transport inhibitor dipyridamole, and was correlated with entry of a normally impermeant solute (sucrose) into infected cells. These data suggest that the system.
Journal of General Virology (1990), 71, 673-679. Printed in Great Britain
673
Effect of herpes simplex virus type 1 infection on nucleoside transport in HeLa $3 cells G. Paid, ~* R. E. Handschumacher, 3 C. Parolin, 1 S. Stefanelli ~ and P. Palatini 2 l Institute of Microbiology, University of Padova Medical School, Via A. Gabelli 63, 35100 Padova, 2Department of Pharmacology, University of Padova Medical School, Largo Meneghetti 2, Padova, Italy and 3Department of Pharmacology, Yale University Medical School, 333 Cedar Street, New Haven, Connecticut, U.S.A.
The initial velocity of thymidine uptake was measured in HeLa $3 cells infected with herpes simplex virus type 1 (HSV-1). The rate of nucleoside influx into the cells was shown to increase from as early as 1 h postinfection (p.i.) up to 8 h p.i. This increased uptake was shown to be attributable to a progressively increasing contribution from passive diffusion superimposed upon normal transport. Thus, the specific nucleoside transport system was still operating with unaltered kinetic parameters 8 h after infection. Despite the inhibition of host cell protein synthesis and its replacement by the synthesis of virus-specified pro-
Introduction The process of nucleoside influx into cells has been shown to be mediated by a mechanism of facilitated diffusion both in erythrocytes and cultured cells (Plagemann & Wohlhueter, 1980; Paterson et al., 1981 ; Young & Jarvis, 1983; Paul et al., 1975; Cass et al., 1974; Jarvis & Young, 1980; Lauzon et al., 1977). Recently, a concentrative carrier-mediated transport of nucleosides has also been found in cells directly isolated from normal tissues (Spector, 1980, 1982, 1985; Schwenk et al., 1984; Jakobs & Paterson, 1986; Darnowski et al., 1987). Passive diffusion does not contribute significantly to nucleoside permeation under physiological conditions (Plagemann et al., 1988), an observation consistent with the hydrophilic nature of the nucleoside molecule. The properties of cell membranes can change quite drastically when cells are infected with viruses, leading to structural alterations of membranes themselves and to modifications in the cells' electric potential (Fritz & Nahmias, 1972; Carrasco & Esteban, 1982; Spear et al., 1970; Farnham & Epstein, 1963; Garry et al., 1979; Francoeur & Stanners, 1978). Moreover, the occurrence of membrane leakiness has been described to account for the entry of normally impermeant metabolites (Carrasco, 1977, 1978; Carrasco & Smith, 1976). Cell leakiness has a 0000-9264 © 1990SGM
teins, the numbers and affinity of the nucleoside transporters in cells 8 h after infection were virtually unchanged. The increased transport of thymidine in infected cultures was resistant to the nucleoside transport inhibitor dipyridamole, and was correlated with entry of a normally impermeant solute (sucrose) into infected cells. These data suggest that the system for the carrier-mediated facilitated diffusion of nucleosides remains intact in HSV-infected cells, but that progressively increasing passive diffusion takes place. Passive diffusion is the major process operating late after virus infection.
number of practical implications, particularly with respect to some aspects of drug metabolism under conditions of viral infection and the use of impermeant compounds as selective antiviral agents (Lacal & Carrasco, 1983; Benedetto et al., 1980). It is reasonable to assume that virus infection and leakiness, in particular, could also affect the uptake of those substances such as nucleosides, which normally only permeate through a transporter. Changes in the permeation of nucleosides could be relevant to the effectiveness of antiviral therapy, which is currently based on nucleoside analogues. The limited evidence that is available suggests a reduced nucleoside transport in cells infected with togaviruses or picornaviruses (Genty, 1975; Castrillo & Carrasco, 1986). However, the majority of these experiments were performed using techniques (filtration and washing) that do not permit measurement of the initial rates of solute influx. With these methods, the contributions from intracellular nucleoside metabolism and redistribution between intraceUular and extraceUular compartments are combined to prevent evaluation of effects on the uptake process. To gain further insight into the effects produced by viral infection on cell nucleoside permeation we have undertaken experiments on the transport of thymidine (TdR) by HeLa $3 cells infected with herpes simplex virus type 1 (HSV-1). TdR was used
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b e c a u s e m o s t a n t i v i r a l a g e n t s are r e l a t e d to it ( D o l i n , 1985) a n d HSV-1 w a s selected as t h e e x p e r i m e n t a l m o d e l b e c a u s e o f its s e n s i t i v i t y to n u c l e o s i d e a n a l o g u e s ( D e C l e r c q , 1982, 1989).
