Download Norovirus and Histo-Blood Group Antigens

Jpn. J. Infect. Dis., 64, 95-103, 2011
Review
Norovirus and Histo-Blood Group Antigens
Haruko Shirato*
Department of Virology II, National Institute of Infectious Diseases, Tokyo 208-0011, Japan
(Received September 8, 2010. Accepted January 12, 2011)
CONTENTS:
1. Introduction
2. Norovirus
3. Histo-blood group antigens (HBGAs)
4. HBGAs and noroviruses
4–1. Volunteer challenge studies
4–2. Distinction between H, A, and B epitopes by
noroviruses
4–3. The importance of terminal residues in the
binding
4–4. Distinction between type 1 and type 2 structures by noroviruses
4–5. Wide HBGA recognition in GII/4 strains
4–6. Putative binding sites on the capsid protein
5. Carbohydrates and caliciviruses
6. Discussion
SUMMARY: Norovirus (NoV), a member of the family Caliciviridae, is a major cause of acute water- and
food-borne nonbacterial gastroenteritis and forms antigenically diverse groups of viruses. Human NoVs are
divided into at least three genogroups, genogroups I (GI), GII, and GIV, which contain at least 15, 18, and 1
genotypes, respectively. Except for a few genotypes, all NoVs bind to histo-blood group antigens (HBGAs),
namely ABH antigens and Lewis antigens, in which carbohydrate core structures (types 1 and 2) constitute
antigenically distinct phenotypes. Volunteer challenge studies have indicated that carbohydrate binding is
essential for genogroup I genotype 1 (GI/1) infection. Non-secretors who do not express FUT2
fucosyltransferase, and consequently do not express H type 1 or Lewis b antigens in the gut, are not infected
after challenge with GI/1. NoV virus-like particles (VLPs), which are recombinant particles that are morphologically and antigenically similar to the native virion, display different ABH and Le carbohydrate-binding
profiles in vitro. Epidemiological studies have shown that individuals with different ABH phenotypes are
infected with NoV strains in a genotype-specific manner. On the other hand, an in vitro binding assay using
NoV VLPs showed a uniform recognition pattern against type 1 and 2 core structures, and bind more tightly
to type 1 carbohydrates than to type 2. Type 1 carbohydrates are expressed at the surface of the small intestine and are presumably targeted by NoV. This property may afford NoV tissue specificity. GII/4 includes
global epidemic strains and binds to more HBGAs than other genogroups. This characteristic may be linked
to the worldwide transmission of GII/4 strains. Although it is still unclear whether HBGAs act as primary
receptors or enhance NoV infectivity, they are important factors in determining tissue specificity and the risk
of transmission.
secretors do not express FUT2 and consequently do not
express H type 1 or Lewis b (Leb) antigens in the gut and
saliva. In an initial study of the interaction between the
prototype strain of NoV, Norwalk virus (NV/68), and
HBGAs, virus-like particles (VLPs) generated with recombinant baculoviruses were used for binding to the tissue
sections (7). The attachment of rNV/68 VLPs to surface
epithelial cells of the gastroduodenal junction was detected,
but only from secretor donors. This attachment was reversed
by fucosidase treatment and by competition with HBGA
trisaccharides or anti-HBGA antibodies. Furthermore,
transfection of cells with FUT cDNA allowed the attachment and internalization of VLPs. Volunteer challenge
studies provided strong evidence that carbohydrate binding is essential for NV/68 infection (9). Using a human
challenge model, it was shown that a non-secretor was fully
penetrant against NV/68 infection as none of these individuals developed an infection after challenge, regardless
of dose. Further informations of HBGA recognition by
NoV were obtained by performing in vitro experiments.
Enzyme-linked immunosorbent assays (ELISAs), salivaVLP binding assays, or carbohydrate-VLP binding assays
have been used to detect the attachment of VLPs to
1. Introduction
Viruses initiate infection by attaching to specific cells
in the target host tissue. Virus receptors are strongly involved in host-, tissue-, and cell-specificity, and carbohydrate molecules are among those used for virus attachment (1). Orthomyxoviruses, polyomaviruses, reoviruses,
coronaviruses, paramyxoviruses, and both murine and
canine parvoviruses use sialic acid for binding, whereas
adenovirus-associated virus 2, herpes viruses, and
flaviviruses recognize heparan sulfate for binding (1).
