Biological Consequences of Neuraminidase ... - Semantic Scholar

Vol. 47, No. 3

JOURNAL OF VIROLOGY, Sept. 1983, p. 385-391 0022-538X/83/090385-07$02.00/0 Copyright C 1983, American Society for Microbiology

Biological Consequences of Neuraminidase Deficiency in Newcastle Disease Virus GLENN W. SMITH AND LAWRENCE E. HIGHTOWER* Microbiology Section, Biological Sciences Group, The University of Connecticut, Storrs, Connecticut 06268 Received 25 March 1983/Accepted 26 May 1983

A second-step revertant (Li) of a temperature-sensitive mutant (Ci) of Newcastle disease virus agglutinated erythrocytes normally but had less than 3% of the

wild-type (strain AV) levels of neuraminidase activity. Revertant LI had seven times more virion-associated N-acetylneuraminic acid (NANA) than strain AV. NANA residues on purified virions were specifically labeled with periodate and tritiated borohydride. Analyses of radiolabeled 1, virions on sodium dodecyl sulfate-polyacrylamide gels showed that most of the virion-associated NANA was in a high-molecular-weight component with an electrophoretic mobility different from that of any known viral protein. NANA was also detected in molecules with the electrophoretic mobility of the viral glycoproteins HN and Fl. Revertant Li had a twofold lower rate constant of attachment to HeLa cells than that of the wild-type. Treatment of Li virions with Vibrio cholerae neuraminidase removed the excess NANA and returned Li attachment kinetics to normal. Revertant Ni, which has 10-fold more neuraminidase activity than Li, penetrated host cells at the same rate as Li. Li was impaired in elution from erythrocytes. Removal of virion-associated NANA exacerbated this defect. Despite a small disadvantage in attachment and a major defect in elution relative to strain AV, revertant Li enjoyed a slight advantage over the wild-type during a single reproductive cycle in cultured chicken embryo cells.

Paramyxoviruses and orthomyxoviruses which have virion-associated neuraminidase activities lack N-acetylneuraminic acid (NANA) residues on their envelopes and recognize NANA-containing receptors on cell surfaces (reviewed in references 3, 6, 8, and 9). The receptor-binding (hemagglutinating) and neuraminidase activities of the avian paramyxovirus Newcastle disease virus (NDV) are carried out by the multifunctional hemagglutinin-neuraminidase (HN) glycoprotein (28). The NDV neuraminidase specifically cleaves NANA residues in oligosaccharides on glycoproteins containing the N-acetylneuraminic acid a2-3 galactose linkage (25) and in oligosaccharides containing this linkage and N-acetylneuraminic acid a2-8 Nacetylneuraminic acid linkages, as shown by Drzeniek with milk oligosaccharides (10). The nature of the biological roles of viral neuraminidases remains an open question. Long-standing hypotheses that neuraminidase activities free paramyxoviruses from respiratory mucins and unfavorable reproductive environments (such as erythrocytes) are still viable. Current thinking about paramyxoviral neuraminidases has been strongly influenced by studies of influenza neuraminidases. Among the most compelling influenza virus experiments are

those which used NANA analogs to inhibit neuraminidase activity (21-23) and temperaturesensitive (ts) mutants with defective neuraminidases (24) to study functions. Results from these studies suggest that a major function of neuraminidase is to eliminate NANA residues from virions. Influenza virions containing NANA residues aggregate at the cell surface, marked reductions in virus yields from cultured cells occur, and presumably the cell-to-cell spread of infection is curtailed. A NANA analog inhibits neuraminidase activity but not hemagglutinating activity of strain AV of NDV; consequently, elution of virus from erythrocytes is blocked (27). Conditional lethal paramyxoviral mutants comparable to the influenza virus ts mutants defective in neuraminidase have not been isolated. However, we recently isolated a nonconditional neuraminidase-deficient mutant of NDV, designated Li, which has provided some insights into the biological roles of paramyxoviral neuraminidases (31). Revertant Li was derived in two steps from RNA' ts mutant Ci (26, 33), which is defective in HN and fusion glycoproteins. The wild-type parent of Ci is the virulent strain AV. Revertant LI has repaired fusion glycoproteins and HN glycoproteins with nor385

