Archives of Virology

Archives of Virology 53, 295----303 (1977)

© by Springer-Verlag 1977

Host Induced Modifications of Neweastle Disease Virus Virion Polypeptides By G. M. TERRY Department of Virology, Royal Postgraduate 3/Iedical School, London, England With 4 Figures Accepted December 7, 1976 Summary The polypeptide composition of Newcastle Disease Virus (NDV) virions grown in two host cell cystems--chorioallantoic membrane (CAM) and BHK-21 cells-was studied. Two strains of virus were compared, one highIy virulent, the other completely avirulent. No significant differences in the polypeptide composition of the two strains of virus could be detected. However, differences were found in virions grown in different hosts, the same differences being found in both strains. An additional polypeptide is found in B H K grown virus which is not present in CAM grown virus and ~his is associated with a decreased relative amount of nucleocapsid protein in B H K grown virus. The possibility of this new poiypeptide being a degradation product of the nucleocapsid protein is discussed. B H K grown virions also contain increased amounts of a polypeptide migrating to a position which might be expected of the F0 precursor gtycoprotein. However, in contrast to the Fo polypeptide, this polypeptide does not appear to be glycosylated. Introduction Newcastle Disease Virus (NDV) has been extensively studied as the type species of the paramyxovirus group and as a result of the close correlation between the virulence of a wide range of well-characterized strains for chicken and the cytopathogenicity of these strains in tissue c~flture (4, 17, 20). Until recently no significant differences in the polypeptide composition of virions of different strains of widely different virulence had been detected by polyacrylamide gel electrophoresis although the amino acid composition of particular polypeptides had been shown to differ (12, 22). Most of this work has been done using virus grown in cells of avian origin. More recently NAGAI st al. (16) have reported that the polypeptide composition of virulent and avirulent NDV strains is similar in virus grown in embryonated eggs or in chorioallantoic membrane cell cultures, but that in virus grown in other cell systems one or both of the virus glycoproteins are present in

296

G . M . TERRY:

the f o r m of i n a c t i v e precursors in a v i r u l e n t strains whereas in v i r u l e n t strains only the fully cleaved active forms are found. I n this s t u d y the results of a comparison of t h e p o l y p e p t i d e composition of a h i g h l y v i r u l e n t (Texas) a n d a c o m p l e t e l y a v i r u l e n t strain (Ulster) of N D V grown in ehorioallantoic m e m b r a n e or B t I K - 2 1 cells are presented. B o t h q u a n t i t a t i v e a n d q u a l i t a t i v e differences were f o u n d in virions g r o w n in different cell systems b u t n o t in virions of different strains g r o w n in t h e same cell system.

Materials and Methods Cell~ and Media BI4K-21 cells were obtained from the ATCC at passage 73 and grown in Eagle's B H K medium supplemented with 10 per cent tryptose phosphate broth (Wellcome Reagents Ltd. Beckenham, England) and 10 per cent calf serum (Flow Laboratories, Irvine, Scotland). Maintenance medium used for virus infected cells was as above but contained only 2 per cent serum. Virus Strains The following strains of N D V were obtained in freeze-dried form from Dr. Alexander (Central Veterinary Laboratories, Weybridge, England): Texas G. B. (virulent), isolated in 1948 near Austen, Texas, U.S.A. (t) and Ulster (avirulent), isolated in 1966 in Northern Ireland (11). Virus stocks were grown in fertile hens eggs. Growth o] Radioactively Labelled Virus 1. N D V (CAM). Virus was grown on the chorioa]lantoic membrane (CAM) of 12 day old de-embryonated eggs as described by H~SLAS~ et al. (5). Each membrane was infected with 6 ml standard medimn (3), containing 10s.5 EIDs0 of virus and usually 40 ~xCi ~I-I-leueine (specific activity of 500--1000 mCi/mmol), or 10 ~Ci 140protein hydrolysate (specific activity of > 4 5 mCi/milliatom carbon), or 40 ixCi 3Hglucosamine (specific activity of 2> I0,000 mCi/mmol). Incubation was at 37°C for 24 hours. The medium was harvested and clarified. 2. N D V (BHK). Virus was produced in 2 litre rolling bottles. Confluent monolayers of BHK-21 cells were infected at 10 EIDs0 per cell in Eagle's basal medium containing 2 per cent calf serum. Isotopes were used at concentrations of 2 ILCi/ml of 14C-protein hydrolysate, or 10 ~zCi/ml of 3H-leueine or 3I-I-glucosamine. Incubation was at 37 ° C for 18--24 hours after which time the medium was harvested and clarified. Virus Puri/ication Both N D V (CAM) and N D V (BHK) were purified by the method of HASLA~ et al. (5), omitting the ammonium sulphate precipitation step. All operations were carried out at 4 ° C. Table 1. Puri]ication CAM-grown Ulster Virus After clarification After sucrose gradient After potassium tartrate gradient

