Effect of tunicamycin on the development of the cytopathic effect in Sindbis virus-infected avian fibroblasts

143, 546-557 (1985)

VIKOLOGY

Effect of Tunicamycin on the Development of the Cytopathic in Sindbis Virus-Infected Avian Fibroblasts EMIN Departm,mt

of

T. ULUG Microbiology,

Received

AND HENRY Untvwsity

Nvuerr~ber

R. BOSE,

of Texas,

13, 1984; uccepted

Austin,

February

Effect

JR.’

Texas

7871%109.5

12, 198.5

In Sindbis virus-infected avian cells the devclopmcnt of the cytopathic effect is correlated with the disruption of plasma membrane function. Sindbis virus inhibits the activity of the Na+K+ATPase, a membrane-associated enzyme complex which regulates intracellular monovalent cation levels. Tunicamycin, which blocks envelope protein glycosylation, prevents inhibition of Na+K+ATPase activity and the development of morphological changes in Sindbis virus-infected cells. Although inhibition of Na+K+ATPase activity is not essential for the termination of host. protein synthesis, membrane-mediated events may favor the selective translation of viral proteins. The termination of host protein synthesis does not contribute to the development of these cytopathic changes in the time frame examined. In tunicamycin-treated, Sindbis virusinfected cells, unglycosylated El is inserted into the plasma membrane but virus release is prevented. In productively infected cells, therefore, the inhibition of Na+K+ATPase activity and the development of the cytopathic effect may result from terminal events in virus assembly and/or virus release. by IWY .tcademic press, rnc.

fluidity (Sefton and Gaffney, 1974; Moore et al., 1976) and permeability to “‘Cr, trypan blue, and metabolic inhibitors (Nozawa and Apostolov, 1982; Peterhans et al., 1979; Contreras and Carrasco, 1979; Carrasco, 1981). The activity of the memhrane-associated Na+K+ATPase in avian cells infected with Sindbis virus is also inhibited (Ulug et al., 1984). The induction of cell death by lytic viruses is presumed to represent the culmination of a variety of metabolic, biosynthetic, and structural lesions within the infected cells. Sindhis virus particles contain two envelope glycoproteins (El and E2) and a nucleocapsid protein (C). The expression of these virion proteins requires the synthesis of a suhgenomic 26 S RNA species (Schlesinger and Kaariainen, 1980). During translation of the 26 S RNA, the nascent capsid protein is cleaved autocatalytically (Aliperti and Schlesinger, 1978; Scupham et al., 1977) and the envelope protein precursors are inserted into the lumen of the rough endoplasmic reticulum

INTRODUCTION

Sindhis virus and Semliki Forest virus are small, enveloped, RNA-containing alphaviruses that replicate in and kill vertebrate cells. The molecular basis for the induction of the cytopathic effect in cultured cells infected by these and other lytic viruses, however, has not been well characterized. The infection of vertebrate cells by alphaviruses results in the suhordination of host macromolecular synthesis, alterations in the intracellular concentrations of metaholites, and modification of the plasma membrane. Plasma memhrane modification in alphavirus-infected cells is characterized by the insertion of envelope proteins and the assembly and release of progeny particles. These events are accompanied by changes in lipid metabolism (Whitehead et al, 1981; Nozawa and Apostolov, 1982), membrane i Author addressed.

004%6822/85 Copyright All rights

to whom requests for reprints

$3.00

Cc: 19% hy Arndem~ PwL/L~, Inc of reproduction in any form reserved.

should be

546

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(Wirth et ab, 1977; Bonatti et al., 1979) and core glycosylated. The glycoproteins are then transferred to the smooth membranes (Erwin and Brown, 1980) where the oligosaccharide chains are trimmed and processed (Bonatti and Cancedda, 1982) and where fatty acids are covalently attached (Schmidt and Schlesinger, 1980). Tunicamycin prevents the initial step in the attachment of these asparagine-linked oligosaccharides (Krag and Robbins, 1977). This prevents the proteolytic processing of PE2 (Leavitt et al, 1977a, b), an envelope protein precursor whose cleavage is necessary for virus release (Jones et al., 1977; Bracha and Schlesinger, 1976). These studies were initiated to define the molecular mechanism by which Sindbis virus induces a cytopathic effect in productively infected cells. In this paper we report that tunicamycin, which inhibits Sindbis virus assembly and particle release, prevents the development of a cytopathic effect in avian fibroblast cultures. Tunicamycin also prevents inhibition of Na+K+ATPase activity by Sindbis virus. The ability of the virus to disrupt membrane integrity may contribute to the development of a cytopathic effect in Sindbis virus-infected cells. MATERIALS

