 5.60
Neu5Ac␣2-6Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc-Sp0 333 3 (terminal Sia) 3.27
Neu5Ac␣2-3Gal1-3GalNAc1-4(Neu5Ac␣2-8Neu5Ac␣2-3)Gal1-4Glc-Sp0 (GD1a-like) 413 0 0.95
Gal1-3GalNAc1-4(Neu5Ac␣2-3)Gal1-4Glc-Sp0 (GM1) 145 0 0.59
a
The strength of binding to glycans with Gal1-4GlcNAc (in boldface and underlined) was much higher than that of GM1- and GD1a-like glycans that are known partners for
some rotavirus strains. The raw data from the glycan array experiments are provided in Table S1 and Fig. S1 in the supplemental material.
b
Average rank of representative glycans for binding with GST-VP8* P[11] from human neonatal strain N155 calculated from 4 different arrays. The rank for each glycan was
calculated using the formula 100 ⫻(test glycan RFU/highest number of RFU in that assay).
Ramani et al.
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hemagglutinated type O and A RBCs but not type B RBCs, while
GST-VP8* P[14] from HAL1166 hemagglutinated only type A
RBCs, as previously shown (7,31). No hemagglutination was ob-
served with GST-VP8* Wa or the GST control (Table 2). How-
ever, glycan expression on RBCs is developmentally regulated (1).
Unbranched polylactosamine chains are more highly expressed
on the surface of embryonic and cord blood erythrocytes, while
the adult levels of branched chains are reached by about 18
months of age. Therefore, hemagglutination assays were also
carried out with pooled umbilical cord blood erythrocytes.
GST-VP8* P[11] hemagglutinated umbilical cord blood eryth-
rocytes. Partial hemagglutination of cord blood erythrocytes
was seen at the highest concentration of Norwalk virus VLPs,
while GST-VP8* P[14], GST-VP8* Wa, and the GST controls
showed no hemagglutination (Table 2). These data are consistent
with the findings of the glycan array that VP8* P[11] can interact
with H type II glycans on cell surfaces.
The VP8* from the SA11-4F and P[11] strains binds to differ-
ent glycans on RBCs for hemagglutination. SA11-4F is known to
bind terminal Sia on cell surfaces, and neuraminidase treatment of
RBCs resulted in a complete loss of hemagglutination. In contrast,
neuraminidase treatment resulted in an increased hemagglutina-
tion titer for VP8* P[11] from N155 and B223 (Table 3), indicat-
ing that in untreated RBCs, Sia may be masking additional sites for
GST-VP8* P[11] binding.
GST-VP8* P[11] binds all CHO cells, but infectivity is en-
hanced with expression of H type II. The specificity and biologi-
cal relevance of glycan array data were further assessed by binding
and infectivity assays on CHO cells that differ in expression of
HBGA. Binding experiments on parental, H-type, and A-type
CHO cells showed similar binding patterns using GST-VP8*
P[11] (Fig. 2). There was no significant difference in the expres-
sion of polylactosamines between these cell lines (Fig. 3). How-
ever, a significant increase in human and bovine G10P[11] titer
was observed in all CHO cells that express the enzyme Fut2, in-
cluding the double-transfectant CHO cells that express Fut2 and
A- or B-type glycosyltransferases, compared to that observed in
the parental cells (Fig. 4). The enzyme Fut2 catalyzes the addition
of a terminal fucose in ␣-1,2 linkage to the LacNAc precursor. The
additional modification of the H type II to A-type or B-type
HBGA did not result in any further significant increase in virus
titer, indicating that the expression of the H type II was the critical
factor for increased infectivity. Terminal Sia binding animal rota-
virus strain SA11-4F, internal Sia binding Wa, and A-type HBGA
binding HAL1166 were included as controls in the infectivity ex-
periments. No difference in infectivity between the cell lines was
observed for SA11-4F and Wa, while HAL1166 showed signifi-
cantly enhanced infectivity on A-type cells. These data confirm
that the interaction of VP8* P[11] with type II glycans on cell
surfaces is biologically relevant.
Neuraminidase treatment of cells results in enhanced bind-
ing on CHO cells. CHO cells express a large number of sialylated
LacNAc glycans (32). Removal of the terminal Sia should there-
fore result in increased binding on these cells. Indeed, neuramin-
idase treatment of CHO cells resulted in significantly enhanced
binding for GST-VP8* P[11], while no significant difference was
observed with GST-VP8* P[14] (Fig. 5).
