A r c h i v e s of Virology 49, 8 1 - - 8 7 (t975) © b y S p r i n g e r - V e r l a g 1975

Effect of DEAE-Dextran on Protein Synthesis in HeLa Cells By J. L. SABOtCIO1, K. J. WIEGERS 2, and G. K o c ~ Roche Institute of Molecular Biology, Nutley, New Jersey, U.S.A. With 3 Figures Accepted June 20, 1975

Summary Concentrations of DEAE-dextran which are currently used to stimulate the interaction between animal cells and natural or synthetic polynucleotides, inhibit protein synthesis in H e L a cells. This inhibition results from the blocking of more than one of the steps in protein synthesis, including transport of amino acids. The results support the hypothesis that enhancement of cellular competence for infection by viral RNA involves interference with initiation of cellular protein synthesis.

Introduction Exposure of tissue culture cells to diethylaminoethyl (DEAE)-dextran is currently used to enhance the competence of cells for infection b y viral I~NA (6, 16) and to stimulate the interaction of animal cells 1. with viral and non-viral natural I~NAs an,/[ 2. with synthetic polynucleotides to induce the synthesis of interferon (I, 17).

In the HeLa

eell-poliovirus system, DEAE-dextran

enhances the infectivity

of virus-specific RNAs up to ten thousand-fold. Part of this effect is due to an increase in the adsorption of the RNAs to the cells (5, 6, 13, 15, 18, 19). I~owever, an increased adsorption alone cannot account for the increment in infectivity, since adsorption is maximal at relatively low concentrations of the polycation (10--20 ~g/ml), while the optimal eonceiltration for plaque formation is ten times higher (4--6, i3, 15, 18, 19). Incubation of virus R N A and DEAE-dextran at 37 ° C results in the formation of complexes which are less infectious than free I~NA (3), while preincubation of the cells with DEAE-dextran is followed b y an increase in R N A infectivity (4--6, 13, 15, 18, 19). Therefore, we postulated that 1 Present. address: ]3epartment of Microbiology, The Wallenberg Laboratory, Dag Hammerskjolds V/~g 21, S-752 37 Uppsala, Sweden. 2 Present address: tIeinrieh Pette Institut fgtr Experimentelle Virologic und Immunologic, Martinistral3e 52, ]3-2000 Hamburg 20, FederM Republic of Germany. Arch. Virol. 49/1

6a

82

J . L . SABORm, K. J. WIEGERS, and G. K o c h :

the p r i m a r y effect of D E A E - d e x t r a n is exerted on the cells r a t h e r t h a n on the I~NAs. I n a s t u d y aimed a t u n d e r s t a n d i n g the m e c h a n i s m b y which D E A E - d e x t r a n enhances the competence of I t e L a cells for infection, we f o u n d t h a t , w i t h i n a certain c o n c e n t r a t i o n range, this p o l y c a t i o n inhibits p r o t e i n synthesis a n d conc o m i t a n t l y enhances cellular competence for infection b y viral RNA. Some characteristics of this i n h i b i t i o n are reported i n this paper.

Materials and Methods Cell Propagation tIeLa cells were grown in suspension in Eagle's minimuJn essential medium (MEM), supplemented with 5 per cent fetal calf serum; cell cultures were maintained at cell densities between 2 4 × 10a eells/ml.

Incorporation Studies Cell cultures were harvested at a density of 4 × 105 eells/ml, centrifuged, and the cells resuspended in MEM at a density of 4 × l0 Geells/ml (concentrated cell suspension). DEAE-dextran and radioactive amino acids were added as described in the legends. All incubations were carried out at 37 ° C. To measure the uptake of labeled amino acids into the cellular acid soluble pool, duplicate 0.5 ml portions of the celt suspension were centrifuged at 3000 × g for five minutes, the supernatant was drained and the pellet carefully rinsed with distilled water. The cell pellets were dissolved with 0.5 ml 0.1 ~ NaOH, a n d aliquots of the solutions neutralized, precipitated with one volume of 10 per cent trichloracetic acid, and centrifuged to separate the acid soluble and acid precipitable fractions. The extracellular phase in the pellets was measured with [14C]labeled inulin. Radioactivity in the acid soluble fraction was measured b y using aquasol as scintillation fluid ; incorporation of radioactive amino acids into protein was measured by the filter paper disc method (9).