AND

METHODS

Cells, viruses, and media. Secondary chick embryo fibroblast (CEF) cultures were prepared from 11-day SPAFAS (Chicago, Ill.) embryos as described previously (Burge and Pfefferkorn, 1966). Cells were plated 36-48 hr prior to experimentation at a density of 4 X lo5 per 35mm 6-well dish, 2 X lo5 per 22-mm 12well dish or 2.4 X lo6 per loo-mm dish. CEF cultures were incubated in minimum essential medium, MEM, containing Earle’s salts (Flow Laboratories) and supplemented with 5% newborn calf serum, NCS, in a humidified 5% CO2 atmosphere at 37”. Cells were mock infected by the addition of MEM or infected with Sindbis virus at a multiplicity of 20-25, unless otherwise indicated. Virus was adsorbed 1 hr at 37” and the cultures were rinsed

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547

and incubated in MEM + 2.5% NCS before the experiments were initiated. For enumerations, cells were removed from the plates with trypsin and counted in a hemocytometer. Tunicamycin treatment of cells. Tunicamycin (Miles Labs) was prepared as a 0.5 mg/ml stock in 95% ethanol. The concentration of ethanol employed in these experiments did not affect cell viability or virus replication. Tunicamycin (0.5 gg/ ml) inhibited virus release by a factor of lo4 when added immediately after the virus adsorption period (data not shown). At this concentration, radioimmune detection of the drug’s effect on envelope protein insertion required approximately one hour (data not shown). Polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDSPAGE) was used to identify and quantitate host and Sindbis virus proteins in cell extracts. For SDS-PAGE analysis, cultures were incubated in methioninefree MEM 1 hr and labeled with 20 &i [35S]methionine (Amersham, 1400 &i/ mmol) in MEM containing 10% the normal methionine concentration (final sp act = 20 Ci/mmol). The medium was removed and cell extracts were prepared by the addition of 2X SDS-PAGE sample buffer (10% glycerol, 0.125 MTris, pH 6.8, 0.001% bromphenol blue, 4% SDS, and 0.1 M dithiothreitol). After heating for 2 min in a boiling water bath, the extracts were subjected to electrophoresis on gradient slab gels (7-15%) in the buffer system described by Laemmli (1970). Coomassie brilliant blue was used to identify molecular weight markers (Bio-Rad). The dried gels were exposed to Kodak X-Omat R film with an intensifying screen for 48 hr. To quantitate the level of viral and host proteins synthesized, the radioactive bands were cut from the gels, solubilized in 30% HzOz overnight, and counted in Bray’s scintillation fluid using a Packard Tricarb 460 C liquid scintillation system. Quantitation of viral RNA synthesis. Viral RNA synthesis was quantitated by monitoring the incorporation of [3H]uri-

