Crystallization and Preliminary X-ray Analyses of Two Neuraminidases from Influenza B Virus Strains BjHong Kong/8/73 and B/Lee/40*

Crystals of neuraminidase heads from two different influenza B virus strains have been grown. Neuraminidase crystals of influenza B/Hong Kong/8/73 were grown from solutions of potassium phosphate. The crystals are tetrqonal prisms, space group 1422; the axes are a = 123 A and c = 166 A. Influenza BILeel40 neuraminidase crystals were grown from solutions of polyethylene glycol 4000. The crystals are tetragonal pyramids, space group P412,2 or its enaqtiomorph P4,Z12; the axes are a = 125 A and c = 282 A.

P4,Z12; the axes are a = 125 A and c = 282 A.
Neuraminidase comprises 5-10% of total influenza Virus protein and is one of two glycosylated surface antigens. The primary antigen is the hemagglutinin which is responsible for the initial attachment of the virion to host cell surface receptors that contain sialic acid residues (1). The neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) catalyzes the cleavage of a-ketosidic linkages between those terminal sialic acid residues and the adjacent sugar residues of cell surface receptors. This cleavage of sialic acids from mucin appears to facilitate viral mobility to and from the site of infection (2,3). Neuraminidase also acts by removing sialic acid residues from the cell surface and from carbohydrate chains of newly synthesized hemagglutinin molecules to prevent self-aggregation of the virions upon budding and to allow subsequent release of matured virions from the host cell membrane (4).
Influenza B neuraminidase molecules are translated from a messenger RNA which encodes two polypeptides, neuraminidase and NB protein, using overlapping reading frames.
There are four potential glycosylation sites contained within the 466-amino acid sequence of BJLeeJ4O neuraminidase (5). Newly translated molecules are firmly anchored into the plasma membrane by a stretch of hydrophobic amino acids near the amino terminus. Neuraminidase molecules have an average monomer molecular weight of 58,000-68,000 (5) and exist as tetramers on the virus surface stabilized by disulfide bridges. The tetrameric neuraminidase "heads" are positioned on the top of a stalk which rises above the virus membrane (6). Biologically active heads can be released from the stalk by trypsin which cleaves the molecule near the top of the stalk.
Because influenza viruses typically exhibit a high degree of * This work was supported by Grants AI-18203, AI-21679, and CA-13148 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
antigenic variation in their surface antigens, strains isolated in a given year may differ greatly from one another and from strains of previous years (7). The result is that current vaccines are unavailing from one year to the next and any vaccine's effectiveness for controlling future influenza eruptions is debatable. However, the activity is conserved, and for this reason, neuraminidase has been selected as one target for the design of specific inhibitors to curtail both the incidence and severity of influenza outbreaks. Several influenza A neuraminidases have been crystallized (8)(9)(10)(11)(12), and the structures of two subtype neuraminidases (N2 and N9) have been deterFined (10,11). The N2 neuraminidase head resolved to 2.9 A resolution is a tetramer consisting of four identical subunits (monomer M, 50,000) arranged with circular 4-fold symmetry. Each monomer is composed of six topographically identical p-sheets arranged in a propeller formation (10). The catalytic sites are located in deep pockets which occur on the upper corners of the box-shaped tetramer. The N9 neuraminidase head shares about 50% amino acid tequence homology with that of N2 neuraminidase and at 3 A resolution (11) is seen to be folded similarly to N2 neuraminidase. There are some differences in the way in which the subunits are organized around the molecular 4-fold axis, and insertions and deletions with respect to N2 neuraminidase occur in four regions.
Influenza B/Lee/40 neuraminidase has less than 25% sequence identity compared with either N2 or N9 neuraminidase in the head region. Conservation of several of the cysteine residues which form disulfide bonds in N2 and N9 neuraminidases and of several amino acid side-chains which Iine the active site pocket suggest that the polypeptides may be similarly folded, but this can only be confirmed by a full structure determination. This is the first report of influenza type B neuraminidase crystals grown reproducibly for single crystal x-ray diffraction analysis. rpm. Under these conditions, the neuraminidase heads sediment as a crystalline pellet; this was dissolved in 0.15 M NaCl and layered onto a 5-20% linear sucrose density gradient. The gradients were spun in a Beckman SW 65 rotor at 58,000 rpm for 7 h. Twenty fractions were collected from the gradient and each was assayed for protein content and neuraminidase activity (9). Active fractions were pooled and dialyzed for 18 h at 4 "C against 0.9% saline and then concentrated by precipitation with 70% saturated ammonium sulfate at 4 "C. This treatment resulted in the purification of truncated neuraminidase molecules, neuraminidase heads, which were enzymatically active and antigenically indistinguishable from intact neuraminidase.
Crystals of the B/Lee neuraminidase suitable for x-ray analysis were obtained by vapor-diffusion equilibration using the hangingdrop method. The droplets consisted of 2 pl of a solution containing 21 mg of protein/ml plus 2 pl of a 30% solution of polyethylene glycol 4000 (PEG-4000) in 0.02 M HEPES' buffer, pH 7.4. An amorphous precipitate formed within 24 h, and crystals arose from this precipitate in 3-5 days. For x-ray studies, crystals were transferred to a stabilizing solution of 30% PEG-4000 in 0.02 M HEPES buffer, pH 7.4. Crystals of B/Hong Kong neuraminidase were also obtained by the vapor-diffusion equilibration technique using hanging drops. Drops consisting of equal volumes of neuraminidase solution (10-15 mg/ml in 0.9% NaCl) and 1.7 M potassium phosphate buffer, pH 6.6, were allowed to equilibrate with a reservoir of 1.9 M potassium phosphate buffer, pH 6.8. Crystals appeared after 24 h at room temperature. Most drops contained masses of micro crystals, but in some drops a few larger crystals grew; the largest dimensions were ' The abbreviation used is: HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. 0.6 X 0.6 X 0.2 mm. crystals were transferred for mounting to a 3 M potassium phosphate stabilizing solution, pH 6.8.
All crystals were mounted in glass capillaries and photographed on a precession camera at 22 "C using nickel-filtered copper radiation from a Rigaku RU-200 rotating anode generator.