Infectivity a 1.03 66.70

(1)

I=IAUb t05

Infect./HAUc (i)

9.81

(64.76)

2000 (19.05)

33.35

250.00 (242.72)

6250 (59.52)

40.00

The infectivity and haemagglutinating activity per mg of protein were determined for the clarified virus used as starting material and for the material obtained from the peaks of infectious virus in the sucrose and potassium tartrate gradients. Figures in brackets indicate the relative increase in specific activity taking the starting material as 1

297

Host Modified NDV Polypeptides

Biological Assays Haemaggqutination activity was measured in standard plastic WHO plates in 0.2 ml volumes using PBS-A as diluent and 0.5 per cent chick red cells. Protein concentrations were measured by the method of LowRr et al. (9}. Infectivity of virus preparations was titrated in 10 day old fertile hen's eggs. The 50 per cent egg infective dose (EIDs0) was estimated by the Karber method (7).

Polyacrylamide Gel Electrophoresis This was carried out essentially as described by MAIZEL (10), for SDS-dise electrophoresis. Samples were dissociated in 2 per cent SDS and 1 per cent 2-mercaptoethanol at 100° C for 1 minute. Electrophoresis was carried out on 7.5 per cent gels (unles~ otherwise stated), at 4 mA per gel until the marker dye had run about 7.5 cm (about 4 hours) and the gels were then stained with Coomassie Brilliant Blue or fractionated using a Savant autogeldivider (Savant Inc. Hicksville, New York), and eluted overnight before counting. Molecular weights were determined by the method of SmaPiRO et at. (21), using the following standard proteins in the purest grades obtainable. ~-galactosidase 135,000 (Sigma Chemical Co., St. Louis, Me. U.S.A.), phosphorylase a 94,000 (Sigma), bovine serum albumin 68,000 (Koch-Light Laboratories Ltd. Colnbrook, England), catalase 60,000 (Sigma), ovMbumin 43,000 (Koch-Light), pepsin 35,000 (Sigma), ehymotrypsinogen a 26,000 (Sigma).

Isotope Counting Technique Radioactivity was measured in an Intertechnique ABAC SL40 liquid scintillation counter. The scintillation fluid contained butyl PBD (Intertechnique Ltd., Portslade, England), 10 g; naphthalene (scintillation grade, Koch-Light, Colnbrook, England) 80 g. ; and toluene : 2-ethoxyethanol (3 : 2 v/v) to 1 litre. Double-labelled samples were corrected for quenching to give disintegrations per minute in the ~H and 14C channels using previously prepared quench curves. l~adioactive isotopes were obtained from Radioehemieal Centre, Amersham, Bucks., England. Results

Virus Puri/ication N D V virions were purified as described in Materials a n d Methods. The purificat i o n procedure was m o n i t o r e d a t each step b y m e a s u r i n g the ratio of i n f e c t i v i t y or h a e m a g g l u t i n a t i n g u n i t s to protein. T y p i c a l results for strain Ulster are given in Table 1. The i n f e c t i v i t y a n d h a e m a g g l u t i n a t i n g a c t i v i t y of purified Ulster (BHK) virus were consistently lower t h a n for Ulster (CAM) virus. Essentially identical results were o b t a i n e d for s t r a i n Texas.

o] N D V Strain Ulster BHK-grown Ulster Virus

Infectivitya

After clarification After sucrose gradient After potassium tartrate gradient a Infectivity b HAU c Infect./HAU

Arch, Virol. 53/4

0.27 (i)

HAU b 430 (1)

Infect./HAU 0.63

6.67 (24.70)

1900 (4.42)

3.51

10.00 (37.04)

3200 (7.44)

3.13

EIDs0/milligram protein × 10 .7 Haemagglutinating units/mg protein Infeetivity/HAU × 10-4

20

298

G . M . TW.RgW:

Polyacrylamide Gel Electrophoresis o~ Puri/ied ND V Three major polypeptide peaks lab, 2+3 and 4 corresponding to the HN, F + I ~ P a n d M proteins (19) a n d two m i n o r peaks A a n d B were observed in