Bovine rotavirus-derived VP4 is critical for increased infec-
tivity. The VP4 in the human neonatal P[11] rotaviruses are of
bovine rotavirus origin (20,24). To further confirm that the in-
creased infectivity with H type II CHO cells was directly mediated
by the bovine rotavirus-derived P[11] VP4, infectivity assays were
carried out on parental and single-transfectant H type II CHO
cells using SA11-4F, bovine G10P[11] rotavirus B223, and six re-
assortants of these two parental viruses. B223 shows nearly 94%
identity in the VP4 gene with the VP4 of the bovine-human reas-
sortant G10P[11] strains. Infections with B223 and reassortant
viruses R-491, R-004, and R-198 that express the bovine P[11]
VP4 resulted in significantly higher virus titers in cell lines ex-
pressing H type II than in parental cells (Fig. 6), confirming that
the bovine-origin P[11] VP4 is the key mediator of this virus-cell
interaction.
DISCUSSION
Rotavirus infections in neonates are clinically and epidemiologi-
cally distinct from infections in older children (11). They are
caused by unusual virus strains that appear to be geographically
restricted and show remarkable strain stability in comparison to
the diversity of strains seen in older children. It has been specu-
lated that the age-dependent restriction of unusual viruses for
neonates could be mediated by the interaction of the spike protein
with maturation-dependent host components (33); however,
TABLE 2 GST-VP8* P[11] hemagglutinates adult and cord blood erythrocytes RBCs
Blood type or
source
Reciprocal HA titer
a
SA11-4F Wa HAL1166 N155 N1509 B223 GST NV VLPs
O⬎128 0 0 ⬎128 ⬎128 ⬎128 0 ⬎128
A1 ⬎128 0 ⬎128 ⬎128 ⬎128 ⬎128 0 ⬎128
A2 ⬎128 0 ⬎128 ⬎128 ⬎128 64 0 ⬎128
B6400⬎128 128 64 0 0
Umbilical cord ⬎128 0 0 ⬎128 ⬎128 ⬎128 0 0
a
Reciprocal hemagglutination (HA) titers of adult human type O, type A, and type B RBCs as well as pooled cord blood erythrocytes by GST-VP8* proteins. No hemagglutination
is indicated by 0. Recombinant Norwalk virus (NV) VLPs, HAL1166 GST-VP8* P[14], and GST were included as controls. GST-VP8* P[11] hemagglutinated all RBCs tested.
TABLE 3 Neuraminidase treatment of RBCs results in increased
hemagglutination with GST-VP8* P[11]
Virus strain
Reciprocal HA titer
a
Without neuraminidase With neuraminidase
SA11-4F 512 0
N155 512 ⬎2,048
B223 256 ⬎2,048
HAL1166 0 0
a
Reciprocal hemagglutination (HA) titers of adult human type O RBCs by GST-VP8*
proteins following neuraminidase treatment. No hemagglutination is indicated by 0.
Neonatal Rotavirus VP8* Binds Type II Precursor
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there has been little conclusive evidence for this to date. This is the
first report demonstrating that the VP8* of a clinically important
neonatal rotavirus strain binds to Gal1-4GlcNAc, the precursor
for type II glycans, and that this binding is biologically relevant for
infection. The binding of P[11] VP8* to precursor glycans raises
important questions about how modification of glycans during
the course of neonatal development may alter susceptibility to
infectious agents.
The repertoire of studies on microbes and their glycan partners
has expanded with recent technological advances in the field of
glycobiology. There is increasing evidence to suggest that glycan-
pathogen interactions play a key role in the pathogenesis of several
infectious agents. This study raises additional questions on
whether developmental modification of glycans may also influ-
ence pathogenesis. Studies in rodents have demonstrated that gly-
cosylation changes in the gut occur throughout the postnatal
phase of development and are controlled by hormonal and dietary
factors through the activity of glycosyltransferases (34). However,
glycan changes in the human neonatal gut have not been exten-
sively studied. Some studies on the developmental regulation of
glycans on human RBCs show that embryonic RBCs have an
abundance of unbranched polylactosamine chains and a relative
lack of branched chains (1). Linear polylactosamine chains are
modified by the activity of the enzyme 1-6 N-acetyl-
glucosaminyltransferase, which transfers N-acetylglucosamine in
1-6 linkage to internal galactose residues in polylactosamine.
This results in the formation of 1-6 N-acetylglucosamine
branches, which may then serve as the substrate for subsequent
LacNAc molecules, thereby forming branched polylactosamine
chains. Adult levels of these branched structures are achieved at
FIG 2 GST-VP8* P[11] binds all CHO cell types. (A and B) Binding of FITC-GST-VP8* P[11] and P[14], respectively, to parental CHO cells (H⫺/A⫺/B⫺, red),
the single-transfectant cells expressing the Fut2 enzyme (H⫹/A⫺/B⫺, purple), and the double-transfectant cells with both Fut2 and A-type glycosyltransferase
(H⫹/A⫹/B⫺, blue).