Norovirus (NoV) appears to recognize and bind to histoblood group antigens (HBGAs), uncharged sugar residues
(2–8) that contain structurally related saccharide moieties,
ABH and Lewis antigens, and so on (Fig. 1). These are
found in saliva and mucosal secretions from intestinal
epithelial cells of secretor individuals who carry the FUT2
gene, which encodes a fucosyltransferase (FUT). Non*Corresponding author: Mailing address: Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen,
Musashi-murayama, Tokyo 208-0011, Japan. Tel: +81-42-5610771, Fax: +81-42-561-4729, E-mail: [email protected]
95
(A)
(B)
Gal 1-3GlcNAc
Gal 1-4GlcNAc
type 2 (precursor)
type 1 (precursor)
1,2FUTs (FUT1, 2)
1,4FUTs (FUT3)
1,2FUTs (FUT1, 2)
1,3FUTs (FUT3 etc)
Gal 1-3GlcNAc 1-2
Fuc
H type 1 (O antigen)
Gal 1-3GlcNAc 1-4
Fuc
Gal 1-4GlcNAc 1-2
Fuc
H type 2 (O antigen)
Gal 1-4GlcNAc 1-3
Fuc
A enzyme
GalNAc 1-3Gal 1-3GlcNAc 1-2
Fuc
A type 1
Lea
Lex
1,4FUTs (FUT3)
1,3FUTs (FUT3 etc)
Gal 1-3GlcNAc 1-4
1-2
Fuc
Fuc
Leb
Gal 1-4GlcNAc 1-3
1-2
Fuc
Fuc
Ley
A enzyme
B enzyme
GalNAc 1-3Gal 1-4GlcNAc 1-2
Fuc
A type 2
Gal 1-3Gal 1-3GlcNAc 1-2
Fuc
B type 1
B enzyme
Gal 1-3Gal 1-4GlcNAc 1-2
Fuc
B type 2
Fig. 1. Diagram of types 1 and 2 carbohydrate structures. ABH and Lewis antigens are synthesized by sequential enzymatic transfer of carbohydrate residues to specific precursor carbohydrate substrates. Types 1 and 2 precursor substrates have different Gal-to-GlcNAc linkages: Galβ1,3-GlcNAcβ- (A) and Galβ1,4-GlcNAcβ- (B), respectively. H
antigens are made by the enzymatic addition of a Fuc residue to the terminal Gal residue in α1,2 linkage with α1,2
fucosyltransferase, FUT1 or 2. FUT3 transfers Fuc to the GlcNAc of types 1 and 2 precursors and H types 1 and 2 in α1,4
and α1,3 linkages, respectively. FUT3 exhibits both α1,3 and α1,4 fucosyltransferase activity, and is the only enzyme
responsible for type 1 Lewis antigens, such as Lea and Leb (A). On the other hand, in type 2 Lewis antigens, not only
FUT3 but also FUT4, 5, 6, and 9 can synthesize Lex and Ley (B). H types 1 and 2 are the terminal moieties expressed in
histo-blood group type O individuals, but in types A, B, and AB individuals the H antigens are further modified by
enzymes that transfer GalNAc (type A), Gal (type B), or either carbohydrate (type AB) to the terminal Gal residue of
an H antigen in α1,3 linkage (A and B). Gal, galactose; GlcNAc, N-acetylglucosamine; Fuc, fucose; GalNAc, Nacetylgalactosamine.
HBGAs (2,4–6,8–10), whereas the association and dissociation kinetics for NoV binding to HBGAs has been measured
using a Biacore assay (8). The NoV recognition sites on
HBGA have been analyzed by enzymatic treatment (for
example, α-1,2-fucosidase, α-N-acetylgalactosaminidase or
α-galactosidase) of HBGAs and by crystallization studies
(4,7,11). Putative binding sites on the NoV capsid protein
have been identified by mutagenesis analyses and computer
modeling (12), evolution trace analysis (13), and crystallization (11,14,15). These studies clarified that HBGAs
are important factors for determining the risk of infection,
although the binding properties of human NoV VLPs to
HBGAs were variable. This paper aims to summarize recent
progress in elucidating the interaction between NoV and
HBGAs.
were hampered until very recently. Although the primary
NoV replication site is unknown, intestinal biopsies from
volunteers who developed illness following oral administration of NoV showed histopathologic lesions involving blunting of the villi of the proximal small intestine (18).