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mal hemagglutinating activity but only 3% of wild-type neuraminidase activity. Revertant Li produces larger-than-normal yields of infectious virus in cultured chicken embryo (CE) cells and is as virulent as the wild-type for CE (31). Because revertant Li has a residual level of neuraminidase activity and is fully capable of reproduction in cultured cells, our experiments were designed as kinetic assays to detect even small changes in rates of biological activities which might depend on neuraminidase activity. Under these conditions, neuraminidase activity need only be rate limiting for a particular function of Li. MATERIALS AND METHODS Viruses and cell culture. NDV strain AV (AustraliaVictoria, 1932) revertant Li and revertant Ni were grown in embryonated chicken eggs and purified as described previously (7, 12). The isolation and preliminary characterization of revertants Li and Ni, which were derived from ts mutant Cl of strain AV, have been described before (31). A purified preparation of the Indiana strain of vesicular stomatitis virus grown in primary CE cells was kindly provided by M. J. Sekellick, The University of Connecticut, Storrs. Secondary cultures of CE cells were grown in Nutrient Colorado Inositol (NCI) medium (GIBCO Laboratories, Grand Island, N.Y.) containing 5% calf serum, as described previously (5, 7). Suspension cultures of HeLa-S3 cells were grown in Joklik modified minimal essential medium supplemented with 5% horse serum. Radioisotopic labeling of NANA on virions. Selective labeling of NANA on virions was carried out by Gahmberg and Andersson (11). Briefly, virions purified in equilibrium density gradients (30) were suspended at a final concentration of 50 ,ug of protein per ml of 1 mM sodium metaperiodate and incubated for 5 min at 0°C. The samples received 0.2 ml of 0.1 M glycerol in phosphate-buffered saline (PBS) and were then diluted into PBS. After concentration by ultracentrifugation, virions were resuspended in a small volume of PBS and reduced with 1.7 mCi of NaB3H4 (Amersham Corp., Arlington Heights, Ill.; 812 Ci/mmol) for 30 min at room temperature. Each sample was diluted with cold PBS and subjected to ultracentrifugation, and the viral pellets were dissolved in polyacrylamide gel sample buffer (30). The [35S]methionine-labeled virions were prepared as described previously (30). Neuraminidase treatment of virions. To quantify virion-bound NANA, egg-grown virions were first purified by equilibrium density gradient ultracentrifugation. Virus samples containing 50 to 100 p.g of protein in 0.1 ml of PBS and control samples lacking virus were mixed with 0.5 U of Vibrio cholerae neuraminidase (grade B; Calbiochem, La Jolla, Calif.) and incubated for 20 h at 37.5°C. One unit of enzyme was defined by the manufacturer as the amount of enzyme required to release 2 nmol of NANA from human acidic al-glycoprotein per min at 37°C and pH 5.5. Released NANA was determined by using the thiobarbituric acid method of Aminoff (1) and was compared to NANA standard curves.

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For attachment and radioisotopic labeling experiments, 100 ,ug of egg-grown virions suspended in 0.1 ml of PBS was mixed with 14 U of neuraminidase and incubated for 1 h at 37.5°C. The mixtures were diluted 50-fold into cold PBS, and the virus was concentrated by ultracentrifugation. The viral pellets were resuspended in 0.1 ml of PBS, and the procedure was repeated with an additional 14 U of neuraminidase. Again, neuraminidase was removed by dilution into PBS followed by ultracentrifugation. Virus suspensions were stored at -70 C. Viral attachment and elution. Viral attachment studies were performed as described by Marcus and coworkers (20). HeLa cell suspensions in NCI medium were precooled on ice, and the number of PFU equal to the number of cells was added. At various intervals, samples were removed and diluted 100-fold into icecold Joklik modified minimal essential medium. After centrifugation to remove cells and attached virus, the supernatants were assayed on CE cells to determine the number of unattached PFU. For elution experiments, bovine erythrocytes (Colorado Serum Co., Denver, Colo.) were washed with PBS and suspended in NCI medium. The number of PFU equal to the number of cells was added, and the mixtures were held on ice for 30 min. Cells were concentrated by centrifugation and washed with icecold NCI medium. Centrifugation was repeated, and the cells were resuspended at their original concentration in the same medium. The amount of infectious virus that attached to erythrocytes was determined by plaque assays of infectious virus in the wash medium. After the washing procedure, the cells were incubated at 37°C. After 5 min of incubation, the suspensions were divided, and V. cholerae neuraminidase was added to one tube to a final concentration of 50 U/ml. At various intervals, samples were taken from each tube, cells were removed by centrifugation for 15 s (at 12,000 x g) in a microfuge, and titers of infectious virus in the supernatants were determined by plaque assays on CE cells at 40.5°C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Radioisotopically labeled virions were dissolved in polyacrylamide gel sample buffer and analyzed on 11.5% polyacrylamide gels, as described previously (17, 30). The fixed gels were prepared for fluorography (2) and then exposed for 3 to 14 days at -70°C to Kodak X-Omat AR film sensitized with an electronic flash (18).

