Crystallization of Newcastle Disease Virus Hemagglutinin-Neuraminidase Glycoprotein

Virology 270, 208–214 (2000) doi:10.1006/viro.2000.0263, available online at http://www.idealibrary.com on

Crystallization of Newcastle Disease Virus Hemagglutinin-Neuraminidase Glycoprotein Toru Takimoto,* Garry L. Taylor,† ,‡ Susan J. Crennell,† Ruth Ann Scroggs,* and Allen Portner* ,1 *Department of Virology and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105; †School of Biology and Biochemistry, University of Bath, Bath BA2 7AY, United Kingdom; and ‡Centre for Biomolecular Sciences, University of St. Andrews, St. Andrews, Fife KY16 9ST, United Kingdom Received December 22, 1999; returned to author for revision February 11, 2000; accepted February 21, 2000 The hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus was isolated by cleaving HN (cHN) from reconstituted virosome with chymotrypsin. N-terminal sequence analysis of the purified cHN showed that chymotrypsin cleavage had occurred at amino acid 123, freeing the C-terminal 454 amino acids. The purified cHN retained its neuraminidase and receptor binding activities and reacted with specific monoclonal antibodies, showing that the isolated cHN was biologically and antigenically functional. The crystals of the cHN were obtained in acetate buffer (pH 4.6) containing polyethylene glycol 3350 and ammonium sulfate and belong to the orthorhombic space group P2 12 12 1 with unit cell dimension of approximately a ⫽ 72 Å, b ⫽ 78 Å, and c ⫽ 198 Å. Crystals of cHN grown in the presence of sialic acid (Neu5Ac) were grown in HEPES buffer (pH 6.2) containing polyethylene glycol 3350 and belong to the hexagonal space groups P6 1 or P6 5 with unit cell dimensions of a ⫽ b ⫽ 137.5 Å and c ⫽ 116.6Å. The orthorhombic crystals produced in this study diffract X rays to at least 2.0-Å resolution, thereby setting the stage for the solution of the three-dimensional structure of the HN glycoprotein of a paramyxovirus. © 2000 Academic Press

the viral glycoproteins in vesicles formed by co-reconstitution of the HN and F glycoproteins is different from that of the conformation of these glycoproteins in either HN or F vesicles or in a mixture of both, suggesting that the viral envelope glycoproteins possess a certain conformation that exists only when they are present within the same membrane (Citovsky et al., 1986). The nucleic acid sequence of the paramyxovirus HN genes predicts a three-domain HN structure: a large hydrophilic carbohydrate-containing domain external to the viral membrane, a small hydrophobic domain spanning the membrane, and a small hydrophilic domain internal to the membrane (Morrison and Portner, 1991; Sakaguchi et al., 1989). The large hydrophilic domain contains the functional sites for cell attachment and neuraminidase activity, as well as the antigenic sites recognized by monoclonal antibodies (MAbs). It is not known whether HN molecule contains combined or separate active sites for hemagglutinin and neuraminidase activities. However, the preponderance of evidence suggests that cell-binding and neuraminidase activities occur at separate sites (Portner, 1981; Portner et al., 1987). Most paramyxovirus HNs form disulfide-linked dimers. In Newcastle disease virus (NDV) HN, a cysteine residue at 123 was identified to be responsible for the disulfidelinked dimer formation (Sheehan et al., 1987). Despite the importance of the diseases caused by paramyxoviruses, the crystallization and subsequent analysis of three-dimensional structure of the HN have not been successful. This structure will advance our

