Human Noroviruses' Fondness for Histo-Blood Group Antigens

Full text

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Bishal K. Singh,a,bMila M. Leuthold,a,bGrant S. Hansmana,b

Schaller Research Group at the University of Heidelberg and the DKFZ, Heidelberg, Germanya

; Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germanyb

ABSTRACT

Human noroviruses are the dominant cause of outbreaks of gastroenteritis around the world. Human noroviruses interact with

the polymorphic human histo-blood group antigens (HBGAs), and this interaction is thought to be important for infection.

In-deed, synthetic HBGAs or HBGA-expressing enteric bacteria were shown to enhance norovirus infection in B cells. A number of

studies have found a possible relationship between HBGA type and norovirus susceptibility. The genogroup II, genotype 4

(GII.4) noroviruses are the dominant cluster, evolve every other year, and are thought to modify their binding interactions with

different HBGA types. Here we show high-resolution X-ray crystal structures of the capsid protruding (P) domains from

epi-demic GII.4 variants from 2004, 2006, and 2012, cocrystallized with a panel of HBGA types (H type 2, Lewis Y, Lewis B, Lewis A,

Lewis X, A type, and B type). Many of the HBGA binding interactions were found to be complex, involving capsid loop

move-ments, alternative HBGA conformations, and HBGA rotations. We showed that a loop (residues 391 to 395) was elegantly

reposi-tioned to allow for Lewis Y binding. This loop was also slightly shifted to provide direct hydrogen- and water-mediated bonds

with Lewis B. We considered that the flexible loop modulated Lewis HBGA binding. The GII.4 noroviruses have dominated

out-breaks over the past decade, which may be explained by their exquisite HBGA binding mechanisms, their fondness for Lewis

HBGAs, and their temporal amino acid modifications.

IMPORTANCE

Our data provide a comprehensive picture of GII.4 P domain and HBGA binding interactions. The exceptionally high

resolu-tions of our X-ray crystal structures allowed us to accurately recognize novel GII.4 P domain interacresolu-tions with numerous HBGA

types. We showed that the GII.4 P domain-HBGA interactions involved complex binding mechanisms that were not previously

observed in norovirus structural studies. Many of the GII.4 P domain-HBGA interactions we identified were negative in earlier

enzyme-linked immunosorbent assay (ELISA)-based studies. Altogether, our data show that the GII.4 norovirus P domains can

accommodate numerous HBGA types.

H

uman noroviruses are responsible for most epidemic

out-breaks of gastroenteritis. There are still no antivirals or

vac-cines approved, despite the discovery of these viruses over 4

de-cades ago (

1

). Noroviruses are genetically and antigenically

diverse (

2

), yet a single genetic cluster (genogroup II, genotype 4

[GII.4]) has dominated over the past decade (

3

). The GII.4

noro-viruses evolve

5% every year and are believed to have a

mecha-nism that allows them to evade the immune system or alter

recep-tor binding profiles (

4–6

). However, immunity to noroviruses is

still poorly understood (

7

).

Human noroviruses interact with histo-blood group antigens

(HBGAs), and this is thought to be important for viral infections

(

8–11

). A recent report showed for the first time that human

no-roviruses infect B cells and that HBGAs (synthetic or from

HBGA-expressing enteric bacteria) can enhance the infection (

12

).

HBGAs are also found as soluble antigens in saliva and are

ex-pressed on epithelial cells. Genetic polymorphisms in genes that

control their synthesis are known to provide intraspecies diversity

(

13

). To date, based on the ABH and Lewis HBGA types, at least

nine different HBGAs have been found to interact with human

noroviruses. Individuals expressing the O type are thought to have

a significantly higher infection rate than those for individuals

with other blood types (

11

). The GII noroviruses are thought to

have preferences for HBGAs in a strain-dependent manner

(

14–19

).

Expression of the norovirus capsid protein in insect cells

re-sults in the formation of virus-like particles (VLPs) that are

anti-genically similar to native virions. The X-ray crystal structure of

prototype (GI.1) norovirus VLPs identified two domains: the shell

(S) and protruding (P) domains (

20

). The S domain forms a

scaf-fold surrounding the viral RNA, whereas the P domain is thought

to contain the determinants for cell attachment and strain

diver-sity. The P domain can be further subdivided into P1 and P2

subdomains, and each subdomain likely has unique functions. In

this study, we determined the X-ray crystal structures of P

do-mains from three epidemic GII.4 variants, from 2004, 2006, and

2012, in complex with a panel of HBGAs in order to elucidate

HBGA binding mechanisms. Our data showed that the GII.4

noroviruses bound numerous HBGA types and that binding

involved complex interactions, including P domain loop

move-ments and alternative HBGA conformations. Importantly, many

of our new findings challenge previous enzyme-linked

immu-Received11 October 2014Accepted21 November 2014

Accepted manuscript posted online26 November 2014

CitationSingh BK, Leuthold MM, Hansman GS. 2015. Human noroviruses’ fondness for histo-blood group antigens. J Virol 89:2024 –2040.

doi:10.1128/JVI.02968-14.

Editor:R. M. Sandri-Goldin

Address correspondence to Grant S. Hansman, g.hansman@dkfz.de. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.02968-14

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nosorbent assay (ELISA)-based studies and reveal interactions

that have not been recognized so far (

4–6

). Altogether, our data

show that the GII.4 noroviruses are capable of binding diverse

HBGA types, which may correlate with a larger proportion of the

human population being susceptible to GII.4 infections.

MATERIALS AND METHODS

Sequence analysis and expression and purification of the P domain.The P domain amino acid sequences from four GII.4 variants, from 1998, 2004, 2006, and 2012 (termed VA387-1998 [PBD entry2OBS], Farm-2004 [GenBank accession number JQ478408], Saga-2006 [accession numberAB447457], and NSW-2012 [accession numberJX459908], re-spectively), were aligned using Clustal X. The Farm-2004, Saga-2006, and NSW-2012 P domains (residues 224 to 538) were expressed inEscherichia coliand purified as previously described (21). Briefly, the codon-opti-mized P domains were cloned into a modified expression vector (pMal-c2X) and transformed intoE. coliBL21 cells for protein expression. Trans-formed cells were grown in LB medium supplemented with 100␮g/ml ampicillin for 4 h at 37°C. Expression was induced with 0.75 mM IPTG (isopropyl-␤-D-thiogalactopyranoside) at an optical density at 600 nm (OD600) of 0.7 for 18 h at 22°C. Cells were harvested by centrifugation at 6,000 rpm for 15 min and disrupted by sonication on ice. A His-tagged P domain protein was purified from a Ni column (Qiagen) and digested with HRV-3C protease (Novagen) overnight at 4°C. Cleaved P domains were separated on the Ni column and dialyzed in gel filtration buffer (25 mM Tris-HCl and 300 mM NaCl) overnight at 4°C. The P domains were purified by size-exclusion chromatography, concentrated to 3 to 7 mg/ml, and stored in gel filtration buffer at 4°C.

Crystallization of norovirus P domains.Crystals were grown in a 1:1 mixture of protein sample and mother liquor for 2 to 6 days at 18°C.

Farm-2004 P domain crystals were grown in 20% polyethylene glycol 3350 (PEG 3350) and 0.2 M magnesium formate, Saga-2006 crystals were grown in 3 M sodium acetate (pH 6.9), and NSW-2012 crystals were grown in 20% PEG 3350 and 0.2 M sodium formate. Farm-2004, Saga-2006, and NSW-2012 formed long, rod-shaped crystals, diamond-shaped crystals, and both diamond- and plate-shaped crystals, respectively. For the P domain and HBGA complexes, we cocrystallized a 30⫻to 60⫻ molar excess of HBGAs (Dextra, United Kingdom). Prior to flash freez-ing, crystals were transferred to a cryoprotectant containing mother li-quor, a 30⫻molar excess of HBGAs, and 30% ethylene glycol or glycerol. Unfortunately, we were unable to produce complex crystals for all P do-mains and HBGAs, and soaking experiments with HBGAs produced crys-tals with high mosaicity and/or the cryscrys-tals dissolved.

