Archives of Virology

Archives of Virology 54, 299--305 (1977)

© by Springer-Verlag 1977

Cell Receptors for Paramyxoviruses By

LILL~ WASSIL~WA Depa.rtment of Biochemistry, Central Veterinary Research Institute, Sofia, Bulgaria With 1 Figure Accepted February 1, 1977

Summary Treatment of chick embryo fibroblasts, calf kidney and B H K cells for 30 minutes with the enzyme neuraminidase from Vibrio cholerae causes an enhancement of the per cent of attached NDV virions. This enhancement does not depend on the multiplicity of infection. The quantity of spontaneously eluted and cellbound virus is two times greater than the quantity of the same virus derived from control cells. N-aeetyl-neuramin lactose inhibits the effect of Vibrio cholerae neuraminidase. After prolonged action of this enzyme, the quantity of adsorbed NDV diminishes. Treatment of the same cells with neuraminidase from influenza virus decreases the per cent of adsorbed NDV with respect to controls. The other p a r a m y x o v i r u s - - b o v i n e parainfluenza 3 virus adsorbs also more intensively on cells treated with Vibrio &olerae neuraminidase. I t is suggested that partial hydrolysis of NANA molecules causes a rearrangement of the cell surface charged groups and thus allows a more effective contact between paramyxoviruses and the cell.

Introduction I t is known that the cell receptors for myxo- and paramyxoviruses are glycoproteins, containing N-acetyl-neuraminic acid (NANA) as the terminal group. The hydrolysis of NANA b y the enzyme n euraminidase diminishes the ability of the cell to adsorb inItuenza virus (7) and NDV (12). The low molecular weight substrate for the neuraminidases--N-acetyl-neuramin lactose does not cause competitive inhibition of virus adsorption (7). The amount of NDV adsorbed onto cells depends on the multiplicity of infection but never exceeds 40 per cent of the input virus (3). The experiments of DRZE?CIEK(4) show that all substances containing NANA can serve as substrates for myxovirus neuraminidase, but the adsorption or elution of the virus from the cells depends on the ability of the

300

LILIA WASSILEWA :

e n z y m e t o s p l i t d i f f e r e n t s u b s t r a t e s . I t w a s s h o w n (9) t h a t for t h e a d s o r p t i o n of m y x o v i r u s e s , a n e n z y m e - s u b s t r a t e i n t e r a c t i o n of v i r u s a n d r e c e p t o r s d o e s n o t p l a y a n i m p o r t a n t r o l e a n d also t h a t t h e h e m a g g l u t i n i n is r e s p o n s i b l e f o r t h e b i n d i n g of t h e v i r u s t o N A N A - c o n t a i n i n g cell r e c e p t o r s . A c c o r d i n g t o LIPKIND a n d URBAKtI (11), t h e s i m u l t a n e o u s o c c u r r e n c e of t h e p r o c e s s of p e n e t r a t i o n a n d e l u t i o n of N D V c a n n o t b e e x p l a i n e d b y t h e h e t e r o g e n e i t y of t h e v i r i o n s a n d t h e cell s u r f a c e p r o p e r t i e s , b u t o n l y b y t h e v a r i o u s p r o b a b i l i t i e s of o c c u r r e n c e of t h e s e p r o c e s s e s i n a u n i f o r m v i r u s - c e l l s y s t e m . T h e g o a t of t h i s c o m m u n i c a t i o n is t o e s t a b l i s h if t h e r e is a n y d i f f e r e n c e i n t h e p r o p e r t i e s of t h e cell s u r f a c e w i t h r e s p e c t t o t h e a t t a c h m e n t of p a r a m y x o v i r u s e s a n d w h e t h e r or n o t o n l y N A N A m o l e c u l e s o n t h e cell s u r f a c e a r e f u n c t i o n a l l y a c t i v e i n t h e a d s o r p t i o n of t h e s e v i r u s e s .