548

ULUG AND BOSE

dine (UdR) into cells in the presence of actinomycin D (AC-D). Cultures were treated 1 hr with 2 pg/ml AC-D (Sigma) prior to virus infection. After virus adsorption, cultures were treated with 2 pg/ ml AC-D in the presence and absence of 500 rig/ml tunicamycin. One hour after virus adsorption, the cultures were labeled with 10 &i [3H]UdR (ICN, 40 Ci/mmol). Four hours after adsorption, ice-cold TCA (0.4 M trichloroacetic acid, 0.02 M sodium pyrophosphate) was added to the cultures. After 1 hr, the cultures were washed with TCA, extracted with NaOH (1 N), and radioactivity was determined as described above. The rate of [3H]UdR incorporation into TCA precipitates was linear for up to 5 hr under these conditions. Na+K+A TPuse assay. Na+K+ATPase activity was monitored by measuring the ouabain-sensitive influx of the K’ tracer “Rb+ (New England Nuclear 1-12.5 Ci/ mg), into cells in the presence of furosemide (Ulug et al, 1984). “RbCl (5 mM, diluted to a sp act of approximately 0.5 Ci/mol with unlabeled RbCl) was added to phosphate-buffered saline (PBS) (Dulbecco and Vogt, 1954) which was prepared free of K+. Cultures were incubated in this medium in the presence and absence of 100 pM ouabain (Sigma) and 1.0 mM furosemide (Hoechst-Roussell) for 10 min in a 37” water bath. The cells were rapidly washed in ice-cold PBS, air dried, and dissolved in NaOH (0.1 N); aliquots were tested for Cerenkov radiation. The difference in the rate of *‘Rb+ uptake in the presence and absence of 100 pM ouabain was used to define Na+ pump activity. Protein levels were determined by the Bio-Rad microassay (Bradford, 1976). The NaOH extracts were neutralized with 0.1 N HCl before protein content was assayed. Purified bovine serum albumin was used as the protein standard. Radioimmune detection of viral proteins using 1”51-labeled staphylococcal protein A. To determine the level of virus envelope protein expression on the surface of infected cells, a radioimmune assay (RIA) system was developed. Heat-inactivated (30 min at 56”) anti-Sindbis virus serum

(Bell et al., 1978) and anti-El and anti-E2 sera (Dalrymple et al., 1976) were absorbed with primary CEF cultures twice for 2 hr. The sera were then clarified by centrifugation (15,000 g for 15 min) in an Eppendorf microfuge. Cultures were washed three times with PBS and exposed to 510% antiserum in PBS 1 hr at 4”. The antiserum was removed and the cultures were exposed to 0.25 &i 12”1-labeled staphylococcal protein A (Amersham, 2 Ci/g) in PBS for 30 min at 4”. The cultures were washed extensively with cold PBS and the level of radioactivity in NaOH extracts was measured in a Beckman 7000 gamma counter. Radioimmune precipitation of viral proteins. Cell surface proteins were iodinated in the presence of lactoperoxidase, immunoprecipitated, and analyzed by SDSPAGE and autoradiography. Monolayer cultures (ea. 5 X lo6 cells) were washed extensively in PBS and exposed to 0.25 mCi Na[““I] (carrier free, Amersham) in the presence of 25 pg lactoperoxidase (Sigma) in 1 ml PBS. Iodination was initiated and maintained by the addition of 10 ~1 HzOz (0.03%) at 5-min intervals for 15 min at 24”. The cultures were washed extensively and extracted for 30 min at 4” with 0.5% Nonidet-P40 (NP-40) in NET (0.2 M NaCl, 5 mM EDTA, 10 mM Tris, pH 8.0) as described by Kessler (1975). The extracts were centrifuged for 15 min at 15,000 g and the supernatant fluids were collected. The extracts were precleared at 4” by the addition of 50 ~1 rabbit serum for 2 hr and 100 ~1 formalin-killed Staphylococcus aureus cells (Pansorbin, Calbiochem-Behring) for an additional hour. After centrifugation (15,000 g for 5 min), 200-p] aliquots of the extracts were exposed to 5 ~1 of immune serum for 2 hr in a final volume of 0.5 ml NET (final NP-40 cone = 0.2%). Immune precipitates were collected, washed four times in NET + O.l%> NP-40 and dissolved in SDSPAGE sample buffer. 35S-labeled virus was prepared by incubating infected cells in the presence of 0.1 mCi [““Slmethionine for 10 hr. The clarified lysates were pelleted through a

INDUCTION

OF CPE BY SINDBIS

15% sucrose cushion in the SW41 rotor (1 hr at 30,000 rpm) and the pellet was dissolved in SDS sample buffer. The dried gels were exposed to Kodak X-Omat-R film for 12 hr at -70”. RESULTS

Tunicamycin Prevents the Development oj a Cytopathic Effect in Sindbis VirusInfected Avian Fibroblasts Avian species represent a major reservoir for alphaviruses in nature (Taylor et al., 1955). Avian fibroblasts rapidly develop a cytopathic effect when productively infected by alphaviruses (Frothingham, 1955). A temporal correlation between vi-