Data collection, structure solution, and refinement.X-ray diffrac-tion data were collected at the European Synchrotron Radiadiffrac-tion Facility, France, at beamlines BM30A and ID23-1, and were processed with XDS (22). Structures were solved using molecular replacement in PHASER (23). The Saga-2006 P domain was determined by molecular replacement using the previously solved GII.10 P domain as a search model (21). The Saga-2006 P domain was then used to determine the structures of the Farm-2004 and NSW-2012 P domains. The Farm-2004 P domain formed crystals in space group P212121, while the Saga-2006 and NSW-2012 P domains were both solved in space group C2. Structures were refined in multiple rounds of manual model building in COOT (24), with subse-quent refinement with PHENIX (25). The HBGAs were added to the models at the final stages of structural refinement in order to reduce bias during refinement. Structures were validated with Molprobity (26) and Procheck (27). HBGA interactions were analyzed using Accelrys Discov-ery Studio (version 4.1), with hydrogen bonding interaction distances of 2.4 to 3.5 Å and hydrophobic interaction distances of 3.4 to 4.5 Å. Figures

FIG 1Amino acid alignment of norovirus GII.4 variants. The P domain amino acid sequences of four GII.4 variants, from 1998, 2004, 2006, and 2012 (termed VA387-1998, Farm-2004, Saga-2006, and NSW-2012, respectively), were aligned using Clustal X. The capsid sequences shared 93 to 95% amino acid identity. The S domain was highly conserved, with only seven amino acid differences (not shown), whereas the P1 (red) and P2 (yellow) subdomains were more variable. The common set of amino acids interacting with HBGAs is shaded in blue (chain A) and green (chain B). Compared to the sequence of the earlier discovered GII.4 variant P domain (VA387-1998), one amino acid insertion was observed in 2004 and remained in 2006 and 2012.

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and protein contact potentials were generated using PyMOL (version 1.12r3pre).

Protein structure accession numbers.Atomic coordinates and struc-ture factors were deposited in the Protein Data Bank (PDB) under acces-sion numbers4OOV,4OOX,4OOS,4X05,4OPS,4WZK,4X07,4X06,

4WZL,4OPO,4WZE,4WZT,4OP7, and4X0C.

RESULTS

Structures of unliganded GII.4 P domains.

Three globally

im-portant epidemic GII.4 noroviruses, from 2004, 2006, and 2012

(Farm-2004, Saga-2006, and NSW-2012 [also known as Sydney

2012], respectively), were selected for P domain and HBGA

bind-ing analysis usbind-ing X-ray crystallography (

Fig. 1

). Most of the

amino acid variations were observed in the P2 subdomains. Data

statistics for GII.4 P domain apo structures are provided in

Table

1

. The P1 subdomains comprised residues 224 to 274 and 418 to

530, whereas the P2 subdomains were between residues 275 and

417 (

Fig. 2

). Similar to that of other human noroviruses, the P1

subdomain comprised

-sheets and one

-helix, while the P2

subdomain contained six antiparallel

-strands that formed a

barrel-like structure. Overall, the GII.4 P dimer structures were

similar, with a maximum root mean square deviation (RMSD)

of 0.52 (

Fig. 2D

). This result corresponded well with the high

sequence identities (93 to 95%) and the amino acid sequence

alignment (

Fig. 1

). In order to follow GII.4 P domain

evolu-tion, amino acid changes from an earlier GII.4 P domain (from

1998) were projected onto the Farm-2004, Saga-2006, and

NSW-2012 P dimer surfaces (

Fig. 3

). Most amino acid changes

were surface exposed, and

50% of these became fixed over the

years. The region immediately beneath the HBGA binding

pocket showed little variation, whereas the surrounding

re-gions contained only a few amino changes. This result

sug-gested that the HBGA pocket is stable and likely contains

im-portant functions.

Structure of 2006 GII.4 P domain–H2-tri complex.

The

HBGAs chosen for this study were involved in a primary

biosyn-thetic pathway and were previously analyzed in ELISA-based

studies (

8

,

10

,

11

,

28–31

). Data collection and refinement

sta-tistics for P domain-HBGA complex structures are provided in

Tables 2

,

3

, and

4

. The H2 trisaccharide (H2-tri) contains a

single ABH fucose moiety. Two H2-tri moieties bound to the

Saga-2006 P dimer. The electron density was well defined for all

three saccharide units, indicating that the HBGA was firmly

held by the P domain (

Fig. 4

). The H2-tri unit was held in place

by a network of hydrophilic and hydrophobic interactions at

the dimeric interface (

Fig. 5A

and

B

). The fucose was held by six

direct hydrogen bonds: two from the side chain of Asp374, two

from the side chain of Arg345, one from the main chain of

Thr344, and one from the main chain of Gly443. A

hydropho-bic interaction was provided from Tyr444. These five amino

acids (Thr344, Arg345, Asp374, Gly443, and Tyr444) were the

common set of residues involved in other GII P-HBGA binding

interactions at this “regular pocket” (

21

). The central galactose

of H2-tri was held by two hydrogen bonds from the side chain

of Ser442, while the terminal

N

-acetylglucosamine was held by

one hydrogen bond from the main chain of Gly392. A number

of water-mediated interactions were also observed for

Saga-2006 –H2-tri.

Comparisons with other GII.4 P domain H2-tri complex

struc-TABLE 1Data collection and refinement statistics for GII.4 P domain apo structuresa

Parameter

Value or description

Farm-2004 (PDB entry 4OOV) Saga-2006 (PDB entry 4OOX) NSW-2012 (PDB entry 4OOS)

Data collection parameters

Space group P212121 C2 C2

Cell dimensions

a,b,c(Å) 62.95, 90.12, 109.21 96.72, 58.94, 62.14 98.48, 55.07, 63.46

␣,␤,␥(°) 90, 90, 90 90, 119.88, 90 90, 120.10, 90

Resolution range (Å) 46.70–1.50 (1.55–1.50) 48.22–1.03 (1.07–1.03) 46.25–1.60 (1.66–1.60)

Rmerge 7.48 (54.74) 3.40 (52.85) 4.43 (24.53)

I/␴I 14.18 (2.37) 15.12 (1.93) 20.63 (5.12)

Completeness (%) 99.24 (97.64) 96.03 (90.00) 98.14 (94.97)

Redundancy 4.9 (4.1) 3.0 (2.7) 3.5 (3.3)

Refinement parameters

Resolution range (Å) 46.70–1.53 27.17–1.20 42.60–1.64

No. of reflections 93,719 92,504 35,573

Rwork/Rfree 14.08/16.41 11.96/14.32 13.77/16.15

No. of atoms 10,090 4,975 5,021

Protein 4,755 2,436 2,406

Ligand/ion 32 40 24

Water 778 295 318

AvgBfactor (Å2)

Protein 13.80 13.90 12.90

Ligand/ion 22.30 22.90 18.80

Water 26.40 26.00 23.30

RMSD

Bond length (Å) 0.009 0.010 0.005

Bond angle (°) 1.29 1.38 1.09

a

The data sets were collected from single crystals. Values in parentheses are for the highest-resolution shell.

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tures were not possible, since the Saga-2006 –H2-tri structure is

the first known GII.4 P–H2-tri complex structure. Superposition

of a GII.4 2004 P dimer-H1 pentasaccharide structure (PDB entry

3SLN

) on the Saga-2006 –H2-tri structure revealed that the first

three saccharides were positioned similarly to H2-tri on the P

dimers (

Fig. 5C

and

D

). However, the

N

-acetylglucosamine in

Saga-2006 –H2-tri was flipped 180° compared to that of the H1

pentasaccharide. The two remaining saccharides of the H1

pen-A: P2

A: P1 B: P1

N C

N

90°

90°

90°

B: P2

N C

N

A: P1

A: P2

C

C

N N

C C

A: P1

A: P2 B: P2

B: P1

B: P2

B: P1

FIG 2X-ray crystal structures of unbound GII.4 P domains. (A) The Farm-2004 P domain apo structure contained one dimer per asymmetric unit. The P domain was subdivided into P1 (chain A in pink and chain B in pale cyan) and P2 (chain A in green cyan and chain B in light magenta) subdomains. (B) The Saga-2006 P domain apo structure contained one monomer per asymmetric unit (a dimer is shown) and was subdivided into P1 (chain A in brown and chain B in yellow-orange) and P2 (chain A in deep teal and chain B in dirty violet) subdomains. (C) The NSW-2012 P domain apo structure contained one monomer per asymmetric unit (a dimer is shown) and was subdivided into P1 (chain A in lime and chain B in marine) and P2 (chain A in blue-white and chain B in teal) subdomains. (D) Superposition of the Farm-2004, Saga-2006, and NSW-2012 P dimers revealed that their overall structures were similar.

