Effects of herpes simplex virus type 1 infection on the

Journal of General Virology(1994), 75, 3337 3344. Printedin Great Britain

3337

Effects of herpes simplex virus type 1 infection on the plasma membrane and related functions of HeLa $3 cells G. Palh, ~* M . A. B i a s o l o , ~ G. Sartor, 4 L. M a s o t t i , s E. Papini, 3 M . Floreani 2 and P. P a l a t i n i 2 Institute of Microbiology 1 and Departments of Pharmacology 2 and Experimental Biomedicine 3, University of Padova Medical School, Via A. Gabelli 63, 35121 Padova, and Institute and Department of Biological Chemistry, Universities of Parma 4 and Bologna 5, Italy

In this study we evaluated modifications of various structural and functional properties of the plasma membrane of HeLa $3 cells following infection by the lytic virus herpes simplex virus type 1 (HSV-1). Na+/K +ATPase activity considerably decreased during the first few hours post-infection (p.i.), whereas Na + and K ÷ concentrations were not significantly affected until a much later period. By 8 h p.i., a partial membrane depolarization in infected cells had occurred, as indicated by a small change in the transmembrane potential. HSV infection induced a time-dependent lipid per-

oxidation of HeLa cell plasma membranes temporally correlated with the progressive reduction in Na+/K ÷ATPase activity. Moreover, a significant decrease of membrane fluidity appeared at a late phase of the viral replicative cycle probably representing cumulative membrane damage. These results demonstrate that HSV-1 infection induced the production of free radicals in nonphagocytic cells. Since lipid peroxidation begins at an early stage of the virus replicative cycle, it may be directly related to viral cytopathicity.

Introduction

Gray et al., 1983), Bunyaviridae (Frugulhetti & Rebello, 1989), Picornaviridae (Carrasco & Smith, 1976; Castrillo & Carrasco, 1986) and Togaviridae (Garry et al., 1979). Although viral gene products have recently been implicated in the selective shut-off of host cell protein synthesis (Wycoff et al., 1992; Kwong & Frankel, 1989), changes in intracellular ion concentrations have been claimed to modulate the function of the eukaryotic ribosome (Carrasco & Smith, 1976; Perez & Carrasco, 1992). Variations in ionic flux during animal virus infection have also been linked to the cytolytic potential of the infectious agent and, more generally, to its ability to permeate cell membranes (Carrasco, 1977; Nair, 1981). On these grounds, an approach to a selective antiviral therapy, based on the uptake of non-permeative cytotoxic molecules by infected cells, has been proposed (Lacal & Carrasco, 1983; Benedetto et al., 1980). In the light of the above, it seemed interesting to investigate whether a correlation exists between viral cytolytic potential and impairment of membrane function crucial to maintaining cell viability. Our attention was focused on membrane effects produced by a strain of herpes simplex virus type 1 (HSV-1) on HeLa $3 cells, a lineage adapted to grow in suspension. In particular, we evaluated alterations in ion permeability, electrical potential and Na+/K +- and Mg2+-ATPase activities in relation to the degree of virus-induced lipid peroxidation and the fluidity of the plasma membrane.

Cytopathogenicity of viruses is related to a series of events starting as soon as the infectious particle has reached the plasma membrane to begin its replicative cycle (Roizman, 1990). Many of the events involved are still unknown, but they are crucial to an understanding of the mechanisms leading to progressive failure of cellular functions, including selective inhibition of the host macromolecular syntheses, changes in cell polarity, permeability and morphology (Carrasco, 1977, 1978; Carrasco & Smith, 1976; Carrasco & Esteban, 1982; Fritz & Nahmias, 1972; Sonenberg, 1990; Etchinson et al., 1982). Since the initial and terminal stages of viral infection are accomplished at the cell membrane (Roizman, 1990), loss of membrane functions such as molecular recognition and transport, signal transduction, or maintenance of selective ionic fluxes could account for cytopathogenic effects induced by the replicating virus. Membrane-related changes following virus infection have been examined in various virus-cell systems. Reversed polarity has been observed in transmembrane potentials of HEp2 and fibroblast cells (Fritz & Nahmias, 1972) and alterations have been reported in the activities of Na +, K * and Ca 2+ pumps of cells infected with Arenaviridae (Rey et al., 1988), Herpesviridae (Hackstadt & Mallavia, 1982), Reoviridae (del Castillo et al., 1991), Rhabdoviridae (Francouer & Stanners, 1978; 0001-2619 © 1994SGM