NoV is a member of the family Caliciviridae, which consists of five genera, namely Norovirus, Sapovirus, Vesivirus,
Lagovirus, and Becovirus (or Nabovirus). A sixth genus,
“Recovirus,” was recently proposed (19) (Table 1). NoV
strains have been isolated not only in humans but also in
pigs, cattle, and mice, and they are classified into the type
species Norwalk virus of the genus Norovirus, thus forming a group of small RNA viruses. NoV is a small, round,
non-enveloped virus with a diameter of 38 nm (Fig. 2) and
contains a single-stranded positive-sense 7.6-kb RNA genome encoding three open reading frames (ORFs). ORF1
encodes a nonstructural polyprotein, whereas ORF2 and
ORF3 encode the major capsid protein VP1 and minor
capsid protein VP2, respectively (Fig. 3) (20). One virus
particle is composed of a copy of the genome RNA, 180
copies of VP1, and a few copies of VP2 (21,22). Expression
of VP1 or both VP1 and VP2 in insect or mammalian cells
results in capsid proteins of approximately 58 kDa that
self-assemble into VLPs (20,23). Although artificial, these
VLPs are morphologically and antigenically similar to those
of their respective native virions (22,24–26). VLPs have
been used to develop ELISAs for serological diagnosis of
NoV infection (24,27). Likewise, antigen detection ELISAs
2. Norovirus
NoV is the major causative agent of acute viral gastroenteritis worldwide. Although NoV usually causes a
short-term, self-limiting disease, oral rehydration and
intravenous replacement of electrolytes are needed when
severe diarrhea is obserbed. Elderly and immunocompromised patients can suffer from severe gastroenteritis,
which sometimes results in death (16,17). As NoV infection occurs only in humans, and since no cell culture system
has yet been developed, molecular analyses to elucidate the
mechanisms underlying infection or productive replication
96
Table 1. Strains representing the four genera of the family Caliciviridae
Genus
Species
Strain
Norovirus
Norwalk virus
Sapovirus
Sapporo virus
Nabovirus
Vesivirus
Newbury-1 virus
Feline calicivirus
Vesicular exanthema of swine virus
European hare syndrome virus
Rabbit hemorrhagic disease virus
Lagovirus
Norwalk, Southampton, Desert Shield, Chiba, BS5 kidneys, etc. (GI*)
Hawaii, Lordsdale, Camberwell, U201, Alphatron, etc. (GII**)
Other strains are Bovine enteric calicivirus, Murine norovirus, Swine
norovirus, etc.
Sapporo, Manchester, Houston, Parkville, etc.
Porcine enteric sapovirus
Newbury-1 virus
Urbana, F9, Japanese F4, etc.
Bovine calicivirus, Primate calicivirus, San Miguel sea lion virus, etc.
GD, etc.
FRG, AST89, BS89, etc.
Most noroviruses and sapoviruses are isolated from humans.
*GI, genogroup I; **GII, genogroup II.
HBGAs are generated by transfer of GalNAc and Gal,
respectively, to an H structure irrespective of the carbohydrate core structure (Figs. 1A and B). The core structures
are classified into four major structures, namely type 1
(Galβ1-3GlcNAcβ), type 2 (Galβ1-4GlcNAcβ), type 3
(Galβ1-3GalNAcα), and type 4 (Galβ1-3GalNAcβ).
FUT1 (H enzyme) and FUT2 (Se enzyme) are α1,2FUTs
that catalyze the transfer of Fuc to the Gal residue of type 1
and 2 chains (Figs. 1A and B), thereby resulting in the
synthesis of H type 1 and H type 2, respectively. FUT1
determines the expression of O-type antigen (H antigen) of
the ABO blood group system on erythrocytes, whereas
FUT2 (Se) determines it in saliva and mucosal secretions,
i.e., secretor status (32). Individuals who have null FUT2
alleles cannot synthesize ABH antigens in secretions and
are therefore termed non-secretors, although they can
express ABH antigens in erythrocytes by FUT1 (33). FUT2
alleles of Caucasian non-secretors are completely inactivated by nonsense mutations, whereas those of Asian nonsecretors are incompletely inactivated by missense mutations (34,35). Thus, Asian non-secretors are incomplete nonsecretors and produce small amounts of ABH HBGAs in
secretions. The FUT3 enzyme is required for Fuc transfer
to type 1 or H type 1 to generate Lewis a (Lea; Galβ1-3
(Fucα1-4)GlcNAc) or Leb (Fucα1-2Galβ1-3(Fucα1-4)
GlcNAc), respectively (Fig. 1A). Moreover, the same enzyme is required for Fuc transfer to type 2 or H type 2 to
generate Lewis x (Lex; Galβ1-4(Fucα1-4)GlcNAc) or
Lewis y (Ley; Fucα1-2Galβ1-4(Fucα1-3)GlcNAc), respectively (Fig. 1B).
Fig. 2. Norovirus GI/4 Chiba407 strain visualized by electron microscopy. The particle is observed as a small, round, and non-enveloped
virus with a diameter of 38 nm.
ORF1
ORF2
ORF3
polyA
VPg
NTPase
(Helicase?)
VPg Protease Polymerase
Capsid
(VP1)
Capsid
(VP2)
sub-genomic RNA
Fig. 3. Genome organization of norovirus. The genome encodes three
ORFs. ORF1 encodes nonstructual proteins, ORF2 encodes capsid
protein VP1, and ORF3 encodes minor capsid protein VP2.
using hyperimmune antisera raised against the VLPs have
been developed to detect NoVs in stools (28–30). VLP
expression has also allowed the cellular receptors or binding molecules for NoV to be identified. Furthermore,
recent genetic studies have enabled human NoVs to be subdivided into at least three genogroups, namely genogroups
I (GI), II (GII), and IV (GIV), which contain at least 15, 18,
and 1 genotypes, respectively (31) (Fig. 4).