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tasaccharide were raised off the P domain, though they were not

held by any residues (

19

). This result shows that the H-type

ori-entation is variable among GII.4 variants or that the longer

pen-tasaccharide and the H type influence the binding orientation.

Structure of 2006 GII.4 P domain–Le

y

-tetra complex.

The

Lewis

y

tetrasaccharide (Le

y

-tetra) contains both an ABH fucose

and a Lewis fucose moiety. Two Le

y

-tetra moieties bound to the

Saga-2006 P dimer. The electron density was well defined for all

four saccharide units (

Fig. 4

). The Lewis fucose of Le

y

-tetra bound

at the regular pocket and was held by the common set of residues

(

Fig. 6A

and

B

). The

N

-acetylglucosamine was held by one

hydro-gen bond from the side chain of Ser442, and the galactose was held

Y250F P504Q -394G, N395A S296T, D298N, I300T

E’340G’ A’346G’

I’300T’, V’365I’, Y’367F’ -394G’, N’395A’ L’375F’, Q’376E’ S’296T’ H’297Q’, D’298N’ P504Q Y250F N395A S355D N407S K329R E346G L375F, Q376E S296T, D298N, I300T V365I, Y367F, T368N

A340G

I389V

N’393S’, -394T’, N’395T’ I’300T’ Y250F P504Q S255G N448D Q397R N393S, -394T, N395T

R’339K’ E’340G’ N’372E’ G’378H’ L’375F’, Q’376E’ H’297R’, D’298N’ T’412N’, G’413V’ V’356A’, H’357P’ S’255G’Q’306L’ T412N T412N, G413V V356A, H257P S255G S352Y G378H R339K L375F, Q376E E340G N372E H297R Y367F, T368S

N393S, -394T, N395T

Q397R P504Q Y250F A346G E340T A346G M333L L375F, Q376E, T377A G378N N310D H297R N372E A294T S364R V356A, H257D T412N, G413T S352Y Y367F, T368E

N393G, -394T, N395T P504Q Q397R Y250F N448D Y250F P504Q Q397R T412N N393G, -394T, N395T

E’340T’

N’310D’

T228S I231V

A’294T’

N’393G’, -394T’, N’395T’ I’300T’

H’297R’, D’298N’

T’412N’,G’413T’ V’356A’, H’357D’ N297R N’372D’, N’373R’

L’375F’, Q’376E’ T’377A’, G’378N’ 90° 90° 90°

B

C

FIG 3Amino acid variations in GII.4 P dimers from 2004, 2006, and 2012 variants. Amino acid changes (red) were highlighted on GII.4 P dimers (side and top views). The changes were numbered according to a change from 1998 to the respective year (labeled once). A cumulative addition of amino acid changes was found. (A) Farm-2004 contained a single amino acid insertion, Gly394, and this remained in 2012. A small number of amino acid changes surrounding the HBGA pocket (black circle), i.e., I389V, L375F, and Q376E, was observed. (B) Saga-2006 contained additional changes, several of which became fixed, e.g., L375F and Q376E. (C) NSW-2012 showed the majority of changes, including several changes in the P1 subdomain.

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by one direct hydrogen bond from the hydroxyl group of Tyr444.

The ABH fucose was not held by any direct hydrogen bonds.

Sev-eral P domain water-mediated interactions with fucose and

galac-tose were also observed. In order for Le

y

-tetra to bind to

Saga-2006, a loop (residues 391 to 394) was shifted from the apo

position to an alternative conformation (

Fig. 6C

).

Other GII.4 P domain–Le

y

-tetra complex structures were not

available; however, comparison with the GII.10 P domain–Le

y

-tetra structure (

21

) revealed different Le

y

-tetra orientations on the

P domains (

Fig. 6D

and

E

). The Lewis fucose of Saga-2006 –Le

y

-tetra bound at the regular pocket, whereas the ABH fucose of

GII.10 P domain Le

y

-tetra bound at the regular pocket. Also, the

terminal saccharides of GII.10 P domain Le

y

-tetra were directed

toward the center of the P dimer, while the terminal saccharides of

Saga-2006 –Le

y

-tetra were leaning toward the edge of the P dimer.

Interestingly, Saga-2006 had fewer direct hydrogen bonds with

Le

y

-tetra than GII.10 did (6 and 10, respectively). Together, these

findings suggest that there is an Le

y

-tetra placement constraint

among the different GII P domains.

Structures of 2004 and 2006 GII.4 P domain–Le

b

-tetra

com-plexes.

The Lewis

b

tetrasaccharide (Le

b

-tetra) contains both ABH

and Lewis fucose moieties. One Le

b

-tetra unit bound to the

Farm-2004 P dimer, whereas two Le

b

-tetra units bound to the Saga-2006

P dimer. The electron density was well defined for all four

saccha-ride units (

Fig. 4

). The ABH fucose of Farm-2004 –Le

b

-tetra

bound at the regular pocket and was held by the common set of

residues (

Fig. 7A

and

B

). The galactose of Farm-2004 –Le

b

-tetra

was held by one hydrogen bond from the side chain of Ser442,

while the

N

-acetylglucosamine was not held by any hydrogen

bonds. The Lewis fucose of Farm-2004 –Le

b

-tetra was held by one

hydrogen bond from the side chain of Asp391 and one hydrogen

bond from the main chain of Gly392. A similar set of direct

hy-drogen bonds was found in the Saga-2006 –Le

b

-tetra structure

(

Fig. 7C

and

D

). Several P domain water-mediated interactions

with the ABH and Lewis fucose moieties were also observed.

In order to better understand Le

b

-tetra binding interactions,

we superpositioned chains A and B of the Farm-2004 apo and

Farm-2004 –Le

b

-tetra structures (

Fig. 7E

). The loop in chain A

interacting with the Lewis fucose of Farm-2004 –Le

b

-tetra

(resi-dues 391 to 394) was in a suitable position to allow direct

hydro-gen bonds with the Lewis fucose. The equivalent loop in chain A of

the Farm-2004 apo structure was in a slightly different

conforma-tion. This result suggested that the loop was repositioned to

sup-port Lewis fucose binding. The loop at the unoccupied HBGA

binding site of Farm-2004 –Le

b

-tetra (chain B) had a

conforma-tion similar to that of the equivalent loop (chain B) of the

Farm-2004 apo structure. The reason that the second Le

b

-tetra unit did

not bind to Farm-2004 was not determined, although steric

hin-drance from the neighboring molecule may have played a role,

as previously discussed (

21

). Nevertheless, these results

high-light the complexity and importance of the flexible loop in

Le

b

-tetra binding.

Structure of 2006 GII.4 P domain–Le

a

-tri complex.

The

Lewis

a

trisaccharide (Le

a

-tri) contains a single Lewis fucose

moi-ety. The electron density was well defined for the Lewis fucose and

less defined for the other two saccharides, which indicated that

these saccharides were only loosely held on the P domain (

Fig. 4

).

Two Le

a

-tri units bound to the Saga-2006 P dimer. The Lewis

fucose bound at the regular pocket and was held by the common

set of residues (

Fig. 8A

and

B

). The

N

-acetylglucosamine was held

by one hydrogen bond from the side chain of Ser442. Galactose

was held by two hydrogen bonds from the hydroxyl group of

Tyr444. Several P domain water-mediated interactions were also

observed with all three saccharides.

Other GII P domain–Le

a

-tri complex structures have yet to be

determined. Superposition of the Farm-2004, Saga-2006, and

NSW-2012 GII.4 apo structures showed that the conformations of

the side chains that interacted with

N

-acetylglucosamine and

ga-lactose (i.e., Ser442 and Tyr444) were similarly orientated. This

result suggests that Farm-2004 and NSW-2012 are also capable of

Le

a

-tri binding (

Fig. 8C

and

D

), although further studies are

re-quired.

Structure of 2012 GII.4 P domain–Le

x

-tri complex.

The

Lewis

x

trisaccharide (Le

x

-tri) contains a single Lewis fucose

moi-ety. Two Le

x

-tri units bound to the NSW-2012 P dimer. The

elec-tron density was well defined for all three saccharide units, which

indicated that the HBGA was firmly held by the P domain (

Fig. 4

).