RESULTS Virion-associated NANA. If the NDV neuraminidase is responsible for removing or excluding NANA from virions, Li virions should contain substantially more NANA than wild-type virions. To test this prediction, purified virions were incubated with V. cholerae neuraminidase, and released NANA was measured as described above. Wild-type virions contained a small but detectable amount of NANA (5.2 ± 3.1 nmol/mg of viral protein), and revertant Ni, which has 30% the normal levels of neuraminidase activity, contained the same amount. In contrast, Li virions contained 34 ± 6.4 nmol (n = 4) of NANA per mg of viral protein. Thus, the neur-

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aminidase-deficient revertant Li contained approximately seven-fold more NANA in virions than the wild type. Additional information on the location of NANA in Li virions was obtained by specifically labeling NANA residues on purified virions by using the periodate-tritiated borohydride method. A low concentration of sodium metaperiodate is used in this technique to selectively oxidize external NANA. The oxidized residues are then radioactively labeled by reduction with tritiated borohydride. The resulting radioactive virions were analyzed by SDS-PAGE (Fig. 1). The usual pattern of [35S]methionine-labeled polypeptides of strain AV is shown in channel A for reference. [3H]NANA-containing components from strain AV and revertant Li virions are shown in channels B and C, respectively. Treatment of Li virions with neuraminidase before radioisotopic labeling eliminated all radioactively labeled components within the limits

a

b c d

e

L

HN

G NP

_

P,F,

FIG. 1. Fluorogram of components from radioactive virions separated by SDS-PAGE. Channel A shows [35S]methionine-labeled polypeptides from strain AV. Major viral proteins are marked. The remaining channels show NANA-containing molecules extracted from purified virions labeled by the periodate-tritiated borohydride method: channel B, strain AV; channel C, revertant Li; channel D, revertant Li treated with neuraminidase before labeling; channel E, vesicular stomatitis virus. The position of the G glycoprotein is marked. Equal amounts of protein were loaded into channels B, C, and D.

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a a

30 20 10 time (minutes) FIG. 2. Attachment of neuraminidase-treated and nontreated virions to HeLa cells. Equal numbers of cells and PFU were mixed and incubated at 0°C for 30 min. At the intervals indicated, portions were centrifuged to remove cells, and the supernatants were assayed for unattached virus. Symbols: 0, strain AV; 0, neuraminidase-treated strain AV; E, revertant Li; O, neuraminidase-treated revertant Li. 0

