Structure and Function of a Membrane Anchor-less Form of the

THE JOURNAL OF BIOLOGICAL CHEMlSTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 28, Issue of October 6, pp. 21425-21431,1993 Printed in U.S.A.

Structure and Function of a Membrane Anchor-less Form of the Hemagglutinin-Neuraminidase Glycoprotein of Newcastle Disease Virus*

(Received for publication, May 26, 1993)

Anne M. MirzaS, John P. SheehanS, Larry W. Hardy$, Rhona L. GlickmanS, and Ronald M. IorioST From the $Department of Molecular Genetics and Microbiology and the $Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01655

The hemagglutinin-neuraminidase (HN) glycoprotein of paramyxoviruses is anchored in the virion membrane near its amino terminus, protruding from the virion surface to mediate attachment to cellular receptors. Solubilization of HN spikes can be achieved by treatment of virions with detergent and high salt concentrations. When the solubilized HN protein from the Australia-Victoria (AV) isolate of the virus is incubated at 37 O C , a chymotrypsin-sensitive site between residues 112 and 113 is exposed.

A chymotrypsin-cleaved soluble form of the protein, named CT-HN, has been prepared using this approach. It is membrane anchor-less, due to removal of a 14- kDa fragment from the NH2 terminus of HN. It retains all potential glycosylation sites and cysteines present in the ectodomain of the native protein. It migrates in nonreducing gels and sediments in sucrose gradients at the rate expected for homodimeric HN. The latter is also consistent with our demonstration by site-directed mutagenesis that cysteine residues at positions 6 and 123, respectively, mediate disulfide-linked homotet- ramer and homodimer formation. CT-HN retains al- most total antigenicity, suggesting that it is conformationally very similar to the intact molecule, as well as receptor recognition function and, at low pH, neuraminidase activity. It should prove to be a useful tool for further studies of the structure and function of this important viral glycoprotein.

The paramyxoviruses are a group of negative-stranded RNA-containing viruses comprised of mumps virus, Newcas- tle disease virus (NDV),’ and the various parainfluenza viruses, including Sendai virus, parainfluenza virus 3 (PIV3), and simian virus 5 (SV5) (1). Paramyxovirions possess two types of glycoprotein spikes which protrude from the cellularly derived virion membrane (2, 3). The hemagglutinin-neuraminidase (HN) glycoprotein spike is responsible for both

* This work was supported by Research Grants AI-12467 and AI- 24770 from the National Institutes of Health and by an institutional Biomedical Research Support Group grant. 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.

IT To whom correspondence should be addressed Dept. of Molecu- lar Genetics and Microbiolom, University of Massachusetts Medical

~ ~~~

School, 55 Lake Ave., North, Worcester, MA 01655. Tel.: 508-856- 5257; Fax: 508-856-5920.

The abbreviations used are: NDV, Newcastle disease virus; HN, hemagglutinin-neuraminidase; CT-HN, a chymotrypsin-cleaved soluble form of the HN protein; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis.

attachment to sialic acid-containing cellular receptors and release from similar moieties catalyzed by its sialidase activity. The fusion glycoprotein spike mediates virus-cell and cell- cell fusion (4). The HN glycoprotein has a stretch of hydrophobic amino acids near its amino terminus that is presumed to be the membrane-spanning domain. The carboxyl terminus of HN has been shown to be exposed on the surface of the virion (5).

NDV is a serotype comprised of hundreds of cross-reactive isolates from all over the world. Disulfide-linked dimeric HN can be demonstrated in virions of some of these isolates. However, for the majority of NDV isolates, no oligomeric form of HN can be identified in SDS-PAGE under nonreducing conditions (6-9). Our laboratory has identified a correla- tion between the existence of disulfide-linked HN and the presence of a cysteine at position 123 of HN (9).