The Lewis fucose bound at the regular pocket and was held by the

common set of residues (

Fig. 9

). The

N

-acetylglucosamine was

held by two hydrogen bonds with the side chain of Ser442, while

the galactose was held by one hydrogen bond from the hydroxyl

group of Tyr444. Interestingly, the terminal galactose of one Le

x

-tri unit was held in two conformations, with an

1.5-Å shift (data

not shown; see the PBD). However, this shift did not result in any

additional binding interactions.

Previous GII P domain and Le

x

-tri complex structures have not

been determined. Nevertheless, the side chains that interacted

TABLE 2Data collection and refinement statistics for Farm-2004 P domain and HBGA complex structuresa

Parameter

Value or description

B-tri

(PDB entry 4X05)

Leb-tetra (PDB entry 4OPS)

Data collection parameters

Space group C2 P212121

Cell dimensions

a,b,c(Å) 175.11, 89.54, 106.73 71.45, 90.11, 91.87 ␣,␤,␥(°) 90, 127.55, 90 90, 90, 90

Resolution range (Å) 19.89–1.96 (2.01–1.96) 47.81–1.75 (1.79–1.75)

Rmerge 15.8 (113.90) 11.20 (53.53)

I/␴I 8.89 (1.29) 9.88 (2.41)

Completeness (%) 99.10 (99.30) 97.30 (95.10)

Redundancy 5.5 (4.7) 5.5 (5.7)

Refinement parameters

Resolution range (Å) 19.89–1.98 47.81–1.76

No. of reflections 90,505 57,869

Rwork/Rfree 16.30/20.31 18.50/21.64

No. of atoms 10,774 9,541

Protein 9,437 4,723

Ligand/ion 132 46

Water 12,045 324

AvgBfactor (Å2)

Protein 22.70 23.20

Ligand/ion 43.00 57.20

Water 31.20 26.80

RMSD

Bond lengths (Å) 0.008 0.013

Bond angles (°) 1.10 1.36

a

The data sets were collected from single crystals. Values in parentheses are for the

highest-resolution shell.

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TABLE 3 Data collection and refinement statistics for Saga-2006 P domain and HBGA complex structures a Parameter Value or description H2-tri (PDB entry 4WZK) A-tri (PDB entry 4X07) B-tri (PDB entry 4X06) Le a-tri (PDB entry 4WZL) Le b-tetra (PDB entry 4OPO) Le y-tetra (PDB entry 4WZE) Data collection parameters Space group C2 C2 C2 C2 C2 C2 Cell dimensions a , b , c (Å) 114.99, 58.81, 98.02 97.96, 58.64, 114.59 113.83, 58.65, 97.31 96.87, 58.83, 124.44 113.66, 58.6, 97.17 97.02, 58.50, 113.86 ␣ , ␤ , ␥ (°) 90, 108.11, 90 90, 105.49, 90 90, 107.34, 90 90, 119.8, 90 90, 107.15, 90 90, 108.1, 90 Resolution range (Å) 46.58–1.47 (1.51–1.47) 48.01–1.28 (1.32–1.28) 48.10–1.22 (1.25–1.22) 49.59–1.57 (1.61–1.57) 46.42–1.38 (1.42–1.38) 48.1 6–1.45 (1.49–1.45) Rmerge 3.091 (48.00) 6.353 (41.88) 3.519 (35.40) 5.205 (48.73) 7.537 (93.06) 2.8 (45.5) I /␴ I 17.31 (2.27) 7.40 (1.96) 13.92 (2.54) 8.11 (1.99) 12.76 (1.71) 10.16 (1.84) Completeness (%) 97.30 (95.10) 98.00 (92.70) 96.70 (90.20) 97.61 (88.60) 99.20 (97.04) 95.17 (93.87) Redundancy 2.9 (2.9) 4.5 (3.6) 2.6 (2.5) 3.0 (2.9) 3.7 (3.6) 2.3 (2.2) Refinement parameters Resolution range (Å) 29.66–1.49 43.18–1.46 32.32–1.22 42.47–1.70 46.42–1.40 31.76–1.46 No. of reflections 99,198 106,175 176,446 65,702 119,485 100,015 Rwork / Rfree 13.88/17.35 16.53/19.30 13.41/16.12 17.03/19.55 15.08/18.54 17.74/21.17 No. of atoms 9,013 10,143 10,037 9,985 10,227 9,826 Protein 4,803 4,842 4,865 4,812 4,850 4,801 Ligand/ion 80 80 140 124 120 100 Water 540 799 716 569 812 542 Avg B factor (Å 2) Protein 24.20 15.50 15.80 17.10 12.90 22.80 Ligand/ion 47.90 36.00 27.90 37.40 30.40 41.70 Water 33.10 27.40 25.60 28.20 25.30 32.30 RMSD Bond lengths (Å) 0.011 0.008 0.010 0.006 0.007 0.033 Bond angles (°) 1.29 1.20 1.36 1.08 1.20 1.73 aThe data sets were collected from single crystals. Values in parentheses are for the highest-resolution shell.

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with

N

-acetylglucosamine and galactose were similarly orientated

in all three GII.4 P domains (

Fig. 8C

and

D

), suggesting that

Farm-2004 and Saga-2006 may also bind Le

x

-tri.

Structures of 2006 and 2012 GII.4 P domain–A-tri

com-plexes.

Two A trisaccharides (A-tri) bound to both Saga-2006 and

NSW-2012 P dimers. The electron density was well defined for all

three saccharide units (

Fig. 4

). The orientations of the A-tri units

in the Saga-2006 and NSW-2012 P domains were similar. Fucose

was held by the common set of residues, while galactose and

N

-acetylgalactosamine were not supported by any direct hydrogen

bonds (

Fig. 10

). Compared to their counterparts in other GII

structures, the A-tri saccharide units were similarly orientated on

the P dimers (

21

).

Structures of 2004, 2006, and 2012 GII.4 P domain–B-tri

complexes.

Two B trisaccharides (B-tri) bound to Farm-2004

(

Fig. 11A

and

B

), Saga-2006 (

Fig. 11C

and

D

), and NSW-2012

(

Fig. 11E

and

F

) P dimers. The electron density was well defined

for all three saccharide units (

Fig. 4

). The fucose was held by the

common set of residues in all complex structures, while the central

and terminal galactose residues were not held by any direct

hydro-gen bonds (

Fig. 11

). Similar to that of NSW-2012–Le

x

-tri, the

terminal galactose of B-tri bound to Saga-2006 was held in two

conformations, with an

1.5-Å shift, and this resulted in several

new water-mediated interactions (

Fig. 11C

and

D

). Interestingly,

the loop described earlier (residues 391 to 394) (

Fig. 8C

and

D

)

was found in two different positions in the Saga-2006 –B-tri

struc-ture. In one conformation, the loop was orientated as in the

Saga-2006 apo structure, while the alternative conformation was

positioned similarly to that in the Saga-2006 –Le

y

-tetra structure

(

Fig. 6C

). The loop movement did not result in any additional

binding interactions but merely indicated that the loop had a

pref-erence for at least two conformations. Compared to their

coun-terparts in other GII structures, the B-tri saccharide moieties were

similarly positioned on the P domains (

21

).

Protein contact potential.

The protein contact potential was

calculated for a panel of GII.4 P dimers in order to better

under-stand the temporal variations in surface charge that might alter

antigenicity and HBGA binding (

Fig. 12

). The region ahead of the

regular pocket and toward the center of the P dimer (binding sites

of A and B types) remained virtually unchanged and was mostly

negatively charged. The regions that contributed to binding of

terminal saccharide moieties of Lewis HBGAs underwent a

mod-ification, i.e., from small areas of negative and positive charges (in

the 1998 variant) to large areas of mostly negative charge. In this

view, it appeared that the more recent GII.4 HBGA binding pocket

became more negatively charged.

