Virology 474 (2015) 181–185
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Brief Communication
Structural analysis of a feline norovirus protruding domain Bishal K. Singh a,b, Sebastian Glatt c, Jean-Luc Ferrer d,e,f, Anna D. Koromyslova a,b, Mila M. Leuthold a,b, Jessica Dunder a,b, Grant S. Hansman a,b,n a
Schaller Research Group at the University of Heidelberg and the DKFZ, Heidelberg 69120, Germany Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg 69120, Germany c EMBL Heidelberg, Structural and Computational Biology Unit, Heidelberg 69117, Germany d University Grenoble Alpes, IBS, F-38027 Grenoble, France e CEA, IBS, F-38027 Grenoble, France f CNRS, IBS, F-38027 Grenoble, France b
art ic l e i nf o
a b s t r a c t
Article history: Received 23 August 2014 Returned to author for revisions 20 September 2014 Accepted 25 October 2014 Available online 19 November 2014
Norovirus infects different animals, including humans, mice, dogs, and cats. Here, we show an X-ray crystal structure of a feline GIV.2 norovirus capsid-protruding (P) domain to 2.35 Å resolution. The feline GIV.2 P domain was reminiscent of human norovirus P domains, except for a novel P2 subdomain α-helix and an extended P1 subdomain interface loop. These new structural features likely obstructed histo-blood group antigens, which are attachment factors for human norovirus, from binding at the equivalent sites on the feline GIV.2 P domain. Additionally, an ELISA showed that the feline GIV.2 was antigenically distinct from a human GII.10 norovirus. & 2014 Elsevier Inc. All rights reserved.
Keywords: Diarrhea Norovirus X-ray crystallography Virus capsid
Introduction Human noroviruses (Caliciviridae family) are the dominant cause of outbreaks of gastroenteritis around the world. Human noroviruses are genetically and antigenically diverse (Hansman et al., 2006). Based on the capsid gene sequences, there are at least six main genogroups of noroviruses, which can be further subdivided into numerous genotypes. Most human noroviruses belong to genogroups I and II (GI and GII). Recently, a feline norovirus causing gastroenteritis in domestic cats was identified (Pinto et al., 2012). Genetic clustering placed the feline norovirus capsid sequence into GIV genotype 2 (GIV.2). Also belonging to GIV are lion, dog, and infrequently detected human noroviruses (Martella et al., 2007, 2009, 2011). Importantly, human norovirus genetic recombination appears to be a common event and the potential for zoonosis has been described in several studies (BankWolf et al., 2010; Bull et al., 2005, 2007;Clarke and Lambden 1997; Eden et al., 2013; Humphrey et al., 1984; Mahar et al., 2013). These
n Correspondence to: CHS Foundation, University of Heidelberg, DKFZ, Norovirus Study Group, Im Neuenheimer Feld 242, Heidelberg 69120, Germany. Tel.: þ 49 6221 42 1520. E-mail address: [email protected] (G.S. Hansman).
http://dx.doi.org/10.1016/j.virol.2014.10.028 0042-6822/& 2014 Elsevier Inc. All rights reserved.
findings highlighted the importance of understanding the differences and similarities between animal and human noroviruses. The X-ray crystal structure of human norovirus virus-like particles (VLPs) reveals two domains, a shell (S) domain and a protruding (P) domain (Prasad et al., 1999). The S domain forms a scaffold surrounding the viral RNA, whereas the P domain, which can be further subdivided into the P1 and P2 subdomains, likely contains the determinants for host recognition. The norovirus P domain can be expressed in Escherichia coli and this can form P dimers analogous to those on VLPs (Hansman et al., 2011). Studies with VLPs and/or P domains have shown that human, bovine, and canine noroviruses bind histo-blood group antigens (HBGAs) (Caddy et al., 2014; Hutson et al., 2002; Zakhour et al., 2009), whereas murine norovirus virions were found to interact with sialic acid (Taube et al., 2009). Little is known about the attachment factors or receptors for feline noroviruses. At least nine different HBGAs have been identified to bind to human norovirus. However, the relatively weak interaction, quality of reagents, and dissimilar ELISA formats have led to conflicting results concerning the specific HBGAs binding to different noroviruses (Caddy et al., 2014; Hansman et al., 2012; Lindesmith et al., 2012; Tan and Jiang 2010). In the study presented here, we show the X-ray crystal structure of a feline GIV.2 norovirus P domain and identified several novel structural features. We also found that the feline
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GIV.2 norovirus P domain was antigenically distinct from a human GII.10 P domain.