4. Histo-blood group antigens and noroviruses
4–1. Volunteer challenge studies
Volunteer challenge studies have provided strong
evidence that carbohydrate binding is essential for NV/68
infection as non-secretors were not infected after challenge
with NV/68. Furthermore, type O secretors were more likely
to be infected with NV/68, whereas type B secretors were
less likely to be infected (9,36). A large-scale infection
experiment including 77 volunteers clearly indicated the
involvement of HBGAs in NoV infection, because all of
the infected individuals appeared to be secretors, and conversely, none of the non-secretors were infected (9).
4–2. Distinctions between H, A, and B epitopes by
noroviruses
ELISA-based binding assays, saliva-VLP binding assays
3. Histo-blood group antigens
HBGAs are carbohydrates that contain structurally
related saccharide moieties (Fig. 1). H antigen (Fuc-α12Gal), i.e., O-type antigen, is generated by fucose (Fuc)
transfer to a galactose (Gal) residue with an α1-2 linkage
(Figs. 1A and B). A antigen (GalNAcα1-3(Fuc-α1-2)
Gal) and B antigen (Galα1-3(Fuc-α1-2)Gal) of ABH
97
GI/1_M87661Norwalk_GI.1
GI/4_AB042808Chiba407_GI.4
GI/5_AJ277614Musgrove_GI.5
GI/9_AB039774SaitamaSzUG1
GI/2_L07418Southampton_GI.2
GI/6_AF093797BS5_GI.6
GI/8_AB081723WUG1
GI/14_AB112100SaitamaT25GI
419
194
0.1
218
899
575
219
996
890
1000
AB187514Otofuke
GI
GI/12_AB058525SaitamaKU19aGI
GI/3_U04469DSV_GI.3
GI/10_AF538679Boxer_GI.8
GI/11_AB058547SaitamaKU8GI
GI/7_AJ277609Winchester_GI.7
GI/13_AB112132SaitamaT35aGI
613
236
157
503
484
GII/1_U07611Hawaii_GII.1
853 GII/12_AB039775SaitamaU1
631
767
924
AJ277618Wortley_GII.12
GII/15_AB058582SaitamaKU80aGII
1000
306
AY772730Neustrelitz260
1000
842
409
958
53
AB045603Gifu96
AB044366Hiroshima
AY502010_Triffin_1999_US_GII.16
AY823304_Sw_OHQW101_GII.18
AY823306_Sw_OHQW170_GII.19
AB126320Sw_Swine43
AB074893SwNoV_Sw918_GII.11
GII/5_AJ277607Hillingdon_GII.5
997
443
AF504671Vietnam026
AF427118Erfurt_GII.10
1000
95
750
987
786 GII/10_AY237415Mc37
803 GII/2_X81879Melksham_GII.2
DQ456824TokyoMK04
AY134748SnowMountain
DQ366347OsakaNI
1000
1000
116
GII/11_AB112221SaitamaT29GII
AY502009_CSE1_GII_17
1000
GII/14_AB078334Kashiwa47
155
AY113106_Fayettevil_GII_13
GII/16_AB112260SaitamaT53GII
GII/18_AB083780YURI_AKITA
1000
GII/4_X86557Lordsdale
X76716Bristol_GII.4
219
192
AY485642Langen1061
738
AY581254OxfordB5S22
38
DQ658413MD-2004_04
158
88AY502023FarmingtonHills
EU310927HoustonTCH186
311
AB220921Chiba_041050
798
DQ369797Guangzhou_NVgz01
824
740
AY741811Dresden174
AB083781YURI32073
515
AY502020CSG12002
378
AY032605MD145_12
AF145896Camberwell
966
1000
131
1000
GII/8_AB067543SaitamaU25
AF195848Amsterdam_GII.8
GII/9_AY054299IdahoFalls
AY038599VA97207_GII.9
909
185
999
67
1000
412
GII/13_AY130761M7
AY130761M7_99_US_GII.14
973 GII/6_AB039776SaitamaU3
1000
AB084071Gifu99
991
AB039777SaitamaU4
974
AJ277620Seacroft_GII.6
GII
1000
1000
333
988
AB039779SaitamaU17
AB067539SaitamaU16
GII/7_AJ277608Leeds_GII.7
AY130762_J23_1999_US_GII.15
GII/19_EF630529_Hokkaido299
1000
999
913
135
GII/3_AB067542SaitamaU201
AB039781SaitamaU18
U02030Tronto_GII.3
1000
AB365435TCH04_577
GII/17_AF195847Alphatron
1000
GV
GIII
1000
GIV?