centrifugation (12000 g for 30 s) through the oil-paraffin mixture into TCA. The microfuge tubes were frozen in dry ice and ethanol and the tube bottom containing the cell pellet in TCA was sliced off. The intracellular incorporation of TdR was measured in a liquid scintillation spectrometer (LKB 1214 RackBeta). For inhibition experiments with dipyridamole HeLa $3 cells were preincubated at 21 °C for 20 min with the inhibitor.
Methods
Sucrose uptake and calculation of intracellular and extracellular space. HeLa $3 cells (5 x 106 cells/ml) were incubated for 2 min with increasing concentrations of [l~C]sucrose (from 0-1 to 1 ktCi), tritiated water (from 0.1 to 1 ~tCi)and [14C]inulin (from 0.1 to 1 p.Ci). At the end of the incubation cells were centrifuged through the oil-paraffin mixture, as previously described. Intracellular volume was calculated by subtracting the inulin space from the water space (Belt, 1983). Sucrose permeation was considered to have occurred when the space occupied by this molecule exceeded the extracellular space, as measured by radioactive inulin.
Radioisotopes and compounds. [14C]Sucrose (>540 mCi/mmol), [3H]TdR (14.2 Ci/mmol), tritiated water (1 Ci/ml) and inulin [14C]carboxylic acid (2 to 10 mCi/mmol) were purchased from Amersham. [3H]NBMPR (4 Ci/mmol; 6-[(4-nitrobenzyl)thio]-9-fl-Dribofuranosylpurine) was purchased from Maravek Biochemicals. NBMPR and dipyridamole were from Aldrich and Sigma, respectively. All reagents used in our experiments were AnalaR grade. Cells and culture conditions. All experiments reported in this paper were performed using HeLa $3 cells, a cell line adapted to growth in suspension. These cells allowed the measurement of the initial rates of nucleoside influx, an approach not feasible with adherent cells. HeLa S3 cells were normally grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% (v/v) foetal calf serum (FCS), 2 mMglutamine, 20 mM-HEPES and antibiotics. Cells were cultured in 25 cm 2 diameter roller bottles at 3.5 r.p.m, in a moist atmosphere of air and 5% CO2, starting with 5 x 104 cells/ml. Virus infection. HeLa $3 cells were infected with the wild-type (wt) strain of HSV-1 (Pall et al., 1988, 1989; Pal6 & Biasolo, 1988) at an m.o.i, of 20 p.f.u./cell. In some control experiments HeLa $3 cells were also infected with the HSV-1 thymidine kinase (TK)-deficient strains R100 (Palfi et al., 1988, 1989) and CL 101-TK 43 (Summers et al., 1983), which multiplied at the same rate in this cell line, as shown by one-step growth measurements. During the adsorption period (1 h) bottles were rotated at 1.5 r.p.m. Cells were then sedimented and resuspended in DMEM with 2% FCS in new bottles, which were again rotated at 3.5 r.p.m. At various intervals after infection cells were harvested and used for studying TdR uptake, titration of NBMPR binding sites and protein synthesis. At the end of each experiment virus growth was measured by titrating the yields of infectious progeny by plaque titrations on susceptible Vero cells (Pahi et aL, 1984). Determination of protein synthesis inhibition. HeLa $3 cells were infected with the wt strain at an m.o.i, of 20 p.f.u./cell and labelled with L-[3sS]methionine (> 1000 Ci/mmol) for each successive 2 h interval during the period from 0 to 8 h post-infection (p.i.). Cultures were labelled in DMEM containing one-tenth the normal amount of Lmethionine and 0.5% dialysed FCS. After the labelling period cells were lysed in Laemmli solution and the lysates separated by 12% SDSPAGE. Following the electrophoretic run, gels were fixed in a 15% TCA and 30% methanol solution, dried and the gel films scanned with an AMBIS automated beta scanning system (Automated Microbiology Systems) to evaluate the relative amount of radioactivity incorporated in each protein band. Determination ofthymidine uptake. Initial rates of thymidine uptake were determined according to the method of Young & Jarvis (1983), as described in detail below. Solutions of the nucleoside were prepared that contained a mixture of unlabelled and radioactive TdR (in a 300 to 1 molar ratio). Equal volumes (100 ~tl)of TdR and cell suspension (5 x 106 cells/ml) were simultaneouslymixed, using a dual syringe (Young & Jarvis, 1983), and dispensed in a series of 500 Ixlmicrohaemocytohaeter (microfuge) tubes placed in an Eppendorf centrifuge 5414. These ~ubes contained 50 Ixlof a (20% w/v) solution of TCA overlaid with 100 ~tlof a mixture of silicone oil and paraffin (density 1.13 g/ml). After various periods of time (0 to 180 s) the cell suspensions were sedimented by