of detection (channel D), indicating that the procedure specifically labeled NANA residues. As an additional control, purified vesicular stomatitis virions were labeled by the periodatetritiated borohydride method. Only the G glycoprotein of this virus contains NANA (4); as expected, only one radioactive band, which had the appropriate mobility for the G glycoprotein, was obtained (channel E). Radioactive NANA residues of strain AV virions were detected in very-high-molecularweight material which did not enter the gel, in a diffuse high-molecular-weight band (M, = 150,000) which did not coincide with any known viral proteins, and in a component with the mobility of the HN glycoprotein. The radioactive pattern from revertant Li was qualitatively similar, except that a small amount of radioactive NANA residues which comigrated with the Fl glycoprotein was also detected. Quantitatively, the pattern of radioactive NANA-containing components from Li virions was dominated by high-molecular-weight material, and the overall levels of incorporation far exceeded those of the wild type, as expected from the NANA assays of virus preparations. Attachment of virions to HeLa cells. Li virions agglutinate erythrocytes (31), suggesting that the attachment function of HN is intact in the revertant. As a more sensitive measure of attachment than hemagglutination, the kinetics of attachment of wild-type and Li virions to HeLa cells were determined (Fig. 2). The velocity constant

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for attachment, k (32), of Li was 1.7 x 10-9 cm3/min, whereas k for wild-type virions was 3.5 X 10-9 cm3/min, approximately twice that of Li. The velocity constant for strain AV was within the range of values reported for attachment of NDV to erythrocytes (20) and CE cells (19). The NDV requirement for cellular receptors containing NANA residues was preserved in revertant Li. Less than 1% of an inoculum of either Li or wild-type virions bound to cells pretreated with neuraminidase. Wild-type and Li virions were treated with neuraminidase to determine whether the removal of NANA affected attachment. Neuraminidase treatment had a negligible effect on the binding of wild-type virions to cells, but it significantly increased the rate of attachment of Li virions. Treated Li virions fully recovered the binding kinetics of strain AV. The presence of NANA residues on Li virions anneared to interfere with attachment to cells, and it was also conceivable that NANA residues inhibited viral neuramini 4ase activity. nidase However, no recovery of viral n activity was detected after treatm4ent of Li vinlons with V. cholerae neuraminidaLse. Penetration of CE cells by virionss. The relative

deurase ieuramLnlidarse

100

0

80 0

-'

time (minutes) FIG. 4. Elution of virions from erythrocytes. mixed Strain AV (0, 0) and revertant Li (N, l) wereincubatwith a suspension of bovine erythrocytes and ed for 30 min at 0°C. Cells were washed, resuspended in fresh medium, and incubated at 37°C. After 5 min of incubation (arrow), each sample was divided, and V.

cholerae neuraminidase was added to one tube. Incubation was continued, and after the indicated intervals, portions from each tube were centrifuged to remove cells. The supernatants were assayed for released PFU. Open symbols represent virus released from neuraminidase-treated samples, and closed symbols represent nontreated samples. The 100o elution level was defined as the amount of infectious virus attached to cells after 30 min at 0°C.

.o . .

a

le

0-

a'4

a

20

L 0

SO time

100 (minutes)

ISO

200

FIG. 3. Penetration of CE cells by strain AV (0),

revertant Li (A), and revertant Ni (O). Virus was adsorbed to cultures at 4°C and then s hifted to 37.50C to initiate penetration. After various pe riods of incubation, antiviral antibody was added to reZplicate cultures to neuralize virus which had not entere4d cells. Surviv-

ing infectious viruses were scored b y plaque assay after the removal of antibody. The da shed line represents the rate of penetration by the par obtained previously (31). The incubati4 en times included the 10-min antibody treatments. IDetailed experimental protocols along with proceduires for antisera preparation have been described prevy iously (31).

rates of penetration of CE cells by strain AV, revertant Li, and revertant Ni were measured as escape from antibody neutralization (Fig. 3). Revertant Ni was originally isolated from revertant Li stocks which were screened for mutants that had regained neuraminidase activity (31). The time required for 50% of attached Li and Ni virions to penetrate (t1/2) was 30 min, which approached wild-type values (t1l2 = 20 min). The rate of penetration (tl/2 = 125 min) of the original ts parent Ci of the revertants is shown in Fig. 3 for reference. Elution of virions from erythrocytes. To measure the capacity of revertant Li to elute from cells relative to strain AV, purified virions were allowed to attach to bovine erythrocytes at 0°C and then were given an opportunity to elute at 37°C. Erythrocytes were used in these experiments because NDV does not readily elute from

host cells at 370C (19, 20). Wild-type virions rapidly eluted from erythro-

cytes (Fig. 4). After 60 min of incubation, all of the initially bound virus was released. Similar elution kinetics have been reported for the Cali-