2,

Fig. 1. Polyacrylamide gel electrophoresis of ~H-leucine-labelled NDV (CAM) with 14C-protein hydrolysate labelled NDV (BHK). • ......... •, 3tI-leucine; o o, ~4Cprotein hydrolysate. Top--Ulster. B o t t o m - - T e x a s Table 2. Molecular weights o/ virion polypeptides o] N D V strains Ulster and Texas Molecular weight (S.D.) × 10-3 Ulster

Polypeptide

CAM

A la lb B 243 4 C

237.40 89.50 80.20 66.45 51.80 36.50 --

Texas

BHK (7.70) (1.80) (0.80) (2.50) (3.30) (1.65)

255.01 89.30 80.80 65.15 52.10 37.40 28.75

CAM (23.1) (4.50) (0.70) (2.00) (2.45) (1.20) (1.70)

230.00 90.50 80.00 64.50 50.20 36.00 --

BHK (5.01) (1.50) (0.50) (1.51) (2.80) (1.00)

250.10 89.80 81.10 65.12 51.50 36.50 27.60

Molecular weights averaged from 5 replicate experiments

(20.50) (3.42) (1.00) (0.78) (2.00) (1.50) (0.80)

Host Modified NDV Polypeptides

299

NDV (CAM) (Fig. 1). The A polypeptide with a molecular weight of around 240,000 (Table 2) corresponds with the high molecular weight protein described by other workers (5, 6), and may be an aggregate of other virus polypeptides although HIGHTOWER and BRATT (6) have suggested by analogy with VSV that it may be involved in the virion I~NA polymerase flmction. Polypeptide 1 was frequently, though not always, seen as a double peak although on 10 per cent gels only a single peak was observed (data not shown). Since glueosamine labelled virus showed the same behaviour of polypeptido i (Figs. 3 and 4), it is possible that different degrees of glyeosylation of the HN polypeptido are responsible. 10BHK

5"

?.

MOLAR -2 RATIO xlO

CAM

10"

5-

i

A

la

Ib B 28.3 POLYPEPTIDE

4

c

Fig. 2. Molar ratios of the polypeptides present in BHK-grown and CAM-grown NDV. Values in shaded columns are for strain Texas, unshaded columns for strain Ulster. The relative number of molecules of each polypeptide present in thevirion is calculated as: dpm in polypeptide peak 1 X X 10G total dpm in virion polypeptide molecular weight of the polypeptide Each of the 4 sets of values is derived from 3 independent preparations of virus labelled with 14C-protein hydrolysate 20*

300

G.M. TEI~Y:

Comparison of the polypeptide composibiou of the two virus strains revealed only slight differences provided that the virus is gro~al in the same host. However, co-electrophoresis of NDV (BHK) and NDV (CAM), (Fig. 1) revealed relatively more polypeptide B and an additional polypeptide C, with a molecular weight of 28,000 (Table 2) in NDV (BHK), irrespective of strain. Further attempts to purify NDV (BHK) by rate zonal or equilibrium centrifugation failed to alter the polypeptide patterns.

Molar Ratios o/Polypeptides o] Purl/led IVD V Virions Figure 2 shows the molar ratios of NDV polypeptides calculated using the molecular weights shown in Table 2. It is again apparent that only small differences exist between the two virus strains. However, NDV (BHK) is now shown to be deficient in polypeptide 2 + 3 compared with NDV (CAM) in both strains. Polypeptides 2 and 3 co-migrate on polyaerylamide gels under the conditions used here, and since their separation when electrophoresed under non-reducing conditions (12) was not quantitative in our hands, it was not possible to determine directly the molar ratios of each polypeptide. An indirect method was therefore 3

'~

1,, 1B ! !

B t

2+3 it

4 ~

c T

2

1

co

2

t

20

F~,^E'r~ON

NO 4 0

6~)

Fig. 3. Co-electrophoresis of 3H-glueosamine-labelled Ulster with 14C-protein hydrolysat~e-labelled Ulster. Top--CAM-grown virus, bottom--BttK grown virus. ~,........... o, 14C-protein hydrolysalbe ; o o, 3H-glueosamine