Using monoclonal antibodies (mAbs), we have identified seven overlapping sites (sites 4-14-1-12-2-23-3) on the HN protein of the Australia-Victoria (AV) isolate of NDV (10- 12). Functional inhibition studies with these mAbs identified those sites important to attachment (all except sites 3 and 4) and neuraminidase (site 23) (11-13). Antigenic variants were selected by escape from neutralizing antibodies to all seven sites. Sequencing the HN genes of several of these variants has made possible the construction of a neutralization map of the antigenic structure of HN and the identification of domains critical to its functions (14-17). All of the antigenically and functionally important residues in HN that have been identified to date are localized in domains carboxyl-terminal to cysteine 123. Similarly, in two isolates having non-disulfide-linked HN, all of the antigenic sites identified thus far are clustered in the carboxyl-terminal two-thirds of the molecule (18, 19). These findings are consistent with the com- monly envisaged structure of the paramyxovirus HN spike as a long stalk-like structure supporting a globular head, which includes the antigenic and functional sites (20).

The availability of a soluble form of HN, lacking the hydrophobic membrane-spanning anchor, would greatly facili- tate further structure/function studies of the molecule. Scheid et al. (21) first showed that protease treatment of the HN of SV5 that has been detergent-solubilized in the presence of high salt produced a membrane anchor-less form of the molecule. Thompson et al. (20) later used this approach to produce a soluble form of the HN of Sendai virus.

As a prelude to the isolation of a soluble form of the HN of NDV useful for structural studies, we have screened the solubilized HN spikes from several isolates of NDV for their susceptibility to proteolytic removal of their hydrophobic amino termini. This susceptibility correlates with a heat- induced unfolding of the HN oligomer. A membrane anchor-

21425

21426 Hemagglutinin-Neuraminidase of Newcastle Disease Virus

less disulfide-linked dimeric form of HN has been prepared from the AV isolate, It retains antigenicity and receptor recognition and neuraminidase activities, although the latter shows a more restricted pH-dependence than the intact protein.

EXPERIMENTAL PROCEDURES

Virus-The AV (1932), B1-Hitchner (1947) (B), Italy-Milano (1945) (IM) and, Ulster 2C (1967) (U) isolates of NDV were grown in the allantoic sac of 10-day-old embryonated hen eggs at 37 "C from a stock of virus one egg passage from cloning. After the death of the majority of the embryos, allantoic fluid was harvested, and virus was purified as follows. After clarification by low-speed centrifugation, the fluid was centrifuged a minimum of 4 h a t 50,000 X g at 5 "C to pellet the virions. Each pellet was resuspended by homogenization in Hanks' balanced salt solution (Life Technology, Inc.) supplemented with 2% donor calf serum (JRH Biosciences, Lenexa, KS) and again clarified.

The supernatant was layered on top of a step gradient comprised of 5 ml of 65% sucrose (Ultrapure; Schwarz/Mann, Cleveland, OH) and 10 ml of 20% sucrose, in standard buffer (10 mM Tris (pH 7.4), 0.1 M NaCl, 2 mM EDTA). After centrifugation at 130,000 X g for 2 h a t 5 "C, virions at the interface of the two sucrose concentrations were collected from the bottom of the tube. This material was further purified by overnight centrifugation under the same conditions through a step gradient comprised of 2545% sucrose solutions at 10% increments in standard buffer. The virus was collected by punc- turing the side of the tube just below the clearly visible band. It was then concentrated at the interface of a smaller gradient identical to the first one described above. Virion protein was quantitated by the method of Lowry et al. (22).

Preparation and Proteolytic Cleavage of Membrane Glycoproteins- Membrane glycoprotein spikes were purified from virions essentially as described by Thompson et al. (20). Virions (10-20 mg protein/ml) were exposed to 1% Triton X-100 (Surfact-Amps X-100; Pierce Chemical Co.) and 1 M KC1 in PB (10 mM sodium phosphate (pH 7.2)) for 45 min at room temperature. Nucleocapsids were pelleted in a Beckman Airfuge in an A-100 fixed angle rotor a t 28 p.s.i. for 75 min in the cold. The supernatant containing the membrane proteins was then dialyzed overnight at 4 "C against PB to precipitate the majority of the membrane (M) protein, which was then pelleted by centrifugation in an Eppendorf centrifuge for 15 min. The supernatant contains the solubilized HN and F spikes and a small amount of the M protein.