INTRODUCTION Paramyxovirus hemagglutinin-neuraminidase (HN) is a multifunctional glycoprotein that plays a central role in viral infection. It is responsible for the virus attachment to sialic acid-containing host cell receptors (Markwell, 1991) and has neuraminidase (sialidase) activity to hydrolyze sialic acid residues, which is likely to function to prevent virus self-aggregation and efficient virus spread by analogy to the role of influenza virus neuraminidase (Palese et al., 1974; Liu and Air, 1993). HN is also required for membrane fusion that is induced by another surface glycoprotein F (Morrison et al., 1991; Sakai and Shibuta, 1989). Significant membrane fusion is observed only when HN and F proteins of the same virus or closely related viruses are expressed in the same cells, suggesting that a virus-type-specific interaction is necessary for the membrane fusion induced by the F protein (Horvath et al., 1992; Hu et al., 1992). Experiments using chimeric and mutated HN proteins indicate that the stalk region as well as head region plays a role in the fusion promotion activity (Bousse et al., 1994, 1995; Deng et al., 1995; Tanabayashi and Compans., 1996; Tsurudome et al., 1998). A physical interaction between HN and F is reported to be required for efficient membrane fusion (Yao et al., 1997; Stone-Hulslander and Morrison, 1997). Circular dichroism studies revealed that the conformation of

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CRYSTALLIZATION OF NDV HN GLYCOPROTEIN

understanding by providing a structural interpretation of how HN binds to a sialic acid-containing receptor on target cell to initiate the virus–cell interaction, how HN plays a role in the fusion process, and how the neuraminidase removes sialic acid from carbohydrates, facilitating the release of progeny virus from infected cells. Previously, we produced rectangular crystals of the HN protein of human parainfluenza virus type-1 (hPIV1) that diffracted X rays weakly to approximately 10 Å (Takimoto et al., 1992). Here, we report the first successful crystallization of the HN protein of NDV that diffract X rays to a resolution beyond 2.0 Å, thereby allowing the determination of the three-dimensional atomic structure of HN. RESULTS AND DISCUSSION Isolation and purification of a soluble form of the HN glycoprotein Paramyxovirus HN is a type II membrane glycoprotein, which is anchored in the viral envelope by a stretch of hydrophobic amino acids near its N terminus. To isolate soluble proteins that are suitable for crystallization, the hydrophobic membrane-spanning region is required to be enzymatically removed (Thompson et al., 1988; Takimoto et al., 1992; Varghese et al., 1983; Wilson et al., 1981). NDV strain Kansas grown in embryonated eggs was purified and subjected to digestion with various proteases. Direct incubation of purified NDV with proteases such as Pronase, trypsin, chymotrypsin, or bromelein was not successful in the isolation of soluble HN molecule (data not shown). It has been reported that NDV HN isolated from virions by detergent was susceptible to chymotrypsin cleavage, suggesting that chymotrypsin was unable to approach its target site on HN when attached to the virion, probably because of its tight oligomer formation or interaction with F proteins. Therefore, as an alternative, we isolated membrane proteins by disrupting virus with Triton X-100 and reconstituted virosomes containing membrane proteins. After sedimentation of the ribonucleoprotein complex, the supernatant containing viral membrane proteins with lipids and Triton X-100 was isolated. Virosomes containing the membrane proteins were reconstituted from the supernatant by withdrawing the detergent using Bio-Beads (Fig. 1, lane 1). To isolate HN, reconstituted virosomes, which contain HN, F, and M proteins, were treated with chymotrypsin at 500 ␮g/mL (Fig. 1, lane 2). Solution containing cHN was purified by filtration through Centricon 100, which resulted in the preparation of pure concentrated cHN (Fig. 1, lane 3). The cHN migrated as a monomer when analyzed by SDS–PAGE under nonreducing condition (data not shown), suggesting that the cHN did not form disulfide-linked oligomers.

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FIG. 1. SDS–PAGE analysis of the cleaved HN stained by Coomassie brilliant blue staining. Lane 1, reconstituted virosome; lane 2, virosome treated with chymotrypsin; and lane 3, purified cHN; 4, purified NDV.