FIG 3 CHO cells do not differ in the levels of polylactosamine. Parental and H-type CHO cells were assayed for polylactosamine by flow cytometry using LEL
and STL lectins. (A and B) Parental H⫺/A⫺/B⫺CHO cells assayed using LEL and STL lectins, respectively; (C and D) H-type H⫹/A⫺/B⫺CHO cells assayed
using LEL and STL lectins, respectively.
Ramani et al.
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about 18 months of age, while unbranched polylactosamine reac-
tivity reaches very low levels. Little is known about such modifi-
cations in the human gut during the course of development. It is
possible that the polylactosamine is abundantly present in the
neonatal gut but is modified during development by the addition
of various glycans that results in changes in glycan length or degree
of branching. This may in part explain the increased neonatal
susceptibility to P[11] rotaviruses and a reduction in the infectiv-
ity of these strains beyond the neonatal age group. Interestingly,
the globally prevalent human rotavirus strains with P[4] and P[8]
VP4 types that infect older children have been identified to bind to
the modified HBGAs and not the precursor units (8,9).
The hemagglutination assays, binding studies, and differential
infectivity on CHO cells corroborate the findings of the glycan
array. GST-VP8* P[11] hemagglutinates both umbilical cord
blood and adult RBCs due to its ability to bind both polylac-
tosamine glycans and H type II HBGA. Neuraminidase treatment
of RBCs results in increased hemagglutination with GST-VP8*
P[11], whereas a complete loss of hemagglutination was seen with
GST-VP8* from SA11-4F on these cells, indicating that Sia masks
or sterically hinders GST-VP8* P[11] binding to untreated RBCs.
In the CHO cell binding experiments, the neonatal G10P[11] vi-
rus was able to bind all cell types tested, including parental cells;
however, the infectivity was enhanced with the expression of H
type II HBGA. The limited infectivity on the parental CHO cells
may not be due to the lack of availability of the LacNAc glycans in
these cells. It has been demonstrated that the parental CHO cells
express an abundance of LacNAc glycans; however, there are few
glycans with greater than 3 repeats, and CHO cells grown on a
monolayer also express a large number of sialylated LacNAc gly-
cans (32). Removal of sialic acid by neuraminidase treatment re-
sults in increased binding on these cells. In addition, lectin bind-
ing studies on the parental and transfected CHO cells showed that
polylactosamine levels did not change between the parental and
transfected cells. This suggests that the increased infectivity ob-
served in the transfected cells is due to the addition of H type II
structures and not due to concomitant modifications in other gly-
can motifs, such as Sia or LacNAc repeats, that could have oc-
curred due to competition between the ␣-1,2-fucosyltransferase
and other glycosyltransferases. As can be seen from Table 1, the
best binding of GST-VP8* P[11] occurs in the presence of multi-
ple units of LacNAc, and terminal sialylation of LacNAc chains
FIG 4 Infectivity of P[11] rotavirus increases with expression of H antigen. The infectivity of rotaviruses in CHO cell lines genetically modified to stably express
different glycans is shown. Infection was carried out in parental CHO cells (H⫺/A⫺/B⫺), the single-transfectant cells expressing the Fut2 enzyme (H⫹/A⫺/
B⫺), and the double-transfectant cells with both Fut2 and A type glycosyltransferase (H⫹/A⫹/B⫺) or B type glycosyltransferase (H⫹/A⫺/B⫹). The rotavirus
strains used (and their titers on CHO cells) include SA11-4F, which binds terminal Sia (4 ⫻105 FFU/ml, where FFU is focus-forming units); Wa, which binds
internal Sia (5.5 ⫻105 FFU/ml); HAL1166, which binds A-type HBGA (7.7 ⫻103 FFU/ml); human neonatal P[11] rotavirus strain N1509 (1.5 ⫻104 FFU/ml);
and bovine P[11] rotavirus strain B223 (5.2 ⫻103 FFU/ml). The yaxis represents the fold difference in comparison to the results for the parental cells. Error bars
represent standard errors of the means of data from quadruplicate wells from at least 3 independent experiments. A Pvalue of ⬍0.001 was considered statistically
significant and is represented by an asterisk.
FIG 5 Neuraminidase treatment of CHO cells results in increased binding to
GST-VP8* P[11]. Binding of FITC-GST-VP8* P[11] or P[14] on H⫺/A⫺/B⫺,
H⫹/A⫺/B⫺, and H⫹/A⫹/B⫺CHO cells was carried out in duplicate with
and without neuraminidase treatment. The change in the median fluorescence
intensity of binding was compared between untreated and neuraminidase-
treated cell lines and expressed as the percent change compared to the results
for untreated cells. The data from all cell lines were pooled for each protein and
treatment group. Error bars represent the standard error of mean change in
binding for all experiments within a group. A Pvalue of ⬍0.05 was considered
statistically significant and is represented by an asterisk.