Materials and Methods Cells P r i m a r y - or s u b c u l t u r e s of c h i c k e m b r y o f i b r o b l a s t s (CEF) calf k i d n e y (CK) a n d B H K line were p r e p a r e d a c c o r d i n g to s t a n d a r d procedures. Viru8e8 T h e a v i r u l e n t s t r a i n ~ u s s e f f of N D V a n d t h e s t r a i n C r e m e n a of b o v i n e p a r a i n f l u e n z a - 3 (PI-3) were used. S t r a i n R u s s e f f c a n b e p r o p a g a t e d o n C E F a n d CIK (15). T h e t i t e r s of t h e viruses, d e t e r m i n e d b y t h e p l a q u e m e t h o d were 4.0 × 10 6 P F U / m l for N D V g r o w n o n C E F , 2.0 × 10~ P F U / m l for N D V g r o w n on B H K , a n d 3.7 × 10 5 P F U / m l for P I - 3 g r o w n o n CK. H e m a g g l u t i n a t i o n was p e r f o r m e d a t r o o m t e m p e r a t u r e w i t h 0.5 p e r c e n t c h i c k a n d 1 p e r c e n t g u i n e a pig e r y t h r o e y t e s .

Treatment el the Cells With Neuraminidases T h e cell m o n o l a y e r s or s u s p e n d e d cells were t r e a t e d w i t h t h e e n z y m e n e u r a m i n i d a s e f r o m Vibrio cholerae ( B e h r i n g w e r k e a n d K o c h L i g h t ) or i n f l u e n z a v i r u s (Calbiochem) a t 37 ° C a n d p H 6.5 for d i f f e r e n t t i m e periods u s i n g a c o n c e n t r a t i o n of 5 u n i t s e n z y m e p e r 106 cells. A f t e r this, t h e cells were w a s h e d t h r e e t i m e s w i t h p h o s p h a t e b u f f e r e d saline (PBS) a n d i n f e c t e d w i t h t h e viruses. T h e m u l t i p l i c i t y of i n f e c t i o n for N D V w a s 2 in C E F or 1 3 t t K cells a n d 0.05 i n C K cells. F o r P I - 3 t h e m u l t i p l i c i t y w a s 0.05. T h e v i r u s e s were a d s o r b e d for 60 m i n u t e s a t r o o m t e m p e r a t u r e or a t 4 ° C. T h e u n a d s o r b e d v i r u s e s were r e m o v e d b y w a s h i n g t h e cells t h r e e t i m e s w i t h P13S. T h e w a s h i n g b u f f e r was collected a n d pooled w i t h t h e u n a d s o r b e d viruses a n d d i l u t e d to a d e f i n i t e v o l u m e . T h e P F U of t h i s s o l u t i o n was d e s i g n a t e d as t h e t i t e r of t h e u n a d s o r b e d virus. F o r t h e e s t i m a t i o n of t h e p e r c e n t of s p o n t a n e o u s l y e l u t e d v i r u s t h e cells o n w h i c h t h e a d s o r p t i o n of t h e viruses w a s m a d e a t 4 ° C were w a s h e d t h r e e t i m e s w i t h P B S h e a t e d to 37 ° C a n d t h e n p l a c e d a t 37 ° C for 60 m i n u t e s w i t h l 0 ml P B S . A f t e r this, t h e t i t e r of t h e e l u t e d v i r u s w a s d e t e r m i n e d b y t h e p l a q u e m e t h o d . Cells n o t t r e a t e d w i t h t h e n e u r a m i n i d a s e s , o n w h i c h N D V or P I - 3 were a d s o r b e d a n d t r e a t e d as a b o v e were u s e d as controls. T h e p e r c e n t of a d s o r b e d viruses was e s t i m a t e d b y t h e t i t e r s of t h e u n a d s o r b e d v i r u s e s f r o m u n t r e a t e d a n d n e u r a m i n i d a s e t r e a t e d cells a n d t h e t i t e r of t h e i n p u t viruses. The Estimation o/Quantity o/Cell-Bound N A N A T h e q u a n t i t y of b o u n d N A N A of t h e cells before a n d a f t e r t r e a t m e n t w i t h t h e e n z y m e n e u r a m i n i d a s e was m a d e b y h y d r o l y s i s of t h e cells w i t h 0.1 mT sulfuric acid a t 80 ° C for 60 m i n u t e s . T h e free N A N A w a s d e t e r m i n e d w i t h t h i o b a r b i t u r i c acid (1). C o u n t i n g of t h e cells w a s m a d e w i t h e r y t h r o s i n t3 (14).