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549

rus release and the induction of a cytopathic effect has been observed (Nozawa and Apostolov, 1982). Since tunicamycin prevents Sindbis virus maturation and release, the effect of tunicamycin on the morphology of Sindbis virus-infected cells was examined. Photomicrographs of uninfected and Sindbis virus-infected avian fibroblast cultures grown in the presence and absence of tunicamycin are presented in Fig. 1. The uninfected chick cultures (panel A) consist of flat, slender fibroblastoid cells arranged in parallel arrays. Four hours after Sindbis virus infection, the cultures exhibited a typical cytopathic effect. The fibroblasts became shriveled, rounded, and refractile (panel B). Cell-

FIG. 1. Effect of tunicamycin on the morphology of uninfected and Sindhis virus-infected cells. {Jninfected and Sindbis virus-infected chick embryo fibroblasts were incubated 4 hr in the presence or absence of 500 rig/ml tunicamycin. The phase-contrast micrographs are of (A), uninfected CEF; (B), Sindbis virus-infected CEF; (C), tunicamycin-treated CEF; (D), tunicamycintreated, Sindbis virus-infected CEF.

550

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AND

cell contact was retained until the rounding cells began to detach from the culture dish. Sindbis virus-infected cells which were treated with tunicamycin after the adsorption period, however, did not exhibit the typical cytopathic appearance observed in productively infected cultures (panel D). Though morphologically unaltered at 4 hr after infection, the tunicamycin-treated, Sindbis virus-infected cells exhibited a fusiform appearance at later times after infection (not shown). Uninfected avian fibroblast cultures treated with tunicamycin (panel C) were morphologically indistinguishable from untreated cultures. Treatment of infected cells with tunicamycin, therefore, prevents the development of morphological changes characteristic of Sindbis virus infection. Efect

of Tunicamycin on Virus-Induced Terminatimz of Host Protein Synthesis

Virus-induced termination of host cell protein synthesis is presumed to play a major role in the development of a cytopathic effect and cell death. Since tunicamycin prevents Sindbis virus from inducing a characteristic cytopathic effect in avian cells, the effect of tunicamycin on host protein synthesis was monitored. Cultures infected with Sindbis virus (m.o.i.

I23456

FIG. 2. Effect of tunicamycin on Sindbis virusdirected protein synthesis. After adsorption, Sindbis virus-infected cells were treated with the following concentrations of tunicamycin (rig/ml): lane (0), lane 2 (2), lane 3 (8), lane 4 (31), lane 5 (125), and lane 6 (500). Four hours after infection, the cultures were labeled with 20 j&i [%]methionine for 30 min. Cell extracts were prepared and analyzed by SDS-PAGE analysis and autoradiography.

BOSE

= 25) and treated with various concentrations of tunicamycin for 4 hr were labeled with [35S]methionine for 30 min. Cell extracts were prepared for SDS-PAGE analysis and autoradiography (Fig. 2). In untreated, Sindbis virus-infected cells (lane 1) the envelope proteins El and E2 and their precursors (PE2 and B) were detected. Treatment of the cultures with increasing concentrations of tunicamycin (lanes 2-6) resulted in little change in the rate of capsid protein synthesis. Elevated levels of the 98K envelope precursor protein “B” and more rapidly migrating, unglycosylated forms of PE2 and El (designated PE2* and El*), however, were observed in cells treated with tunicamycin. Although the rate of PE2* and El* synthesis was reduced approximately 60% as a consequence of tunicamycin treatment, the overall rate of viral protein synthesis was not significantly affected. In the presence and absence of tunicamycin, viral proteins accounted for 8595% of the total proteins synthesized. Although host protein synthesis was arrested in Sindbis virus-infected cells treated with tunicamycin, these cultures did not exhibit typical morphological changes by 4 hr after infection. It is, therefore, unlikely that the inhibition of host protein synthesis, in itself, causes the rapid development of the cytopathic effect in productively infected cells. It has been proposed that plasma membrane modification contributes to the termination of host protein synthesis in cells infected with lytic viruses (Carrasco, 1977). To define the contribution of plasma membrane modification on the competition of host and viral mRNA translation, the effect of tunicamycin on protein synthesis in cultures infected with various multiplicities of virus was determined (Fig. 3). Cultures were mock infected (lane 1) or infected with 5-40 PFU/cell of Sindbis virus (lanes 2-5) and incubated in the absence (panel A) or the presence (panel B) of 500 rig/ml tunicamycin for 4 hr. Protein synthesis was monitored by pulsing the cultures 30 min with [35S]methionine. Two multiplicity-dependent effects