3338

G. P a l l t a n d o t h e r s

Methods Radioisotopes and chemicals'. Tritiated water (1 Ci/ml) and inulin [14C]carboxylic acid (2 to 10mCi/mmol) were purchased from Amersham. diS-Ca-(5) (100 gM stock solution in DMSO) was a kind gift of A. Waggoner (Amherst College, Mass., U.S.A.); 3-(p-(6phenyl)- 1,3,5-hexatrienyl)phenylpropionic acid (DPH-PA) was obtained from Molecular Probes Inc. (Eugene, Oreg., U.S.A.) and kept as a 1 mM stock solution in tetrahydrofuran. All reagents used in our experiments were AnalaR grade. Cells, virus, infection and culture conditions. HeLa $3 cells, a cell line adapted to grow in suspension, were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% (v/v) fetal calf serum (FCS), 2 mM-glutamine, 20 mM-HEPES and antibiotics. Cells were grown in 25 cm diameter roller bottles at 3.5 r.p.m, in a moist atmosphere of air and 5 % CO 2, starting with 5 x 104 cells/ml. HeLa $3 cells were infected with the wild-type (wt) strain of HSV-1 (Palfi & Biasolo, 1988; Palfi et al., 1988, 1990) or the F prototype strain of HSV1 at a multiplicity of infection (m.o.i.) of 20 plaque forming units (p.f.u.)/cell. Both viruses multiplied at the same rate in this cell line, as shown by one-step growth measurements. In particular, kinetics of growth were comparable to those observed in Vero cells with a transition from early to late phase of protein synthesis beginning at 5 to 6h post-infection (p.i.) (Palfi et al., 1990). Infection was always productive and characterized by an infectious cycle of 16 to 18 h (not shown). During the absorption 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. Cells were harvested at various intervals after infection and cell volumes, monovalent cation concentrations, transmembrane potential, lipid peroxidation and membrane fluidity were determined. Cell volumes were determined by using [14C]inulin and 3H20 as previously distributed (Palfi et al., 1990). The basal value for uninfected cells was 0.8+_0.15gl/10 ~ cells. Cell volume progressively increased during infection reaching a maximum value of about 1.1 +0.2 gl/10 ~ cells at 24 h p.i. For measurement of ATPase activities, a partially purified membrane preparation was used (see below). Purification of HeLa $3 cell plasma membranes. Purification of plasma membranes was accomplished essentially as described by Atkinson & Summers (1971). Briefly, 1"0 x l0 s HeLa $3 cells, harvested by centrifugation and washed in Earle's solution, were resuspended in 20 vols 10 mM-Tri~HC1, pH 8 and ruptured with few strokes of a Dounce homogenizer. After adjustment of the homogenate to 3 mMMgClz, 10 mu-NaC1, nuclei were removed by centrifugation and supernatants layered onto discontinuous sucrose gradients (15 ml 30 %, w/w, sucrose over 5 ml 45 %, w/w, sucrose). Gradients were centrifuged at 9000 g for 10 min in a SW27 rotor, Ghosts were collected from the 30 to 45 % sucrose interface, diluted in Tris buffer and washed by centrifugation at 9100 g for 5 rain before use. All operations were carried out at 4 °C. Determination o f ATPase activities. Na+/K +- and Mg2+-ATPase activities were determined essentially as previously described (Finotti et al., 1990). Briefly, mock-infected and infected HeLa $3 cells (40 to 50 lag proteins) were incubated (at 37 °C for 30 min) in the absence and in the presence of 0.2 mM-ouabain in 1 ml of the following medium: 100mM-NaCl; 20mM-KCI; 3 mM-MgCI2; 3 mM-ATP; 36mMimidazole histidine buffer pH 7.1; 0-1 mM-EGTA; 2mM-phosphoenolpyruvate plus 30 gg pyruvate kinase as an ATP regenerating system, and 0.32 mg/ml sodium deoxycholate. The addition of a lyric agent such as deoxycholate is necessary for full expression of the Na+/K+-ATPase activity of vesicular membrane preparations, since free access of substrate and activators to their respective binding sites does not occur unless vesicular structures are opened (Jorgensen & Skou, 1971). At 0.32 mg/ml deoxycholate, which was found to be the