Source o/Materials L-[Sill-labeled amino acid mixture was obtained from Schwarz Bioreseareh; L-[5-3H[arginine with a specific activity of 8.9 Ci/mmole, and L-[U-14C]glutamic acid with a specific activity of 285 mCi/mmole were obtained from Amersham/Searle; [carboxy-14C] inulin with a specific activity of 1.1 mCi/g was obtained from Calbiochem. DEAE-dextran with a molecular weight of 2 × 106 was a product of Pharmacia; aquasol was a product of New England Nuclear.

Results Figure 1 shows the effect of four different c o n c e n t r a t i o n s of D E A E - d e x t r a n on the i n c o r p o r a t i o n of labeled a m i n o acids into HeLa cell protein. A t a concentration of 15 [xg D E A E - d e x t r a n / m l , labeled a m i n o acids are incorporated a t a rate corresponding to 78 per cent of the control t h r o u g h o u t the e x p e r i m e n t . . A t 60, 120 a n d 180 ~g D E A E - d e x t r a n / m l , the rate of i n c o r p o r a t i o n is the same d u r i n g the first t e n m i n u t e s of i n c u b a t i o n , representing 60 per cent, of the control; after t e n m i n u t e s , there is a change in the rate for all three concentrations, the new rate being 30 per cent of the control i n the ease of 60 ~g D E A E - d e x t r a n / m l , a n d 16 per cent of the control for b o t h 120 a n d 180 ~g D E A E - d e x t r a n / m l . As shown in Table 1, when D E A E - d e x t r a n a n d labeled a m i n o acids are added to the cells at the same time, there is a n i n h i b i t i o n in the i n c o r p o r a t i o n of

Effect of D E A E - D e x t r a n on P r o t e i n Synthesis

83

labeled amino acids into the eelhdar acid soluble pool, although this inhibition is lower than the inhibition in the incorporation into protein. The differences in the extent of inhibition of both amino acid transport and protein synthesis are more pronounced when the amino acids are added 15 minutes after the addition of

4.0

'~ 3.0 O_ X

E

~2.o 1.0

I

I 20

I

1 40 MINUTES

t

I 60

Fig. 1. Effect of D E A E - d e x t r a n on the incorporation of radioactive amino acids into H e L a cell protein Aliquots of 0.9 m l of a c o n c e n t r a t e d suspension of t t e L a cells were m i x e d with 0.1 mI of saline solution (0.15 ~ NaC1, 0.03 lvi Tris/tIC1, pI-I 7.2) containing 5 ~Ci of [3H]labeled a m i n o acid m i x t u r e and D E A E - d e x t r a n to o b t a i n various final concentratinos. A t the times indicated, duplicate 50 ~I samples were t a k e n to measure incorporation of r a d i o a c t i v i t y into protein. , - - , - - - , no D E A E - d e x t r a n ; ~-a-a, 15 ~g D E A E - d e x t r a n / m l ; =--D--~, 60 ~.g D E A E - d e x t r a n / m l ; o - - o - - o , 120 ~g D E A E - d e x t r a n / m l ; A---A--A, 180 ~g D E A E . d e x t r a n / m l Table 1. EHect o] DEAE-dextran on the transport o/ labeled amino acid,s into the acid soluble pool in HeLa cells and on the incorporation o/ labeled amino acids into protein Concentration of

Incubation time

DEAE-dextran

5 minutes

~g/Inl

cpm

0.0 15.0 60.0 120.0

Ae. sol. 5757 (100) 4763 (82.7) 4140 (71.9) 4120 (71.5)

15 minutes

Protein 549 (100) 381 (69.3) 312 (56.8) 300 (54.6)

Ae. sol. 7200 (100) 5960 (82.7) 4470 (62.0) 4420 (61.3)

Protein 1428 (t00) 1080 (75.6) 649 (45.4) 609 (42.6)

P o r t i o n s of 2.7 m l of a c o n c e n t r a t e d suspension of R e L a cells were m i x e d w i t h 0.3 m l of saline solution containing 15 ~C [SH]-labeled amino acid m i x t u r e plus different a m o u n t s of D E A E - d e x t r a n . 5 a n d 15 minutes after t h e additions, duplicate samples were t a k e n in order to measure the incorporation of labeled amino acids into protein and the acid soluble pool as described under " M e t h o d s " . All figures represent the r a d i o a c t i v i t y corresponding to 50 y.1 of cell suspensions. Figures in parentheses indicate percent of control Arch. Virol. 49/1