INDUCTION A I2345

OF CPE BY SINDBIS

B I2345

FIG. 3. Effect of tunicamycin on the selective termination of host protein synthesis. Cultures were infected with the following multiplicities of virus (PFU/cell): lane 1 (O), lane 2 (5), lane 3 (lo), lane 4 (20), lane 5 (40). After the adsorption period, the cultures were incubated 4 hr in the absence (panel A) or the presence (panel B) of 500 rig/ml tunicamycin. The cultures were pulsed 30 min with [?S]methionine and cell extracts were analyzed by SDS-PAGE and autoradiography.

of tunicamycin on host protein synthesis were observed. During low multiplicity infection (lane 2 in panels A and B), tunicamycin reduced the overall rate of viral protein synthesis. The rate of capsid protein synthesis under these conditions was reduced approximately 40% as a consequence of tunicamycin treatment. This effect is due, in part, to a reduction in virus-directed RNA synthesis in the tunicamycin-treated cultures. Actinomycin D resistant [3H]uridine incorporation was reduced as a consequence of tunicamycin treatment during low multiplicity infection (Fig. 4). At elevated multiplicities of infection (lo-40 PFU/cell), total viral protein synthesis was either not affected or stimulated by tunicamycin treatment, Under these conditions, the rate of host protein synthesis was elevated two- to threefold in cultures treated with tunicamycin (compare lanes 3, 4, and 5 in panels A and B). This effect was most evident when actin (AC) synthesis was examined. Because actin (43K) is resistant to treatments which affect host protein synthesis (Garry et ah, 1979, 1982), its synthesis represents a stringent measure of the

551

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selective termination of host protein synthesis by lytic viruses. Under all multiplicities of infection examined, the rate of actin synthesis was elevated two- to fivefold as a consequence of tunicamycin treatment. In lane 3, for example, the rate of actin synthesis was reduced 89% by productive virus infection (7.87 vs 69.3 pmol [35S]methionine incorporated/30 min/mg cell protein) and only 55% in virus-infected cells treated with tunicamycin (38.7 vs 88.0 pmol [35S]methionine incorporated/30 min/mg cell protein). Although tunicamycin inhibited virusdirected RNA synthesis during low multiplicity infection, the level of viral RNA synthesis at elevated multiplicities in tunicamycin-treated cells was identical or elevated in comparison with untreated cells (Fig. 4). Under these conditions, equivalent levels of virus-directed RNA synthesis did not result in an equivalent degree of termination of host protein synthesis. Plasma membrane modification, therefore, may contribute to the selective inhibition of host translation during productive virus infection.

0 MULTIPLICITY

IO

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INPUT

FIG. 4. Effect of tunicamycin on viral RNA synthesis. Actinomycin D treated cultures were infected at various multiplicities for 1 hr and incubated in the presence (closed symbols) or absence (open symbols) of 500 rig/ml tunicamycin. The cultures were labeled with 10 &i [3H]uridine between 1 and 4 hr p.i. and precipitated with TCA. Error between triplicate determinations did not exceed 5%.

552

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AND

I 0

2

8

TUNICAMYCIN

31

125

500

hCj/l’d)

FIN. 5. Effect of tunicamycin on Nat pump activity in uninfected and Sindbis virus-infected chick cells. Triplicate cultures of uninfected (open symbols) and Sindbis virus-infected cells (closed symbols) were treated with various concentrations of tunicamycin after the addition of virus. Four hours later, they were exposed to 5 mM s6RbCI in the presence and absence of ouabain. Na+ pump activity is expressed as the difference in the rate of 86Rb’ uptake in the presence and absence of ouabain. Error between replicate determinations did not exceed 5%‘.