For proteolytic cleavage of the membrane glycoproteins, supernatants containing both HN and F were treated with 0.10 volume of protease (200 pg/ml in PB) for 30 min at 37 "C. All proteases were obtained from Sigma. For evaluation of the protease-treated proteins, they were separated by SDS-PAGE (23) under nonreducing conditions and electroblotted onto nitrocellulose (Schleicher & Schuell) for 16 h at 100 mA, as described previously (10). After washing with phosphate-buffered saline containing 0.3% Tween 20 for a total of 1 h and once briefly with deionized water, the protein bands were visualized with colloidal gold total protein stain (Bio-Rad). This procedure made possible the visualization of the F protein, which in some isolates is not detected in gels by conventional stains.

Sucrose Gradient Sedimentation Analyses-The HN- and F-containing supernatants were centrifuged in a SW-41 rotor at 35,000 rpm for 16 h at 10 "C through linear gradients of 7.5-22.5% sucrose in PB with a 0.5-ml pad of 65% sucrose. For analysis of intact proteins, 0.1% Triton X-100 was added to the gradient buffers to prevent aggregation. Fractions (0.25 ml) were collected and aliquots from alternate ones subjected to SDS-PAGE under nonreducing conditions. Western blotting was performed as described previously (17), using mAb 14r (11).

NH2-terrninal Sequencing-To identify the new amino terminus on the cleaved HN glycoprotein, 500 pmol of chymotrypsin-treated solubilized membrane glycoproteins were electrophoresed as above through 3-mm-thick gels in the absence of reducing agent. The proteins were transferred to Immobilon (Millipore, Bedford, MA) membranes, stained with Coomassie Blue and sequenced in the Protein Chemistry Facility at the university, using an Applied Bio- systems 470A Protein Sequencer, essentially as described by Matsu- daira (24).

mAbs and ELZSA Assays-The isolation and initial characteriza- tion of all mAbs have been described previously (10-12). ELISA

assays for the recognition of solubilized or proteolytically cleaved HN by mAbs was performed as described previously (12) with 1 pg of antigen adsorbed to the wells.

Receptor Recognition and Neuraminidase Assays-The retention of the ability to recognize cellular receptors in the proteolytically cleaved form of HN was confirmed by immunofluorescence. The protease-treated HN (100 pg) was adsorbed to monolayers of chicken embryo cells for 1 h. After washing with PB, adsorbed HN was detected with a mixture of anti-HN mAbs followed by fluorescein isothiocyanate-conjugated rabbit anti-mouse antibody (Kirkagaard and Perry Labs, Gaithersburg, MD).

The amount of N-acetylneuraminic acid released from fetuin (Sigma) by preparations of proteolytically cleaved HN was determined colorimetrically (25).

Site-directed Mutagenesis-The construction of the HN expression vector SVL-HN has been described previously (26). With reference to the published sequence (27), the oligonucleotides 99-GCGCAGT- TAGCCAAGTT-115 (to introduce the Cys to Ser substitution at position 6 (C6S-mutated protein)) and 452-AGCGGGTGGGGGGC ACC-469 (to introduce the Cys to Trp substitution at position 123 (C123W-mutated protein)) were purchased from Oligos, Etc., Inc. (Wilsonville, OR). The protocols for the introduction of the desired mutations into the HN gene and the preparation of plasmid DNA were described previously (26). The wild type and mutated HN proteins were expressed from the SVL-HN vectors transfected into COS-7 cells, using the DEAE-dextran method (28). Cells were seeded at 5 X 106/60-mm plate 1 day prior to transfection with 1.5 pg of supercoiled DNA. The dimethyl sulfoxide shock treatment was not used.

To determine the effect of the mutation(s) on the oligomeric structure of HN, lysates of [36S]methionine-labeled transfected COS cell lysates were reacted with a mixture of mAbs specific for HN and the immunoprecipitates subjected to SDS-PAGE under nonreducing conditions. Briefly, cells were labeled for 2 h at 37 "C with 100-150 pCi of EXPRE35S36S Labeling Mix (Du Pont-New England Nuclear). The medium was removed and the cells were lysed in 0.5 ml of 0.5% deoxycholate, 1% Triton X-100 in phosphate-buffered saline, containing 1 mM phenylmethylsulfonyl fluoride. Following removal of the nuclei by centrifugation, the extracts were incubated with a mixture of mAbs specific for HN and the complexes immunoprecipitated with rabbit anti-mouse beads (Bio-Rad) (13) and analyzed by SDS-PAGE. The gels were fixed in glacial acetic acid, soaked in 22% 2,5-diphenyloxazole, rinsed with water, dried, and exposed to Kodak X-Omat AR film.

