VIROLOGY

183, 74-82 (199 1)

Synthesis

of Semliki Forest Virus RNA Requires Continuous

Lipid Synthesis

LUIS PEREZ, ROSARIO GUINEA, AND LUIS CARRASCO’ Centro de Biologia Molecular

(CSIC-lJAM)/I), Universidad

Received November

Aut6noma,

26, 1990; accepted

Canto Blanco, 28049 Madrid, Spain

March

11, 199 1

The involvement of lipid biosynthesis in the replication of Semliki Forest virus (SFV) in HeLa cells has been analyzed by the use of cerulenin, an inhibitor of lipid synthesis. The presence of this agent from the beginning of infection blocks the appearance of viral proteins. However, when the antibiotic is added at later stages of infection it has no effect on protein synthesis, the cleavage of viral proteins and their acylation by palmitic acid. Cerulenin is a powerful inhibitor of viral RNA synthesis, as analyzed by [3H]uridine incorporation, incorporation of [32P]phosphate into viral replication complexes, or Northern blot analysis of viral RNAs hybridized with minus- or plus-stranded riboprobes. Finally, analysis of phospholipids made in SFV-infected cells indicates that viral infection clearly stimulates the synthesis of phosphatidyl choline and modifies the membrane formed as analyzed by sucrose gradient centrifugation. Cerulenin blocks the synthesis of phospholipids and inhibits the formation of new membranes. These results show that, when the synthesis of lipids is blocked by cerulenin, SFV RNA replication is hampered, suggesting that the synthesis of viral RNAs needs continuous lipid synthesis and membrane formation. o 19% Academic PWSS. IW.

INTRODUCTION

glycoproteins E3 and E2. Late during infection the synthesis of (+) 42 S RNA predominates in order to provide genomic RNAs that are encapsidated in new virion particles (Strauss and Strauss, 1988). Synthesis of the three species of viral RNA: (+) 42 S RNA, (-) 42 S RNA, and 26 S mRNA takes place in special structures located in the cytoplasm, in close connection with membranes (Acheson and Tamm, 1967; Grimleyeta/., 1968; FroshaueretaI., 1988). Replication of viral genomes in association with membrane complexes is a rather widespread phenomenon in animal viruses (Har-ford et al., 1966; Acheson et a/., 1967; Caliguiri and Tamm, 1970). Thus, the synthesis of picornavirus RNA is tightly associated with cytoplasmic vesicles that proliferate throughout infection (Dales et a/., 1965; Girard et al., 1967; Butterworth et al., 1970; Bienz et a/., 1990). The proliferation of these newly made membranous vesicles is accompanied by the stimulation of phospholipid synthesis (Cornatzer et al., 1961; Penman, 1965; Mosser et a/., 1972; Vance et al., 1980; Guinea and Carrasco, 1990). The consequences that the inhibition of lipid synthesis has on the growth of some animal viruses has been investigated by means of the antibiotic cerulenin (Schlesinger and Malfer, 1982; lkuta and Luftig, 1986; Gallo and Sarngadharan, 1988; Guinea and Carrasco, 1990). Treatment of animal virus-infected cells with cerulenin blocks the maturation of some viral proteins. Thus, the presence of cerulenin inhibited the acylation of the HIV 1 protein Pr 53 gag (Gallo and Sarngadharan, 1988) the VSV G glycoprotein and some Sindbis virus glycoproteins (Schlesinger and Malfer, 1982; Kotwal and Ghosh, 1984). Under the conditions used, Kotwal and

Semliki Forest virus (SFV), together with Sindbis virus are the best characterized members of the Togaviridae family at the molecular level (Schlesinger and Schlesinger, 1990). The SFV 42s viral RNA genome of 11,442 bases has been sequenced and encodes at least nine proteins (Garoff et al., 1980a,b; Takkinen, 1986). Four of them are expressed during the early phase of infection; they are made from a polyprotein precursor synthesized from the 5’ two-thirds of the 42 S RNA genome that acts as the single early mRNA known (Takkinen, 1986). Expression of these early polypeptides known as nsP1, nsP2, nsP3, and nsP4 are required for viral RNA replication (Strauss and Strauss, 1988). Once full-length negative copies of the 42 S RNA are made, they serve as templates to synthesize more copies of genomic RNA and the 26 S mRNA. This subgenomic 26 S mRNA corresponds to one-third of the (+) 42 S RNA, encodes the 3’end of this RNA and is synthesized by internal initiation of transcription (Strauss and Strauss, 1988). The 26 S mRNA is translated in the late phase of infection to give rise to another polyprotein precursor that is cleaved to the four structural proteins, i.e., C, p62 (E3 plus E2), and El, plus a small hydrophobic protein of 6K encoded between glycoproteins E2 and El (Schlesinger and Schlesinger, 1990). This 6K protein is associated with membranes and its function remains unknown (Welch and Sefton, 1980; Gaedigk-Nitscho et a/., 1990). Further cleavage of p62 in trans-Golgi vesicles renders the ’ To whom reprint requests should be addressed. 0042.6822/91

$3.00

Copynght 0 1991 by Academic Press, Inc. All rights of reproduction I” any form reserved.