DISCUSSION

There is considerable debate on norovirus GII.4 evolution and the

corresponding interactions with different HBGA types (

4–6

,

8

,

11

,

28–31

). In this study, we determined the X-ray crystal structures

of three P domains from epidemic GII.4 variants, from 2004,

2006, and 2012, with a panel of HBGAs. The exceptionally high

resolutions of our structures allowed us to accurately define

HBGA interactions, several of which were not previously

deter-mined for GII.4 P domains (i.e., H2-tri, Le

y

-tetra, Le

a

-tri, and

Le

x

-tri). A common set of conserved residues (i.e., Asp374,

Arg345, Thr344, Tyr444, and Gly443) firmly held both the ABH

TABLE 4Data collection and refinement statistics for NSW-2012 P domain and HBGA complex structuresa

Parameter

Value or description

A-tri (PDB entry 4WZT) B-tri (PDB entry 4OP7) Lex-tri (PDB entry 4X0C)

Data collection parameters

Space group P41212 P41212 P41212

Cell dimensions

a,b,c(Å) 104.74, 104.74, 190.85 104.61, 104.61, 190.54 104.83, 104.83, 191

␣,␤,␥(°) 90, 90, 90 90, 90, 90 90, 90, 90

Resolution range (Å) 48.26–1.85 (1.90–1.85) 48.19–1.90 (1.97–1.90) 45.95–1.70 (1.75–1.70)

Rmerge 8.758 (98.50) 11.31 (115.40) 6.856 (128.60)

I/␴I 16.73 (1.60) 10.70 (1.09) 15.62 (1.20)

Completeness (%) 98.80 (98.30) 93.80 (93.70) 99.20 (97.60)

Redundancy 6.5 (6.5) 4.3 (4.2) 3.6 (3.6)

Refinement parameters

Resolution range (Å) 48.26–1.91 48.19–1.92 40.14–1.72

No. of reflections 81,872 76,345 112,461

Rwork/Rfree 15.37/17.91 18.47/20.81 15.96/17.95

No. of atoms 10,006 9,760 10,085

Protein 4,803 4,762 4,802

Ligand/ion 88 76 142

Water 718 492 747

AvgBfactor (Å2)

Protein 26.30 26.90 23.10

Ligand/ion 42.80 37.90 43.60

Water 34.50 31.50 34.70

RMSD

Bond lengths (Å) 0.008 0.007 0.013

Bond angles (°) 1.13 1.11 1.32

a

The data sets were collected from single crystals. Values in parentheses are for the highest-resolution shell.

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H2-tri Saga-2006

A-tri Saga-2006

A-tri NSW-2012

B-tri Farm-2004

B-tri Saga-2006

B-tri NSW-2012

Le

a

-tri Saga-2006

Le

y

-tetra Saga-2006

Le

b

-tetra Farm-2004

Le

b

-tetra Saga-2006

Le

x

-tri NSW-2012

FIG 4Representative simulated annealing difference omit maps. The omit maps (blue) were contoured between 2.5 and 2.0␴. H2-tri is an␣-L -fucose-(1-2)-␤-D-galactose-(1-4)-N-acetyl-␤/␣-D-glucosamine, A-tri is an␣-L-fucose-(1-2)-␤-D-galactose-(3-1)-N-acetyl-␣-D-galactosamine, B-tri is an␣-L -fucose-(1-2)-␤/␣-D-galactose-(3-1)-␣-D-galactose, Ley-tetra is an-L-fucose-(1-2)--D-galactose-(1-4)-N-acetyl-/-D-glucosamine-(3-1)--L-fucose, Leb-tetra is an-L -fucose-(1-2)-␤-D-galactose-(1-3)-N-acetyl-␤-D-glucosamine-(4-1)-␣-L-fucose, Lea-tri is a

-D-galactose-(1-3)-N-acetyl-␤/␣-D-glucosamine-(4-1)-␣-L-fucose (the electron density was well defined for fucose and partially defined for theN-acetylglucosamine and galactose), and Lex-tri is a-D-galactose-(1-4)-N -acetyl-␤/␣-D-glucosamine-(3-1)-␣-L-fucose. Underlining represents the position of the reducing end hydroxyl group, which was fixed in the␣position in the crystal structures.

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and Lewis fucose moieties. The GII.4 variants were capable of

binding numerous Lewis HBGA types, and we discovered that

the Lewis HBGA binding mechanisms, particularly those for

Le

y

-tetra and Le

b

-tetra, involved more complex interactions

than the A-tri and B-tri binding interactions. A flexible loop

(residues 391 to 395) on the P dimer appeared to be versatile

and acted as a helping hand with Lewis HBGA tetrasaccharides.

In one example, the loop was cleverly repositioned to allow

Le

y

-tetra binding (

Fig. 6C

). In another example, the loop

pro-vided direct hydrogen- and water-mediated bonds with Le

b

-tetra after a slight repositioning (

Fig. 7

). This flexible loop

likely modulates binding of Lewis HBGAs, although

in vivo

interactions may involve additional mechanisms.

In comparing the sequences of the variant GII.4 P domains, we

found that most amino acid changes were surface exposed and

that

50% became fixed (

Fig. 3

). The region immediately beneath

the HBGA binding pocket showed few amino acid changes. On the

other hand, the regions that contributed to binding of terminal

saccharides of Lewis HBGAs underwent a more noticeable

modi-fication, i.e., from small areas of negative and positive charges (in

the 1998 variant) to larger areas of mostly negative charge (

Fig.

12

). The amino acid variations likely corresponded to temporal

changes in antigenicity, as previously described (

32

), but how

these changes related to apparent alterations in HBGA binding

remains unclear.

Even though not all complex structures could be determined,

we considered that these three GII.4 P domains were capable of

binding to all HBGA types examined, since binding interactions

were similar and only a few amino acid changes surrounding the

HBGA pocket were observed. We previously showed that the

rarely detected GII.10 strain also firmly bound a number of HBGA

types (H2-tri, A-tri, B-tri, and Le

y

-tetra) but only weakly bound

Le

b

-tetra and was unable to bind Le

a

-tri and Le

x

-tri (

21

).

There-fore, it is tempting to speculate that the GII.4 P domains are better

adapted to bind numerous HBGA types, whereas the rarely

de-tected GII.10 virus is less capable, which may also convey to the

ABH-fucose

ABH-fucose

N-acetylglucosamine

C

D

N-acetyl-

FIG 5Saga-2006 P dimer binding interactions with H2-tri. (A) Closeup surface and ribbon representation of the Saga-2006 –H2-tri complex structure, showing the hydrogen bonds (black lines) with H2-tri (cyan sticks) and water-mediated interactions (red spheres). (B) Saga-2006 P dimer and H2-tri binding interactions. FUC,␣-fucose; GAL,␤-galactose; NDG,␣-N-acetylglucosamine. The black lines represent the hydrogen bonds, the red line represents the hydrophobic interaction with the hydroxyl group of Tyr444, and the sphere represents water. (C) The ABH fucose of Saga-2006 –H2-tri bound at the regular pocket. The acetyl group ofN-acetylglucosamine was leaning toward the edge of the P dimer. (D) The ABH fucose of TCH05-2004 –H1-pentasaccharide bound at the regular pocket. The acetyl group ofN-glucosamine was leaning toward the center of the P dimer.

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apo loop position

Saga-2006-Le

y

-tetra loop position

C

D

E

Lewis-fucose

ABH-fucose

ABH-fucose

Lewis-fucose

FIG 6Saga-2006 P dimer binding interactions with Ley-tetra. (A) Closeup surface and ribbon representation of the Saga-2006 –Ley-tetra complex structure, showing the hydrogen bonds with Ley-tetra (green sticks) and water-mediated interactions. (B) Saga-2006 P dimer and Ley-tetra binding interactions. FUC, ␣-fucose; NDG,␣-N-acetylglucosamine; GAL,␤-galactose. (C) A loop in the Saga-2006 P2 subdomain (residues 391 to 394) was repositioned from the apo position (gray) to an alternative position (deep teal) for Ley-tetra binding. (D) The Lewis fucose of Saga-2004 –Ley-tetra bound at the regular pocket on the P domain and was leaning toward the edge of the P dimer. (E) The ABH fucose of GII.10 P domain Ley-tetra bound at the regular pocket and was orientated toward the center of the P dimer.

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Farm-2004 apo-chain A

Farm-2004 Le

b

-chain A (bound)

Farm-2004 Le

b

-chain B (unbound)

Farm-2004 apo-chain B

A

E

B

C

D

Lewis fucose

FIG 7Farm-2004 and Saga-2006 P dimer binding interactions with Leb-tetra. (A) Closeup surface and ribbon representation of the Farm-2004 –Leb-tetra complex structure, showing the hydrogen bonds with Leb-tetra (marine sticks) and water-mediated interactions. (B) Farm-2004 P dimer and Leb-tetra binding interactions. FUC,␣-fucose; GAL,␤-galactose; NAG,␤-N-acetylglucosamine. The black lines represent the hydrogen bonds, the red line represents the hydrophobic interaction with the hydroxyl group of Tyr444, and the sphere represents water. (C) Closeup surface and ribbon representation of the Saga-2006 – Leb-tetra complex structure, showing the hydrogen bonds with Leb-tetra and water-mediated interactions. (D) Saga-2006 P dimer and Leb-tetra binding interactions. (E) Superposition of both the A and B chains of the Farm-2004 apo (gray and black) and Farm-2004 –Leb-tetra (cyan and pink) structures.