RESULTS

Identification of the Cysteine Residues Involved in Disulfide Bond Formation between HN Monomers and Dimers-The presence of disulfide-linked tetrameric and dimeric HN in virions of NDV correlates with the presence of a cysteine residue at positions 6 and 123, respectively (9). To establish that the presence of these cysteine residues and disulfide- linked HN in virions are causally related, we have used site- directed mutagenesis to introduce C6S and C123W mutations into the HN molecule from the AV isolate. These substitutions are the same as the amino acid residues occupying these positions in the majority of non-disulfide-linked HN proteins of other isolates (9,29). We have evaluated the effect of these substitutions on the oligomeric structure of the protein.

Lysates of radioactively labeled transfected COS cells were immunoprecipitated with a mixture of mAbs specific for HN and analyzed by SDS-PAGE under nonreducing conditions (Fig. IA). Analysis of immunoprecipitates of wild type-transfected cells (lane 3 ) shows three bands comigrating with those immunoprecipitated from virus-infected cells (lane 1 ). These correspond to monomer and disulfide-linked dimer and tetramer HN. The immunoprecipitate from cells transfected with the vector alone (lane 2 ) does not contain a protein migrating at the rate of any form of HN. The immunoprecipitate from cells expressing the C6S-mutated protein is shown in lane 4. The predominant bands in this immunoprecipitate migrate in the gel at approximately the rate of monomeric and dimeric HN. Tetrameric HN present in the infected and wild type-

Hemagglutinin-Neuraminidase of Newcastle Disease Virus 21427

A 1 2 3 4 5 . . . ._ .. . ..-

/

/

- 200 K

-92.5 K

8 - 69 K / . HN,

FIG. 1. Site-directed mutagenesis of the cysteines involved in homo-oligomer formation. COS cells were infected with AV (lane 1 ) or transfected with SVL ( l a n e 21, SVL-HNwt ( l a n e 3), the vector encoding the C6S (lane 4 ) , or C6S-Cl23W ( l a n e 5) mutated protein. At 42-44 h post-transfection, the cells were labeled for 2 h and lysed. Infected cells were labeled for 1 h at 6 h post-infection. The HN protein was immunoprecipitated with a mixture of mAbs and electrophoresed under nonreducing ( A ) or reducing ( B ) conditions. The position of molecular weight markers on each gel are also indicated.

transfected lysates cannot be detected. The replacement of cysteine at position 6 with serine has eliminated the disulfide- linked tetrameric form of the molecule.

When the C123W mutation is added to the C6S-mutated protein, the predominant band in the immunoprecipitate (lane 5 ) is a band migrating at the same rate as monomeric HN. No band comigrating with either dimeric or tetrameric HN present in cells transfected with the wild type can be detected. The replacement of the cysteine at position 123 with tryptophan eliminated the disulfide-linked dimeric form of the molecule, confirming our original prediction based on sequence homology (9). These results confirm that disulfide- linked tetramer formation in the HN spike of the AV isolate is mediated by Cys-6 in the cytoplasmic domain of the molecule and that disulfide-linked dimer formation is mediated by the membrane-proximal Cys residue in the ectodomain, at position 123.

The monomeric form of HN in the CGS-mutated protein migrates slightly slower than its counterpart in the wild type- transfected cells (lanes 3 and 4 in Fig. hi). However, when the same immunoprecipitates were electrophoresed under reducing conditions (Fig. lB) , all forms of HN migrated at the same rate. This suggests that the difference in migration observed under nonreducing conditions is due to a disulfide- related difference in folding.

Given that other members of the NDV serotype also lack one or more of these cysteines (29), it is not surprising that both of the mutated proteins are expressed at the cell surface, recognized by a mixture of anti-HN mAbs, and able to he- madsorb chicken erythrocytes. This indicates that substitution at either cysteine residue does not grossly alter HN conformation, although subtle effects on its functions have not been ruled out.