74

75

RNA REPLICATION REQUIRES LIPID SYNTHESIS

A

I

Hela

I

tOh

P97 ~62 El.2

-p97 -p62-

E1,2-

c

E

60

'C ;I =

40

010 0.00 0,os 0,io o, O,i5

Cerulenin

(mM)

FIG. 1. SFV protein synthesis and acylation in the presence of cerulenin. Confluent HeLa cell monolayers were infected with SFV at a m.o.i. of 50 PFU/cell. After 60 min of adsorption at 37’ the medium was replaced and monolayers were incubated with fresh DMEM supplemented with 2% calf serum. At the times indicated the cells were labeled with 10 &i/ml [35S]methionine or 50 rCi/ml [3H]palmitic acid. After 1 hr, samples were harvested and analyzed by polyacrylamide gel electrophoresis as described under Materials and Methods. (A) [?S]Methionine labeling: 0.08 mM cerulenin was added at the times indicated. (B) [35S]Methionine (left) and [3H]palmitic (right) labeling: The period of labeling was from 5 to 6 hr p.i. 0.1 mMcerulenin was added at 3 hr p.i. (+). (C) Measurement of [3H]uridine (0) and [35S]methionine (0) incorporation into HeLa cells under different concentrations of cerulenin. Cells were labelled during 60 min after 5 hr in the presence of cerulenin.

Ghosh (1984) showed that cerulenin only affected Gprotein acylation during VSV growth. However a more detailed analysis of the effects of cerulenin on VSV or Sindbis virus indicated that the reduction of virus production by cerulenin could be partially explained by inhibition of virus RNA synthesis (Schlesinger and Malfer, 1982). Interestingly, the growth of Sindbis virus in a CHO mutant cell line that does not synthesize phospholipids is restricted in a still unidentified step

subsequent to virus entry and uncoating (Kuge er al., 1989). Recently, we found that the inhibition of phospholipid synthesis potently blocked the formation of poliovirus RNA, suggesting that continuous phospholipid synthesis is required for picornavirus genome replication (Guinea and Carrasco, 1990). We have now analyzed the synthesis of lipids in SFV-infected cells and the consequences that the inhibition of lipid synthesis have for the virus replication cycle.

76

PEREZ, GUINEA, AND CARRASCO 5ooc

z d d z z

loaded on a 15% polyacrylamide gel and electrophoresed overnight at 90 V. Acylation of SFV proteins

4ooc

Cell monolayers were labeled for 1 hr with 50 &i/ml [9,1 O-3H]palmitic acid (40-60 Ci/mmol; Amersham) dissolved in 2% calf serum DMEM. After labeling, the samples were prepared and analysed as described above for [35S]methionine labeled samples.

3000

z F .-i

2000

Estimation of uridine incorporation in RNA

I :s L 3

1000

s

0 -l-

. , . , . , . , . , . , . , . , . , .

012345678910 h p.i. FIG. 2. Activity of cerulenin on SFV RNA synthesis. HeLa cells grown in 24-well plates were infected at a m.o.i. of 50 PFU per cell, as described in the legend of Fig. 1. After the adsorption period, the cells were incubated in the presence of 5 pglml actinomycin D, and labeled for 1 hr with 10 &i/ml [3H]uridine. Each point was plotted in the middle of the incubation time, i.e., incubation from 0 to 1 hr was plotted at 0.5 hr and so on. Samples were precipitated with 5% TCA and harvested with 0.1 N NaOH containing 1% SDS. Solid line: control of infection. Dashed lines: 0.08 mMcerulenin added at the times indicated by the arrows.

MATERIALS AND METHODS Cells and viruses HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% calf serum, and incubated at 37” in a 5% CO, atmosphere. SFV virus was grown in HeLa cells in DMEM with 2% calf serum. The concentration of virus was estimated by plaque assay. Analysis of proteins by polyacrylamide gel electrophoresis HeLa cells were incubated in methionine free DMEM and labelled for 1 hr with lo-20 &i/ml of [35S]methionine (1450 Ci/mmol; Amersham) at the times indicated in each experiment. At the end of the labeling period the medium was removed and monolayers were dissolved in 200 ~1of buffer containing 62.5 mM TrisHCI, pH 6.8, 2% SDS, 0.1 n/r dithiothreitol, 17% glycerol, and 0.024% bromophenol blue. Samples were