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lower prevalence of GII.10 strains in the general population (

21

)

and the worldwide distribution of GII.4 viruses.

The affinities between noroviruses and HBGAs are weak and in

the high micromolar range (

33

). We previously showed that the

GII.10 P domain bound H2-tri with an affinity of 390

M (

33

).

Similarly, the Saga-2006 P domain had weak affinities for HBGAs

(

100

M) by saturation transfer difference-nuclear magnetic

resonance (STD-NMR) analysis (A. Mallagaray, G. S. Hansman,

C

D

Tyr444

Ser442

Tyr444

Ser442

Loop 391-394

Chain A

Loop 391-394

Chain B

Chain A

Chain B

FIG 8Saga-2006 P dimer binding interactions with Lea-tri and superposition of GII.4 P domains. (A) Closeup surface and ribbon representation of the Saga-2006 – Lea-tri complex structure, showing hydrogen bonds with Lea-tri (camel-colored sticks) and water-mediated interactions. (B) Saga-2006 P dimer and Lea-tri binding interactions. FUC,␣-fucose; NDG,␣-N-acetylglucosamine; GAL,␤-galactose. (C) Superposition of apo and HBGA-bound Farm-2004, Saga-2006, and NSW-2012 P dimer structures (with HBGAs removed from the structures). The circles represent the HBGA binding pocket. Farm-2004 P1 subdomains (chain A in pink and chain B in pale cyan) and P2 subdomains (chain A in green cyan and chain B in light magenta), Saga-2006 P1 subdomains (chain A in brown and chain B in yellow-orange) and P2 subdomains (chain A in deep teal and chain B in dirty violet), and NSW-2012 P1 subdomains (chain A in lime and chain B in marine) and P2 subdomains (chain A in blue-white and chain B in teal) are indicated by color coding. (D) Close-up of the P2 subdomain flexible loop (residues 391 to 394). In the case of H2-tri, Ley-tetra, Lea-tri, and Lex-tri, theN-acetylglucosamine was held by the side chain of Ser442, while the galactose was held by a hydrogen bond from the hydroxyl group of Tyr444. The loop required for the Lewis HBGA-tetrasaccharide interactions was found in multiple conformations on both A and B chains.

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FIG 9NSW-2012 P dimer interaction with Lex-tri. (A) Closeup surface and ribbon representation of the NSW-2012–Lex-tri complex structure, showing the hydrogen bonds with Lex-tri (salmon-colored sticks) and water-mediated interactions. (B) NSW-2012 and Lex-tri binding interactions. FUC,-fucose; NDG, ␣-N-acetylglucosamine; GAL,␤-galactose. The black lines represent the hydrogen bonds, the red line represents the hydrophobic interaction with the hydroxyl group of Tyr444, and the sphere represents water.

C

D

D

FIG 10Saga-2006 and NSW-2012 P dimer interactions with A-tri. (A) Closeup surface and ribbon representation of the Saga-2006 A-tri complex structure, showing the hydrogen bonds with A-tri (yellow sticks) and water-mediated interactions. (B) Saga-2006 and A-tri binding interactions. FUC, ␣-fucose; GLA,␣-galactose; A2G,␣-N-acetylgalactosamine. The black lines represent the hydrogen bonds, the red line represents the hydrophobic interaction with the hydroxyl group of Tyr444, and the sphere represents water. (C) Closeup surface and ribbon representation of the NSW-2012–A-tri complex structure, showing the hydrogen bonds with A-tri and water-mediated interactions. (D) NSW-2012 and A-tri binding interactions.

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and T. Peters, unpublished data). In addition, a recent study

found that a GII.4 P domain (VA387 strain) had comparable

af-finities for different HBGA types

in vitro

(

34

). Based on the

num-ber of direct hydrogen bonds and water-mediated interactions,

small changes in P domain affinities for HBGAs may exist, and

these may be important

in vivo

.

The precise roles of HBGAs in a norovirus infection are still

poorly understood, although synthetic HBGAs or

HBGA-express-C

E

D

F

FIG 11Farm-2004, Saga-2006, and NSW-2012 P dimer interactions with B-tri. (A) Closeup surface and ribbon representation of the Farm-2004 –B-tri complex structure, showing hydrogen bonds with B-tri (pink sticks) and water-mediated interactions. (B) Farm-2004 P dimer and B-tri binding interactions. FUC,␣-fucose; GLA,␣-galactose. The black lines represent the hydrogen bonds, the red line represents the hydrophobic interaction with the hydroxyl group of Tyr444, and the sphere represents water. (C) Closeup surface and ribbon representation of the Saga-2006 –B-tri complex structure, showing hydrogen bonds with B-tri and water-mediated interactions. The galactose was found in two different conformations (gray and pink sticks). (D) Saga-2006 P dimer and B-tri binding interactions, showing newly formed hydrogen bonds (blue lines) with the alternative galactose position. (E) Closeup surface and ribbon representation of the NSW-2012–B-tri complex structure, showing hydrogen bonds with B-tri and water-mediated interactions. (F) NSW-2012 P dimer and B-tri binding interactions.

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ing enteric bacteria were found to enhance human norovirus

in-fection in B cells (

12

). Interestingly, the synthetic HBGA (H-type

disaccharide) in the infection experiment was conjugated to

pol-yacrylic acid (PAA). Several studies have found that conjugated

linkers may affect and/or influence HBGA binding interactions

(

35

,

36

). Further structural studies with norovirus VLPs in

com-plex with HBGAs may help to explain the possible binding

mech-anisms

in vivo

.

Many of our newly determined HBGA binding results

chal-lenged previous ELISA-based findings (

4–6

). We found that

Farm-2004 bound Le

b

-tetra and B-tri, whereas an ELISA study

showed that GII.4 VLPs with an identical P domain sequence

(termed 2002) did not bind Le

b

-tetra and only weakly bound B-tri

(

6

). We also found that Saga-2006 bound H2-tri, A-tri, B-tri, Le

a

-tri, Le

b

-tetra, and Le

y

-tetra, while several ELISA studies showed

that GII.4 VLPs with an almost identical P domain sequence

(termed 2006) did not bind to Le

y

-tetra (

5

), A-tri (

5

), H2-tri (

4

),

or Le

a

-tri (

4

). Finally, we showed that NSW-2012 bound A-tri,

B-tri, and Le

x

-tri, whereas a recent ELISA study showed that GII.4

2012 VLPs with an identical P domain sequence (termed

GII.4-2012) did not bind to Le

x

-tri (

4

). Certainly,

in vivo

interactions

may be different from the results of X-ray crystallography and

ELISA-based studies. Nevertheless, these new data provide a new

focal point for improving HBGA binding assays in order to

in-crease our understanding of norovirus and HBGA interactions.

ACKNOWLEDGMENTS

The funding for this study was provided by the CHS foundation and the Helmholtz-Chinese Academy of Sciences.

G.S.H. designed the research, M.M.L. and Anne-Kathrin Herrmann performed initial Farm-2004 structural refinement, and G.S.H. and B.K.S. finalized all structures.

We acknowledge the European Synchrotron Radiation Facility (beamlines ID23-1 and BM30A) for provision of synchrotron radiation facilities. We thank Thomas Peters and Alvaro Mallagaray for performing the STD-NMR experiments (unpublished data). We also thank members of the Norovirus Study Group, Joel Sussman, and Henri-Jacques Dele-cluse for critical comments on the manuscript.

REFERENCES

1.Kapikian AZ, Wyatt RG, Dolin R, Thornhill TS, Kalica AR, Chanock RM.1972. Visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis. J Vi-rol10:1075–1081.

2.Hansman GS, Natori K, Shirato-Horikoshi H, Ogawa S, Oka T, Katay-ama K, Tanaka T, Miyoshi T, Sakae K, Kobayashi S, Shinohara M, Uchida K, Sakurai N, Shinozaki K, Okada M, Seto Y, Kamata K, Nagata N, Tanaka K, Miyamura T, Takeda N. 2006. Genetic and antigenic diversity among noroviruses. J Gen Virol87:909 –919.http://dx.doi.org /10.1099/vir.0.81532-0.