Results and discussion In order to better understand the structural implications between animal and human norovirus capsids we solved the X-ray crystal structure of a recently identified feline GIV.2 norovirus P domain (Pinto et al., 2012). A single crystal of the feline GIV.2 P domain diffracted to 2.35 Å resolution (Fig. 1a). The structure was solved using molecular replacement with the GII.10 P domain as a search model (Hansman et al., 2011). Molecular replacement indicated a dimer in space group P21. Refinement of GIV.2 P domain led to an Rwork value of 0.214 and Rfree ¼0.268, with a well-defined electron density for most of the dimer (Table 1). Based on the GII.10 P domain structure, the GIV.2 P domain could be subdivided into P1 (224–277 and 438–569) and P2 (278–437) subdomains (Fig. 1). Analogous to
other human noroviruses, the GIV.2 P1 subdomain contained an
α-helix and seven β-strands, while the P2 subdomain contained six anti-parallel β-strands that formed a barrel-like structure. However,
compared to the GII.10 capsid sequence, the feline GIV.2 P2 subdomain contained a large insertion between residues 295 and 329 (Fig. 1b). The electron density of this insertion was disordered in chain A between residues 297 and 327 and in chain B between residues 297 and 319. Unfortunately, the disordered regions could not be modeled into the P domain structure. However, part of the insertion in chain B (residues 320–327) had clearly defined electron density and was found to be a short α-helix (Fig. 1a), although the α-helix had higher B-factors as compared to nearby residues and protein average, which suggested that the insertion was flexible. Interestingly, secondary structure prediction of the feline GIV.2 P domain indicated that the insertion actually formed a long α-helix structure (data not shown). The location of the feline GIV.2 P2 subdomain α-helix corresponded to an extended loop on the GII.10 P2 subdomain (Fig. 2a)
Fig. 1. The X-ray crystal structure of GIV.2 feline norovirus P domain and an amino acid sequence alignment. (A) The GIV.2 P domain dimer was colored according to monomers (chains A and B) and P1 and P2 subdomains. Chain A: P1 (yellow–orange), chain A: P2 (fire-brick), chain B: P1 (deep-blue), and chain B: P2 (lime-green). Chain A residues 297–327 and chain B residues 297–319 were not modeled into the structure due to poor electron density. Part of the P2 subdomain (B chain residues 320–327) contained an α-helix structure. (B) The capsid amino acid alignment sequences of feline GIV.2 and human GII.10 noroviruses were aligned using ClustalX. The feline GIV.2 P2 subdomain contained an insertion in the P2 subdomain between 295 and 329. The P1 and P2 subdomains were colored deep-blue and lime-green, respectively. The S domain was not labeled. The GII.10 P domain residues involved directly or indirectly with HBGAs (Hansman et al., 2011) were highlighted, side-chain interactions (red), backbone interactions (blue), and hydrophobic interaction (purple). The asterisks show conserved residues.
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Table 1 Data collection and refinement statistics.
Data collection Space group Cell dimensions a, b, c (Å) α, β, γ (1) Resolution (Å) Rmerge I/σI Completeness (%) Redundancy Refinement Resolution (Å) No. reflections Rwork/Rfree No. atoms Protein Ligand/ion Water B-factors Protein Ligand/ion Water R.m.s. deviations Bond lengths (Å) Bond angles (1)
Feline apo (no glycan) (4QUZ)
Feline A tria (unbound glycan) (4QV2)
Feline GNAa (unbound glycan) (4QVA)
Feline sialic acid (unbound glycan) (4QVJ)
P21
P21
P21
P 21 21 21
57.74, 99.92, 59.61 90, 97.92, 90 49.63-2.10 (2.16-2.10)b 20.4 (76.5)b 5.26 (1.38)b 96.64 (74.6)b 3.0 (2.6)b
57.48, 99.38, 59.89 90, 97.54, 90 49.4-1.63 (1.67-1.63)b 14.74 (1.20)b 9.45 (1.28)b 99.20 (93.97)b 4.1 (3.7)b
57.53, 99.38, 69.34 90, 111.04, 90 45.6-2.10 (2.16-2.10)b 13.2 (52.0)b 6.97 (2.17)b 92.70 (90.50)b 2.8 (2.7)b
57.70, 99.60, 129.92 90, 90, 90 49.9-2.10 (2.15-2.10)b 17.32 (63.4)b 7.32 (2.16)b 98.30 (85.10)b 4.7 (4.0)b
49.63-2.35 27,538 0.214/0.268 5013 4787 20 206
27.39-1.68 75,647 0.160/0.183 10,125 4797 20 770
35.53-2.18 35,321 0.196/0.228 9640 4720
49.93-2.26 35,550 0.213/0.271 5141 4738
404
403
38.90 43.10 32.60
16.90 22.80 27.70
30.10
32.70
30.40
34.10
0.004 0.83
0.004 0.923
0.006 0.992
0.005 0.893
Each data set was collected from single crystals, respectively. a b
GNA ¼N-glycolylneuraminic acid and A tri¼ A-trisaccharide. Values in parentheses are for highest-resolution shell.