DQ093067Alpha23
606
AY228235MuNoV_1
DQ223042MuNoV_3
DQ911368MuNoV_Berlin
AJ011099BoNoV_Jena
AY126474BoNoV_Dumfries
1000
Fig. 4. A phylogenetic dendrogram based on the ORF2 gene of NoV. A representative strain of each genotype is shown.
Reproduced with permission from Dr. Kazuhiko Katayama.
98
or carbohydrate-VLP binding assays, are commonly used
to detect and quantify NoV VLP attachment to HBGAs.
The VLPs derived from NV/68 bind to HBGAs in saliva
from secretor individuals. They preferentially bind to H type
1 and Leb synthetic carbohydrates (2,4–6,9). Although NV/
68 VLPs bind to type A antigens in saliva and synthetic
type A carbohydrates, they do not bind to either type B
synthetic carbohydrates or the majority of type B antigens
in saliva (2,4,5,9). However, other NoV VLPs display
different ABH and Lewis carbohydrate-binding profiles
(Table 2) (2–5,8,10). Indeed, a recent epidemiological study
showed that some NoV strains could infect individuals with
different ABH or secretor phenotypes (37). Each genotype
therefore seems to recognize different HBGAs. For example, the VLPs from the GII/2 BUDS strain bind A and B
antigens but not H antigen, whereas the VLPs from GII/16
OIF strain bind only Lea antigen (Table 2).
4–3. The importance of terminal residues in binding
The recognition sites on HBGA by the GI/1 NV/68 and
GII/4 VA387 strains have been analyzed in detail. The
attachment of NV/68 VLPs that bind to H, A, and Le-b
antigens depends on the presence of α1,2-fucose (α-Fuc)
or N-acetylgalactosamine (α-GalNAc). This conclusion is
based on three observations: (i) NV/68 VLPs do not bind
to H antigen precursors that lack α-Fuc (2,8); (ii) α1,2fucosidase treatment abolishes the attachment of NV/68
VLPs to surface epithelial cells of the gastroduodenal junction (7); and (iii) crystallization studies have suggested that
α-Fuc or α-GalNAc interact with NV/68 capsid protein
(14). On the other hand, the attachment of VA387 VLPs,
which bind to H, A, B, Leb, and Ley antigens, depends on
the presence of α-Fuc, α-GalNAc, or galactose (α-Gal).
This conclusion is based on two observations: (i) the binding of VA387 VLPs to A and B antigens can be reversed by
treatment with α-N-acetylgalactosaminidase or α-galactosidase (4); and (ii) crystallization studies have suggested
that α-Fuc, α-GalNAc, and α-Gal interact with VA387
capsid protein (11). It has been suggested recently that other
genotypes that bind to H, A, and/or B antigen also require
α-Fuc for binding. Although GI/1, GI/2, GI/3, GII/3, GII/
6, and GII/7 VLPs bind to H type 1, type 2, and/or type 3
carbohydrates, none of these VLPs bind to type 1, type 2,
or type 3 carbohydrates, thus suggesting that the terminal
α-Fuc on these H trisaccharides may govern the binding
between NoV and the H antigen (8). GI/1, GI/3, GI/8, GII/
4, and GII/5 VLPs that bind to A and/or B trisaccharides
(Table 3) do not bind to A and B disaccharides (Table 3)
(8). α-Fuc therefore appears to govern NoV binding not
only to H antigens but also to A and B antigens.
4–4. Distinction between type 1 and type 2 structures
by noroviruses
Type 1 core structures are widely expressed in
endodermally derived tissues such as lining and glandular
epithelia (38). In contrast, type 2 core structures are found
mainly in ecto- or mesodermally derived tissues, including
skin and erythrocytes (38–40). In the human gastroduodenal
junction, type 1 structures are found exclusively at the level
of the surface epithelia, whereas type 2 structures are
preferentially found at the glandular level (41). Immunohistochemical analysis has shown that the binding of rNV/
68 to the gastroduodenal junction is correlated with the presence of H type 1 antigen but not H type 2 antigen (7). In
ELISA experiments, the strength of rNV/68 VLP bind to
synthetic H carbohydrates has been reported to follow the
order H type 1 trisaccharides > H type 2 trisaccharides > H
disaccharides (6). Meanwhile, the results of Biacore experiments (8) showed that: (i) GI/3 and GII/4 bind more
efficiently to H type 2 than to H type 1 tetrasaccharides;
(ii) GI/1, GI/2, GI/3, GI/4, GI/8, and GII/4 bind more
efficiently to A type 2 than to A type 1 pentasaccharides;
(iii) GI/8 and GII/4 bind more efficiently to B type 2 than
to B type 1 pentasaccharides; and (iv) the dissociation of
GII/4 r104 is slower in B type 1 than B type 2. These
results indicate that NoV VLPs are able to distinguish between type 1 and type 2 carbohydrates and bind more tightly
to type 1 carbohydrates than to type 2.