Host Modified NDV Polypeptides

30t

e m p l o y e d . Since p o l y p e p t i d c s 1 a n d 2 are g l y c o s y l a t e d (14), it was possible, b y co-eleetrophoresing 3H-glueosamine a n d 14C-protein h y d r o l y s a t e labelled N D V p r e p a r a t i o n s (Figs. 3 a n d 4), to show t h a t t h e y are p r e s e n t in a p p r o x i m a t e l y e q u i m o l a r p r o p o r t i o n s in b o t h N D V (CAM) a n d N D V ( B H K ) (Table 3). I t is

Table 3. Relative amounts o/the two glycoproteins present in N D V virions Molar ratio polypeptide 2 potypeptide 1 a -k 1 b

Virion Texas

CAM-grown BI-IK-grown

Ulster

1.124

1.076

1.386

0.806

Molar ratios were determined from glueosamine-labelled virion polypeptide patterns (Figs. 3, 4) in the manner described in the legend to Fig. 2. The figures thus obtained for polypeptide 1 was set to 1 and the corresponding value for polypeptide 2 is shown above 3

A v

1,,, 18 f

~

r

2+3 T

4 f

2

2

1

•

~a~c~loN

NO

Fig. 4. Co-eleetrophoresis of aH-glueosamine-labelled Texas with 14C-protein hydrolysate-labelled Texas. Top--CAM-grown virus, bott~om--BItK grown virus. • . . . . . . ., 14C-protein hydrolysate; o ~ o , sI-I-glueosamine

302

G.M. T E ~ Y :

assumed that the molecular weight of each glycoprotein is close to the value determined b y SDS gel eleetrophoresis and t h a t each is glyeosylated to the same extent (2, 8). Since both NDV (CAM) and NDV (BI-IK) contain similar amounts of pol3rpeptide 1 (Fig. 2) and hence polypeptide 2, there must be a reduced amount of p o l ~ e p t i d e 3 in NDV (BHK). The results presented in Figures 3 and 4 also demonstrate that p o l ~ e p t i d e s 1 and 2 are the only glycoproteins present in either NDV (BI-IK) or NDV (CAM). Polypeptide ]3 does not, therefore, appear to correspond with F0, the glycoprotein precursor of the F protein found in NDV infected cells (18).

Discussion Although only small differences in polypeptide composition of a virulen~ and an avirulent strain of N D V grown in either CAM or B H K cells were detected, there are significant differences between N D V (CAN) and NDV (BHK). These differences m a y be summarised as follows: NDV (BHK) contains 1. relatively more of polypeptide ]3 than NDV (CAM). 2. An additional polypeptide C and 3. reduced amounts of polypeptide 3, the nucleocapsid protein. The identity of polypeptide B is not yet clear. Its molecular weight and its greater abundance in NDV (BHK) (especially in Ulster strain) suggest tha~ it m a y be the F0 glyeoprotein which is the precursor of the F protein (polypeptide 2) (16). However, we have consistently failed to detect any incorporation of SH-glucosamine into polypep~ide 13. A further obstacle to the identification of this polypeptide with the F0 protein is that, in contrast to the results reported b y NAGAI et M. (16), similar molar ratios of the F protein and the H N protein (polypeptides 2 and 1, respectively) occur in virions of the avirulent strain (Ulster) even when greta1 in B H K cells. Since polypeptide 13 is present in both NDV (BHK) and N D V (CA~) and since it can be detected in purified plasma membrane preparations from NDV infected but not uninfected B H K cells (manuscript in preparation), it is probably a virus induced polypeptide and not a host contaminant. Since polypeptide C is Mso found only in plasma membrane of NDV infected B t t K cells, it is also considered to be a virus specific polypeptide. However, its absence in NDV (CAM) suggests t h a t it m a y not be a p r i m a r y gene product. Since its oecurenee coincides with a reduced amount of nueleocapsid protein (polypeptide 3) it m a y be a cleavage product of this protein. ]V[OUNTCASTLEet al. (13, 15) have shown t h a t paramyxovirus nucleocapsid proteins are suseeptible to proteolytie cleavage and that the product is still able to associate with viral RNA to form nucleocapsids. Work is underway to examine the tryptie digests of N D V nucleocapsid protein and polypeptide C to establish a possible relationship.

Acknowledgments This work was supported by the ?¢Iedieal Research Council and the Royal Postgraduate Medical School in the laboratory of Professor A. P. Waterson. I would like to thank Professor Waterson for his support and interest in this work.

References 1. BO~E'Z, W. A. : The isolation of a neurotropie strain (GB) of Newcastle disease virus. S. Western Veterinarian 5, 19--21 (1951).