Susceptibility of the HN from Different NDV Isolates to Proteolytic Cleavage-As is the case for the HN of other paramyxoviruses (20,21), with a single known exception (30), the surface glycoprotein spikes of intact virions of NDV were impervious to treatment with proteases (data not shown). Therefore, solubilized membrane glycoproteins from several NDV isolates were screened for protease sensitivity. Solubi- lized HN spikes from any of several NDV isolates tested were resistant to proteolysis at either 25 or 30 "C over a wide pH range (data not shown). Only when the solubilized spikes were incubated at 37 "C at high pH could proteolysis be demonstrated and then only for some isolates.

The results of treatment of the membrane glycoprotein preparations from three different isolates with three different proteases are shown in Fig. 2. Only that from the AV isolate is susceptible to all three of the proteases (lanes 2 4 ) , as evidenced by its conversion to faster migrating forms of differing sizes (collectively indicated by the arrow in Fig. 2) after treatment with each protease. The HN proteins of two isolates, IM (lanes 5-8) and U (lanes 9-12), are resistant to cleavage by any of the three proteases. The non-disulfide- linked HN from the B isolate is also cleaved by the protease, but the neuraminidase activity of the cleaved protein is minimal (data not shown), and this form of cleaved HN was not characterized further. The F proteins of the AV and U isolates appear also to be cleaved by each of the proteases, resulting in a slight increase in the rate of migration in SDS-PAGE, whereas that of the IM isolate is apparently degraded (Fig. 2).

Chymotrypsin-cleaved HN from the AV Isolate Is a Mem- brane Anchor-less Disulfide-linked Dimer-Although solubilized HN from the AV isolate is cleaved by all three proteases, we chose to characterize the chymotrypsin-cleaved form. This protease cleaved HN efficiently and reproducibly, always gen- erating a single band in SDS-PAGE. Thermolysin sometimes did not cleave efficiently and trypsin often resulted in two distinct bands on SDS-PAGE.

Solubilized chymotrypsin-treated HN from the AV isolate is hereafter referred to as CT-HN. SDS-PAGE analysis (Fig. 2, lane 2 ) shows that HN no longer electrophoreses as a tetramer, but only as a form, slightly smaller (120 kDa) than intact dimeric HN (148 kDa). The sedimentation rate of CT- HN in sucrose gradients relative to size markers confirms that it is, indeed, a dimer (Fig. 3R) . Moreover, it fails to aggregate despite the absence of detergent in the gradients. Under these conditions, intact HN spikes aggregate and sediment to the dense sucrose pad at the bottom of the gradient (Fig. 3A).

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

F

M

FIG. 2. Protealytic cleavage of oolubilized membrane proteins of NDV isolates. Solubilized membrane proteins of the AV (lanes 1 4 ) . IM (lunes .5-8), and U (lanes 9-12) isolates were electrophoresed under nonreducing conditions without proteaw treatment (lanes 1, 5, and 9 ) and following treatment with 0.10 volume of 200 pg/ml of chymotrypsin (lanes 2, 6, and IO), thermolynin (lanes3, 7. and IZ), or trypsin (lanes 4.8, and 12). The cleaved forms of the HN from the AV isolate are indicated by the arrow.


A m

HN4

31

-Triton 21 11 3 1

B rn 31 23 21 19

- Triton 11 I

FIG. 3. CT-HN is a membrane anchor-less dimer. Intact ( A ) and chymotrypsin-treated ( B ) glycoproteins from the AV isolate were sedimented through 7.5-22.5% sucrose gradients. Alternate fractions (0.25 ml) were electrophoresed under nonreducing conditions and H N detected by Western blot analysis.

CT Cleavage SI'..

FIG. 4. NHderminal sequencing of CT-HN. The sequence at the amino terminus of CT-HN was determined as described by Matsudaira (24) and the protease cleavage site deduced from the known amino acid sequence of the HN of the AV isolate (27).