4–5. Wide histo-blood group antigen recognition in
GII/4 strains
In an infection experiment with a human GII/4 strain,
pigs that expressed either A or H antigen on duodenal
tissue had higher rates of diarrhea and seroconversion, and
the number of viruses shed into feces was also higher (42),
thereby suggesting that the interaction between NoV and
HBGAs may determine the susceptibility to NoV infection
in both pigs and humans. It has been shown that GII/4 VLPs
bind to HBGAs more broadly and strongly than VLPs from
other strains (4,5,8) (Table 2). Indeed, GII/4 is known to be
a global epidemic genotype. NoV outbreaks occurred in
236 Japanese healthcare facilities for the elderly during the
winter of 2004–2005, causing 13 deaths in seven facilities.
Three NoV strains associated with the fatal cases were
isolated from three geographically separate facilities and
analyzed genetically. This analysis showed that all three
isolates belonged to GII/4 (43). It has been hypothesized
that the broad HBGA recognition of GII/4 strains may be
linked to the strength of the transmission.
4–6. Putative binding sites on the capsid protein
Putative binding sites on the NoV capsid protein have
been identified by mutagenic analyses and computer modeling (12), evolution trace analysis (13), and cocrystallization and X-ray analysis of recombinant P proteins with
synthetic type A or B trisaccharides (11). The amino acid
residues 267N, 291R, 292G, 293T, 300N, 322D, 327D,
329H, 331N, 333T, 334Q, 335F, 337H, 338S, 339S, 341T,
363G, 368N, 373L, 374S, 375W, 377S, 378P, 380S, 429G,
430A, and 431Y (NV/68 numbering) on the P2 domain were
predicted to be important for HBGA binding (11–13) (Table
2). Mutagenic analyses using GII/4 strain VA387 and GII/
5 strain MOH have suggested residues 291R, 292G, 293T,
300N, 335F, and 368N as putative carbohydrate binding
sites (12). Evolution trace analysis has shown that the residues 267N, 322D, 327D, 329H, 331N, 333T, 334Q, 341T,
373L, 374S, 375W, and 377S, which are known to bind
carbohydrate and sugar molecules, are located near the P2
domain cavity at the dimeric interface of rNoV or lie in the
vicinity of this cavity (13). The residues 337H, 338S, 339S,
363G, 429G, 430A, and 431Y were predicted to be important for GII/4 binding to HBGAs by cocrystallization and
X-ray analysis of recombinant P proteins of VA387 with
synthetic type A or B trisaccharides (11). In contrast,
residues 327D, 329H, 338S, 377S, 378P, and 380S were
predicted to be important for GI/1 binding to HBGAs (14).
The cocrystal structure revealed that the A trisaccharide
binds to the NV/68 P domain by interacting with these
residues in a different manner to that reported for the VA387
P-domain-A-trisaccharide complex.
99
Table 2. Summary of carbohydrate binding patterns and genotype-specific residues of the putative binding site
Residue no.
Genogroup Genotype
VLP
Binding pattern (a)
267b 291c 292c 293c 300c 322b 327bd 329bd 331b 333b 334b 335c 337e 338de 339e 341b 363e 368c 373b 374b 375b 377bd 378d 380d 429e 430e 431e
100
GI
1
1
2
2
3
3
3
4
8
10
rNV/68
r124
r258
C59
r645
DSV
VA115
rCV
rW18
Boxer
H type 1, 2, 3, A, Leb
H type 1, 2, 3, A, Leb
H type 1, 3, A, Lea
H, A
H type 2, A, Lea
no binding
no binding
H type 1/2, A, Lea, Leb
H type 1/2, A, B, Lea, Leb
Leb, Ley
N
N
N
N
N
N
N
N
N
N
R
R
R
R
R
R
R
R
R
R
G
G
G
G
G
G
G
G
G
G
T
T
K
K
S
K
S
R
K
R
N
N
N
N
N
N
T
N
N
N
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
H
H
H
H
H
H
H
H
H
H
N
N
R
R
E
S
E
E
N
T
T
T
S
S
S
T
S
S
T
V
Q
Q
K
K
P
A
P
K
F
K
F
F
T
T
T
T
T
I
Q
I
H
H
G
G
N
T
D
N
Q
T
S
S
D
D
T
G
T
N
D
G
S
S
P
P
G
S
G
P
P
D
T
T
R
R
V
N
V
V
Q
S
G
G
E
E
G
N
G
E
N
D
N
N
P
P
S
–
S
–
H
D
L
L
I
I
L
L
L
I
L
L
S
S
E
E
G
S
G
Q
G
T
W
W
W
W
W
W
W
W
W
W
S
S
S
S
S
S
S
S
S
T
P
P
Q
Q
P
P
P
P
A
W
S
S
S
S
S
S
S
S
S
S
G
G
N
N
G
G
G
N
G
N
A
A
N
N
T
V
A
Q
Q
P
Y
Y
R
R
N
N
N
S
P
–
GII
1
1
2
3
3
3
3
4
4
4
5
5
6
6
7
9
12
14
16
r485
rHV
BUDS
r18-3
r336
PiV
Mexico
r104
VA387
Grimsby
r754
MOH
r7k
r445
r10-25
VA207
r76
r47
OIF
no binding
no binding
