Genotype-dependent glycan specificity in rotaviruses – evolution of a new

paradigm.

B. V. V. Prasad1,2 , L. Hu1, , S. E. Crawford2, R. Czako2, S. Ramani2, N. Cortes-Penfield2, D. F. Smith3, G. Kang4, J.

Le Pendu5, and M. K. Estes2

1Departments of Biochemistry and Molecular Biology and 2Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX,

3Department of Biochemistry, Emory University School of Medicine, GA, 4Christian Medical College, Vellore, India, 5NSERM, Nantes, France.

The key event of initial cell attachment of rotavirus is mediated by VP8*domain of the VP4 spike

VP4 (VP5*+VP8*)

VP7

VP2

VP1/VP3

VP6

VP8*

VP5*

Prasad et al., Nature (1996); Li et al., JVI (2009); Settembre et al., EMBO J. (2010)

Classification of rotavirus strains

• Rotaviruses exhibit enormous genetic and strain diversity

• Point mutations, gene rearrangements, and genetic reassortment contribute to the expanding diversity

• Based on neutralization specificity to VP7 and VP4, rotaviruses are classified into G (VP7) and P (VP4) serotypes

• Based on VP4 sequences, rotaviruses are classified into > 35 P genotypes

Sialidase sensitive rotaviruses • Virus infectivity decreases by sialidase treatment of cells • VP8* binds to glycans with terminal sialic acid (Sia) (e.g. GD1a)

Sialidase insensitive rotaviruses • Virus infectivity is not affected by sialidase treatment of cells • VP8* binds to glycans with internal sialic acid (Sia) (e.g. GM1)

Structures of VP8* of sialidase sensitive and insensitive strains

Simian RRV P[3]

Sensitive Terminal Sia

Narrow

Porcine CRW-8 P[7]

Sensitive Terminal Sia

Narrow

Sia

Sia

Human DS-1 P[4]

Insensitive Internal Sia

Wide

Human Wa P[8]

Insensitive Internal Sia

Wide

Do all sialidase-insensitive HR genotypes recognize sialoglycans?

Sialidase Insensitive Human Rotavirus HAL1166 P[14] VP8*

• This virus was first Isolated from an infant with diarrhea in Finland

• The P[14] rotavirus strains are being increasingly documented globally

• Phylogenetically distinct from the previously reported VP8*s

• These human rotaviruses are thought have jump from animal to human hosts

X-ray structure of P[14] VP8* not compatible

with Sia binding

The galectin-like fold: • Two twisted β-sheets (green & blue)

separated by a shallow cleft

Resolution: 1.5Å Space group: P21 R= 17.8%, Rfree=21.9%

Glycan array of GST-P[14] VP8*

N-terminal GST tag

611 glycans :

Sialylated glycans with

terminal or internal Sia,

Non-sialylated glycans

P[14] VP8*

N

C

Anti-GST Ab

Glycan#

Structure

Average RFU

%CV

367

GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ1-2Manα1-3(GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp20

2703

4

332 GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-Sp0 1892 6

89 GalNAcα1-3(Fucα1-2)Galβ-Sp18 1574 18

85 GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ-Sp8 1324 11

88 GalNAcα1-3(Fucα1-2)Galβ-Sp8 867 16

86 GalNAcα1-3(Fucα1-2)Galβ1-4Glcβ-Sp0 807 26

390 GalNAcα1-3(Fucα1-2)Galβ1-3GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ-Sp0 681 17

141 Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ-Sp0 [GM1 with internal Sia] 5 184

409

Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ-Sp0

[“GD1a like” with terminal Sia] 1 272

Human rotavirus P[14] VP8* specifically binds to A-type histo-blood group antigen (HBGA)

* Relative Fluorescence Units (RFU) % Coefficient of Variation (%CV) =100Std.Dev/Mean

Sialidase-insensitive human rotavirus HAL1166 P[14] VP8* specifically binds to a non-sialylated glycan, A-type HBGA

HBGA Sia

Simian P[3] Sensitive

Terminal Sia

Narrow

Human P[14] Insensitive

HBGA

Narrow

• VP8* of a sialidase-insensitive human rotavirus also has a narrow cleft

• HBGA binding site in the P[14] VP8* remarkably overlaps with that of the Sia binding site in animal sialidase sensitive strains

Dormitzer et al., EMBO J (2002); Hu et al., Nature (2012)

Subtle changes within the same structural framework lead to altered receptor specificity

• Conformational change of R101

• Insertion of S187

• Switch the side chain orientations of Y188 and Y189

Simian RRV, P[3], sialidase-sensitive Human HAL 1166, P[14], sialidase-insensitive

Sia HBGA

Is the interaction with HBGA biologically relevant?

P[14] VP8* specifically recognizes A-type HBGA

• Dose-dependent abrogation of HAL1166 infectivity by anti-A-type HBGA antibody

• HAL1166 infectivity increases in CHO cells expressing type A HBGA

P[9]

Hu et al., Nature (2012)

P[14] P[1] P[1] P[14]

Anti-A Isotype ctrl

Glycan specificity varies between VP8* of different P genotypes

Sia

P[14], Insensitive, Narrow

Correlates with wide cleft

Glycan binding site

HBGA GM1 or other glycan?

P[3], Sensitive, Narrow P[8], Insensitive, Wide

?