Titration of NBMPR binding sites. HeLa $3 cells were incubated at 21 °C for 20 min with an increasing concentration of [3H]NBMPR (from 0-1 to 20 nM). After this time cells were centrifuged, as above, and radioactivity was measured in the cell pellets. Specific binding was estimated as the difference between [3H]NBMPR binding in the presence and absence of a 1000-fold excess of unlabelled NBMPR.
Results Inhibition o f protein synthesis A f t e r virus i n f e c t i o n , s y n t h e s i s o f cellular p o l y p e p t i d e s d e c l i n e d p r o g r e s s i v e l y f r o m 2 h o n w a r d s , as d e p i c t e d i n Fig. 1 a n d c o n f i r m e d b y a c o m p u t e r i z e d r a d i o m e t r i c a n a l y s i s o f r a d i o a c t i v e p o l y a c r y l a m i d e gels (not shown). A d r a s t i c i n h i b i t i o n o f t h e host cell p r o t e i n s y n t h e s i s i n H S V - l - i n f e c t e d cells b e g a n f r o m 4 h p.i., at w h i c h t i m e cells were still i n t a c t , as j u d g e d b y t r y p a n b l u e exclusion. A l t h o u g h t h e r e w a s a n a l m o s t 100~o r e d u c t i o n i n t h e s y n t h e s i s o f s o m e cellular p r o t e i n s , c o m p l e t e i n h i b i t i o n was n o t p r o d u c e d , e v e n at 8 h p.i. A c t i n s e e m e d to b e t h e least affected o f t h e w h o l e set o f cellular p r o t e i n s .
Transport studies Z e r o - t r a n s flux e x p e r i m e n t s were p e r f o r m e d at 1, 4, 8 a n d 24 h p.i. A t 24 h p.i. t h e m a j o r i t y o f cells c o u l d n o t be s e d i m e n t e d t h r o u g h t h e s i l i c o n e oil m i x t u r e , b u t rem a i n e d at the i n t e r f a c e b e t w e e n the m e d i u m a n d the l i p o p h i l i c solvent. T h e u p t a k e p r o c e s s was t h e r e f o r e o n l y m e a s u r e d at i n t e r v a l s f r o m 0 to 8 h p.i. I n b o t h i n f e c t e d a n d u n i n f e c t e d H e L a $3 cells t h e process o f T d R influx i n c r e a s e d l i n e a r l y i n t h e p e r i o d f r o m 2 to 20 s a f t e r a d d i t i o n o f t h e n u c l e o s i d e to t h e i n c u b a t i o n m e d i u m . T h e r e a f t e r the rate o f u p t a k e d e c l i n e d , r e a c h i n g a p l a t e a u after a b o u t 40 s (Fig. 2a). A d d i t i o n o f a large excess o f u n l a b e l l e d T d R a b o l i s h e d t h e l i n e a r u p t a k e t h a t occurred from 2 s onwards, but did not prevent the very r a p i d i n c r e a s e i n r a d i o a c t i v i t y o b s e r v e d w i t h i n t h e first
Nucleoside uptake in HSV-l-infected cells 5
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Fig. 1. Autoradiographic profile of proteins from uninfected (lane 1) and infected (lanes 2 to 5) cells. Cells were labelled for 2 h intervals from 0 to 2 h p.i. (lane 2), 2 to 4 h p.i. (lane 3), 4 to 6 h p.i. (lane 4) and 6 to 8 h p.i. (lane 5). 2 s. Therefore, it seems likely that a non-specific T d R association with the plasma m e m b r a n e , rather than a carrier-mediated uptake, was responsible for the increase in radioactivity measured during the initial 2 s. Accordingly, initial rates o f uptake were determined as the difference between the uptake at 15 and 5 s. The rates of T d R influx are expressed as the c h a n g e in the intracellular concentration of nucleoside (molarity) per s. As shown in Fig. 2(b) a threefold increase in the influx rate occurred after 8 h o f viral infection, although the increase in T d R influx was already evident as early as 1 h p.i.