Neonatal Rotavirus VP8* Binds Type II Precursor
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results in a reduction in the strength of binding to GST-VP8*
P[11] compared to that for fucosylated LacNAc (H type II). It
should be noted that long polylactosamines terminating with fu-
cose or Sia are not present in the glycan array, and this precludes
making a definitive assessment of the strength of their binding to
GST-VP8* P[11] in the array compared to that of large polylac-
tosamine glycans lacking these monosaccharides.
In this study, it was seen that bovine rotavirus G10P[11] B223
showed a glycan binding profile and hemagglutination properties
similar to those of the human neonatal viruses. G10P[11] rotavi-
rus infections have been widely reported to cause diarrhea in cattle
in many parts of the world (13,15,35,36). These strains contain
all gene segments of bovine rotavirus origin. The human neonatal
G10P[11] infections are, however, caused by bovine-human reas-
sortant strains; these viruses possess a VP4 of bovine rotavirus
origin (20,24). To confirm that the presence of the bovine P[11]
VP4 was key for the increased infectivity on H type II CHO cells,
infectivity assays were carried out with a bovine G10P[11] rotavi-
rus strain B223 and reassortants derived using B223 and SA11-4F
that differ in their genetic makeup. B223 and all reassortant vi-
ruses possessing the bovine P[11] VP4 (R-491, R-004, and R-198)
showed increased infectivity on H type II cells. The availability of
reasssortant rotavirus strains thus allowed confirmation that the
P[11] VP4 of bovine rotavirus origin is the key mediator of the
interaction with type II glycans described in this study. It should,
however, be noted that the magnitude of the fold change was the
highest for B223 in comparison to that for the reassortant viruses,
indicating a role for other genes and gene combinations in infec-
tivity.
The findings of this study are highly relevant in the context
of current rotavirus vaccines in developing countries. Rotavi-
rus diarrhea results in nearly half a million deaths annually;
over 80% of these deaths occur in the developing countries of
Asia and Africa (37). Currently licensed vaccines that are highly
efficacious in developed countries do not appear to be as effec-
tive in the developing world (38–40). This has led to the eval-
uation of new vaccines and alternate immunization schedules,
including maternal and neonatal immunizations. One of the
new vaccines being evaluated in India is 116E, which is based
on an asymptomatic human neonatal G9P[11] rotavirus. This
strain also possesses a P[11] VP4 gene of bovine rotavirus ori-
gin and has been found to be highly immunogenic (21,24). The
findings of this study suggest that neonatal immunization with
this neonatal P[11] virus may prove to be effective, as these
strains may be able to replicate more efficiently in the neonatal
gut and thus result in better vaccine take. However, this also
leads to questions of whether vaccine uptake and response may
differ at different ages of vaccination depending on the VP4
genotype of the vaccine virus and glycan profile of the vacci-
nated infant. These questions will be addressed through future
studies involving characterization of glycan interactions for
additional rotavirus VP4 types, including the globally preva-
lent genotypes and those present in vaccine viruses.
ACKNOWLEDGMENTS
We thank Monica McNeal, Cincinnati Children’s Hospital and Medical
Center, Cincinnati, OH, for help with adapting the human neonatal
G10P[11] virus to cell culture. We also thank Jézabel Rocher, INSERM,
Université de Nantes, Nantes, France, for her expertise with the prepara-
tion of CHO transfectants and flow cytometry analysis.
This study was supported by NIH grants R01 AI080656, R01 AI36040,
and P30 DK56338, which funds the Texas Medical Center Digestive Dis-
FIG 6 Increased infectivity on H-antigen-expressing CHO cells is mediated by VP4. The infectivity of SA11, B223, and laboratory-derived reassortant rotaviruses
in parental CHO cells (H⫺/A⫺/B⫺) and cells expressing H antigen (H⫹/A⫺/B⫺) is shown. The origins of the VP4, VP7, and remaining genes in virus strains
are listed below the strain. Pvalues of ⬍0.001 represent significant differences in virus infectivity between the parental and single-transfectant cell lines and are
represented by asterisks.
Ramani et al.
7262 jvi.asm.org Journal of Virology
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eases Center, and a grant from Robert Welch Foundation (Q1279). Gly-
can array analyses were provided by the Protein-Glycan Interaction Re-
source of the Center for Functional Glycomics, which was supported by
GM62116 and GM98791. Flow cytometry studies were carried out at the
Cytometry and Cell Sorting Core at the Baylor College of Medicine with
funding from the NIH (NIAID P30AI036211, NCI P30CA125123, and
NCRR S10RR024574) and the assistance of Joel M. Sederstrom.
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