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301

Treatment o/the Cells With Trypsin C E F cells in monolayer were treated with 0.007 per cent trypsin (Difco) for~30 minutes at 37 ° C. After this the cells were washed 3 times with PBS, infected with NDV, and then treated as described above for determination of unadsorbed virus.

Statistical Evaluation Statistical evaluation of the results was made by the Student-Fisher method.

Results

The El]oct o/Neuraminidase Treatment o/the Cells on the Adsorptio~ o / N D V and P]-3 T h e t r e a t m e n t of C E F , B H K or CK cells for 30 m i n u t e s w i t h Vibrio choleras n e u r a m i n i d a s e (VCN) caused a b o u t 20 per cent e n h a n c e m e n t of t h e a d s o r p t i o n of the i n p u t N D V or P I - 3 (Table 1). T r e a t m e n t of t h e same cells w i t h influenza virus n e u r a m i n i d a s e (IVN) diminished t h e per cent of adsorbed virus. Table 1. The e//ect o/30 minutes treatment with di/]erent neuraminidases on the adsorption

o / N D V and PI.3 Unadsorbed virus

Cells

Treatment with

Virus

0.6 ml of the dilution

Number of plaques~

Per cent adsorption of the input~

Cb

Dc

10a l0 a 103 104 104 104 t02 t02 10~

103=~5 59=t=5 170_!=6 92±5 55±5 186 ± 9 51~:3 30±3 80~:8

54±2.0 72-f-2.0 25~-2.2 50=]=2.3 70=t=2.8 26 ± 3.0 53~3.0 72±3.0 27~1.0

-7 8 -l0 11 -12 9

-18 29 -20 24 -t9 26

Significance

CEF CEF CEF BHK BHK BtIK CK CK CK

-VCN IVN

IVN

NDV NDV NDV NDV NDV NDV NDV NDV NDV

CK

--

PI-3

102

44~4

50±3.0

--

--

CK CK

VCN IVN

PI-3 PI-3

102 102

24±3 64±3

73±3.0 26±3.0

12 12

23 24

--

VCN IVN -VCN

Mean value and standard error from 3 experiments b Coefficient for 95 per cent statistical significance c The difference between the per cent of adsorbed virus on neuraminidase-treated and control cells T h e d a t a in t h e table show t h a t t h e differences in t h e per cent e l adsorbed virus on control a n d n e u r a m i n i d a s e t r e a t e d cells are significant. T h e t i t e r of e x t r a c e l l u l a r N D V from V C N - t r e a t e d cells was 6 . 0 4 - 0 . 2 X 106 P F U / m l a n d t h a t of c o n t r o l s - - l . 0 _-t=0.2× 106 P F U / m l . T h e t i t e r of cell-bound N D V f r o m t r e a t e d cells was 6 . 0 ± 0 . 1 0 × 1 0 7 P F U / m l a n d t h a t of c o n t r o l - 3.0±0.10×107pFU/ml. T h e t i t e r of s p o n t a n e o u s l y eluted virus from Vibrio choleras n e u r a m i n i d a s e t r e a t e d ceils was two times higher t h a n t h e t i t e r of t h e same virus in control cells (6.1 - i 0 . 1 0 × 103 P F U / m l a n d 3.6 ~ 0 . 1 0 × 103 P F U / m l ) .

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LII~IA WASSILEWA:

The results o b t a i n e d with B H K cells in suspension showed t h a t the a m o u n t of virus adsorbed on VCN ~reated cells also in this system was a b o u t 25 per cent higher t h a n t h a t of control cells.