and a short α-helix on a vesivirus P2 subdomain, which is also a member in Caliciviridae family (Chen et al., 2006). Interestingly, the feline GIV.2 P domain contained a P1 subdomain interface loop, which was shown to contain essential residues for GII norovirus binding to HBGAs (Hansman et al., 2011). However, the GIV.2 P1 subdomain interface loop was found to be longer than other known GII noroviruses and was partially extended out from the surface of the P2 subdomain (Fig. 2b). In order to determine a possible feline GIV.2 P domain interaction with HBGAs and other ligands we performed co-crystallization experiments with A-trisaccharide, N-glycolylneuraminic acid, and sialic acid. Structural analysis of over 30 different crystal complexes with varying concentrations of ligands (15–60 M excess of protein) and crystallization conditions failed to show any bound HBGAs (Table 1). The HBGA binding pockets on human GI and GII noroviruses are distinct (Bu et al., 2008; Cao et al., 2007; Choi et al., 2008). The GI noroviruses bind HBGAs on a single P domain monomer, whereas GII noroviruses bind HBGAs at a dimeric interface. The human norovirus HBGA binding interaction is usually coordinated by a conserved set of residues in each genogroup and includes a network of hydrogen bonds and water-mediated interactions (Bu et al., 2008; Cao et al., 2007; Choi et al., 2008; Hansman et al., 2011). In order to better understand the reasons why these HBGAs did not bind to the feline GIV.2 P domain, we superpositioned GI.1 and GII.10 P domain A-trisaccharide complex structures on the unbound feline GIV.2 P domain structure. At the equivalent GI.1 HBGA binding pocket on the GIV.2 P domain, the A-trisaccharide clashed with a GIV.2 P2 subdomain loop and the P2 subdomain αhelix (Fig. 2c). At the equivalent GII.10 HBGA binding pocket on the GIV.2 P domain, the A-trisaccharide clashed with the extended P1 subdomain interface loop (Fig. 2d). The GI.1 and GII.10 residues that typically interacted with the HBGAs were not conserved in the feline GIV.2 capsid sequence or the corresponding P domain
structure (Fig. 1b and data not shown). In light of these findings, the feline GIV.2 norovirus might not bind HBGAs at the equivalent GI or GII norovirus HBGA pockets on the GIV.2 P domain or they bind other attachment factors. A recent ELISA-based study showed that canine GIV.2 and GVI.3 norovirus VLPs were unable to bind synthetic A-trisaccharides (Caddy et al., 2014). However, the study showed that canine GIV.2 and GVI.3 norovirus VLPs bound to canine tissue samples expressing A and H antigens of HBGAs. The authors acknowledged that additional factors may have contributed to the binding in tissue samples and that their ELISA-based assays had certain limitations (Caddy et al., 2014). Nevertheless, the negative ELISA result using synthetic A-trisaccharide resembled our X-ray crystallography finding that the GIV.2 noroviruses were unable to bind A-trisaccharides. Similar to feline GIV.2 norovirus, the GI and GII norovirus residues interacting with the HBGAs were also not conserved in canine GIV.2 and GVI.3 noroviruses (Caddy et al., 2014). Interestingly, feline GIV.2 and canine GIV.2 shared 90% amino acid identity and the capsid protein was identical in length. Likely, a canine GIV.2 P domain structure had comparable features to the feline GIV.2 structure, including an extended P1 subdomain interface loop and P2 subdomain α-helix. Therefore, our X-ray crystallography data provided a possible explanation of the GIV.2 noroviruses inability to bind synthetic A-trisaccharides, i.e., steric clashes on the P domain at the equivalent GI and GII HBGA pockets. It would be interesting to discover the additional factors that contributed to the canine GIV.2 VLP binding in tissue samples (Caddy et al., 2014). The HBGA binding interactions among antigenically diverse noroviruses are still unclear, and little is known about strains that do not interact with HBGAs (Hansman et al., 2010). At least two human norovirus strains, Hawaii virus (GII.1) and Snow Mountain Virus (GII.2), were found not to bind or only weakly bind HBGAs, respectively (Huang et al., 2005; Lindesmith et al., 2005). Further studies with other noroviruses, including non-HBGA binders, will be
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P1 subdomain interface loop GIV.2 P2 subdomain -helix
GII.10 extended loop
GIV.2 GII.10 GI.1
A-trisaccharide clash
A-trisaccharide clash
Fig. 2. Feline norovirus P2 subdomain α-helix and P1 subdomain interface loop. (A) Superposition of GII.10 P dimer showed an extended loop (salmon) was at a similar location to the feline GIV.2 norovirus α-helix and both were connected to the P2 subdomain β-strands. (B) The feline GIV.2 P1 subdomain interface loop (GIV.2 numbering: 469–484) was longer than human GI.1 (GI.1 numbering: 425–431) and human GII.10 (GII.10 numbering: 445–456) P1 subdomain interface loops. For GII human noroviruses, the interface loop plays a critical role in HBGA binding. (C) Superposition of a GI.1 P domain A-trisaccharide complex structure (2ZL7) onto the feline GIV.2 norovirus structure (colored as in Fig. 1a) showed the A-trisaccharide (cyan sticks) clashed with a feline GIV.2 P2 subdomain loop and P2 subdomain α-helix. (D) Superposition of a GII.10 P domain A-trisaccharide complex structure (3PA1) onto the feline GIV.2 norovirus structure showed the A-trisaccharide (yellow sticks) clashed with the feline GIV.2 P1 subdomain interface loop.
Conclusions The overall structure of the feline GIV.2 P domain was similar to human norovirus, except for the novel P2 subdomain α-helix and extended P1 subdomain interface loop. These new structural features were located near the HBGA binding pockets in human GI and GII noroviruses. The functions of these new structural features are currently unknown; however they appeared to inhibit HBGAs from binding at the equivalent GI and GII HBGA pockets on the feline GIV.2 P domains.
4.00
GII.10 P domain and rabbit antiserum GII.10 P domain and guinea pig antiserum GIV.2 P domain and rabbit antiserum GIV.2 P domain and guinea pig antiserum
3.50 3.00 OD490nm
2.50 2.00 1.50 1.00 0.50
80 0 16 00 32 00 64 00 12 80 0 25 60 0 51 20 10 0 24 0 20 0 48 0 40 0 96 00
0 20
0
0.00 40
important for a better understanding of the HBGA role(s) in norovirus infections. In order to determine possible cross-reactivities and common epitopes between feline GIV.2 and human GII.10 P domains, we performed an antibody ELISA using polyclonal antisera raised against GII.10 VLPs. The GII.10 antisera detected GIV.2 P domain at a dilution of 1600, whereas the GII.10 antisera detected GII.10 P domain at dilutions greater than 409,600 (Fig. 3). The low dilution level of the antisera that cross-reacted against GIV.2 indicated that GIV.2 and GII.10 were antigenically distinct, which was supported by the low ( 38%) amino acid P domain sequence identity. The low level of cross-reactivity suggested that the GIV.2 and GII.10 P domains had different immunoreactive and/or immunodominant epitopes. Similar low levels of cross-reactivities were usually found among different human norovirus genogroups (Hansman et al., 2011).