3.Siebenga JJ, Vennema H, Renckens B, de Bruin E, van der Veer B, Siezen RJ, Koopmans M.2007. Epochal evolution of GGII.4 norovirus capsid proteins from 1995 to 2006. J Virol81:9932–9941.http://dx.doi.org /10.1128/JVI.00674-07.

4.Debbink K, Lindesmith LC, Donaldson EF, Costantini V, Beltramello M, Corti D, Swanstrom J, Lanzavecchia A, Vinje J, Baric RS. 2013. Emergence of new pandemic GII.4 Sydney norovirus strain correlates with escape from herd immunity. J Infect Dis208:1877–1887.http://dx.doi.org /10.1093/infdis/jit370.

5.Lindesmith LC, Debbink K, Swanstrom J, Vinje J, Costantini V, Baric RS, Donaldson EF.2012. Monoclonal antibody-based antigenic mapping of norovirus GII.4-2002. J Virol86:873– 883.http://dx.doi.org/10.1128 /JVI.06200-11.

6.Lindesmith LC, Donaldson EF, Lobue AD, Cannon JL, Zheng DP, Vinje J, Baric RS.2008. Mechanisms of GII.4 norovirus persistence in human populations. PLoS Med5:e31.http://dx.doi.org/10.1371/journal .pmed.0050031.

7.Debbink K, Lindesmith LC, Donaldson EF, Baric RS.2012. Norovirus immunity and the great escape. PLoS Pathog8:e1002921.http://dx.doi .org/10.1371/journal.ppat.1002921.

VA387-1998

TCH05-2004

Farm-2004

Saga-2006

NSW-2012

FIG 12Surface representation of protein contact potential of GII.4 P dimers. The protein contact potential (where red represents a negative charge, white represents a neutral charge, and blue represents a positive charge;⬃⫺55 to⫹55 kT/e) was calculated for VA387-1998 (PDB entry2OBT), TCH-2004 (PDB entry3SLD), Farm-2004, Saga-2006, and NSW-2012 (top views [left] and close-ups of the HBGA pocket [right]). Leb-tetra of the Farm-2004 –Leb-tetra structure (marine sticks) was modeled into the VA387, TCH-05, Saga-2006, and NSW-2012 struc-tures. B-tri (pink sticks) and A-tri (yellow sticks) were complex strucstruc-tures. The regions surrounding the regular ABH fucose binding pocket remained mostly unchanged and negatively charged. The regions binding terminal saccharides of Lewis HBGAs changed from small patches of negative/positive charge to larger areas of negative charge.

on November 7, 2019 by guest

http://jvi.asm.org/

(17)

8.Huang P, Farkas T, Marionneau S, Zhong W, Ruvoen-Clouet N, Morrow AL, Altaye M, Pickering LK, Newburg DS, LePendu J, Jiang X.

2003. Noroviruses bind to human ABO, Lewis, and secretor histo-blood group antigens: identification of 4 distinct strain-specific patterns. J Infect Dis188:19 –31.http://dx.doi.org/10.1086/375742.

9.Huang P, Farkas T, Zhong W, Tan M, Thornton S, Morrow AL, Jiang X.2005. Norovirus and histo-blood group antigens: demonstration of a wide spectrum of strain specificities and classification of two major bind-ing groups among multiple bindbind-ing patterns. J Virol79:6714 – 6722.http: //dx.doi.org/10.1128/JVI.79.11.6714-6722.2005.

10. Harrington PR, Lindesmith L, Yount B, Moe CL, Baric RS. 2002. Binding of Norwalk virus-like particles to ABH histo-blood group anti-gens is blocked by antisera from infected human volunteers or experimen-tally vaccinated mice. J Virol76:12335–12343.http://dx.doi.org/10.1128 /JVI.76.23.12335-12343.2002.

11. Rockx BH, Vennema H, Hoebe CJ, Duizer E, Koopmans MP.2005. Association of histo-blood group antigens and susceptibility to norovirus infections. J Infect Dis191:749 –754.http://dx.doi.org/10.1086/427779. 12. Jones MK, Watanabe M, Zhu S, Graves CL, Keyes LR, Grau KR,

Gonzalez-Hernandez MB, Iovine NM, Wobus CE, Vinje J, Tibbetts SA, Wallet SM, Karst SM.2014. Enteric bacteria promote human and mouse norovirus infection of B cells. Science346:755–759.http://dx.doi.org/10 .1126/science.1257147.

13. Ayukekbong JA, Fobisong C, Tah F, Lindh M, Nkuo-Akenji T, Berg-strom T.2014. Pattern of circulation of norovirus GII strains during natural infection. J Clin Microbiol52:4253– 4259.http://dx.doi.org/10 .1128/JCM.01896-14.

14. Tan M, Jiang X. 2005. Norovirus and its histo-blood group antigen receptors: an answer to a historical puzzle. Trends Microbiol13:285–293.

http://dx.doi.org/10.1016/j.tim.2005.04.004.

15. Tan M, Jiang X.2011. Norovirus-host interaction: multi-selections by human histo-blood group antigens. Trends Microbiol19:382–388.http: //dx.doi.org/10.1016/j.tim.2011.05.007.

16. Cao S, Lou Z, Tan M, Chen Y, Liu Y, Zhang Z, Zhang XC, Jiang X, Li X, Rao Z.2007. Structural basis for the recognition of blood group tri-saccharides by norovirus. J Virol81:5949 –5957.http://dx.doi.org/10.1128 /JVI.00219-07.

17. Bu W, Mamedova A, Tan M, Xia M, Jiang X, Hegde RS.2008. Structural basis for the receptor binding specificity of Norwalk virus. J Virol82:

5340 –5347.http://dx.doi.org/10.1128/JVI.00135-08.

18. Kubota T, Kumagai A, Ito H, Furukawa S, Someya Y, Takeda N, Ishii K, Wakita T, Narimatsu H, Shirato H.2012. Structural basis for the recognition of Lewis antigens by genogroup I norovirus. J Virol86:11138 – 11150.http://dx.doi.org/10.1128/JVI.00278-12.

19. Shanker S, Choi JM, Sankaran B, Atmar RL, Estes MK, Prasad BV.

2011. Structural analysis of histo-blood group antigen binding specificity in a norovirus GII.4 epidemic variant: implications for epochal evolution. J Virol85:8635– 8645.http://dx.doi.org/10.1128/JVI.00848-11. 20. Prasad BV, Hardy ME, Dokland T, Bella J, Rossmann MG, Estes MK.

1999. X-ray crystallographic structure of the Norwalk virus capsid. Science

286:287–290.http://dx.doi.org/10.1126/science.286.5438.287.

21. Hansman GS, Biertumpfel C, Georgiev I, McLellan JS, Chen L, Zhou T, Katayama K, Kwong PD.2011. Crystal structures of GII.10 and GII.12 norovirus protruding domains in complex with histo-blood group anti-gens reveal details for a potential site of vulnerability. J Virol85:6687– 6701.http://dx.doi.org/10.1128/JVI.00246-11.

22. Kabsch W.1993. Automatic processing of rotation diffraction data from

crystals of initially unknown symmetry and cell constants. J Appl Crystal-logr26:795– 800.http://dx.doi.org/10.1107/S0021889893005588. 23. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC,

Read RJ.2007. Phaser crystallographic software. J Appl Crystallogr40:

658 – 674.http://dx.doi.org/10.1107/S0021889807021206.

24. Emsley P, Lohkamp B, Scott WG, Cowtan K.2010. Features and devel-opment of Coot. Acta Crystallogr D Biol Crystallogr66:486 –501.http: //dx.doi.org/10.1107/S0907444910007493.

25. Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung L-W, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH. 2010. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr66:213–221.http://dx.doi.org/10.1107/S090744490905 2925.

26. Chen VB, Arendall WB, 3rd, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC. 2010. MolProbity: all-atom structure validation for macromolecular crystal-lography. Acta Crystallogr D Biol Crystallogr66:12–21.http://dx.doi .org/10.1107/S0907444909042073.

27. Morris AL, MacArthur MW, Hutchinson EG, Thornton JM. 1992. Stereochemical quality of protein structure coordinates. Proteins12:345– 364.http://dx.doi.org/10.1002/prot.340120407.