Influx of sucrose The entry o f sucrose was measured in order to determine whether infected cells b e c a m e leaky to this normally
Fig. 2. (a) Kinetics of TdR uptake in mock-infected (Ak)and infected HeLa $3 cells at 8 h p.i. (0). TdR concentration was 500 ~tM. (b) Comparative rates of TdR ( ) and sucrose (---) entry into HeLa $3 cells at various times p.i.
i m p e r m e a n t molecule. In contrast to the negligible u p t a k e o f sucrose by uninfected cells ( < 0-1 ~tmol/5 x 10 s cells), sucrose uptake in cells at 8 h p.i. corresponded to 1-5 ~tmol per 5 x 10 s cells (0.2 ~tl sucrose per ~tl intracellular space, Fig. 2b). The increased uptake o f sucrose clearly paralleled the increased uptake o f T d R by infected cultures and clearly indicates a c h a n g e in the permeability o f infected cells.
Titration of NBMPR binding sites The results of N B M P R binding to uninfected H e L a cells and cells 8 h after virus infection are presented in Fig. 3(a) and (b) as Scatchard plots. Virtually the same maximal binding occurred in mock-infected and infected cells. This indicates that, even after 8 h, virus infection did not cause a reduction in the n u m b e r o f those nucleoside transporters capable of binding N B M P R (9.5
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Fig. 4. TdR uptake by HeLa $3 cells as a function of TdR concentration (a). Numbers in the figure (h p.i.) represent the time of cell exposure to HSV-I before uptake determination, which was measured between 5 and 15 s, as described in the text. Within this time the uptake was linear at all concentrations used. (b) Double-reciprocal plot of the data shown in panel (a).
x l 0 s a n d 8-9 x 105 per cell in u n i n f e c t e d a n d infected cells, respectively). P r e l i m i n a r y e x p e r i m e n t s h a d indicated t h a t the m a x i m a l i n h i b i t i o n o f T d R u p t a k e by N B M P R was 65 ~o, a n o b s e r v a t i o n t h a t is consistent with previous results ( P l a g e m a n n et al., 1988). T h e curvature of the b i n d i n g isotherms suggests the presence of at least two i n d e p e n d e n t b i n d i n g sites with different affinities for N B M P R . D i s s o c i a t i o n c o n s t a n t s for N B M P R , e v a l u a t e d from b i n d i n g isotherms by c o m p u t e r fitting, as
previously described ( W o h l h u e t e r et al., 1983), indicated that the overall affinity of N B M P R for the t r a n s p o r t e r was n o t c h a n g e d significantly by viral infection (Kd for the high affinity site was 1.3 x 10 -1° a n d 1.4 x 10 -1° M i n u n i n f e c t e d a n d infected cells, respectively).