Serial dilution of antisera
Fig. 3. An antibody ELISA showing the GIV.2 and GII.10 P domain cross-reactivities. The GIV.2 and GII.10 P domains (5 μg/ml) were each coated on ELISA plates. Polyclonal antisera (rabbit and guinea pig) raised against human GII.10 VLPs were serial diluted and added to triplicate wells. Standard deviation was negligible and omitted from the figure. A cutoff of OD490 nm 40.2 was used to determine the minimal reactive dilution (dashed line). The GII.10 P domain reacted against rabbit antiserum (blue line) and guinea pig antiserum (orange line) at dilutions greater than 409,600, whereas GIV.2 P reacted against rabbit antiserum (green line) and guinea pig antiserum (purple line) at a dilution of 1600.
Materials and methods An amino acid alignment of capsids from the feline GIV.2 norovirus (Pinto et al., 2012) and a human GII.10 norovirus was used to predict the GIV.2 P domain and design a construct for expression in E.coli
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(Hansman et al., 2011). The feline GIV.2 P domain sequence was longer than the GII.10 and contained a large insertion in the P2 subdomain (Pinto et al., 2012). A construct containing the feline GIV.2 P domain (amino acids 225–565) was expressed and purified as previously described (Hansman et al., 2011). Approximately 100 μg of GIV.2 P domain per liter was purified and this was concentrated to 3 mg/ml for crystallization. Crystals were grown using a hanging drop method in a 1:1 mixture of protein sample and crystallization mother liquor (19% w/v PEG4000, 0.095 M sodium citrate pH5.6, 5% glycerol, and 19% v/v 2-propanol) at 18 1C for 7 days to produce thin plate-like crystals. For co-crystallization experiments with HBGAs, an equal volume of P domain, mother liquor, and 30–60 M excess of A-trisaccharide, Nglycolylneuraminic acid (feline blood group A antigen), or sialic acid (Dextra, UK) were added. Prior to flash freezing, crystals were transferred to a cryoprotectant containing mother liquor, 10–30 M excess of HBGA (for complexes), and 30% ethylene glycol. X-ray diffraction data were collected at beamline BM30A at the European Synchrotron Radiation Facility, France and processed with XDS (Kabsch 1993). Structures were solved using molecular replacement in PHASER (McCoy et al., 2007). Structures were refined in multiple rounds of manual model building in COOT (Emsley et al., 2010) and refined with PHENIX (Adams et al., 2010). Structures were validated with Procheck (Morris et al., 1992) and Molprobity (Chen et al., 2010). Secondary structure predictions were determined using GOR server (Garnier et al., 1996). Figures were generated using PyMOL (Version 1.12r3pre). The cross-reactivities between feline GIV.2 and human GII.10 P domains were performed using an antibody ELISA with rabbit and guinea pig polyclonal antiserum raised against GII.10 VLPs as previously described (Hansman et al., 2004, 2005). Acknowledgments This work was funded by the C.H.S. Foundation. The funding source had no influence with this research. G.S.H. designed the research; G.S.H., B.K.S., and S.G. analyzed the structural data; G.S.H. and A.K. analyzed the ELISA data; and G.S.H., wrote the paper, on which all authors commented. We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities and we would like to thank BM30A staff for assistance in using the beamline. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2014.10.028. References Adams, P.D., Afonine, P.V., Bunkóczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.-W., Kapral, G.J., Grosse-Kunstleve, R.W., McCoy, A.J., Moriarty, N.W., Oeffner, R., Read, R.J., Richardson, D.C., Richardson, J.S., Terwilliger, T.C., Zwart, P.H., 2010. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221. Bank-Wolf, B.R., Konig, M., Thiel, H.J., 2010. Zoonotic aspects of infections with noroviruses and sapoviruses. Vet. Microbiol. 140, 204–212. Bu, W., Mamedova, A., Tan, M., Xia, M., Jiang, X., Hegde, R.S., 2008. Structural basis for the receptor binding specificity of Norwalk virus. J. Virol. 82, 5340–5347. Bull, R.A., Hansman, G.S., Clancy, L.E., Tanaka, M.M., Rawlinson, W.D., White, P.A., 2005. Norovirus recombination in ORF1/ORF2 overlap. Emerg. Infect. Dis. 11, 1079–1085. Bull, R.A., Tanaka, M.M., White, P.A., 2007. Norovirus recombination. J. Gen. Virol. 88, 3347–3359. 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. Cao, S., Lou, Z., Tan, M., Chen, Y., Liu, Y., Zhang, Z., Zhang, X.C., Jiang, X., Li, X., Rao, Z., 2007. Structural basis for the recognition of blood group trisaccharides by norovirus. J. Virol. 81, 5949–5957. Chen, R., Neill, J.D., Estes, M.K., Prasad, B.V., 2006. X-ray structure of a native calicivirus: structural insights into antigenic diversity and host specificity. Proc. Natl. Acad. Sci. USA 103, 8048–8053.