Hydrolysis o/the Cell Associated N A N A D e t e r m i n a t i o n of the b o u n d N A N A before a n d after 30 m i n u t e s t r e a t m e n t of the cells with VCN showed t h a t the enzyme split 2 5 - - 3 0 per cent of the b o u n d N A N A (Table 2). Table 2. Splitting o/cell-bound NANA by Vibrio eholerae neuraminidase Bound NANA ( ~g/106 cells) Cells

Before enzyme treatment

After enzyme treatment

Per cent of splitting

CEF BHK CK

1.66-t-0.10 t.40~-0.12 2.60±0.10

1.15~0.07 1.05:[: 0.06 1.90~0.12

30~2 25±3 27~i

The Influence o/Vibrio eholerae Neuraminidase Exposure Time on ND V Adso~Ntion The C E F were treated for different time periods with Vibrio cholerae neuraminidase. The per cent of u n a d s o r b e d virus was increased after a prolonged action of the enzyme (Fig. 1).

100

>

o 80 LU m

g m Q

=z Go z

m a-

40

20

TIME

OF

TREATMENT

(rnin)

:Fig. I. The per cent of unadsorbed virus from neuraminidase t,re~ted (. control cells (. .............................) -~ Standard error

• ) and

Cell Receptors for Paramyxoviruses

303

Speeqicity o/the Neuraminidase Effect on Virus Adsorption T h e cells were t r e a t e d 30 m i n u t e s w i t h a m i x t u r e of 5 units Vibrio cholerae n e u r a m i n i d a s e a n d 200 ~xg N - a e e t y l - n e u r a m i n lactose (a p r o d u c t of Sigma, conraining 75 p e r cent 2 - - 3 ' isomer a n d 25 p e r cent 2 - - 6 ' isomer). T h e r e a f t e r t h e cells were t r e a t e d as described in Materials a n d Methods. N - a e e t y l - n e u r a m i n lactose i n h i b i t e d t h e effect of Vibrio cholerae n e u r a m i n i d a s e on virus a d s o r p t i o n (Table 3).

The Effect o] Trypsin Treatment o[ the Cells on the Adsorption o / N D V T r e a t m e n t of the C E F m o n o l a y e r with 0.007 p e r cent t r y p s i n caused a red u c t i o n in t h e a m o u n t of i n p u t virus a d s o r b e d from 54 per cent to 24 p e r cent. This difference is s t a t i s t i c a l l y significant. Table 3. Inhibition o] the effect o/Vibrio cholerae neuraminidase on the adsorption o/ N D V Unadsorbed virus~, Cells CEF CEF CEF BHK BHK BHK

Treatment with

Per cent of the inpu~

PFU/ml 1.6=L0.20 × 9.84- 0.08 × 1.6 ~=0.20 × 1.8=t=0.10 × 1.2±0A3 × 1.7 ~- 0.15 ×

- -

VCN b VCN-~-NANL c - -

VCN VCN q- N A N L

105 10a 105 10~ 10~ 106

48-t-2 28± 2 48~ 2 44~= 2 30~ 3 43 =t=3

a Mean value and standard error from 3 experiment,s b Vibrio cholerae neuraminidase c N-aeetyl-neuramin lactose