The wide cleft correlates with a deletion at position 136, and a significant change at position 101

The amino acids at the known glycan binding site change significantly between VP8* of narrow cleft and wide cleft

?

Representative structures of VP8*: Class A: P[14] HAL1166; Class C: P[3] RRV; Class D: P[8] Wa

Structure-based classification of VP8*s

P[14] HAL1166 A-type HBGA

P[9] K8 A-type HBGA

Class A Human RV, s-i, narrow cleft

Class B Animal RV, s-i, narrow cleft

P[5] Bovine UK ?

P[12] Equine H2 ?

P[16] Murine Eb ?

P[3] Simian

RRV Sia

P[7] Porcine CRW8

Sia

Class C Animal RV, s-s, narrow cleft

Class D Human RV, s-i, wide cleft

P[4] DS-1 ?

P[8] Wa ?

P[6] McN13 ?

P[11] N155 ?

How do genotypic variations affect glycan specificity in VP8* with a wide cleft?

• What glycans do they bind? • Do glycans bind to the same region as that in the narrow cleft?

Structures of P[11] VP8* of neonate-specific sialidase insensitive strain N155

Human N155 P[11] VP8* Human Indian G2P[4] VP8*

Human N155 P[11] VP8* Cell dimensions: a=36.53Å, b=71.94Å, c=58.05Å, α=90°, β=90.02°, γ=90° Resolution: 1.66 Å Space group: P 1 21 1 Rwork= 15%, Rfree=17%

0

2

4

6

8

10

0

2

4

6

8

10

H-/A- H+/A- H+/A+

Fo

ld

d

iffe

re

nc

e in

vira

l tite

r

fro

m p

are

nta

l c

ell lin

e

H-/A- H+/A- H+/A+

P[11] VP8* specifically recognizes HBGA H type II precursor

Fo

ld

d

iffe

re

nc

e in

vira

l tite

r

fro

m p

are

nta

l c

ell lin

e

G10P[11] infectivity increases in CHO cells expressing type A or H HBGA

SA114F (G3P[1]) N155 (G10P[11])

* Relative Fluorescence Units (RFU); % Coefficient of Variation (%CV) =100Std.Dev/Mean

Glycan Structure Ave. RFU

Std % CV

Fucα1-2Galβ1-4(Fucα1-3)GlcNAcβ1-3GalNAcα-Sp14 (H-type and Lewisy)

458 20 4

Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ-Sp0 (GM1with internal Sia)

10 1 6

Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ-Sp0 (GD1a-like with terminal Sia)

30 14 46

VP8* of sialidase-insensitive human rotavirus G2P[4] (wide cleft) binds to H-type and Lewisy HBGAs.

Glycan array of G2P[4] VP8*

• Interactions with sialo-glycans for initial cell attachment is not an

obligatory requirement as previously thought.

• Rotaviruses exhibit genotype-dependent variations in glycan

specificity which may have implications in host specificity, tissue

tropism, susceptibility, pathogenesis and interspecies

transmission.

• Specific recognition of A-type HBGA may be the basis for inter-

species transmission observed in P[14] rotaviruses.

• Glycan binding site in globally dominant P[4] VP8* is distinct; in

addition to H-type P[4]VP8* can bind to Lewis HBGA – basis for

increased prevalence?

• VP8* of neonate-specific P[11] strain shows significant structural

alterations and specifically interacts with H type II precursor -

what is the significance?

Conclusions

Thank you!

We acknowledge the support from NIH grants AI36040 (to B.V.V.P.), AI 080656 and P30 DK56338 (to M.K.E.), GM62116 (to the Consortium for Functional Glycomics), and the Robert Welch foundation (Q1279) to B.V.V.P.

We thank Dr. Robert Atmar and Dr. Sreejesh

Shanker for helpful discussions and BCM X-ray core facility for data collection.

Acknowledgements

Precursor FUT3 FUT2

Lea

Leb

H type 1

FUT3 Enzyme A Enzyme B

A type 1 B type 1

A Leb B Leb

FUT3 FUT3

Keys:

Gal

GlcNAc

Fuc

GalNAc

FUT: Fucosyltransferase FUT2: Secretor Enzyme FUT3: Lewis Enzyme

1 - 3

1 - 3 1 - 3

1 - 3 1 - 3 1 - 3

1 - 3 1 - 3

2 1

2 1

2 1

2 1

2 1

4 1

4 1

4 1

4 1

2 1

1 - 3 1 - 3

1 - 3 1 - 3

Glycan#

Structure

Average RFU

%CV

367

GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ1-2Manα1-3(GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp20

2703

4

332 GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-Sp0 1892 6

89 GalNAcα1-3(Fucα1-2)Galβ-Sp18 1574 18

85 GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ-Sp8 1324 11

88 GalNAcα1-3(Fucα1-2)Galβ-Sp8 867 16

86 GalNAcα1-3(Fucα1-2)Galβ1-4Glcβ-Sp0 807 26

390 GalNAcα1-3(Fucα1-2)Galβ1-3GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ-Sp0 681 17

141 Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ-Sp0 [GM1 with internal Sia] 5 184

409

Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ-Sp0

[“GD1a like” with terminal Sia] 1 272

Human rotavirus P[14] VP8* specifically binds to A-type histo-blood group antigen (HBGA)

* Relative Fluorescence Units (RFU) % Coefficient of Variation (%CV) =100Std.Dev/Mean