Cloning and sequence of NDV Kansas HN cDNA To obtain the protein sequence of NDV strain Kansas HN and to characterize the functions of the protein, a cDNA clone of the HN gene of NDV Kansas strain was constructed by reverse transcription–polymerase chain reaction (RT–PCR) from mRNA isolated from NDV-infected cells. Primers used for PCR were synthesized from the information of other NDV strain HN genes sequenced previously (Sakaguchi et al., 1989). The constructed cDNA was cloned into vector pTF1 at HindIII and KpnI sites. Sequencing of the cDNA indicated that the HN gene encodes a protein of 577 amino acids with six potential glycosylation sites (Fig. 2). The molecular mass of unglycosylated HN is predicted to be 63,069 Da from sequence data. Sequence comparison with other published strains (Sakaguchi et al., 1989) indicates that Kansas strain is most closely related to strain Beaudette C (99% homologous). A previous report showed that the cysteine residue 123 was responsible for the formation of disulfide-linked dimers in some strains of HN (Sheehan et al., 1987). SDS–PAGE analysis under reducing and nonreducing conditions showed that the Kansas HN does not form disulfide-linked dimers. Sequence analysis of strain Kansas indicated that this strain has Trp instead of Cys at position 123. N-terminal sequence analysis of c-HN To identify the precise cleavage site, we sequenced the N-terminal amino acids of the cHN by automated Edman degradation. The sequence obtained was GAPIHDPDFIGGIGK. By comparison with the complete protein sequence predicted from NDV HN cDNA, we found that chymotrypsin cleaved the HN molecule at the site between Trp123 and Gly124 (Fig. 2). This result confirmed that the cHN no longer contained the hydrophobic anchor region (residues 27–50) and was consistent with the migration of the cHN on SDS–PAGE. Biological activities of cHN We characterized the cHN to ensure it retains the structure and functions of intact HN protein. Neuramini-

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FIG. 2. Predicted amino acid sequence of HN protein of NDV strain Kansas. Predicted transmembrane region is shown in bold. Six potential glycosylation sites are underlined. The arrow shows chymotrypsin cleavage site on HN.

dase activity of cHN was measured and compared with the intact HN protein on purified virus. The neuraminidase activity of the purified cHN (A 549–0.949) was almost identical to that of the same amount (3 ␮g) of intact HN protein (A 549–0.961), establishing that cleavage and purification had not altered the level of NA function. Receptor binding activity of the cHN was determined by the hemagglutination inhibition test. Our results indicated that chicken red blood cells preincubated with more than 3 ␮g/ml cHN were not agglutinated by 4 HA units of purified NDV, showing that the cHN retains the receptor binding activity and blocked all the receptors on the red blood cells. Further evidence that the cHN retained its native structure was the ability of cHN to bind conformationally sensitive MAbs to the same extent as intact HN (data not shown).

To obtain crystals of a putative cHN–sialic acid complex, we incubated the cHN (12 mg/ml) with sialic acid at the final concentration of 10 mM. The complex crystallized in a precipitant that contained 20% PEG 3350 in 0.1

Crystallization of cHN Crystallization of the cHN was carried out using vapor diffusion in hanging drops. We obtained a number of different crystal forms, including bipyramidal and rhomboid (Fig. 3A), in a precipitant that contains polyethylene glycol (PEG) 3350 and 0.2 M ammonium sulfate in 0.1 M citrate buffer (pH 4.6). Crystals varied in size, but the largest grew to maximum dimensions of 0.5 ⫻ 0.2 ⫻ 0.3 mm. The crystals were washed, dissolved in PBS, and analyzed by SDS–PAGE. We detected a strong band of protein that migrated to the position of purified cHN on SDS–PAGE, showing that the crystals were made of cHN. In addition, cHN from the dissolved crystals retained neuraminidase activity (data not shown).

FIG. 3. Crystals of NDV cHN (A) and cHN-Neu5Ac complex (B). Bipyramidal and rhomboid crystals were grown from solutions of PEG3350. Maximum dimensions of these crystals were 0.2–0.5 mm.

CRYSTALLIZATION OF NDV HN GLYCOPROTEIN

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FIG. 4. Diffraction from a 0.5-degree rotation of crystals of cHN obtained on synchrotron beamline X11 at DESY, Hamburg. (A) Orthorhombic crystal form where the edge of the diffraction pattern corresponds to 1.98-Å resolution. (B) Hexagonal crystal form of the cHN–sialic acid complex, where the edge of the diffraction pattern corresponds to 2.65-Å resolution.