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Virology journal homepage: www.elsevier.com/locate/yviro
Brief Communication
Structural analysis of a feline norovirus protruding domain Bishal K. Singh a,b, Sebastian Glatt c, Jean-Luc Ferrer d,e,f, Anna D. Koromyslova a,b, Mila M. Leuthold a,b, Jessica Dunder a,b, Grant S. Hansman a,b,n a
Schaller Research Group at the University of Heidelberg and the DKFZ, Heidelberg 69120, Germany Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg 69120, Germany c EMBL Heidelberg, Structural and Computational Biology Unit, Heidelberg 69117, Germany d University Grenoble Alpes, IBS, F-38027 Grenoble, France e CEA, IBS, F-38027 Grenoble, France f CNRS, IBS, F-38027 Grenoble, France b
art ic l e i nf o
a b s t r a c t
Article history: Received 23 August 2014 Returned to author for revisions 20 September 2014 Accepted 25 October 2014 Available online 19 November 2014
Norovirus infects different animals, including humans, mice, dogs, and cats. Here, we show an X-ray crystal structure of a feline GIV.2 norovirus capsid-protruding (P) domain to 2.35 Å resolution. The feline GIV.2 P domain was reminiscent of human norovirus P domains, except for a novel P2 subdomain α-helix and an extended P1 subdomain interface loop. These new structural features likely obstructed histo-blood group antigens, which are attachment factors for human norovirus, from binding at the equivalent sites on the feline GIV.2 P domain. Additionally, an ELISA showed that the feline GIV.2 was antigenically distinct from a human GII.10 norovirus. & 2014 Elsevier Inc. All rights reserved.
Keywords: Diarrhea Norovirus X-ray crystallography Virus capsid
Introduction Human noroviruses (Caliciviridae family) are the dominant cause of outbreaks of gastroenteritis around the world. Human noroviruses are genetically and antigenically diverse (Hansman et al., 2006). Based on the capsid gene sequences, there are at least six main genogroups of noroviruses, which can be further subdivided into numerous genotypes. Most human noroviruses belong to genogroups I and II (GI and GII). Recently, a feline norovirus causing gastroenteritis in domestic cats was identified (Pinto et al., 2012). Genetic clustering placed the feline norovirus capsid sequence into GIV genotype 2 (GIV.2). Also belonging to GIV are lion, dog, and infrequently detected human noroviruses (Martella et al., 2007, 2009, 2011). Importantly, human norovirus genetic recombination appears to be a common event and the potential for zoonosis has been described in several studies (BankWolf et al., 2010; Bull et al., 2005, 2007;Clarke and Lambden 1997; Eden et al., 2013; Humphrey et al., 1984; Mahar et al., 2013). These
n Correspondence to: CHS Foundation, University of Heidelberg, DKFZ, Norovirus Study Group, Im Neuenheimer Feld 242, Heidelberg 69120, Germany. Tel.: þ 49 6221 42 1520. E-mail address: [email protected] (G.S. Hansman).
http://dx.doi.org/10.1016/j.virol.2014.10.028 0042-6822/& 2014 Elsevier Inc. All rights reserved.
findings highlighted the importance of understanding the differences and similarities between animal and human noroviruses. The X-ray crystal structure of human norovirus virus-like particles (VLPs) reveals two domains, a shell (S) domain and a protruding (P) domain (Prasad et al., 1999). The S domain forms a scaffold surrounding the viral RNA, whereas the P domain, which can be further subdivided into the P1 and P2 subdomains, likely contains the determinants for host recognition. The norovirus P domain can be expressed in Escherichia coli and this can form P dimers analogous to those on VLPs (Hansman et al., 2011). Studies with VLPs and/or P domains have shown that human, bovine, and canine noroviruses bind histo-blood group antigens (HBGAs) (Caddy et al., 2014; Hutson et al., 2002; Zakhour et al., 2009), whereas murine norovirus virions were found to interact with sialic acid (Taube et al., 2009). Little is known about the attachment factors or receptors for feline noroviruses. At least nine different HBGAs have been identified to bind to human norovirus. However, the relatively weak interaction, quality of reagents, and dissimilar ELISA formats have led to conflicting results concerning the specific HBGAs binding to different noroviruses (Caddy et al., 2014; Hansman et al., 2012; Lindesmith et al., 2012; Tan and Jiang 2010). In the study presented here, we show the X-ray crystal structure of a feline GIV.2 norovirus P domain and identified several novel structural features. We also found that the feline
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GIV.2 norovirus P domain was antigenically distinct from a human GII.10 P domain.