D i s e u s s i o n

T r e a t m e n t of living cells w i t h t h e e n z y m e n e u r a m i n i d a s e removes N - a c e t y t n e u r a m i n i e acid from t h e cell surface. This d i r e c t a c t i o n of t h e e n z y m e c a n n o t e x p l a i n our results, because t h e r e c e p t o r p r o p e r t y of a g l y e o p r o t e i n is d e t e r m i n e d o n l y b y t h e t e r m i n a l N A N A a n d t h e rest of the g l y c o p r o t e i n molecule does n o t p l a y a n y essential role (9). Besides the direct action of the enzyme, t h e r e is ind i r e c t action, which has n o t been well studied. The e n z y m e r e m a i n s on t h e surface of t h e t r e a t e d cells even if t h e y are well washed (16) a n d can split new molecules, which c o n t a i n N A N A ; t h e t r e a t e d cells show a n a l t e r a t i o n in t h e t r a n s m e m b r a n e m o v e m e n t of ions, sugars a n d a m i n o acids which s t i m u l a t e cell g r o w t h a n d division (17); t h e r e m o v a l of sialie acid m a y cause e o n f o r m a t i o n a l changes in cell surface g l y c o p r o t e i n s (19). This i n d i r e c t effect of t h e e n z y m e m a y e x p l a i n also t h e o b s e r v e d e n h a n c e m e n t of N D V a d s o r p t i o n o n t o cells a f t e r t r e a t m e n t w i t h VCN. A c c o r d i n g to W E i s s (20), N A N A p r o b a b l y is a r r a n g e d in clusters on a single p r o t e i n m o l e c u l e - - 3 0 N A N A residues for one g l y c o p h o r i n molecule (13). These clusters h a v e a poisson d i s t r i b u t i o n a n d r e p r e s e n t regions w i t h a high dens i t y of anionic groups such as f o u n d on mierovilli (20). I t is known, t h a t for t h e i n t e r a c t i o n of virus w i t h t h e cell, e l e c t r o s t a t i c forces (2, 18) are v e r y i m p o r t a n t , t h u s regions w i t h a high d e n s i t y of anionic groups would be m o r e a c t i v e in t h e

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LILIA WASSILEWA:

adsorption of virus. Brief t r e a t m e n t of the cells with VCN probably diminishes the density of anionic groups in the clusters b y removal of some N A N A residues. Transmembrane m o v e m e n t of ions in VCN-treated cells (17) m a y cause the appearance of new groups on the surface of cells, which m a y participate in virus adsorption. According to HUA~'G (8) the myxoviruses (fowl plaque virus) have positively and negatively charged groups on their surfaces and can be adsorbed on cationic and anionic surfaces. I n natural membranes, there are several charged groups (phosphate, amino and q u a r t e r n a r y amino, guanido, earboxyl groups) which in addition to neuraminie acid, are also potential receptors for the myxoviruses. Our results are in agreement with the above findings and show t h a t for the paramyxoviruses the ionic charge of the cell surface and the arrangement of the charged groups are the main factors in determining the efficacy of virus adsorption. Our results t h a t N D V adsorption is decreased after prolonged action of VCN are also in agreement with this suggestion because the main charged groups of the cell s u r f a c e - - M A M A m o l e c u l e s - - a r e removed. The inability of the influenza virus neuraminidase to " u n m a s k " other charged groups in our studies perhaps depends on the specificity of the virus enzyme preferentially to split 2-3-1inked N A N A (5), which splitting p r o b a b l y is incomplete and cannot cause rearrangement of the charged groups on the cell surface. I t must, be taken into account, t h a t the commercial nenraminidase from influenza virus contains small amounts of proteolytic enzymes as impurities deriving from the methods of preparation of the virus enzyme (6). The dat~ t h a t mild trypsin t r e a t m e n t of the cells diminishes the adsorption of the paramyxoviruses due to the release of glycoproteins from the cell surface (10) support this possibility. The data concerning the increased q u a n t i t y of spontaneously eluted N D V and the higher titer of extracellular and cellbound N D V after t r e a t m e n t of the celts with Vibrio cholerae neuraminidase are in agreement with the suggestion (11 ) t h a t the a m o u n t of eluted, penetrated and cell-associated virus depends on the q u a n t i t y of the a t t a c h e d virus. References 1. ASIINOF~', D.: Methods for" quantitative estimation of N-acetyl neuraminie acid and their application to hydrolysates of sialomueoids. Biochem. J. 81, 384---392 (1961). 2. ALLISO~¢,A. C., VALEnTIne,, R. C.: Virus particle adsorption. I I I . Adsorption of viruses by cell monot~yers and effects of some variables on adsorption. Biochim. biophys. Acta 40, 400--410 (1960). 3. BAI~rgY, R. D. : Factors which affect the release of Newcastle disease and Sendai viruses from infected allantoie cells. J. gen. Mierobiol. 39, 229--238 (1965). 4. DRZENIEK, R. : Differences in splitting capacity of virus and V. cholerae neuraminidase on sialie acid type substrates. Biochem. biophys. Res. Commun. 26,631 638 (1967). 5. DRZENIEK, R. : Viral and bacterial neuraminidases. Curt. Top. Mierobiol. Immunol. 59, 34--74 (1972). 6. DP~ZENIEI~,R. : Neuraminidases of myxoviruses. Behring Inst. Mitt,. 55, 1--10 (1974). 7. HA~r~',R. F., STEWART,R. C. : Role of siMic acid receptors in adsorption of influenza virus to chick embryo cells. J. Immunol. 94, 842--851 (1965). 8. I-IUA~O, R. T. C. : Adsorption of influenza virus to charged groups on natural and artificial surfaces. Med. Mierobiol. Immunol. 159, 129--135 (1974). 9. H~rANe, ~. T. C., ROTT, R., KnE~-K, tt.-D. : On the receptor of influenza viruses. I. Artificial receptors for influenza virus. Zeitsehr. Naturforsch. 28 C, 342 345 (1973).