optimal concentration, Na+/K+-ATPase activity increased on average by 100%, whereas Mg2+-ATPase activity remained essentially unchanged, as previously observed (Jorgensen & Skou, 1971). The ATPase reaction was started by the addition of membrane suspension and stopped by the addition of 0.25 ml cold 50 % trichloroacetic acid. Inorganic phosphate (P0 released in the supernatant was measured according to the method of Fiske & Subbarow (1925). Product formation was linear with time for the duration of the assay. Mg2+-ATPase activity was taken as the activity in the presence of 0-2 mM-ouabain. Preliminary experiments had shown that this activity was equal to that measured in the absence of sodium and potassium. Na+/K+-ATPase activity was calculated as the difference between the activity obtained in the absence and that obtained in the presence of ouabain. Proteins were measured by the Lowry method.

Determination o f intracellular Na + and K + concentration. Mockinfected and infected HeLa $3 cells were harvested at different times p.i. and washed three times in a Na +- and K+-free solution (choline-HC1 buffer). Pellets (10 r cells) were resuspended in a lyric solution containing 4.5 ml double-distilled, deionized (ddd) water and 0.5 ml 0.5 % Triton X-100. Samples were either frozen at - 2 0 °C or immediately analysed. Appropriate dilutions of the cell lysates were made in ddd water and Na ÷ and K + levels were determined using a Varian atomic absorption spectrophotometer model AA1475 and reference standards. In some experiments, before harvesting and washing, cells were seeded in fresh medium at a concentration of 5 × 105 cells/ml and incubated for 10 min at 37 °C with 5 gg/ml gramicidin D. The use of this ionophore allowed evaluation of the intracellular concentrations of Na + and K + under conditions where the plasma membrane does not maintain ionic gradients. Measurements o f transmembrane potential of HeLa $3 cells'. The measurement of transmembrane potential was performed by recording the decay of the fluorescence intensity of the probe diS-Ca-(5), which is incorporated into cells as a function of the transmembrane potential (Waggoner, 1976; Tsien & Hladky, 1978; Rink et al., 1980). Mock-infected and infected HeLa $3 cells, at 8 h p.i., were washed twice in a buffer containing 123 mM-NaC1, 6 mM-KCI, 0.8 mN-MgC12, 1.5 mM-CaCI2, 5 mM-sodium phosphate, 5 mM-citric acid, 5-6 mM glucose, pH 7.4, and resuspended at a concentration of 107 cells/ml. Cells were diluted directly in the thermostatically controlled cuvette (37 °C) of a 650-40 Perkin-Elmer spectrofluorometer and the probe was added at a final concentration of 200 riM. In some cases the fluorescent probe was present without cells in order to measure the spontaneous decay of fluorescence. After an initial instantaneous rise in the fluorescence quantum yield (excitation 620 nm; emission 660 nm), a decrease was recorded which corresponded to the cellular incorporation of the probe, a process dependent on the transmembrane potential. In a control experiment, partial membrane depolarization was obtained by bringing the KCI concentration of the buffer up to 60 mM. Once the fluorescence signal was stable, 1 gg/ml gramicidin D was added in order to fully depolarize the membranes and so obtain maximal recovery of the fluorescent probe: the higher the transmembrane potential the higher the fluorescence recovery that follows gramicidin D addition. Measurements o f membrane fluidity o f HeLa $3 cells. Membrane fluidity was measured by a time-resolved fluorescence technique that enables us to establish the speed of rotation of the fluorescent probe DPH-PA in the plane of the membrane bilayer. Time-resolved fluorescence experiments were carried out by adding 1 laN-DPH-PA to a suspension of 0.4x 10¢ cells/ml in a fluorescence 1 cm pathlength cuvette. The absorbance at 337 nm was less than 0.15 and remained constant during all experiments. A time-correlated single photon counting fluorometer, equipped with an Edinburgh F 199 pulsed lamp, Philips XP2020Q fast photomultiplier, Tenelec fast N1M electronics

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Fig. 1. Time course of the effect of HSV infection on the Na+/K +- (a) and Mg 2+- (b) ATPase activities of HeLa $3 cell membranes. Each point is the mean of four separate experiments performed in duplicate with different cell preparations. Mean _+S.l). control activities of Na÷/K ~and Mg~+-ATPase were 1.19+0-25 and 329_+0.70 lamol PJmg protein/hour, respectively.