Results and discussion In order to better understand the structural implications between animal and human norovirus capsids we solved the X-ray crystal structure of a recently identified feline GIV.2 norovirus P domain (Pinto et al., 2012). A single crystal of the feline GIV.2 P domain diffracted to 2.35 Å resolution (Fig. 1a). The structure was solved using molecular replacement with the GII.10 P domain as a search model (Hansman et al., 2011). Molecular replacement indicated a dimer in space group P21. Refinement of GIV.2 P domain led to an Rwork value of 0.214 and Rfree ¼0.268, with a well-defined electron density for most of the dimer (Table 1). Based on the GII.10 P domain structure, the GIV.2 P domain could be subdivided into P1 (224–277 and 438–569) and P2 (278–437) subdomains (Fig. 1). Analogous to
other human noroviruses, the GIV.2 P1 subdomain contained an
α-helix and seven β-strands, while the P2 subdomain contained six anti-parallel β-strands that formed a barrel-like structure. However,
compared to the GII.10 capsid sequence, the feline GIV.2 P2 subdomain contained a large insertion between residues 295 and 329 (Fig. 1b). The electron density of this insertion was disordered in chain A between residues 297 and 327 and in chain B between residues 297 and 319. Unfortunately, the disordered regions could not be modeled into the P domain structure. However, part of the insertion in chain B (residues 320–327) had clearly defined electron density and was found to be a short α-helix (Fig. 1a), although the α-helix had higher B-factors as compared to nearby residues and protein average, which suggested that the insertion was flexible. Interestingly, secondary structure prediction of the feline GIV.2 P domain indicated that the insertion actually formed a long α-helix structure (data not shown). The location of the feline GIV.2 P2 subdomain α-helix corresponded to an extended loop on the GII.10 P2 subdomain (Fig. 2a)
Fig. 1. The X-ray crystal structure of GIV.2 feline norovirus P domain and an amino acid sequence alignment. (A) The GIV.2 P domain dimer was colored according to monomers (chains A and B) and P1 and P2 subdomains. Chain A: P1 (yellow–orange), chain A: P2 (fire-brick), chain B: P1 (deep-blue), and chain B: P2 (lime-green). Chain A residues 297–327 and chain B residues 297–319 were not modeled into the structure due to poor electron density. Part of the P2 subdomain (B chain residues 320–327) contained an α-helix structure. (B) The capsid amino acid alignment sequences of feline GIV.2 and human GII.10 noroviruses were aligned using ClustalX. The feline GIV.2 P2 subdomain contained an insertion in the P2 subdomain between 295 and 329. The P1 and P2 subdomains were colored deep-blue and lime-green, respectively. The S domain was not labeled. The GII.10 P domain residues involved directly or indirectly with HBGAs (Hansman et al., 2011) were highlighted, side-chain interactions (red), backbone interactions (blue), and hydrophobic interaction (purple). The asterisks show conserved residues.
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183
Table 1 Data collection and refinement statistics.
Data collection Space group Cell dimensions a, b, c (Å) α, β, γ (1) Resolution (Å) Rmerge I/σI Completeness (%) Redundancy Refinement Resolution (Å) No. reflections Rwork/Rfree No. atoms Protein Ligand/ion Water B-factors Protein Ligand/ion Water R.m.s. deviations Bond lengths (Å) Bond angles (1)
Feline apo (no glycan) (4QUZ)
Feline A tria (unbound glycan) (4QV2)
Feline GNAa (unbound glycan) (4QVA)
Feline sialic acid (unbound glycan) (4QVJ)
P21
P21
P21
P 21 21 21
57.74, 99.92, 59.61 90, 97.92, 90 49.63-2.10 (2.16-2.10)b 20.4 (76.5)b 5.26 (1.38)b 96.64 (74.6)b 3.0 (2.6)b
57.48, 99.38, 59.89 90, 97.54, 90 49.4-1.63 (1.67-1.63)b 14.74 (1.20)b 9.45 (1.28)b 99.20 (93.97)b 4.1 (3.7)b
57.53, 99.38, 69.34 90, 111.04, 90 45.6-2.10 (2.16-2.10)b 13.2 (52.0)b 6.97 (2.17)b 92.70 (90.50)b 2.8 (2.7)b
57.70, 99.60, 129.92 90, 90, 90 49.9-2.10 (2.15-2.10)b 17.32 (63.4)b 7.32 (2.16)b 98.30 (85.10)b 4.7 (4.0)b
49.63-2.35 27,538 0.214/0.268 5013 4787 20 206
27.39-1.68 75,647 0.160/0.183 10,125 4797 20 770
35.53-2.18 35,321 0.196/0.228 9640 4720
49.93-2.26 35,550 0.213/0.271 5141 4738
404
403
38.90 43.10 32.60
16.90 22.80 27.70
30.10
32.70
30.40
34.10
0.004 0.83
0.004 0.923
0.006 0.992
0.005 0.893
Each data set was collected from single crystals, respectively. a b
GNA ¼N-glycolylneuraminic acid and A tri¼ A-trisaccharide. Values in parentheses are for highest-resolution shell.