RESULTS AND DISCUSSION
Neuraminidase molecules have previously been isolated from influenza B viruses by trypsin digestion (15) and were characterized by electron microscopy but not chemically (16).
To determine the site(s) of trypsin cleavage, the trypsinreleased heads from B/Lee neuraminidase were S-carboxymethylated and sequenced using an Applied Biosystems 47QA gas-phase sequencer. A single sequence was obtained Glu-

Met-Thr-Phe-Pro-Pro-Pro-Glu-Pro-Glu-Trp-Thr-Tyr-Pro-
Arg--Ser-CMCys-Gln-Gly- Comparison with the complete protein sequence translated from the gene sequence of B/Lee neuraminidase (5, 14) showed that the heads were released by cleavage a t Lys-69, and there was no detectable heterogeneity at the amino terminus. The unidentified residues a t cycles 16 and 21 were Leu and Ser, respectively. Two available alternative sites, Arg-84 and Lys-94, were not cleaved by trypsin.
Comparison of the nucleotide sequence coding for B/Hong Kong neuraminidase* with that of B / h e neuraminidase (5) shows 94% amino acid sequence homology over the whole molecule. Much of this variation is in the stalk, and in the trypsin-released heads used for the crystallographic studies, the homology is 95%.
The B/Lee neuraminidase crystals exhibit either tetragonal, pyramidal, or rectangular morphology. Most of the crystals are tetragonal pyramids, but sometimes the rectangular plates grow in the same drop (Fig. lA). Crystals grow in the pH range 6.2-8.2, but the largest grow around pH 7.4 to maximum dimensions of 0.4 X 0.2 X 0.06 mm. X-ray precession photographs taken on a Rigaku RU-200 rotating anode indicate that the crystals are tetragonal and belong to the Laue group 4/mmm. Fig. 2A shows a zero level precession photograph taken with the x-ray beam parallel to the 4-fold axis. The space group P4,212 or its enantiomorph is specified by the systematic absence of reflections 001 with 1 p 4n and h00 yith h # 2n. Unit cell parameters are a = 125 A and c = 282 A. The crystals are stable-to x-rays at room temperature for 5 days and diffract to 3.0-A resolution.
Using an average subunit molecular weight of 50,000, calculated values of V, (17) for one, two, or four subuni!s/ crystallographic asymmetric unit are 10.94,5.47, and 2.74 A3/ dalton; the corresponding solvent volume fractions are 89, 78, and 55%, respectively. Either of the latter two values are in the range found for other proteins, although the V , for two subunits would indicate a higher solvent content than is usually seen for protein crystals grown from PEG-4000. It appears that there is either a complete tetramer or half of a tetramer in the crystallographic asymmetric unit.
In contrast, B/Hong Kong neuraminidase crystals grow as thick rectangles or as wedges with both morphologies appearing in the same drop, although the wedge morphology dominates the population. These crystals grow with average dimensions of 0.3 X 0.22 X 0.08 mm and were the crystals used for the preliminary space group determination (Fig. 1B). Left undisturbed for 6 weeks, beautiful tetragonal prisms are grown to maximum dimensions of 0.6 X 0.6 X 0.2 mm at room temperature.
The B/Hong Kong neuraminidase crystals diffract to 3.0-A resolution, but are relatively unstable. The average crystal lifetime in the x-ray beam on a Rigaku rotating anode generator operated at 40 kV X 50 mA is about 12 h. X-ray precession photographs taken on a rotating anode indicate that the B/Hong Kong neuraminidase crystals are also tetragonal and belong to the Laue group 4/mmm. Fig. 2B shows a zero level precession photograph taken with the x-ray beam parallel to the 4-fold axis. The space group I422 is specified by the systematic absence of reflections hklewith h + k + 1 # 2n. The unit cell dimensions are a = 123 A and c = 165 A.
Calculated V, valup for one or two subunits/asymmetric unit are 3.12 and 1.56 A3/dalton. The corresponding solvent velumes are 61% and 21%, respectively. The value of 3.12 A3/ dalton lies within the range consistent for that of most other proteins. Thus, there is one subunit in the asymmetric unit. Presently the B/Hong Kong neuraminidase crystals are more likely to furnish a complete native data set required for a full influenza B neuraminidase structure determination, and efforts are currently underway to obtain high-resolution structural information from these crystals.