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fornia strain of NDV (20). In contrast, Li virions were much less effective than wild-type virions in eluting from erythrocytes. Only about 40% of the attached Li virions were recovered in the medium as PFU after a 60-min incubation period. Elution of Li virions increased dramatically after the addition of V. cholerae neuraminidase to the virus-cell complexes. As expected, elution of wild-type virions was not affected by this treatment. Approximately 70% of the cell-bound Li was released from erythrocytes within 10 min of incubation with neuraminidase. The remaining attached virus had either initiated further stages of penetration or was inactivated at the cell surface, since longer periods of incubation with neuraminidase did not remove additional infectious virus. Also, very little additional Li was released when neuraminidase was added to virus-cell complexes after 30 min of incubation at 37°C. To evaluate the effects of Li virion-bound NANA on elution, Li virions were treated with neuraminidase before attachment to erythrocytes at 0°C. The yield of released virus after a 60-min incubation at 37°C was reduced by 35% relative to untreated Li virions, suggesting that NANA residues on Li facilitated elution. Single reproductive cycles of virions in CE cells. Single-cycle growth curves of strain AV and revertant Li at 37.5°C are shown in Fig. 5. Reproduction and release of infectious virus into the culture medium were not impaired by the lack of neuraminidase in Li. In fact, Li progeny virions accumulated in the medium at a reproducibly faster rate than that of wild-type progeny. Similar curves were obtained for Li reproduction at 42.5°C, the nonpermissive temperature for the ts parent of Li; however, reproduction of wild-type virions was even less efficient at this temperature (data not shown). DISCUSSION The major effects of a neuraminidase deficiency in revertant Li were (i) impaired elution of virions from erythrocytes, (ii) a sevenfold increase in virion-associated NANA residues, and (iii) a twofold decrease in the velocity constant for attachment of Li to HeLa cells. NANA residues were detected on virions of strain AV by the thiobarbituric acid and the periodatetritiated borohydride methods. In earlier studies of paramyxovirus SV 5, no detectable NANA residues were found on virions by either the thiobarbituric acid method or colloidal iron staining for electron microscopy (14-16). Therefore, paramyxoviruses may differ in their residual levels of virion-associated NANA. Potential attachment sites for NDV-associated NANA residues include viral glycoproteins, host glyco-

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o

3

3

6 9 hours post -infection

12

FIG. 5. Single reproductive cycles of strain AV (0) and revertant Li (U) in CE cells at 37.5°C. Virions were added to cells at an input multiplicity of 3 PFU/cell and allowed to adsorb for 1 h at 4°C. Cultures were washed five times with prewarmed Joklik modified minimal essential medium containing 5% calf serum and were then incubated in this medium. At the intervals indicated, portions of the medium were collected, and infectious virus was quantified by plaque assay at 37.5°C.

proteins, and host glycolipids. Our analyses by SDS-PAGE suggest that NANA may be associated with HN in wild-type virions and with both Fi and HN in revertant Li virions. More detailed biochemical analyses will be needed to substantiate this observation. The diffuse [3H]NANA-labeled material centered about 150,000 daltons coincided with a diffuse band of Coomassie blue staining material (unpublished observation). Since no viral proteins of this size are known, it is possible that these NANA residues were associated with host glycoproteins. We have no information on the possible association of NANA with virion glycolipids. In general, the gel patterns of [3H]NANA-containing molecules from virions indicated that the NANA content of molecules which contained low levels of NANA in the wild type increased in Li virions, but no major new species were detected, with the possible exception of Ft. The additional NANA residues on Li appeared to be responsible for its difficulties in attachment, since neuraminidase treatment returned the attachment rate of Li to the wild-type rate. The effect of NANA may have been to increase electrostatic repulsion between Li virions carrying more negative charges and the negatively charged cell surface. Alternatively,