6b

J . L . SABORIO,K. J. WIEGERS,and G. K o e ~ :

84

D E A E - d e x t r a n (Fig. 2 a). Experiments were performed in the presence of DEAEdextran to test if a positive or a negative charge of the labeled amino acid had a n y influence on transport and on the incorporation of label into protein. When labeled glutamic acid was used, the results were qualitatively similar to the ones obtained with the amino acid mixture (Fig. 2b); however, when arginine was the labeled amino acid, incorporation of radioactivity into the acid soluble pool was not inhibited, although the rate of incorporation into protein was inhibited about 50 per cent b y both 60 and 120 [zg DEAE-dextran/m] (Fig. 20). b

I00

80

"E 6O

8

40

ZO

,

[

30

f

I

60

l

I

90

L

I

I

I~'0

~

I

-~0 6 0

I

I

90

I

I

120

1 ~ I I I I I 30 6 0 9 0 t20

fitg DEAE d s x t r a n / m f

Fig. 2. Effect of D E A E - d e x t r a n on the i n c o r p o r a t i o n of radioactive amino acids into protein and the acid soluble pool in I-IeLa cells A l i q u o t s of 2.0 ml of a consent.rated suspension of H e L a cells were i n c u b a t e d for 15 m i n u t e s w i t h 0.2 ml saline solution (control cells), or w i t h different concentrations of D E A E - d e x t r a n , prior to the a d d i t i o n of : a) 5~ Ci [3H] -labeled amino acid m i x t u r e / m l , b) 2.5 i~Ci [U-laC] g]utamic acid/ml, and c) 10 ,~Ci [5-~I-I[ arginine/ml. F i v e m i n u t e s after the a d d i t i o n of the labeled amino acids, duplicate 0.5 ml samples were t a k e n to measure r a d i o a c t i v i t y in the acid soluble pool ; the rate of incorporation into protein was m e a s u r e d as indicated in F i g u r e 1. o---o---o, acid soluble r a d i o a c t i v i t y ; ~ - - = - - n , rate of i n c o r p o r a t i o n of labeled a m i n o acids into protein

To study the reversibility of the inhibition produced b y DEAE-dextran, cells were incubated for 15 minutes with DEAE-dextran, collected by centrifugation, resuspended in fresh medium and incubated to measure the incorporation of labeled amino acids into protein and the acid soluble pool. As shown in Figure 3, a complete recovery of both amino acid transport and protein synthesis was obtained with the cells incubated with 15 ~g DEAE-dextran/ml, whereas only a partial recovery was obtained in the case of 60 and 120 [zg DEAE-dextran/ml. Discussion The results reported in this paper indicate that DEAE-dcxtran inhibits the transport of labeled amino acids into the cellular acid soluble pool, although the extent of inhibition of transport differs depending on the amino acid tested (Table 1,

Effect of DEAE-Dextran

on Protein Synthesis

85

I00

8O

c

oo

6O

4O

m

I

0

30

=

1

i

60

I

120

~g DEAE-dextran/rnl

Fig. 3. Reversibility of the effect of D E A E - d e x t r a n on the incorporation of labeled amino acids into protein and acid soluble pool in t I e L a cells Aliquots of 10.0 ml of a concentrated suspension of H e L a cells were incubated for 15 minutes with 1 ml saline solution (control cells), or with 15, 60 or 120 y,g D E A E dextran/ml ; 40 ml of cold MEM were added to each sample, and the cells collected b y centrifugation. The celt pellets were resuspended in t0.0 ml of warm MEM, and the suspension incubated for 20 minutes at 37 ° C. Portions of 2.0 ml of these suspensions were used to measure incorporation of radioactive amino acids into protein and into the acid soluble pool as indicated in Figure 2 a o---c--o, aeid soluble radioactivity; - - D - - ~ - - , rate of incorporation of labeled amino acids into protein. The values obtained with control ceils, no~ centrifuged, are represented by the filled symbols