NH2-terminul Sequencing of CT-HN-On the basis of our determination that disulfide bonds between H N monomers involve cys-123 and that CT-HN is a nonhydrophobic dimer, we predicted that the protein was cleaved somewhere between the membrane and cys-123. In order to identify the actual protease cleavage site in HN, the sequence at the NH, terminus of gel-separated CT-HN electroblotted onto transfer membrane was determined. The analysis yielded two overlapping sequences, a predominant one 2 residues longer than a minor sequence (Fig. 4). This is consistent with proteolysis a t two sites that are only 2 residues apart in the linear amino acid sequence of the molecule. Comparison to the sequence of the HN of the AV isolate (27) indicates that chymotrypsin cuts between Tyr-112 and Gln-113, as well as to a lesser extent between Leu-110 and Ser-111.

CT-HN Is Antigenically Similar to the Intact Protein-To determine if the proteolytic cleavage of HN has affected the

TABLE I Effect of heat-induced unfolding and proteolytic cleavage of the

solubilized H N glycoprotein spike on the mAb b i n d i q titpr (X IO") in ELISA assays

mAb site Solubilized Solubilized apecificity H N H N (37 ' C ) CT-HN

~~ ~~ ~~ "

4 15 27 17 14 9 13 10

1 10 6 R 12 22 30 30 2 5 I 6

23 9 10 2 3 6 I 10

"

c

antigenic structure of the molecule, we determined the ability of mAbs to seven antigenic sites on the intact molecule to recognize the partially unfolded HN and CT-HN (Table I) . After incubation a t 37 "C, the molecule retains total antigenicity relative to the intact protein, suggesting that exposure of the protease-sensitive site is not accompanied by a gross alteration of its conformation. The same can be said for CT- HN, with the possible exception of antigenic site 23, to which antibody binding appears to be slightly reduced as a result of the removal of the NH2 terminus of the protein.

Receptor Recognition by CT-HN-The retention of receptor recognition activity by CT-HN was demonstrated by its ability to bind to monolayers of chicken embryo cells, as detected by indirect immunofluorescence (Fig. SA). Fig. 5R shows the background fluorescence obtained when the anti-HN mAbs are omitted.

Although CT-HN has no hemagglutinating (HA) activity, its ability to recognize receptors for the virus on chicken

Hemagglutinin-Neuraminidase of Newcastle Disease Virus 21429

FIG. 5. Receptor recognition by CT-HN. One-hundred pg of CT-HN was adsorbed to a 60-mm Petri dish containing a confluent monolayer of chicken embryo fibroblasts for 1 h a t room temperature. After washing with PB, adsorbed CT-HN was detected ( A ) with a mixture of anti-HN mAbs and fluorescein isothiocyanate-conjugated goat anti-rabbit second antibody, as described previously (26). B shows the control in which the primary antibodies were omitted.

1.4

1.21

1.0 m ; 0.8 a 0 . 6 0

0 . 4

0.2

pH 4 . 5 5 6 7

FIG. 6. Neuraminidase activity of CT-HN as a function of pH. The neuraminidase activity of solubilized H N (solid bars), solubilized HN which has been incubated a t 37 "C for 30 min (open bars), and CT-HN (hashed bars).

erythrocytes was confirmed by its inhibition of the hemagglutinating activity of the intact virus.

Neuraminidase Activity of CT-HN-The neuraminidase activity of CT-HN was compared with that of the solubilized protein both untreated and after incubation a t 37 "C for 30 min, mimicking the proteolytic cleavage conditions (Fig. 6). CT-HN retains neuraminidase activity, but only at low pH. At pH 4.5, CT-HN possesses 75% of the neuraminidase activity of untreated HN, very similar to that of the 37 "C- incubated material. However, even at the slightly increased pH of 5, CT-HN begins to demonstrate decreased activity, especially relative to the untreated HN protein spike (45%). The two continue to diverge at the higher pH values of 6 and 7,'partially because CT-HN demonstrates lower activity as the pH is raised and because intact HN has a pH optimum near 6. At neutral pH, the cleaved protein has only about 15% of the neuraminidase activity of untreated HN. Thus, CT- HN has a lower pH optimum than the intact protein.

Susceptibility to Proteolytic Cleavage Correlates with a Heat- induced Unfolding of the HN Spike-The resistance to proteolysis of HN from most isolates of the virus is not due to sequence divergence at the protease-sensitive site. The sequence of amino acids in this region of the HN molecule, from residues 103 through 119, is completely conserved in 13 NDV isolates (29). This includes the U isolate, which is not suscep-

tible to the proteases. To try to understand the molecular basis for the differential sensitivity to proteolysis, we determined the effect of incubation at 37 "C on the sedimentation rate through sucrose gradients of solubilized HN prepared from the AV and U isolates.