A, B
H type 3, A, B
H tyep 3, A, B
A, B, Leb
A, B, Leb
H type 1, 2, 3, A, B, Leb
H type 1, 3, A, B, Leb, Ley
H, A, B
A, B
A, B
H type 2, 3, B, Lea, Leb
H type 1/2, 3
H type 1/2, 3, B, Lea, Leb
Lex, Ley
B
no binding
Lea
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
R
R
K
R
R
R
R
R
R
R
R
R
R
R
K
K
R
Q
R
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
R
R
Q
T
T
V
T
D
D
D
K
K
T
T
E
T
R
R
M
Q
Q
T
Q
Q
Q
Q
N
N
N
E
E
Q
Q
E
Q
Q
Q
H
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
I
I
V
V
V
V
V
I
I
I
V
V
V
V
L
L
L
L
L
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
T
T
I
A
A
A
A
L
L
L
L
L
A
A
A
A
L
L
L
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
R
R
R
R
R
R
R
T
T
T
R
R
R
R
R
R
R
D
–
N
N
D
N
D
N
N
T
T
T
N
N
D
D
N
G
D
N
N
T
T
A
T
T
T
T
S
S
S
A
A
A
A
A
A
A
A
A
C
C
N
T
T
T
T
T
T
T
N
N
T
T
T
T
C
K
A
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
N
R
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
K
Q
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
N
D
–
H
Q
Q
Q
Q
K
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
L
L
L
I
I
I
I
V
V
V
L
L
M
M
V
I
L
V
V
D
N
D
V
V
V
V
Q
Q
Q
E
E
D
D
G
I
E
V
V
G
G
G
G
G
G
G
S
S
S
G
G
G
G
G
S
G
L
Q
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
Q
G
T
T
F
R
R
R
R
Y
Y
Y
F
F
Y
Y
H
H
V
G
G
a: Summary of the saliva- or carbohydrate-binding assay results reported in Refs 2, 5, 6, 8, and 10.
b: These residues are located near the only P2 domain cavity or lie in the vicinity of the cavity and therefore are thought to play roles in NoV-HBGAs binding (Ref 13).
c: These residues have been proven to be important for NoV binding to HBGAs by mutagenic analyses with VA387 and MOH strains (Ref 12).
d: These residues were predicted to be important for NoV binding to HBGAs by X-ray crystallographic analysis with NV/68 strains (Ref 14).
e: These residues were predicted to be important for NoV binding to HBGAs by X-ray crystallographic analysis with VA387 strain (Ref 11).
–: One amino acid deletion.
Table 3. Structure of carbohydrates used in in vitro binding assays
Structure
type 1
type 2
type 3
H (di)
H type 1
H type 2
H type 3
Lea
Leb
A (di)
B (di)
A (tri)
B (tri)
A type 1
B type 1
A type 2
B type 2
Galb1-3GlcNAcb-R
Galb1-4GlcNAcb-R
Galb1-3GalNAca-R
Fuca1-2Galb-R
Fuca1-2Galb1-3GlcNAcb-R
Fuca1-2Galb1-4GlcNAcb-R
Fuca1-2Galb1-3GalNAca-R
Galb1-3(Fuca1-4)GlcNAcb-R
Fuca1-2Galb1-3(Fuca1-4)GlcNAcb-R
GalNAca1-3Galb-R
Gala1-3Galb-R
GalNAca1-3(Fuca1-2)Galb-R
Gala1-3(Fuca1-2)Galb-R
GalNAca1-3(Fuca1-2)Galb1-3GlcNAcb1-3Galb-R
Gala1-3(Fuca1-2)Galb1-3GlcNAcb1-3Galb-R
GalNAca1-3(Fuca1-2)Galb1-4GlcNAcb1-3Galb-R
Gala1-3(Fuca1-2)Galb1-4GlcNAcb1-3Galb-R
Disaccharide
Disaccharide
Disaccharide
Disaccharide
Trisaccharide
Trisaccharide
Trisaccharide
Trisaccharide
Tetrasaccharide
Disaccharide
Disaccharide
Trisaccharide
Trisaccharide
Pentasaccharide
Pentasaccharide
Pentasaccharide
Pentasaccharide
Glc, glucose; Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; Lac, lactose;
GalNAc, N-acetylgalactosamine.
cluded that there was no correlation between the binding
patterns and the genogroup (5). The reason for this contrasting conclusion remains unclear as each study group
used different strains and because a single amino acid
change of the P domain has been shown to result in a change
in the HBGA binding pattern (12). On the other hand, strains
GI and GII show the same recognition pattern against
type 1 and 2 core structures, binding more tightly to type 1
carbohydrates than to type 2. Tan et al. speculated that
HBGAs could be an important factor in convergent NoV
evolution as the functional convergence of NoV strains with
the same HBGA targets would subsequently result in the
acquisition of analogous HBGA binding interfaces in the
two genogroups despite their differing amino acid compositions (49). Since NoV forms many antigenically diverse
groups, identification of the common NoV binding epitopes
on host cells, if any, would be useful for the development
of possible virus detection kits or antiviral agents.