Figs. 2a, b, e). F r o m these e x p e r i m e n t s , it is n o t y e t possible to d e t e r m i n e to w h a t e x t e n t t h e i n h i b i t i o n of p r o t e i n synthesis b y D E A E - d e x t r a n is due to the r e d u c e d t r a n s p o r t of a m i n o acids. The results o b t a i n e d w i t h l a b e l e d arginine (Fig. 2c) clearly show t h a t some other step(s) of p r o t e i n synthesis is i n h i b i t e d also b y the p o l y c a t i o n , since in this ease i n e o r p o r a t i o n of labeled amino acids into p r o t e i n is inhibited, whereas no effect on its t r a n s p o r t into t h e acid soluble pool was d e t e c t e d . A n analysis of t h e effect of D E A E - d e x t r a n on the r i b o s o m a l profiles was carried o u t in a n a t t e m p t to i d e n t i f y t h e step(s) of p r o t e i n synthesis b l o c k e d b y t h e p o l y cation. Control e x p e r i m e n t s , however, showed t h a t i n c u b a t i o n of c y t o p l a s m i c e x t r a c t s (from u n t r e a t e d cells) with 50 b~g D E A E - d e x t r a n / m l , in vitro, results in non-specific p r e c i p i t a t i o n of polysomes, ribosomes, 60S r i b o s o m a l subunits, b u t n o t 4~0S s u b u n i t s when a n a l y z e d b y zonal eentrifugation. I n c u b a t i o n of H e L a ceils w i t h 15 or 150 Bg of D E A E - d e x t r a n / m l for 10 m i n u t e s a t 37 ° C a n d s u b s e q u e n t analysis of t h e c y t o p l a s m i c e x t r a c t s b y zonal centrifugation r e v e a l e d a decrease in t h e a m o u n t of polyribosomes, ribosomes a n d 60S r i b o s o m a l subunits. The 40S r i b o s o m a l subnnits r e m a i n e d unaffected. I t is conceivable t h a t D E A E - d e x t r a n b o u n d to the cell m e m b r a n e during i n c u b a t i o n w i t h cells r e m a i n s m e m b r a n e - b o u n d (18) a n d t h e n complexes w i t h ribosomes a n d p o l y r i b o s o m e s d u r i n g t h e p r e p a r a t i o n of cell extracts. 6b*

86

J . L . SABOam, K. J. ~¢VIEGER8,and G. KocH:

F u r t h e r experiments are necessary to clarify the mechanism b y which D E A E d e x t r a n inhibits protein synthesis in H e L a cells. The inhibition of protein synthesis exerted b y exposure of 4 × 106 cells/ml to either 60 or 120 ~tgDEAE-dextran/ ml is not readily reversible (Fig. 3). However, the exposure of 1 × 107 cells/ml to t0 per cent dimethylsulfoxide and 400 ~xg D E A E - d e x t r a n / m l for 6 minutes at 37 ° C prior to infection with poliovirus does not interfere with virus replication a n d does not result in a decreased yield of infectious virus particles (11). Therefore, the sensitivity of the synthesis of cellular and virM proteins to inhibitors of protein synthesis was recently compared in our laboratory. Experiments with H e L a cells at 2.5 hours after infection with poliovirus have revealed t h a t the s3mthesis of poliovirus proteins is less sensitive t h a n the synthesis of cellular proteins to inhibition b y D E A E - d e x t r a n and to specific inhibitors of initiation of protein synthesis, such as m e d i u m hypertonieity (10, 15), T P C K (L-1-Tosylamido-2phenylethyl chloromethyl ketone) (12) and cytochalasin B (8), b u t not to inhibitors of elongation, such as p u r o m y e i n and eycloheximide (manuscript in preparation). The exposure of cells to medium hypertonieity (15), dimethy]sulfoxide (14), ethanol (7), cytoehalasin B (8) or D E A E - d e x t r a n rapidly inhibits the initiation of cellular protein synthesis, b u t affects the rate of I~NA synthesis only after a lag of 2 0 - - 3 0 minutes (manuscript in preparation). Only a brief incubation of cells with either one of these inhibitors of protein synthesis is required to increase the competence of the cells for infection b y viral R N A . Therefore, we suggest t h a t the inhibition of initiation of cellular protein synthesis, b u t not of R N A synthesis, might be a prerequisite for an efficient enhancement of eellnlar competence for infection b y viral R N A .