Native solubilized HN spikes from the AV isolate sediment in fractions 11-13 (Fig. 7A). However, when this preparation is incubated at 37 "C for 30 min prior to centrifugation, it sediments significantly slower, in fractions 13-19 (Fig. 7B), although it still migrates in the gel as both tetramer and dimer. The simplest explanation for the altered sedimentation rate of HN is a heat-induced unfolding of the molecule.

When solubilized HN spikes from the U isolate, the monomer of which is larger than AV's (29), are subjected to the same treatment, no change in the sedimentation rate is detectable. The majority of the HN sediments in fractions 13- 17, whether it is heated (Fig. 8B) or not (Fig. 8A). Thus, tetrameric HN from the U isolate is more stable to heating and apparently does not undergo the conformational change observed with AV's HN.

mAbs to Site 23 Detect a pH-dependent Change in the Neuraminidase Active Site-Previous findings have suggested

m 3 21 19 I7 IS 13 11 1

A

B

.FIG. 7. Sucrose gradient sedimentation analysis of the solubilized virion glycoproteins of the AV isolate. Solubilized virion glycoproteins from the AV isolate were sedimented through 7.5-22.5% sucrose gradients containing 0.1% Triton X-100. A is untreated proteins and R protein that has been incubated for 30 min a t 37 "C prior to sedimentation. The proteins in alternate fractions weke separated on nonreducing SDS-PAGE and detected by Western blot analysis.

1 rn 31 21 19 17 15 13 11

A

HN

HN

FIG. 8. Sucrose gradient sedimentation analysis of the solubilized virion glycoproteins of the U isolate. Solubilized virion glycoproteins from the U isolate were sedimented through 7.5-22.5% sucrose gradients containing 0.1% Triton X-100. A is untreated proteins and B protein that has been incubated for 30 min a t 37 "C prior to sedimentation. The proteins in alternate fractions were separated on nonreducing SDS-PAGE and detected by Western blot analysis.


that mAbs to antigenic site 23 may bind at or near the neuraminidase active site in native HN (16). Since the neuraminidase activity of CT-HN demonstrates different pH re- quirements than the native protein, it was of interest to compare the ability of anti-HN mAbs to inhibit neuraminidase at low and neutral pH. After treatment of solubilized HN with mAb 23d at neutral pH, only 21% of its neuraminidase activity remains. Neuraminidase inhibition assays could not be performed at neutral pH with CT-HN, since it has only minimal neuraminidase activity at this pH to begin with. However, at pH 5 the antibody does not inhibit the neuraminidase activity of either intact HN (104% activity remaining) or CT-HN (95% activity remaining). This is despite the fact that site 23 mAbs, as well as all other mAbs tested, bind in ELISA assays to both proteins at this pH (data not shown). Furthermore, the change in HN at low pH is reversible, since returning the molecule to neutral pH results in a restoration of the neuraminidase-inhibiting activity of the site 23 mAb. Thus, failure of antibody 23d to inhibit the neuraminidase activity of CT-HN is unrelated to the proteolytic removal of the NH2-terminal domain of the molecule.

DISCUSSION

The HN glycoprotein spikes of several isolates of NDV, solubilized from the virion membrane with detergent and a high concentration of salt, have been screened for susceptibility to proteolysis. Our goal is the isolation and character- ization of a membrane anchor-less form of the protein suitable for further studies of its structure and function. A soluble form of the HN glycoprotein of NDV has been produced from the tetrameric spike of the AV isolate of the virus. The protein has been rendered membrane anchor-less by the removal of its amino terminus, including the hydrophobic membrane- spanning sequence, by treatment with chymotrypsin at elevated temperature.