and Silena BS27N multichannel analyser, was used. Time resolved anisotropy [r(t~]was determined by measuring the decays of parallel and perpendicular exciting beams at 337 nm and monitoring the emission at 430 nm as previously reported (Zannoni et al., 1983). Anisotropy decays were analysed using the sum and difference method of Chen et al. (1977) allowing calculation of the following parameters: the rotational correlation time, q~, which is inversely related to the membrane fluidity since it is dependent on the packing density of the lipid molecules, and the infinite time anisotropy, r~o. From r~ the second rank order parameter was derived (Zannoni et al., 1983). The order parameter represents the distribution of molecular orientations relative to a reference axis (in this case the axis perpendicular to the bilayer plane) and it is also inversely related to membrane fluidity, since the spatial order of the phospholipid bilayer decreases as fluidity increases.

sensitive to viral infection, its residual activity being 75 % o f the control. Since b o t h activities were expressed in r e l a t i o n to the p r o t e i n c o n t e n t o f p l a s m a m e m b r a n e s purified f r o m w a s h e d cell pellets (see M e t h o d s ) , the o b s e r v e d decreases reflect a real r e d u c t i o n o f A T P a s e activities, n o t m e r e l y a n effect due to cell loss. T h e c o n d i t i o n s o f low speed c e n t r i f u g a t i o n used to o b t a i n cell pellets after e x p o s u r e to HSV-1 were such as to a v o i d c o n t a m i n a t i o n b y m e m b r a n e debris or o t h e r n o n - n u c l e a r c o m p o n e n t s . Values o b t a i n e d t h e r e f o r e m a i n l y r e p r e s e n t the activity o f i n t a c t cells.

Determination of lipid peroxidation. Membrane lipid peroxidation was assessed by the measurement of malondialdehyde (MDA) by reaction with thiobarbituric acid (TBA) as described by Buege & Aust (1978). Aliquots of cell suspensions containing 200 gg proteins were incubated for 20 min at 37 °C in 0.5 ml 100 mM-Tris HC1 (pH 7.4), 3 mM-MgC12and 8 mM-KC1, and then boiled for 15 min after addition of 1 ml of stock solution containing 0.375 % TBA, 15 % trichloroacetic acid, 025 M-HC1 and 0-01% butylated hydroxytoluene (BHT). The addition of BHT was made in order to prevent peroxidation during the assay. After cooling, the samples were centrifuged at 1500 g for 10 min. The MDA content of the supernatants was determined from the absorbance at 532nm by using an extinction coefficient of 1.56 x 105 M-1 cm-1 (Buege & Aust, 1978).

Effect o f viral infection on intracellular Na + and K + concentrations

Results Effect o f virus infection on membrane A TPases Because m e m b r a n e A T P a s e s p l a y a crucial role in the m a i n t e n a n c e o f cell v i a b i l i t y b y r e g u l a t i n g selective ionic fluxes t h r o u g h the p l a s m a m e m b r a n e , these enzymic activities were c h o s e n as a key p a r a m e t e r for assessment o f the m o d i f i c a t i o n o f m e m b r a n e f u n c t i o n after infection by HSV-1. A s shown in Fig. 1, the activity o f N a + / K ÷A T P a s e o f H e L a $3 cells was significantly r e d u c e d d u r i n g the first 8 h p.i. Thereafter, the activity d e c r e a s e d m o r e slowly, r e a c h i n g a p l a t e a u b e t w e e n 12-24 h p.i. A t 24 h p.i., infected cells m a i n t a i n e d a b o u t 40 % o f the original N a + / K + - A T P a s e activity, a l t h o u g h viability was c o m p r o m i s e d ( ~ 30 % o f the cells s t a i n e d with t r y p a n blue). A similar p a t t e r n was also o b s e r v e d with M g 2+A T P a s e . H o w e v e r , this e n z y m e a p p e a r e d to be less