Saturation kinetics experiments Fig. 4 shows the kinetics of T d R u p t a k e by infected a n d
Nucleoside uptake in HSV-l-infected cells uninfected cells as a function of TdR concentration. Consistent with the exclusive presence of a carriermediated influx, the uptake process reached a plateau in uninfected cells. In infected cells the transport kinetics underwent a dramatic change and the saturation curve did not reach a plateau. This alteration in the uptake process was evident as early as 1 h p.i. and was the major uptake process by 8 h p.i. Kinetic parameters of the influx process were determined by computer fitting (Roos & Pfleger, 1972) of the experimental data to the following equation: Y = (I'm x S/Km + [S])+ k x [S], where Y is TdR uptake, [S] is TdR concentration, Vmand Km are maximum uptake and Michaelis constant of the saturable component, respectively, and k is the firstorder rate constant for passive permeation. This analysis showed that k increased dramatically with the infection time, whereas Km and Vm showed little variation. In particular, these values before and after 8 h of cell exposure to HSV-I were 203 + 18 and 185 + 25 ~tMfor Kin; 0"10 + 0"01 and 0.11 + 0.01 IXMper second for Vm and0.01 x 10-4 + 0.002 x 10-4and2.1 x 10-4 _ 0.2 x 10-4 per second for k, respectively (means + S.D. of four determinations). The same data are represented in Fig. 4(b) as double-reciprocal plots. It can be seen that the plot is linear, as expected from pure Michaelis-Menten kinetics, only for uninfected cells. After infection the plots curved downwards, a result consistent with the presence of a passive diffusion component (Roos & Pfleger, 1972).
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Inhibition studies Further evidence for development of a carrier-independent entry of TdR was obtained by inhibition experiments with dipyridamole. This inhibitor specifically prevents the carrier-mediated uptake of nucleosides (Cass et al., 1981; Paterson et al., 1980; Eilam & Cabantchik, 1977; Aaronow et al., 1986), whereas the passive diffusion component should be unaffected. Dipyridamole was preferred to NBMPR for these experiments, since HeLa cells proved to be more sensitive to the former inhibitor (maximal inhibition was 90% at 1 IxM-dipyridamole, as previously observed; Paterson et al., 1980). Fig. 5(a) shows that 100 nMdipyridamole produced 67% inhibition of nucleoside uptake in uninfected cells. At progressively later times after virus infection, the proportion of uptake that was inhibited by dipyridamole decreased, indicating that the dipyridamole-sensitive component of the total uptake was becoming less and less important as the viral infection progressed. Fig. 5(b) is a representative experiment of TdR uptake in the presence and absence of 1 txM-dipyridamole at 8 h p.i. In the presence of a saturating concentration of the inhibitor the nucleoside
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Fig. 5. (a) Percentage inhibition of TdR uptake by 100 rtMdipyridamole in HeLa $3 cells at different times p.i. Uptake was determined between 5 and 15 s using a concentration of 500 IxM-TdR. (b) Concentration dependence of TdR uptake by HeLa $3 cells 8 h p.i. in the presence (closed circles) and absence (closed triangles) of 1 ~tMdipyridamole.
uptake was relatively resistant to the inhibitor and residual uptake was almost linear, again indicating the presence of a major contribution from a passive diffusion process.