M HEPES buffer (pH 6.2 or 6.4). cHN alone would not form crystals under similar conditions, suggesting that some conformational changes were induced by complex formation with sialic acid. The crystals grew as hexagonal pyramids or a combination of hexagonal prisms and pyramids (Fig. 3B). X-ray diffraction analysis The pH 4.6 cHN crystals diffracted to about 2.7 Å on the in-house X-ray source and to beyond 2.0 Å on the synchrotron source (Fig. 4A). The crystals belong to the orthorhombic space group P2 12 12 1 as judged by systematically absent axial reflections. The asymmetric unit is likely to contain a dimer of 50-kDa monomers, as this gives a solvent content of 57%; however, a self-rotation function did not reveal any obvious noncrystallographic symmetry axis (Fig. 5A). The crystals were protected with 10% glycerol (v/v) added to the crystallization buffer before flash freezing in a N 2 gas stream at 100 K. The putative cHN–sialic acid complex crystals belong to the space group P6 1 or its enantiomorph P6 5, as judged by systematically absent reflections. The crystals diffracted to 2.7 Å at the synchrotron and were cryoprotected by dipping the crystals into crystallization solution containing 20% glycerol (v/v) (Fig. 4B). Table 1 gives the data collection statistics. The asymmetric unit is again likely to contain two monomers, giving a solvent content of 62%, and the self-rotation function reveals a noncrystallographic twofold normal to the sixfold screw axis (Fig. 5B).

Concluding remarks This study is the first report of the crystallization of a paramyxovirus HN that diffracts X rays to a high resolution (⬍2.0 Å), thereby setting the stage for the solution of the HN three-dimensional structure. The determination of the HN structure at atomic resolution is likely to provide new insights into the antigenic structure of HN and the molecular mechanism of virus attachment, entry, and release. In addition, knowledge of the HN structure should provide a stimulus for future investigation and suggest strategies for the development of antiviral drugs that prevent and/or ameliorate paramyxovirus infections. MATERIALS AND METHODS Viruses and cells NDV was grown in 10-day-old chick embryos and purified by sucrose density gradient centrifugation (Portner et al., 1975). Baby hamster kidney (BHK) cells were maintained in Dulbecco’s modified Eagle’s medium with 5% fetal calf serum. Reconstitution of virosome and isolation of proteolytically cleaved HN Membrane proteins were isolated by treatment of purified NDV with 1% Triton X-100 in PBS. Insoluble materials were sedimented by centrifugation at 35,000 rpm for 2 h at 4°C in a Beckman SW55 rotor. Supernatant containing membrane proteins were collected, and Triton

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FIG. 5. Self-rotation function plots. Stereographic projections of the ␬ ⫽ 180 degrees section, showing the orientation of twofold axes. In both cases, data within the resolution range of 8–4 Å was used, with a 20-Å radius of integration. (A) The orthorhombic crystal form. (B) The hexagonal crystal form showing clear noncrystallographic twofold axes normal the crystallographic sixfold screw axis at ␾ ⫽ 21, 51, 81, 111, 141, and 171 degrees.

X-100 was removed by incubation with Bio-Beads SM-2 Adsorbent (Bio-Rad, Hercules, CA), which resulted in the reconstitution of virosomes containing membrane proteins. The virosomes were incubated with chymotrypsin (0.5 mg/ml) overnight at room temperature. TPCK (60 ␮g/ml) was added to terminate chymotrypsin digestion, and the soluble cHN was separated from virosome by ultracentrifugation at 37,000 rpm for 2 h at 4°C in a Beckman SW55 rotor. The cHN in the supernatant was further purified and concentrated by filtration through Centricon-100 (Amicon). Cloning and sequencing of HN gene Cloning of NDV HN cDNA was performed by RT–PCR. Briefly, mRNAs were isolated from NDV-infected BHK