T h e effect o f HSV-1 infection on the i n t r a c e l l u l a r c o n c e n t r a t i o n s o f N a + a n d K + was i n v e s t i g a t e d in r e l a t i o n to the o b s e r v e d i n h i b i t i o n o f N a + / K + - A T P a s e activity. As d e p i c t e d in Fig. 2 (a), a significant decrease o f i n t r a c e l l u l a r K + ( a b o u t 2 5 % , f r o m 125 mM to 95 tory0 was a p p a r e n t s h o r t l y after virus a b s o r p t i o n to the cell surface. This decrease was a c c o m p a n i e d b y an increase in N a + c o n c e n t r a t i o n ( a b o u t 38 %, f r o m 18 mM to 29 mM). K + a n d N a + values r e t u r n e d to the c o n t r o l level at 4 h p.i. F r o m this time o n w a r d s , K + b e g a n to decline to reach a m i n i m u m o b s e r v e d value o f 40 mM at 24 h p.i., the last m e a s u r e m e n t . A t this time, the fall in K + was a c c o m p a n i e d b y an increase ( a b o u t 3 4 % , f r o m 18 mM to 27 raM) o f i n t r a c e l l u l a r N a +. T h e K + c o n c e n t r a t i o n , however, never r e a c h e d the value in g r a m i c i d i n t r e a t e d cells (2-5 mM). A s with the A T P a s e m e a s u r e m e n t s , values r e p o r t e d reflect those in i n t a c t cells.

Modification o f the transmembrane potential Because t r a n s m e m b r a n e p o t e n t i a l is r e g u l a t e d b y the i n t r a c e l l u l a r c a t i o n c o n c e n t r a t i o n , p a r t i c u l a r l y b y the conc e n t r a t i o n o f K +, this p a r a m e t e r was also e v a l u a t e d . A n indirect m e a s u r e m e n t o f the t r a n s m e m b r a n e p o t e n t i a l was o b t a i n e d b y d e t e r m i n i n g the fluorescence d e c a y o f the p r o b e diS-C3-(5), w h o s e kinetics o f i n c o r p o r a t i o n

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Fig. 2. Na+ (O, O) and K+ (1, D) concentrationsin HSV-1 infected(filledsymbols)and mock infected(open symbols)HeLa $3 cells at various intervals followinginfection. Each point represents the mean of three separate experimentsperformed in duplicate. Fig. 3. Transmembranepotential of HeLa $3 cells, measured as diS-Ca-(5) incorporation, 8 h after HSV-1 infection. For details see Methods. into cells depends on their transmembrane potential. Fig. 3 shows that probe uptake by infected cells at 8 h p.i. was reduced as compared to mock infected cells over a period of measurement of more than 2 min. Moreover, the rate of probe accumulation in infected cells was lower than that in uninfected cells, as indicated by the slope of the fluorescence signal. In addition, normalization of the signal in infected cells took place over a longer period of time (not shown). Under these conditions, the addition of gramicidin produced a recovery that was 85 % of that of control cells (not shown). Fig. 3 also indicates that the addition of KC1 to mock-infected ceils caused a reduction in the uptake process. Taken together these results are consistent with an alteration of the cell membrane potential as if infected cells were partially depolarized. Nevertheless, a residual potential must be operating in infected cells to allow probe accumulation, although over a longer period than in control cells.

Modifications of membrane fluidity Because membrane-bound enzymes depend critically on the physical state of the membrane for their activity, the occurrence of possible changes in plasma membrane fluidity was investigated as a possible mechanism of inhibition of Na+/K+-ATPase activity in infected HeLa $3 cells. Membrane fluidity was evaluated by means of time-resolved fluorescence techniques with the fluorescent probe DPH-PA. This probe does not cross the phospholipid bilayer but rotates in the outer layer of the membranes. The parameters that can be calculated by this kind of measurement, rotational correlation time (@)

and second rank order parameter ( < P 2 > ) , are both inversely related to membrane fluidity since, as previously stated, the packing density and the spatial order of the phospholipid bilayer decrease as fluidity increases. A steep increase in the rotational correlation time (over 100 %), as reported in Fig. 4(b), was observed between 5 and 8 h after infection. Also, the order parameter increased from a value of 0-75 at 5 h p.i. to a maximum value of 0.85 at 12 h p.i. From these experiments it appeared that reduction in membrane fluidity was a late event, becoming evident only 5 h after infection.