It is clear that HBGAs are important factors for determining the tissue specificity of NoV. Avian and equine
influenza viruses are known to preferentially bind to the
terminal SAα2-3Gal linkage, whereas human influenza
viruses preferentially bind to the SAα2-6Gal linkage, significantly affecting host specificity in influenza virus infection (50–52). A similar relationship may exist between
NoV carbohydrate recognition and its tissue specificity as
the binding of NoV VLP to the gastroduodenal junction
has been reported to be correlated with the presence of H
type 1 antigen but not H type 2 (7). Furthermore, ELISAbased in vitro binding assays have shown that NoV VLPs
are able to distinguish between type 1 and type 2 carbohydrates and that they bind more tightly to type 1 than to
type 2 (8). Moreover, carbohydrates are important factors in
determining the host specificity of caliciviruses. For example,
the α-Gal epitope recognized by bovine NoV (NB2) is
absent from all human tissues since the human gene encoding
an enzyme required for its synthesis has been inactivated
by mutations during evolution of the Hominidaea lineage.
Although this sugar motif is present in other mammals, such
as pigs, it is not expressed at the right location to allow
5. Carbohydrates and caliciviruses
Among caliciviruses, human NoV and rabbit hemorrhagic
disease virus (RHDV) bind to HBGAs. Indeed, RHDV in
the genus Lagovirus was the first calicivirus shown to bind
to HBGAs, thereby prompting us to study the interaction
between NoV and HBGAs. Ruvoën-Clouet et al. observed
that RHDV binds to a trisaccharide (Fucα1-2Galβl4GlcNAcβ1-R) present on the surface epithelial cells of the
upper respiratory and digestive tracts, which are natural
entry points for the virus (44). Feline calicivirus (FCV),
bovine NoV, and murine NoV also bind to carbohydrates.
FCV infects the upper respiratory tract by attaching to
α2,6-linked sialic acids (SA) and using the junctional
adhesion molecule-1 for internalization (45,46). Murine
NoV (strain MNV-1) binds to SA moieties on cultured and
primary murine macrophages (47). In particular, binding
to terminal SA on the ganglioside GD1a is important
during the attachment phase in the viral life cycle. Bovine
NoV attaches to a sugar motif resembling B blood group
antigen. Zakhour et al. reported that the prototype of the
bovine NoV, namely, Bo/Newbury2/76/UK (NB2), which
is a cow-specific strain, binds very specifically to the α-Gal
epitope present on the surface of the small intestine of the
cow (48).
6. Discussion
Although the binding properties of human NoV VLPs to
HBGAs were variable, strains in the same genotype show
a marked tendency to exhibit the same HBGA binding
patterns (Table 2). They also indicate that identical amino
acids on putative binding sites are well conserved (Table
2). Moreover, these amino acid residues are relatively conserved within each genogroup (Table 2) (15), thus suggesting that strains in the same genogroup have similar HBGA
binding patterns (15). Indeed, we have previously found
that the majority of GI strains have high binding abilities to
the Lea antigen and, conversely, that no strain binds strongly
to the Lea antigen in GII (8), although another study con101
infection: the surface of the small intestine. The bovine virus
should not therefore infect humans or pigs. In contrast, the
human GII/4 virus is able to infect pigs that express HBGAs
on their intestinal mucosa (40). Caliciviruses are suspected
to cause a wide spectrum of diseases, including gastroenteritis (human, pigs, calves, cats, dogs, and chickens),
vesicular lesions and reproductive failure (pigs and sea
lions), respiratory infections (cats and cattle), and a fatal
hemorrhagic disease (rabbits and hares). As the distribution of saccharide motifs depends on the species, tissue,
and cell, this difference might be the cause of the different
cell- and tissue-specificities of caliciviruses.
17.
18.
19.
20.
21.
Acknowledgments I wish to thank Dr. Naokazu Takeda of the Research
Collaboration Center on Emerging and Re-emerging Infections, Thailand,
for a critical reading of the manuscript.
This study was supported in part by a grant for Research on Food Safety
from the Ministry of Health, Labour and Welfare of Japan.
22.
23.
24.
Conflict of interest None to declare.
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