cells and then reverse transcribed into cDNA by avian myoblastosis virus reverse transcriptase. The cDNA was subject to PCR amplification using NDV HN-specific primers that hybridize the 5⬘ and 3⬘ noncoding regions of the HN gene. The primers were designed from the sequence information published previously (Sakaguchi et al., 1989). The specific primers include unique restriction sites HindIII and KpnI, respectively. The NDV HN cDNA was subcloned into vector plasmid pTF1 at HindIII and KpnI sites (Takahashi et al., 1992). The cloned HN cDNA was sequenced by the dideoxy chain termination method using the Sequenase version 2.0 T7 DNA polymerase (Amersham Life Science). The sequence of the NDV Kansas HN gene described here was deposited in GenBank under Accession No. AF212323.

TABLE 1 X-ray Data Collection Statistics Dataset

Native 1

Native 2

Sialic acid complex

Temperature (K) Space group Unit cell (Å), a, b, c Resolution (Å) No. of observations No. unique R merg (%) Completeness (%)

293 P2 12 12 1 73.3, 78.0, 202.6 3.0 172104 20022 9.3 83

100 P2 12 12 1 72.3, 77.9, 199.2 2.0 623166 68217 4.9 86

100 P6 1/P6 5 137.7, 137.5, 116.6 2.7 498619 38673 5.2 94

i i Note. R merg ⫽ ⌺ hkl ⌺ i 兩⌺ I hkl ⫺ 具I hkl典/⌺ hkl ⌺ i 具I hkl 典, where the sum i is overall separate measurements of the unique reflections hkl. Native 1 is room temperature native collected in-house. Native 2 and the putative sialic acid complex were collected at the Hamburg synchrotron.

CRYSTALLIZATION OF NDV HN GLYCOPROTEIN

Characterization of the cHN The N-terminal sequence of cHN was determined by automated Edman degradation on a gas-phase sequencer (Applied Biosystems). Neuraminidase activity of cHN was determined according to the method of Aminoff (1961). Cell binding activity of cHN was assayed by inhibitory effect of cHN to hemagglutination by intact virus. Purified cHN was incubated with cRBC at 4°C for 1 h, and then 4 HA units of intact virus (NDV) were added to the mixture. After 1-h incubation at 4°C, the hemagglutination inhibition was measured. Reactivity of cHN with MAbs sensitive to the HN structure was determined by enzyme-linked immunosorbent assay (Portner et al., 1987) Crystallization procedure Vapor-diffusion crystallization was performed in hanging drops. An equal mixture of cHN (10 mg/ml) and precipitant (25% PEG 3350, 200 mM ammonium sulfate, 100 mM citrate buffer, pH 4.6) was drop-suspended on a glass plate over the precipitant. For the crystallization of the putative cHN–sialic acid complex, 9 volumes of cHN (12 mg/ml) were incubated with 1 volume of 100 mM Neu5Ac (Sigma) at room temperature for 1 h and then crystallized as described earlier. We used 20% PEG 3350 in 0.1 M HEPES buffer (pH 6.2) as precipitant. X-ray diffraction analysis X-ray diffraction data were collected from cHN crystals either at room temperature, using quartz capillary mounts, or at 100 K, using crystals mounted in cryo-loops with a suitable cryoprotectant to avoid icing. Data were collected on in-house rotating anode sources operating typically at 50 kV and 100 mA or at beamline X11 at DESY (Hamburg, Germany) using image plate detectors. Data were processed using the HKL (Otwinowski and Minor, 1997) data reduction package. Self-rotation functions were calculated using the GLRF software package (Tong and Rossman, 1997). ACKNOWLEDGMENTS The work of A.P. was supported by National Institute of Allergy and Infectious Disease Grants AI-11949 and AI-38956, Cancer Center Support (CORE) Grant CA-21765, and the American Lebanese Syrian Associated Charities (ALSAC) of St. Jude Children’s Research Hospital. G.L.T. was funded by a Wellcome Trust Grant. Access to the synchrotron was possible through an EU TMR/LSF grant. The beamline scientists at DESY are thanked for their assistance. We acknowledge Graeme Laver for his interest and encouragement.

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