Lipid peroxidation induced by virus infection The possibility that viral infection directly generates reactive oxygen species (ROS) in the same cell that sustains viral replication was estimated by evaluating lipid peroxidation in infected cells. The time course of lipid peroxidation in infected HeLa $3 cells, as assessed indirectly by M D A formation, is shown in Fig. 5(a). M D A formation appeared to increase immediately after infection took place and reached a 100 % increment after 24 h. M D A generation by mock-infected cells did not change with time during the same period (not shown). There is a highly significant negative correlation (r = - 0 ' 9 9 ; P < 0-01) between cell lipid peroxidation and Na+/K+-ATPase activity, as shown in Fig. 5 (b).

Discussion Infection of cultured eukaryotic cells by animal viruses can alter several membrane functions crucial for the

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maintenance of cell homeostasis. In an attempt to shed light on the modifications which occur at the membrane level during the course of viral infection, we have analysed a number of parameters which, taken together, should provide a comprehensive picture of the structural and functional state of the membrane. As an experimental model we have adopted a human cell line, HeLa $3 cells, infected by HSV-1, the prototype of enveloped D N A viruses causing lytic infection. HeLa $3 cells have the advantage of growing in suspension and, consequently, of allowing biochemical and biophysical variables to be measured directly. Similar measurements would not be possible with adherent cells without trypsinization of the monolayers, a procedure most likely to alter some membrane properties. As reported in the present paper, the effect of HSV-1 infection on HeLa $3 cells is not as dramatic as would be expected from a cytolytic virus. The membrane functions evaluated here are only gradually impaired with time in a manner quite distinct from the abrupt alterations caused, for example, by picornaviruses (Carrasco & Smith, 1976). This is especially true of the changes occurring in intracellular

cation concentrations. In fact, although Na+/K+-ATPase is progressively reduced during the first 8 h p.i., the intracellular levels of monovalent cations show a significant decrease for K + and a significant increase for Na + coinciding with the virus adsorption period and at late times of infection (24 h). While the early reversible ion perturbation does not appear to be related to a decrease in the activity of the Na+/K + pump and might be the result of an alteration of membrane permeability following fusion of the viral envelope with the cytoplasmic membrane, the late K + drop and Na + increase (24 h p.i.) probably reflect a terminal irreversible stage of pump damage prior to cell death. It follows that a residual pump function sufficient to maintain an ion flux compatible with cell viability must be operating in HeLa $3 cells late in viral replication. This function prevents the attainment of high Na + concentrations as reported for Bunyaviridae and Picornaviridae (Frugulhetti & Rebello, 1989; Carrasco & Smith, 1976; Castrillo & Carrasco, 1986; Nair, 1981). The possibility that the observed phenomena are an expression of the average activity of infected and

3342

G. Palh and others

uninfected cells or of asynchronously infected cells is quite unlikely considering the high m.o.i, used. Moreover, immunofluorescence experiments, which were routinely performed as a control of the efficiency of the infection (24h p.i.), always showed that 100% of the cells were infected (data not shown). The measured modifications are, therefore, the sum of the effects driven by infection in each single cell throughout a round of viral replication. At 8 h p.i., when pump function is reduced by only 50% but cell viability is not dramatically affected ( ~ 10 % by trypan blue exclusion) since the virus has not yet completed its replicative cycle, measurement of transmembrane potential by diS-C3-(5 ) cell incorporation indicates a decrease in this parameter that parallels the alteration in Na+/K + intracellular concentration. These data are in apparent contrast with those of Fritz & Nahmias (1972) who demonstrated, by a classical impilation technique, the occurrence of reverse polarity in HSV-infected cells at 8 h p.i. However, these authors used HEp-2 cells that were detached from a plastic surface by vigorous shaking with glass beads, a procedure that can be particularly harmful to the membranes of virus-infected cells. Consequently, the reported changes in polarity may have been the result of structural damage to an already labile membrane. The fluorescence measurements performed here to assess the physical state of the membrane have provided clear-cut evidence of a decrease in membrane fluidity. However, such modification takes place after the reduction of Na+/K+-ATPase activity has already become prominent, indicating that reduced membrane fluidity is not responsible for the decline of Na+/K+-ATPase activity. Although in earlier studies a correlation was often found between lipid fluidity and activity of membrane-embedded enzymes (see e.g. Sinesky et al., 1979), according to more recent investigations there is no evidence that the fluidity of the surrounding lipid has any significant effect on enzyme activity (Lee et al., 1989; Zakim et al., 1992). Analogous considerations apply to Mg2+-ATPase activity. Free radical production may provide a more convincing explanation for the decline of these enzyme activities. It is known that oxygen free radicals are generated by phagocytic leukocytes following viral infection (for reviews see Oda et al., 1989; Muller, 1992; Revillard et al., 1992; Maeda & Akaike, 1991 ; Hurst & Barrette, 1989; Kehrer, 1993). These reactive oxygen intermediates play an important role in killing infecting agents but can also cause cellular damage since lipids, proteins and nucleic acids are all targets for free radical injury (Kehrer, 1993). Because of the ease with which it can be determined, lipid peroxidation is the most widely used index of free radical formation (Kehrer, 1993). Our