Alterations in intracellular monovalent cation levels is a common result of infection by several lytic viruses and is presumed to contribute to the cytopathic effect seen in the infected cells (Pasternak and Micklem, 1981). In Sindbis virus-infected cells, a reduction in intracellular K+ levels is due to an inhibition of the activity of the ouabain-sensitive Na+K’ATPase (Ulug ef ul., 1984). The Na+K+ATPase (or Na+ pump) is an integral membrane enzyme complex employed by cells to maintain a high intracellular concentration of Kf and a low level of intracellular Na’ (Glynn and Karlish, 1975; Kaplan, 1978). To determine if altered Na+ pump activity contributes to the development of the cytopathic effect in Sindbis virus-infected cultures, the effect of tunicamycin on Na’ pump activity was

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monitored. Infected and uninfected cultures were exposed to varying concentrations of tunicamycin for 4 hr and then pulsed with *‘Rb+ (a K+ tracer) in the presence and absence of ouabain (Fig. 5). In uninfected cultures (open circles), the rate of ouabain-sensitive “jRb+ uptake was reduced in the presence of increasing concentrations of tunicamycin. This reduction in activity may reflect a toxic effect of tunicamycin on the heavily glycosylated p subunit of the Na+K+ATPase (Fambrough and Bayne, 1983). In the absence of tunicamycin, Sindbis virus infection of chick cells resulted in a 35-40s reduction in the rate of ouabain-sensitive “Rb+ uptake (closed symbols). In the presence of increasing concentrations of tunicamycin, the infected cells exhibited elevated levels of Na+K+ATPase activity. Tunicamycin, therefore, prevents the inhibition of Nat pump activity by Sindbis virus. A correlation, therefore, exists between the ability of the virus to disrupt ion transport and to induce a cytopathic effect.

The El Envelope Protein Is Expressed ow the Surface of’ Tunicumycin-Treated Cells Tunicamycin has been reported to interfere with the glycosylation, processing, and transport of Sindbis virus envelope proteins (Leavitt et nl., 1977a, b). Sindbis virus envelope proteins, however, have been detected in the plasma membranes of tunicamycin-treated, Sindbis virus-infected cells (Scheefers et d., 1980; Mann ef rrl., 1983). The lack of CPE development in tunicamycin-treated, Sindbis virus-infected cells may result from the failure of sufficient quantities of one or both of the envelope proteins to be inserted into the plasma membrane. To define the time course and level of envelope protein expression on the surface of tunicamycintreated, Sindbis virus-infected cells, a radioimmune assay employing anti-Sindbis virus serum was performed. After a l-hr adsorption period, tunicamycin (500 ng/ ml) was added to one half of the cultures.

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x f If K c

50.

: L

0

I

2

HOURS

AFTER

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4

5

INFECTION

FIG. 6. Expression of Sindbis virus proteins on the surface of untreated and tunicamycin-treated cells. Mock-infected or Sindbis virus-infected cultures were incubated in the presence or absence of 500 rig/ml tunicamycin for the times indicated. Surface expression of virus determinants was monitored in triplicate by exposing the cells to 8% antivirus serum 1 hr at 4” and monitoring the level of bound antibody using ‘“51-labeled staphylococcal protein A. O-uninfected CEF, l -Sindbis virus-infected CEF, n -tunicamycin treated, Sindbis virus-infected CEF. Error between replicate determinations did not exceed 10%.

At various times after infection, the cultures were exposed to antiviral serum and ““I-labeled staphylococcal protein A was used to monitor the level of antibody