This form of HN, called CT-HN, sediments in sucrose gradients and migrates in nonreducing SDS-PAGE at the rate expected of a species smaller than homodimeric HN. This is consistent with the site of the proteolytic cleavage, predomi- nantly between residues 111 and 112, as determined by NH2- terminal sequencing, and with the results of a mutational analysis of HN expressed in COS cells from an SV-40-based vector. The intermolecular bonds in disulfide-linked tetrameric and dimeric HN are mediated, respectively, by cysteine residues at position 6, in the cytoplasmic domain of the protein, and at position 123. This supports our original prediction based on sequence analyses of HN from several different isolates of the virus. We had shown previously that position 6 is occupied by a cysteine residue in AV but by a serine residue in two isolates (IM and U), which sediment as tetramers, but migrate in gels as dimers (9). Thus, CT-HN is a dimer, in which the intermolecular bonds are disulfide bridges between Cys-123 residues.

AV is the only NDV isolate identified thus far for which a tetrameric, as well as dimeric, form of the molecule can be demonstrated under the denaturing conditions of nonreducing SDS-PAGE. Thus, the oligomeric structure of AV's HN is analogous to that of the HN of SV5, which apparently exists as disulfide-linked dimers that form a mixture of disulfide- linked and non-disulfide-linked tetramers (31). This is also similar to the oligomeric structure of the HN spike of Sendai virus (6, 20). However, solubilized HN glycoprotein spiles from this virus can be separated into two species on sucrose gradients and proteolytic cleavage in the stalk region of this protein results in a mixture of dimers and tetramers (20). These results suggest that HN may exist on the surface of

Sendai virions as a mixture of dimers and tetramers. Contrary to this, the disulfide-linked forms of the AV isolate of NDV and SV5 (31) cosediment, suggesting that all of the HN in virions exists as tetramers, with only a fraction held together by intermolecular disulfide bonds.

Also, the HN oligomers of SV5 partially dissociate under the centrifugation conditions, unless the pH is maintained at 5 throughout lysis and sedimentation in sucrose gradients (31). No such dependence on pH for oligomer stability could be demonstrated for any of the forms of the disulfide-linked HN of NDV (data not shown).

The possibility remains that the disulfide-linked tetramer is merely an artifact of the lysis conditions; i.e. the disulfide bond proposed to exist between Cys-6 residues on HN mole- cules in a pair of dimers may form only upon lysis of the virion and exposure of these domains to oxidizing conditions. However, we have been unable to block its formation by inclusion of alkylating reagents in the lysis buffer (data not shown).

The differential susceptibility of HN from different isolates cannot be explained by a simple presence or absence of the protease site in this region of HN. The sequence in this region is completely conserved in several isolates that are either sensitive or resistant to treatment with chymotrypsin. Al- though we cannot rule out the possibility that it is related to differential utilization of the N-linked glycosylation site at residues 119-121, it seems more likely that the differential susceptibility is due a difference in the strength of the intermolecular forces holding monomers together as tetramers. Susceptibility to proteolysis correlates closely with a low- heat-induced conformational change in HN, detectable by a slightly altered sedimentation pattern in sucrose gradients. Our hypothesis is that exposure to elevated temperature in- duces an unfolding of the stalk region of the HN tetramer, resulting in the exposure of a previously inaccessible domain of the molecule in which resides the protease-sensitive site. Apparently, the HN tetramer in the U isolate is maintained by stronger intermolecular forces that are resistant to this treatment.

Within the limitations imposed by the use of mAbs to seven overlapping antigenic sites on HN, six of which bind to conformationally dependent epitopes, CT-HN appears to re- tain the antigenic structure of the intact HN tetramer. The only mAbs which show a significant decrease in binding to the membrane anchor-less form of HN are those to site 23. A representative mAb to this site demonstrates a 5-fold decrease in binding to CT-HN relative to the solubilized intact HN tetramer. The finding that mAbs to this site also detect a pH- dependent change in the neuraminidase active site may be related to the narrow pH optimum in this range for the neuraminidase activity of CT-HN. The receptor recognition activity of HN is apparently intact, as evidenced by its ability to bind to chicken embryo cell monolayers and to inhibit the hemagglutinating activity of the intact virus. Thus, CT-HN should prove a useful tool for further structure/function studies of HN.

Acknowiedgnents-We thank Dr. John Mole for performing the NH2-terminal amino acid sequencing. We also thank Michael Bratt for critical reading of the manuscript and acknowledge the excellent technical assistance of Nathaniel Averill and Jeff Barhon.

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Structure and Function of a Membrane Anchor-less Form of the