and a short α-helix on a vesivirus P2 subdomain, which is also a member in Caliciviridae family (Chen et al., 2006). Interestingly, the feline GIV.2 P domain contained a P1 subdomain interface loop, which was shown to contain essential residues for GII norovirus binding to HBGAs (Hansman et al., 2011). However, the GIV.2 P1 subdomain interface loop was found to be longer than other known GII noroviruses and was partially extended out from the surface of the P2 subdomain (Fig. 2b). In order to determine a possible feline GIV.2 P domain interaction with HBGAs and other ligands we performed co-crystallization experiments with A-trisaccharide, N-glycolylneuraminic acid, and sialic acid. Structural analysis of over 30 different crystal complexes with varying concentrations of ligands (15–60 M excess of protein) and crystallization conditions failed to show any bound HBGAs (Table 1). The HBGA binding pockets on human GI and GII noroviruses are distinct (Bu et al., 2008; Cao et al., 2007; Choi et al., 2008). The GI noroviruses bind HBGAs on a single P domain monomer, whereas GII noroviruses bind HBGAs at a dimeric interface. The human norovirus HBGA binding interaction is usually coordinated by a conserved set of residues in each genogroup and includes a network of hydrogen bonds and water-mediated interactions (Bu et al., 2008; Cao et al., 2007; Choi et al., 2008; Hansman et al., 2011). In order to better understand the reasons why these HBGAs did not bind to the feline GIV.2 P domain, we superpositioned GI.1 and GII.10 P domain A-trisaccharide complex structures on the unbound feline GIV.2 P domain structure. At the equivalent GI.1 HBGA binding pocket on the GIV.2 P domain, the A-trisaccharide clashed with a GIV.2 P2 subdomain loop and the P2 subdomain αhelix (Fig. 2c). At the equivalent GII.10 HBGA binding pocket on the GIV.2 P domain, the A-trisaccharide clashed with the extended P1 subdomain interface loop (Fig. 2d). The GI.1 and GII.10 residues that typically interacted with the HBGAs were not conserved in the feline GIV.2 capsid sequence or the corresponding P domain
structure (Fig. 1b and data not shown). In light of these findings, the feline GIV.2 norovirus might not bind HBGAs at the equivalent GI or GII norovirus HBGA pockets on the GIV.2 P domain or they bind other attachment factors. A recent ELISA-based study showed that canine GIV.2 and GVI.3 norovirus VLPs were unable to bind synthetic A-trisaccharides (Caddy et al., 2014). However, the study showed that canine GIV.2 and GVI.3 norovirus VLPs bound to canine tissue samples expressing A and H antigens of HBGAs. The authors acknowledged that additional factors may have contributed to the binding in tissue samples and that their ELISA-based assays had certain limitations (Caddy et al., 2014). Nevertheless, the negative ELISA result using synthetic A-trisaccharide resembled our X-ray crystallography finding that the GIV.2 noroviruses were unable to bind A-trisaccharides. Similar to feline GIV.2 norovirus, the GI and GII norovirus residues interacting with the HBGAs were also not conserved in canine GIV.2 and GVI.3 noroviruses (Caddy et al., 2014). Interestingly, feline GIV.2 and canine GIV.2 shared 90% amino acid identity and the capsid protein was identical in length. Likely, a canine GIV.2 P domain structure had comparable features to the feline GIV.2 structure, including an extended P1 subdomain interface loop and P2 subdomain α-helix. Therefore, our X-ray crystallography data provided a possible explanation of the GIV.2 noroviruses inability to bind synthetic A-trisaccharides, i.e., steric clashes on the P domain at the equivalent GI and GII HBGA pockets. It would be interesting to discover the additional factors that contributed to the canine GIV.2 VLP binding in tissue samples (Caddy et al., 2014). The HBGA binding interactions among antigenically diverse noroviruses are still unclear, and little is known about strains that do not interact with HBGAs (Hansman et al., 2010). At least two human norovirus strains, Hawaii virus (GII.1) and Snow Mountain Virus (GII.2), were found not to bind or only weakly bind HBGAs, respectively (Huang et al., 2005; Lindesmith et al., 2005). Further studies with other noroviruses, including non-HBGA binders, will be
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P1 subdomain interface loop GIV.2 P2 subdomain -helix
GII.10 extended loop
GIV.2 GII.10 GI.1
A-trisaccharide clash
A-trisaccharide clash
Fig. 2. Feline norovirus P2 subdomain α-helix and P1 subdomain interface loop. (A) Superposition of GII.10 P dimer showed an extended loop (salmon) was at a similar location to the feline GIV.2 norovirus α-helix and both were connected to the P2 subdomain β-strands. (B) The feline GIV.2 P1 subdomain interface loop (GIV.2 numbering: 469–484) was longer than human GI.1 (GI.1 numbering: 425–431) and human GII.10 (GII.10 numbering: 445–456) P1 subdomain interface loops. For GII human noroviruses, the interface loop plays a critical role in HBGA binding. (C) Superposition of a GI.1 P domain A-trisaccharide complex structure (2ZL7) onto the feline GIV.2 norovirus structure (colored as in Fig. 1a) showed the A-trisaccharide (cyan sticks) clashed with a feline GIV.2 P2 subdomain loop and P2 subdomain α-helix. (D) Superposition of a GII.10 P domain A-trisaccharide complex structure (3PA1) onto the feline GIV.2 norovirus structure showed the A-trisaccharide (yellow sticks) clashed with the feline GIV.2 P1 subdomain interface loop.
Conclusions The overall structure of the feline GIV.2 P domain was similar to human norovirus, except for the novel P2 subdomain α-helix and extended P1 subdomain interface loop. These new structural features were located near the HBGA binding pockets in human GI and GII noroviruses. The functions of these new structural features are currently unknown; however they appeared to inhibit HBGAs from binding at the equivalent GI and GII HBGA pockets on the feline GIV.2 P domains.
4.00
GII.10 P domain and rabbit antiserum GII.10 P domain and guinea pig antiserum GIV.2 P domain and rabbit antiserum GIV.2 P domain and guinea pig antiserum
3.50 3.00 OD490nm
2.50 2.00 1.50 1.00 0.50
80 0 16 00 32 00 64 00 12 80 0 25 60 0 51 20 10 0 24 0 20 0 48 0 40 0 96 00
0 20
0
0.00 40
important for a better understanding of the HBGA role(s) in norovirus infections. In order to determine possible cross-reactivities and common epitopes between feline GIV.2 and human GII.10 P domains, we performed an antibody ELISA using polyclonal antisera raised against GII.10 VLPs. The GII.10 antisera detected GIV.2 P domain at a dilution of 1600, whereas the GII.10 antisera detected GII.10 P domain at dilutions greater than 409,600 (Fig. 3). The low dilution level of the antisera that cross-reacted against GIV.2 indicated that GIV.2 and GII.10 were antigenically distinct, which was supported by the low ( 38%) amino acid P domain sequence identity. The low level of cross-reactivity suggested that the GIV.2 and GII.10 P domains had different immunoreactive and/or immunodominant epitopes. Similar low levels of cross-reactivities were usually found among different human norovirus genogroups (Hansman et al., 2011).