Discussion The process of virus infection produces gradual alterations of the permeability and transport properties of the
678
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cell membrane. For example, certain molecules that do not enter the cell under normal conditions can cross the membrane barrier after viral infection. Conversely, some molecules that are actively transported into normal cells are no longer accumulated in infected cells (Lacal & Carrasco, 1983). The exact consequences of viral infection on the entry of nucleosides into cells have not yet been elucidated. In the present paper we have shown that the transport of TdR in HeLa $3 cells is greatly affected by infection with HSV-1. The first important observation is the increase in the net rate of TdR uptake that occurs after infection. As the initial rates of TdR influx were always measured, this phenomenon is probably a direct effect of modifications to the properties of the membrane and not secondary to intracellular changes in the activity of enzymes encoded by either the cell or the virus, such as nucleoside kinases, phosphorylases or phosphatases. Similar results were obtained using the HSV-1 TK-deficient strains R100 and CL 101TK 43, or after depleting the cells of ATP by arsenate treatment (Paterson et al., 1981 ; Wohlhueter et al., 1979) (not shown). After infection, as illustrated in Fig. 4, passive diffusion was gradually superimposed upon facilitated transport. Accompanying the increase of passive diffusion of TdR is the entry of sucrose, a normally impermeant molecule with approximately the same molecular size as the nucleoside. Inulin (6000K), a much larger polymer, is still excluded by the intracellular compartment under the same conditions. The increase in passive diffusion caused by HSV-1 infection correlates temporally with the virus-induced inhibition of cell protein synthesis. Saturation kinetics experiments provided evidence that the affinity of the nucleoside transporter for thymidine is not significantly changed by viral infection and that the maximum velocity of the carrier-mediated transport process is unaltered. This latter result is consistent with the observation that the total number of transporter molecules, as deduced from the maximum value of NBMPR binding, is not decreased in infected cells. These results suggest that the half-life of the transporter is quite long (certainly more than 8 h) and that the facilitated diffusion route is still operating in HSV-infected cells. Our results on the transport of TdR in HSV-l-infected HeLa $3 cells are somewhat at variance with data previously reported by other authors (Genty, 1975; Castrillo & Carrasco, 1986), showing a decrease in total nucleoside uptake after virus infection. However, these studies were carried out with different viruses (vesicular stomatitis virus and encephalomyocarditis virus). In addition, the experimental conditions were such that actual differences in transport could not be measured,
since uptake was assessed from data on incorporation and variation of nucleoside pools at times longer than 1 min. Influx of normally impermeant substances, especially low Mr aminoglycosides, has previously been reported in virus-infected cells (Lacal & Carrasco, 1983; Benedetto et al., 1980). This property led to the suggestion of a new approach to an antiviral chemotherapy with substances that are made permeant by virus infection. For those antiviral nucleosides which are transported via the facilitated diffusion system, it could be suggested that prior blockade with dipyridamole would prevent entry of cytotoxic agents into uninfected cells, but allow entry into the infected cell population. Further investigation into the nature of the damage produced by HSV-1 infection upon cell structures that are directly or indirectly involved in transport functions is certainly warranted. Such information may bear a potential relationship to viral pathogenicity and may also contribute to dissection of some functional properties of carriers whose structure has not been defined. The authors are indebted to Dr W. P. Summers for help and suggestions and to Mr M. Guida for technical assistance. This work was supported by grants from CNR, MPI, MS progetto A.I.D.S 1989, the U.S.P.S. (CA 45303 and 08341) and the American Cancer Society (CH67).
References AARONOW, B., TOLL, D., PATRICK, J., MCCARTAN, K. & ULLMAN, B. (1986). Dipyridamole insensitive nucleoside transport in mutant murine T lymphoma cells. Journal of Biological Chemistry 261, 14467-14473. BELT, J. A. (1983). Heterogeneity of nucleoside transport in mammalian ceils. Two types of transport activity in L1210 and other cultured cells. Molecular Pharmacology 24, 479-484. BENEDETTO, A., ROSSI, G. B., AMICI, C., BELARDELLI, F., CIOE, L., CARRUBA, G. & CARRASCO, L. (1980). Inhibition of animal virus produced by means of translation inhibitors unable to penetrate normal cells. Virology 106, 123-132. CARRmCO, L. (1977). The inhibition of cell functions after viral infection: a proposed general mechanism. FEBS Letters 76, 11-15. CARRASCO, L. (1978). Membrane leakiness after viral infection and a new approach to the development of antiviral agents. Nature, London 272, 694-699. CARRASCO, L. & SMITH, A. E. (1976). Sodium ions and the shut-offof host cell protein synthesis by picornaviruses. Nature, London 264, 807-809. CARRASCO, L. & ESTEBAN, N. (1982). Modification of membrane permeability in vaccinia-virus infected cells. Virology 117, 62-69. CASS, C. E., GAUDETTE,L. A. & PATERSON, A. R. P. (1974). Mediated
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Nucleoside uptake in H S V - I - i n f e c t e d cells
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(Received 7 September 1989; Accepted 20 November 1989)