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10. KAPEY,LEI~, M., GAL-0Z, R., G~OV~R, N. B., DOL$ANSKI, F. : Natural shedding of carbohydrate-containing macromolecules from cell surfaces. Exp. Cell Res. 79, 152--158 (1973). i 1. LIPKIND, M. A., URBAKtt, V. YU. : Quantitative studies on the dependance of NDV penetration and elution on the attachment multiplicity. Arch. ges. Virusforsch. 44, 1--13 (1974). 12. MAt~CUS, P. J., SALB, J. M., SCttWAI¢ZTS, V. Cx.: Nuclear surface N-acetyl neuraminie acid terminating receptors for myxovirus attachment. Nature 208, 1122-1124 (1965). t3. NICOLSON, G. L., PAINTER, R. G. : Anionic sites of h u m a n erythrocyte membranes. II. Antispeetrin-induced transmembrane aggregation of the binding sites for positively charged colloidM particles. J. Cell Biol. 59, 395--406 (1973). 14. PmLLIPS, H. J., TEa~.¥BEaR¥, J. E. : Counting actively metabolizing tissue cultured cells. Exp. Cell Res. 113, 341---347 (1957). 15. RUSSEFF, Cm: Abwandlung des ]?seudogeflfigelpest-Virus dureh fortlaufende Passagen in Gewebekulturen. Zbl. Bake. I Orig. 184, 403--411 (1962). 16. SEDLACEK, ~I. H., SEILEt~, t ~. R. : Demonstration of Vibrio-cholerae neuraminidase (VCN) on the surface of VCN-treated cells. Behring Inst. Mitt. 55, 254--257 (1974). 17. }L~xttERI, A., RUOSLAItTI, E., NOODLING, S. : Neuraminidase stimulates division and sugar uptake in density-inhibit~ed cultures. Nature (New Biol.) 238, 211--212 (1972). 18. VAJ,EN~IN~, R. C., ALLISON, A. C. : Virus particle adsorption. I. Theory of adsorption and experiments on the attachment of particles to non-biologicM surfaces. Biochim. biophys. Acta 34, 10--23 (1959). 19. WARaEN, L., BUell, C. A. : The synthesis and repair of the cell surface. Behring Inst. Mitt. 55, 224--230 (1974). 20. WEIss, L. : The topography of cell surface sialic acids and their possible relationship to specific cell interactions. Behring Inst. Mitt. 55, 185--193 (1974). Author's address: Dr. LILIA W~tSSILEWA, Central Veterinary Research Institute, Boul. P. Slavejkov 15, Sofia 1606, Bulgaria. Received February 9, 1976

Cell receptors for paramyxoviruses.

Archives of Virology Archives of Virology 54, 299--305 (1977) © by Springer-Verlag 1977 Cell Receptors for Paramyxoviruses By LILL~ WASSIL~WA Depa...
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