bound to the surface of the infected cells. As illustrated in Fig. 6 (closed circles), viral proteins were detected on the surface of infected cells by 1.5 hr after virus adsorption. By 4 hr after adsorption, the expression of viral envelope proteins on the surface of the infected cells was maximal. In contrast, the tunicamycin-treated, virus-infected cultures exhibited a reduced expression of viral determinants. By 5 hr after infection, the level of antibody that bound to infected cells treated with tunicamycin was reduced approximately 55Y0 (open circles). Tunicamycin-treated, uninfected cultures did not bind significant amounts of lz51-labeled staphylococcal protein A after exposure to antiviral serum. These results indicate that insertion of the envelope proteins into the plasma membrane is not, in itself, responsible for the development of a cytopathic effect. To determine the nature of the viral proteins present on the surface of tunicamycin-treated, Sindbis virus-infected cultures, a radioimmune assay was performed using sera directed against El and E2. El and E2 are antigenically distinct polypeptides (Dalrymple et ah, 1976). The results, presented in Table 1, indicate that only the unglycosylated El (El*) envelope protein was expressed on the surface of tunicamycin-treated, Sindbis virus-infected cells. The level of anti-Sindbis and anti-El antibodies that bound to the in-

TABLE SURFACE

DETECTION

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OF SINDBIS VIRUS ENVELOPE PROTEINS AFTER TUNICAMYCIN RADIOIMMUNE BINUINC OF STAI’HYLO~OCCAL PROTEIN A

Sindbis virus-infected

TREATMENT

RY

cells”

Antiserum*

4 hr pi.

4 hr p.i. + tunic.

0 hr p.i.

Anti-SB Anti-El Anti-E2

123.0 * 6.7” 240.5 f 17.3 110.9 + 4.2

102.1 + 8.3 141.7 -+ 3.0 91.4 * 2.7

87.9 f 2.5 82.7 f 3.2 91.0 * 8.3

Uninfected

cells

68.7 k 6.8 61.8 + 4.3 61.0 f 4.5

“Triplicate cultures were infected 1 hr at 37” and either incubated in the presence or absence of tunicamycin (500 rig/ml) for 4 hr or immediately processed (0 hr pi.). *The cultures were washed 3X with PBS and incubated 1 hr at 4” in the presence of PBS containing a 1:20 dilution of the indicated antiserum. ‘The results are expressed as nanograms of ia5I-labeled protein A bound per milligram cell protein.

554

ULUG

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fected cells after tunicamycin treatment was reduced approximately 60% in agreement with results shown in Fig. 6. Unglycosylated PE2 (PE2*) could not be detected on the surface of tunicamycintreated, Sindbis virus-infected cells. The level of anti-E2 which bound to tunicamycin-treated, Sindbis virus-infected cells was identical to the level bound to infected cells which have not yet expressed surface envelope proteins. The antisera presumably detected virions which were bound to cells but not endocytosed. The failure to detect PE2* on the surface of infected cells was probably not due to the inability of the antisera to recognize this protein. When [35S]methionine-labeled cell extracts were prepared from tunicamycin-treated, Sindbis virus-infected cells, a polypeptide corresponding to PE2* was precipitated with anti-Sindbis serum, although with reduced affinity (data not shown). The reduced binding of antisera to tunicamytin-treated cells may, however, also reflect a lower affinity of the antibody for the unglycosylated forms of the envelope proteins. Conclusive evidence demonstrating that El* is inserted into the plasma membrane was provided by studies in which this protein was immunoprecipitated from 123

El E2-

-El*

cFIG. 7. Radioimmune precipitation of “%labeled El* and El. Sindbis virus infected cells were incubated in the absence (lane 2) or presence (lane 3) of 500 n&ml tunicamycin for 4 hr following virus adsorption. The cultures were labeled with izsI in the presence of lactoperoxidase, immune precipitated with anti-El serum, and processed for SDS-PAGE and autoradiography. %-labeled virion proteins were used as standards in lane 1.

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‘%I-labeled cells (Fig. 7). Infected cultures, incubated 4 hr in the absence (lane 2) or the presence (lane 3) of 500 rig/ml tunicamycin, were iodinated in the presence of lactoperoxidase. Under conditions where cell surface proteins were specifically labeled, El* was immunoprecipitated from tunicamycin-treated cells with antiEl serum. DISCUSSION