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additional NANA residues on HN may have directly or indirectly altered receptor-binding sites. The fact that the wild type and neuraminidase-treated Li had the same velocity constants for attachment but differed dramatically in neuraminidase activity further substantiates our previous conclusion (29, 31) that receptor binding and neuraminidase activities have been successfully uncoupled in revertant Li. NANA residues on Li also appeared to affect elution from erythrocytes. The 97% inhibition in neuraminidase activity of Li resulted in a 60% reduction in the amount of released infectious virus. Neuraminidase treatment of Li before attachment resulted in a further 35% reduction in the amount of released virus. The facilitation of elution by virion-associated NANA could be due to the same factors responsible for the reduced attachment rate of Li to HeLa cells. It is interesting to compare the properties of the ts mutants of influenza virus (24) defective in neuraminidase at the nonpermissive temperature to those of Li. Most of the phenotypical changes in the influenza mutants derived from increased NANA content of virions assembled at the nonpermissive temperature. Influenza virus-bound NANA apparently serves as a receptor for the influenza hemagglutination glycoprotein, causing aggregation of virions. The aggregated virions fail to cause hemagglutination, and this capacity, but not infectivity, can be recovered by treating virions with exogenous neuraminidase. Aggregation of progeny influenza mutants at the cell surface results in 103- to 104-fold decreases in titers of infectious virus. Like the influenza mutants, Li virions contained much larger amounts of NANA than wild-type virions, and they were deficient in neuraminidase activity. However, unlike the influenza mutants, LI virions were capable of wild-type hemagglutinating activity, and progeny released from cultured cells accumulated at a faster rate than that of the wild type and to similar final titers. Our results indicate that Li virions did not aggregate significantly. Either the NANA residues on Li virions were not recognized as receptors or the 3% residual neuraminidase activity was sufficient to prevent aggregation. Prompted by a recent report (13) that liposomes containing myxo- and paramyxoviral glycoproteins require the presence of viral neuraminidase for fusion with cells, we tested the ability of revertant Li to penetrate CE cells. Assuming that escape from antibody neutralization after viral attachment reflects the fusion of the NDV envelope with the cellular membrane, we concluded that revertant Li has nearly normal rates of penetration. Revertant Ni, which has levels of neuraminidase activity sufficient to reduce virion NANA levels to normal, penetrat-

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ed cells at the same rate as Li. Therefore, additional NANA residues on Li virions were not responsible for the small differences in penetration between Li and the wild type. This difference was probably due to the incomplete repair of the fusion glycoprotein of Li (31). We have previously shown that revertant Si, which has the same fusion glycoprotein as Li, penetrates CE cells faster (t1/2 = 60 min) than the ts parent (t1/2 = 125 min) (31). In the present study, revertant Li, which was derived from Si, was shown to have a t1j2 for penetration of 30 min, approaching the wild-type rate (t1/2 = 20 min). Revertant Li virions have approximately three times more HN glycoproteins than do SI virions but i6-fold less neuraminidase activity (31). The increased capacity of Li to form additional attachments through HN, bringing viral and cellular membranes into closer apposition for subsequent fusion, could explain the increased rate of penetration of Li relative to that of Si. We have not found support for the idea that neuraminidase is required for penetration. Although we cannot rule out roles for the NDV neuraminidase in functions like penetration (for which 3% residual activity may not be rate limiting), we can conclude that attachment to cellular receptors and elution from erythrocyte receptors are among the viral functions most sensitive to a neuraminidase deficiency. Revertant Li and its companion set of mutants, Ci, Si, and Ni, should be valuable in future studies of the pathogenesis of Newcastle disease in birds. The uncoupling of receptor binding and neuraminidase activities in revertant Li also adds to a growing number of recent observations based on mutant analysis, monoclonal antibody studies, and a NANA analog, suggesting that the enzymatic site(s) and receptor-binding site(s) on the HN glycoprotein may be different (27). ACKNOWLEDGMENTS We thank M. A. K. Markwell for suggesting the periodatetritiated borohydride technique. This work was supported by Public Health Service grant HL23588 from the National Institutes of Health and by National Science Foundation grants PCM 78-08088 and PCM 81-18285. We benefited greatly from the use of a cell culture facility supported by Public Health Service grant CA 14733 from the National Cancer Institute.

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