28. Tan M, Hegde RS, Jiang X.2004. The P domain of norovirus capsid protein forms dimer and binds to histo-blood group antigen receptors. J Virol78:6233– 6242.http://dx.doi.org/10.1128/JVI.78.12.6233-6242 .2004.

29. Harrington PR, Vinje J, Moe CL, Baric RS.2004. Norovirus capture with histo-blood group antigens reveals novel virus-ligand interactions. J Virol

78:3035–3045.http://dx.doi.org/10.1128/JVI.78.6.3035-3045.2004. 30. Tan M, Huang P, Meller J, Zhong W, Farkas T, Jiang X.2003.

Muta-tions within the P2 domain of norovirus capsid affect binding to human histo-blood group antigens: evidence for a binding pocket. J Virol77:

12562–12571.http://dx.doi.org/10.1128/JVI.77.23.12562-12571.2003. 31. Lindesmith L, Moe C, Marionneau S, Ruvoen N, Jiang X, Lindblad L,

Stewart P, LePendu J, Baric R.2003. Human susceptibility and resistance to Norwalk virus infection. Nat Med9:548 –553.http://dx.doi.org/10 .1038/nm860.

32. Allen DJ, Gray JJ, Gallimore CI, Xerry J, Iturriza-Gomara M.2008. Analysis of amino acid variation in the P2 domain of the GII-4 norovirus VP1 protein reveals putative variant-specific epitopes. PLoS One3:e1485.

http://dx.doi.org/10.1371/journal.pone.0001485.

33. Hansman GS, Shahzad-Ul-Hussan S, McLellan JS, Chuang GY, Geor-giev I, Shimoike T, Katayama K, Bewley CA, Kwong PD.2012. Struc-tural basis for norovirus inhibition and fucose mimicry by citrate. J Virol

86:284 –292.http://dx.doi.org/10.1128/JVI.05909-11.

34. Han L, Kitova EN, Tan M, Jiang X, Pluvinage B, Boraston AB, Klassen JS.1 October 2014. Affinities of human histo-blood group antigens for norovirus capsid protein complexes. Glycobiologyhttp://dx.doi.org/10 .1093/glycob/cwu100.

35. Caddy S, Breiman A, le Pendu J, Goodfellow I.2014. Genogroup IV and VI canine noroviruses interact with histo-blood group antigens. J Virol

88:10377–10391.http://dx.doi.org/10.1128/JVI.01008-14.

36. Rademacher C, Krishna NR, Palcic M, Parra F, Peters T.2008. NMR experiments reveal the molecular basis of receptor recognition by a calicivirus. J Am Chem Soc130:3669 –3675.http://dx.doi.org/10.1021 /ja710854r.

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Figure

FIG 1 Amino acid alignment of norovirus GII.4 variants. The P domain amino acid sequences of four GII.4 variants, from 1998, 2004, 2006, and 2012 (termedVA387-1998, Farm-2004, Saga-2006, and NSW-2012, respectively), were aligned using Clustal X

FIG 1

Amino acid alignment of norovirus GII.4 variants. The P domain amino acid sequences of four GII.4 variants, from 1998, 2004, 2006, and 2012 (termedVA387-1998, Farm-2004, Saga-2006, and NSW-2012, respectively), were aligned using Clustal X p.2
TABLE 1 Data collection and refinement statistics for GII.4 P domain apo structuresa

TABLE 1

Data collection and refinement statistics for GII.4 P domain apo structuresa p.3
FIG 2 X-ray crystal structures of unbound GII.4 P domains. (A) The Farm-2004 P domain apo structure contained one dimer per asymmetric unit

FIG 2

X-ray crystal structures of unbound GII.4 P domains. (A) The Farm-2004 P domain apo structure contained one dimer per asymmetric unit p.4
FIG 3 Amino acid variations in GII.4 P dimers from 2004, 2006, and 2012 variants. Amino acid changes (red) were highlighted on GII.4 P dimers (side and topviews)

FIG 3

Amino acid variations in GII.4 P dimers from 2004, 2006, and 2012 variants. Amino acid changes (red) were highlighted on GII.4 P dimers (side and topviews) p.5
TABLE 2 Data collection and refinement statistics for Farm-2004 Pdomain and HBGA complex structuresa

TABLE 2

Data collection and refinement statistics for Farm-2004 Pdomain and HBGA complex structuresa p.6
TABLE 4 Data collection and refinement statistics for NSW-2012 P domain and HBGA complex structuresa

TABLE 4

Data collection and refinement statistics for NSW-2012 P domain and HBGA complex structuresa p.8
FIG 4 Representative simulated annealing difference omit maps. The omit maps (blue) were contoured between 2.5 and 2.0�� �

FIG 4

Representative simulated annealing difference omit maps. The omit maps (blue) were contoured between 2.5 and 2.0�� � p.9
FIG 5 Saga-2006 P dimer binding interactions with H2-tri. (A) Closeup surface and ribbon representation of the Saga-2006–H2-tri complex structure, showingthe hydrogen bonds (black lines) with H2-tri (cyan sticks) and water-mediated interactions (red sphere

FIG 5

Saga-2006 P dimer binding interactions with H2-tri. (A) Closeup surface and ribbon representation of the Saga-2006–H2-tri complex structure, showingthe hydrogen bonds (black lines) with H2-tri (cyan sticks) and water-mediated interactions (red sphere p.10
FIG 6 Saga-2006 P dimer binding interactions with Leshowing the hydrogen bonds with Ledomain and was leaning toward the edge of the P dimer

FIG 6

Saga-2006 P dimer binding interactions with Leshowing the hydrogen bonds with Ledomain and was leaning toward the edge of the P dimer p.11
FIG 7 Farm-2004 and Saga-2006 P dimer binding interactions with Lecomplex structure, showing the hydrogen bonds with Leinteractions

FIG 7

Farm-2004 and Saga-2006 P dimer binding interactions with Lecomplex structure, showing the hydrogen bonds with Leinteractions p.12
FIG 8 Saga-2006 P dimer binding interactions with LeLeLeinteractions. FUC,a-tri and superposition of GII.4 P domains

FIG 8

Saga-2006 P dimer binding interactions with LeLeLeinteractions. FUC,a-tri and superposition of GII.4 P domains p.13
FIG 9 NSW-2012 P dimer interaction with Lehydrogen bonds with Lex-tri. (A) Closeup surface and ribbon representation of the NSW-2012–Lex-tri complex structure, showing thex-tri (salmon-colored sticks) and water-mediated interactions

FIG 9

NSW-2012 P dimer interaction with Lehydrogen bonds with Lex-tri. (A) Closeup surface and ribbon representation of the NSW-2012–Lex-tri complex structure, showing thex-tri (salmon-colored sticks) and water-mediated interactions p.14
FIG 10 Saga-2006 and NSW-2012 P dimer interactions with A-tri. (A) Closeup surface and ribbon representation of the Saga-2006 A-tri complexstructure, showing the hydrogen bonds with A-tri (yellow sticks) and water-mediated interactions

FIG 10

Saga-2006 and NSW-2012 P dimer interactions with A-tri. (A) Closeup surface and ribbon representation of the Saga-2006 A-tri complexstructure, showing the hydrogen bonds with A-tri (yellow sticks) and water-mediated interactions p.14
FIG 11 Farm-2004, Saga-2006, and NSW-2012 P dimer interactions with B-tri. (A) Closeup surface and ribbon representation of the Farm-2004–B-tri complexstructure, showing hydrogen bonds with B-tri (pink sticks) and water-mediated interactions

FIG 11

Farm-2004, Saga-2006, and NSW-2012 P dimer interactions with B-tri. (A) Closeup surface and ribbon representation of the Farm-2004–B-tri complexstructure, showing hydrogen bonds with B-tri (pink sticks) and water-mediated interactions p.15
FIG 12 SurfacerepresentationofproteincontactpotentialofGII.4Pdimers.Theproteincontactpotential(whereredrepresentsanegativecharge,whiterepresentsa neutral charge, and blue represents a positive charge; ��55 to �55 kT/e) wascalculated for VA387-1998 (PDB ent

FIG 12

SurfacerepresentationofproteincontactpotentialofGII.4Pdimers.Theproteincontactpotential(whereredrepresentsanegativecharge,whiterepresentsa neutral charge, and blue represents a positive charge; ��55 to �55 kT/e) wascalculated for VA387-1998 (PDB ent p.16