Referenees 1. COLBY, C., CH,~iV£BEI~LAIN,1~I. J. : The specificity of interferon induction in chick embryo cells by helicM RNA. Proe. Nat. Aead. Sci. U.S.A. 63, 160--167 (1969). 2. DEMAEYER-GuIGNARD, J., DEIVIAEYEI¢,:E., MONTAGNIER,L. : Interferon messenger R N A : translation in heterologous cells. Proe. Nat. Aead. Sci. U.S.A. 69, 1203--1207 (1972). 3. ttAVLIZA, D., KOCH, G. : Complex formation between poliovirus R N A and polycations. Arch. Biochem. Biophys. 147, 85 91 (t971). 4. KocH, G. : Stability of polycation-induced cell competence for infection, by viral I~NA. Virology 45, 8 4 1 - 8 4 3 (1971). 5. KocH, G. : Interaction of poliovirus-speeifie RNAs with I-IeLa cells and E . coli. In : Current Topics in Mierobiol. and Immunol., 62, 89--138. Berlin-HeidelbergNew York: Springer 1973. 6. KocH, G., BISHOP, J. M. : The effect of polyeations on the interaction of viral R N A with mammalian cells: studies on the infectivity of single- and double-stranded poliovirus RNA. Virology 35, 9--17 (t968). 7. KocH, F., KocH, G. : l~eversible inhibition of maeromoleeular synthesis in HeLa cells by ethanol. Res. Comm. in Chem. Pathol. and Pharmacol. 9, 291--298 (1974). 8. KOCH, G., OP~'EI~I~fA2~:N,I-I. : Sensitization of I.ieLa cells for viral RNA infection by eytochMasin B. Virology 63, 395--403 (1975). 9. MANs, J. I~., NOVELLI, G. D. : Measurement of the incorporation of radioactive amino acids into protNn by a filter-paper disc method. Arch. Bioehem. Biophys. 147, 85--91 (1961). 10. Nuss, D. L., O~PEEMANN, H., KOCH, G.: Selective blockage of initiation of host protein synthesis in I~NA-virus infected ceils. Proc. Nat. Aead. Sci. U.S.A. 72, 1258--1262 (1975).

:Effect of D E A E - D e x t r a n o:rl Protein Synthesis

87

11. OPPERMAIglg,I-I., KOCH, G. : Kinetics of poliovirus replication in H e L a cells infected by isolated RNA. Bioehem. Biophys. Res. Comm. 52, 638--640 (1973). t2. PosrG, S.-S., Nuss, D. L., KooI~, G.: InhiMtion of Initia*ion of protein synthesis in mammalian tissue culture cells by L-l-tosyI-amido-2-phenyleLhyl ehloromethyl ketone. J. biol. Chem. 250, 2 4 0 2 4 5 (1974). 13. SABOI~IO,J. L., ZARucI~I, T., K o c g , G. : /Zeversible inhibition of protein synthesis in H e L a cells and its role in sensitization of cells for infection by viral RNA. ASM Proceedings, No. V- i78 (1973). 14. SABORIO, J. L., Koctt, G. : Reversible inhibition of protein synthesis in H e L a cells by dimethylsulfoxide. J. biol. Chem. 248, 8343--8347 (1973). i5. SABO•IO, J. L., Pos-G, S.-S., Koc~t, G.: Selective and reversible inhibition of initiation of protein synthesis in mammalian cells. J. tool. Biol. 85, 195--211 (1974). 16. VA~IEgI, A., PACA~O, J. S. : Observations on the assay of infeetious viral ribonueleie acid: effects of DMSO and D E A E - d e x t r a n . Virology 32, 84---92 (I965). 17. VILCEI4, J., BAt~K~AX, S. L., HAVELL, E. A.: Control of interferon synthesis: effect of diethyl-aminoethyl-dextran on induction by polyinosinie-polyeytidylie acid. J. Virol. 10, 614--621 (1972). 18. ~ENTZKY, P., KOCH, G.: Influence of polyeations on the interaction between poliovirus multistranded ribonueleie acid and H e L a eetls. J. Virol. 8, 35--40 (1971). I9. WIEGEI~S, K. J., KocI~, G.: Interaction of poliovirus-induced double-stranded I~NA with I-IeLa cells. Arch. Bioehem. Biophys. 148, 89--96 (1972). Authors' address: Dr. G. KocH, Roche Institute of Molecular Biology, Nutley, NJ07110, U.S.A. Received J u n e 12, 1975