Host Modified NDV Polypeptides

303

2. BRETSCH~t, M. S. : Major h u m a n erythrocyte glycoprotein spans the cell membrane. Nature (Hew Biology) 231, 229--232 (1971). 3. FAZE~AS DE ST. GROT~, S., WroTE, I). O. : An improved assay for the infectivity of influenza viruses. J. I~yg. 56, 151--162 (1958). 4. GRANOFF,A. : The isolation of a plaque-type m u t a n t from Newcastle disease virus (NDV). Bacteriol. Prec. (See. Amer. Bacteriologist), 74, 74 (1955). 5. HASLA~, E. A., CI~EYNE, I. M., W'~ITE, D. O. : The structural proteins of Newcastle disease virus. Virology 39, 1 t8--129 (1969). 6. HmHTOWER, L. E., BRA:C$, M. A. : Protein synthesis in Newcastle disease virus infected chicken embryo ceils. J. Virol. 13, 788--800 (1974). 7. K£RBE~, G.: Beitrag zur kollektiven Behandlung pharmakologiseher Reihenversuche. Arch. exp. Pathol. Pharmakol. 162, 480--483 (1931). 8. KLE~K, H.-D., CALIFUIgI, L. A., CHoPPix, P. W. : The proteins of the Parainfluenza virus SV 5. II. The carbohydrate content and glyeoproteins of the virion. Virology 42, 473--481 (1970). 9. LOWRY, O. H., ROSESrGaO~rGH, N. J., FAg~, A. L., RANDALL, R. J.: Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265--270 (1951). 10. MAIZEL, J. V. : I n : MARAMOICSC~,K., KOPROWSKI, H. (eds.), Methods in Virology, Vol. 5, 179--246. New York: Academic Press 1971. i l . MOFEtU~AN,J. B., GOI~DON,W. A. M., FI~I~AY, J. T. T.: A n outbreak of subclinicM Newcastle disease in Northern Ireland. Vet. Rec. 82, 589--592 (1968). 12. MOOSE, N. F., BUI~KE,D. C," Characterisation of the structural proteins of different strains of Newcastle disease virus. J. gen. Virol. 25, 275--289 (1974). 13. MOVNTCASTLE,W. E., CONPAXS, R. W., CAnmuI~I, L. A., CHoPPIN, P. W. : Nucleocapsid protein subunits of Simian virus 5, Newcastle disease virus and Sendai virus. J. Virol. 6, 677--684 (1970). 14. MO~'N~0ASTLE,W. E., COMPAlVS,R. W., CHOPPIN,e . W. : Proteins and glycoproteins of Paramyxoviruses; A comparison of Simian virus 5, Newcastle disease virus and Sendal virus. J. Virol. 7, 47--52 (1971). 15. MOUNTCASTLE, W. E., COMPANS, I~. W., LACKLAND, H., CHOPPIN, P. W. : Proteolytie cleavage of subunits of the nucleoeapsid of the Paramyxovirus, Simian virus 5. J. Virol. 14, 1253--1261 (1974). 16. NAGAI,Y., KLENK, H.-D., ROT~, R. : Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle Disease Virus. Virology 72, 494 to 508 (1976). 17. REEVE, P., ALEXANDER,D. J. : Plaque formation, cell fusion and haemadsorption by Newcastle disease virus. Cytobios 5, 55--57 (1970). 18. SA~ISON, A. C. g . , F o x , C. F.: Precursor protein for Newcastle disease virus. J. Virol. 12, 579--587 (1973). 19. SCHEID,A., CHOPPIN,P. W. : Identification of biological activities of Paramyxovirus glycoproteins. Activation of cell fusion, haemolysis and infectivity by proteolytie cleavage of a n inactive precursor protein of Sendal virus. Virology 57, 475--490 (i974). 20. SCHLOER, G. M., HANSON, 1%. P. : Relationship of plaque size and virulence for chickens of 14 representative Newcastle disease virus strains. J. Virol. 2, 40--47 (1968). 21, S~AP~t~o, A. L., VrN~E~A, E,, MA~ZEL, J. V.: Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. biophys. Res. Commun. 28, 815--820 (1967). 22. S~Ae~O, S. C., BRAT~, M. S. : Proteins of four biologically distinct strains of Newcastle disease virus. Prec. Soc. exp. Biol. Med. 136, 834--838 (1971). Author's address: Dr. G. M. TEm~Y, Wellcome FMDV Laboratory, Pirbright, Woking, Surrey, England. Received October 13, 1976