The infection of vertebrate cells with alphaviruses results in the inhibition of host macromolecular synthesis (Wengler, 1980), disruption of intracellular metabolite levels (Cassels and Burke, 1973; Whitehead et ccl., 1981) and alterations in plasma membrane composition (Nozawa and Apostolov, 1982) and function (Ulug et al., 1984; Gray et ah, 1983). These events are presumed to result in the death of the infected cells. The development of a cytopathic effect in Sindbis virus-infected cells has been reported to correlate with the release of progeny virus, increased 51Cr permeability, and alterations in fatty acid saturation (Nozawa and Apostolov, 1982). The mechanism, however, by which the cytopathic effect is induced by Sindbis virus has not been described. In this investigation, tunicamycin was employed to block viral envelope protein maturation. Terminal events in the assembly and release of progeny virus particles were thereby blocked. These events appear to be necessary for the development of a cytopathic effect in Sindbis virus-infected cells. Metabolic and biosynthetic lesions in Sindbis virus-infected cells do not lead to the development of a cytopathic effect in the time frame examined. The enhanced rate of hexose uptake detected in alphavirus-infected cells (Gray et al., 1983) was not selectively affected by tunicamycin (data not shown). Although tunicamycin inhibited virus-directed RNA synthesis during low multiplicity infection, equivalent or elevated levels of viral RNA and protein were synthesized in tunicamycintreated cultures infected at higher mul-

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tiplicities. Tunicamycin-treated cells infected at these multiplicities, however, synthesized two- to threefold more host protein than did untreated, Sindbis virusinfected cells. Furthermore, under these conditions, the rate of capsid protein synthesis was not appreciably altered. These results are not consistent with the concept that capsid protein selectively inhibits host translation, as suggested by in vitro experiments reported by Van Steeg et al. (1984). The selective termination of host protein synthesis may be favored, in part, by disruption of plasma membrane integrity. During high multiplicity infection, however, very little host protein was synthesized in the presence of tunicamycin. The termination of host protein synthesis, therefore, cannot in itself account for the rapid development of the cytopathic effect in Sindbis virus-infected avian cells. A correlation exists between the ability of Sindbis virus to alter the integrity of the plasma membrane and to induce the development of a cytopathic effect. Infection of avian cells by Sindbis virus results in reduced intracellular K+ levels and a concomitant reduction in cell volume. These altered salt levels result, at least in part, from a reduction in Na+ pump turnover (Ulug et al., 1984). The inhibition of Nat pump activity may, therefore, directly account for the altered morphology of the infected cells. Alternatively, Sindbis virus may affect the integrity of the cytoskeletal system, leading to the development of the cytopathic effect. The extent of cytoskeletal changes in Sindbis virusinfected cells has, however, not been evaluated. The development of a cytopathic effect in Sindbis virus-infected cells requires terminal events in the assembly of virus, or virus release. Insertion of unglycosylated El* into the surface membrane is, in itself, insufficient to induce the development of the cytopathic effect or to inhibit Na+ pump activity. Tunicamycin treatment of Sindbis virus-infected cells does, however, result in stimulation of (Na+K+Cll) cotransport activity, suggesting that El* insertion results in a func-

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tional modification of the plasma membrane in Sindbis virus-infected, tunicamycin-treated cells (manuscript in preparation). The precise stage in the morphogenesis of Sindbis virions which leads to the development of a cytopathic effect is not known. PE2 cleavage, E2 insertion, or virus release could be involved in the development of the cytopathic effect. The selective expression of viral envelope sequences from molecularly cloned DNA may facilitate identification of the event(s) which induces the development of the cytopathic effect. ACKNOWLEDGMENTS The authors express their gratitude to Dennis T. Brown, Robert E. Johnston, and Robert F. Garry for their helpful comments during the preparation of this manuscript. We gratefully acknowledge the gift of furosemide from Hoechst-Roussell, antisera from J. M. Dalrymple, and tunicamycin from D. T. Brown. This investigation was supported by Public Health Service Grants CA 26169 and CA 27003, awarded by the National Cancer Institute. E.T.U. is a postdoctoral fellow of the Robert A. Welch Foundation (Grant F-849). REFERENCES ALIPERTI, G., and SCHLESINGER, M. J. (1978). Evidence for an autoprotease activity of Sindbis virus capsid protein. Virology SO, 366-369. BELL, J. W., GARRY, R. F., and WAITE, M. R. F. (1978). Effect of low NaCl medium on the envelope glycoproteins of Sindbis virus. J. Vim!. 25, 764769.

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