results have shown that viral infection can elicit free radical generation from a non-phagocytic cell type also. An increase in the cellular level of free radicals may either result from activation of free radical-generating mechanisms or from impairment of anti-oxidant defense systems (Yu, 1994). The strict correlation observed here between increase in lipid peroxidation and reduction in Na+/K+-ATPase activity suggests that virus-induced generation of free radicals is responsible for the progressive decline of the enzyme activity. This conclusion is supported by the observations that Na+/K+-ATPase activity is quite sensitive to the action of free radicals (Thomas & Reed, 1990; Rohn et al., 1993). In principle, this inhibitory effect may either result from a direct interaction of free radicals with the enzyme protein or may be secondary to lipid peroxidation, which reduces the fluidity of the membrane bilayer. The former explanation is in keeping with the demonstration that certain free radicals inhibit Na+/K+-ATPase by interacting with essential sulphydryl groups of the enzyme (Akera & Brody, 1970). However, the latter possibility appears unlikely, since we observed no temporal correlation between variations in Na+/K+-ATPase activity and membrane fluidity. Our previous results (Palfl et al., 1990), obtained using the same virus strain and the same cell line, clearly showed that viral infection causes a progressive inhibition of protein synthesis and an increase in membrane leakiness, reaching their maxima at 4 or 8 h p.i., respectively. These modifications are also likely to be the result of virus-induced generation of free radicals. Recent in vivo and in vitro experiments have shown that free radical formation causes a significant increase in nuclear DNA single-strand breaks, with consequent decrease in protein synthesis, and produces damage to the plasma membrane resulting in leakiness to otherwise nonpermeative solutes (Bagchi et al., 1993; Sawada et al., 1992; Block, 1991). Based on our previous and present results, the pattern of impairment of various cellular functions during viral infection can now be traced. (1) Protein synthesis and Na+/K+-ATPase activity decline through the early phase of viral growth before any significant changes in ion gradients and plasma membrane fluidity occur. (2) Disruption of ionic gradients is observed only at late times p.i., when the Na+/K+-ATPase is severely impaired. (3) Membrane leakiness becomes prominent only after a sharp reduction of fluidity occurs (from 5 h p.i. onwards). Although increase in lipid peroxidation begins as soon as viral infection takes place, it is possible that its effect on the viscosity of the phospholipid bilayer becomes detectable only above a certain degree of peroxidation. Alternatively, a relative increase in the membrane cholesterol content may be responsible for

HSV-1 infection modifies cell plasma membrane reduced fluidity. It has been shown that peroxidative damage is accompanied by an increase in the cholesterol/phospholipid ratio (Diani et al., 1991), a modification known to reduce membrane fluidity (Sinesky et al., 1979). On the basis of the above considerations, the suggestion of Local & Carrasco (1983) that cell leakiness might be exploited for an antiviral strategy based upon normally non-permeative poisons does not appear feasible, since leakiness takes place to a significant extent only at a late stage of viral growth (Palfl et al., 1990), when virus release from infected cells is already operating and cells are irreversibly damaged. Investigations of cell response to viral infection using different cell lines and tissues as well as different virus strains is now necessary in order to ascertain whether free radical generation is a phenomenon limited to our experimental model or represents a general response of the infected cell to a lytic virus. The authors acknowledge the valuable support of Professor F. Dabbeni-Sala for suggestions regarding lipid peroxidation. The technical contribution of Dr Concei Dos Santos to some of the experiments is also recognized. This work was financially supported by an AIDS grant no. 820491 from ISS.

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(Received 5 May 1994; Accepted 12 August 1994)