Serial dilution of antisera
Fig. 3. An antibody ELISA showing the GIV.2 and GII.10 P domain cross-reactivities. The GIV.2 and GII.10 P domains (5 μg/ml) were each coated on ELISA plates. Polyclonal antisera (rabbit and guinea pig) raised against human GII.10 VLPs were serial diluted and added to triplicate wells. Standard deviation was negligible and omitted from the figure. A cutoff of OD490 nm 40.2 was used to determine the minimal reactive dilution (dashed line). The GII.10 P domain reacted against rabbit antiserum (blue line) and guinea pig antiserum (orange line) at dilutions greater than 409,600, whereas GIV.2 P reacted against rabbit antiserum (green line) and guinea pig antiserum (purple line) at a dilution of 1600.
Materials and methods An amino acid alignment of capsids from the feline GIV.2 norovirus (Pinto et al., 2012) and a human GII.10 norovirus was used to predict the GIV.2 P domain and design a construct for expression in E.coli
B.K. Singh et al. / Virology 474 (2015) 181–185
(Hansman et al., 2011). The feline GIV.2 P domain sequence was longer than the GII.10 and contained a large insertion in the P2 subdomain (Pinto et al., 2012). A construct containing the feline GIV.2 P domain (amino acids 225–565) was expressed and purified as previously described (Hansman et al., 2011). Approximately 100 μg of GIV.2 P domain per liter was purified and this was concentrated to 3 mg/ml for crystallization. Crystals were grown using a hanging drop method in a 1:1 mixture of protein sample and crystallization mother liquor (19% w/v PEG4000, 0.095 M sodium citrate pH5.6, 5% glycerol, and 19% v/v 2-propanol) at 18 1C for 7 days to produce thin plate-like crystals. For co-crystallization experiments with HBGAs, an equal volume of P domain, mother liquor, and 30–60 M excess of A-trisaccharide, Nglycolylneuraminic acid (feline blood group A antigen), or sialic acid (Dextra, UK) were added. Prior to flash freezing, crystals were transferred to a cryoprotectant containing mother liquor, 10–30 M excess of HBGA (for complexes), and 30% ethylene glycol. X-ray diffraction data were collected at beamline BM30A at the European Synchrotron Radiation Facility, France and processed with XDS (Kabsch 1993). Structures were solved using molecular replacement in PHASER (McCoy et al., 2007). Structures were refined in multiple rounds of manual model building in COOT (Emsley et al., 2010) and refined with PHENIX (Adams et al., 2010). Structures were validated with Procheck (Morris et al., 1992) and Molprobity (Chen et al., 2010). Secondary structure predictions were determined using GOR server (Garnier et al., 1996). Figures were generated using PyMOL (Version 1.12r3pre). The cross-reactivities between feline GIV.2 and human GII.10 P domains were performed using an antibody ELISA with rabbit and guinea pig polyclonal antiserum raised against GII.10 VLPs as previously described (Hansman et al., 2004, 2005). Acknowledgments This work was funded by the C.H.S. Foundation. The funding source had no influence with this research. G.S.H. designed the research; G.S.H., B.K.S., and S.G. analyzed the structural data; G.S.H. and A.K. analyzed the ELISA data; and G.S.H., wrote the paper, on which all authors commented. We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities and we would like to thank BM30A staff for assistance in using the beamline. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2014.10.028. References Adams, P.D., Afonine, P.V., Bunkóczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.-W., Kapral, G.J., Grosse-Kunstleve, R.W., McCoy, A.J., Moriarty, N.W., Oeffner, R., Read, R.J., Richardson, D.C., Richardson, J.S., Terwilliger, T.C., Zwart, P.H., 2010. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221. Bank-Wolf, B.R., Konig, M., Thiel, H.J., 2010. Zoonotic aspects of infections with noroviruses and sapoviruses. Vet. Microbiol. 140, 204–212. Bu, W., Mamedova, A., Tan, M., Xia, M., Jiang, X., Hegde, R.S., 2008. Structural basis for the receptor binding specificity of Norwalk virus. J. Virol. 82, 5340–5347. Bull, R.A., Hansman, G.S., Clancy, L.E., Tanaka, M.M., Rawlinson, W.D., White, P.A., 2005. Norovirus recombination in ORF1/ORF2 overlap. Emerg. Infect. Dis. 11, 1079–1085. Bull, R.A., Tanaka, M.M., White, P.A., 2007. Norovirus recombination. J. Gen. Virol. 88, 3347–3359. 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. Cao, S., Lou, Z., Tan, M., Chen, Y., Liu, Y., Zhang, Z., Zhang, X.C., Jiang, X., Li, X., Rao, Z., 2007. Structural basis for the recognition of blood group trisaccharides by norovirus. J. Virol. 81, 5949–5957. Chen, R., Neill, J.D., Estes, M.K., Prasad, B.V., 2006. X-ray structure of a native calicivirus: structural insights into antigenic diversity and host specificity. Proc. Natl. Acad. Sci. USA 103, 8048–8053.
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