HeLa cells grown in 24-well plates were infected with SFV at a m.o.i. of 50, in medium containing actinomycin D (5 pg/mI). Every hour after infection 10 pCi/ml [5’-3H]uridine (25-30 Ci/mmol; Amersham) were added. The labeling medium was removed after 1 hr and the cell monolayer treated with 0.5 ml 5% trichloroacetic acid, washed twice with ethanol, dried under an infrared lamp, and dissolved in 200 ~1of 0.1 N NaOH/ 1% SDS. Samples of 150 ~1 were counted in a liquid scintillation spectrometer. Northern blot analysis of RNA Total cytoplasmic RNA was isolated from HeLa cells grown in 60-mm dishes and infected with SFV at a m.o.i. of 50. The indicated concentration of cerulenin was added after adsorption of the virus. At the times indicated the cells were lysed in a buffer containing 10 ml\/lTris-HCI, pH 7.8, 1 rnM EDTA, 150 mll/l NaCI, and 0.65% Nonidet P-40. After removing nuclei by low speed centrifugation, supernatants were mixed with an equal volume of a buffer containing 20 mM TrisHCI, pH 7.8, 350 mll/l NaCI, 20 mM EDTA, and 1% SDS. Samples were extracted with a mixture of phenol:chloroform:isoamyl alcohol (24:24: 1 (v/v/v)) and RNA was precipitated with 2 vol of ethanol. A 5-119 sample of denatured RNA was subjected to electrophoresis in a 1.2% agarose/formaldehyde gel and transferred to nitrocellulose filters as previously described (Guinea and Carrasco, 1990). RNA samples immobilized on nitrocellulose filters were hybridized under standard conditions (Guinea and Carrasco, 1990) to plus- or minus-strand-specific 26 S RNA probes generated by in vitro transcription of the SFV 26 S cDNA plasmid pGEM 1-SFV (kindly provided by Drs. H. Garoff and P. LiljestrUm). Bands obtained in the films were quantitated with computing densitometer (Model 300A, Molecular Dynamics). Estimation of glycerol incorporation into lipids HeLa cells grown in 24-welt plates were infected with SFV at a m.o.i. of 50 PFU per cell. Every hour after

RNA REPLICATION 2-4

o-2 c

28s

-

18s

-

-

+

7

6-8 7

77

h.p.i. CRL

a

8

FIG. 3. Analysis of the SFV RNAs synthesized in the presence or absence of cerulenin. Monolayers of HeLa cells infected with SFV (50 PFUkell) were incubated in DMEM supplemented with 2% calf serum and 5 fig/ml actinomycin D. At the times indicated, the cells were labelled with 20 &i/ml [32P]POi- for 2 hr. 0.08 mM cerulenin (CRL) was added as indicated during the labeling period. RNA was extracted as described under Materials and Methods. 3 pg RNA per sample was electrophoresed in a 0.89/o agarose gel at 80 V. The gel was dried and exposed with a X-ray film.

infection cells were incubated in medium containing 10 &i/ml of [3H]glycerol (l-3 Ci/mmol) at 37” for 1 hr. Afterward, the labeling medium was removed, and the cells were harvested in 200 ~1 of distilled water. The lipid fraction was obtained by extraction with chloroform:methanol(2:1) and washed with 0.99/o NaCkmethanol:chloroform (47:48:3). The chloroform fraction was dried under vacuum and counted in Bray’s scintillation fluid. Phospholipid

analysis

by thin-layer

chromatography

HeLa cells grown in 60-mm dishes were infected with SFV at a m.o.i. of 50 PFU per cell. Lipids were labeled by incubating the cells for 2 hr at 37” in a medium containing 10 &i/ml of [3H]glycerol. In some instances, the inhibitor cerulenin was present in the medium at a concentration of 0.08 mM. Lipid fractions were extracted as described previously (Guinea and Carrasco, 1990). Samples were redissolved in 100 ~1 chloroform and applied to activated silica gel thin-layer plates. The plates were developed in chloroform:methanol:acetic acid:water (25:15:4:2), dried, and visualized with iodine prior to autoradiography. Densitometric scanning was performed in a computing densitometer. Sucrose gradient

centrifugation

of membranes

Cells grown in 60-mm plates were infected with SFV at 50 PFU per cell and labeled with 20 &i/ml of [3H]glycerol at 37” for 1 hr. After labeling, cells were centrifuged for 5 min at 2500 rpm washed with phosphate

buffer saline, resuspended in 1 ml of RSB-Mg2+ and disrupted in a Potter-Elvehjem homogenizer (Thomas Scientific). Nuclei and cell debris were removed by centrifugation (5 min at 1500 rpm) and the cytoplasmic extract was placed on top of a discontinous sucrosedensity gradient with the following composition: 2.5 ml 60% sucrose in RSB, 3.5 ml 45%, 3.5 ml 40% 3.5 ml 30%, and 3.5 ml 25%. Gradients were centrifuged at 25,000 rpm for 20-22 hr in a Sorvall AH-627 rotor. Fractions (1 ml) were collected using an ISCO density gradient fractionator and extracted with chloroform: methanol (2: 1). Radioactivity was determined by scintillation counting. RESULTS Effects of cerulenin

on SFV protein synthesis

Cerulenin an antibiotic synthesized by Cephalosporium caerulens (Omura, 1976) is a potent and selective inhibitor of lipid synthesis in HeLa cells (Guinea and Carrasco, 1990). Addition of cerulenin to SFV-infected HeLa cells after virus entry drastically blocks the appearance of late proteins (Fig. 1A), whereas if the antibiotic is added later, 2 or 4 hr after the removal of the virus inoculum, there is no effect on viral protein synthesis. On the other hand, the presence of cerulenin does not block the shut-off of host translation induced by SFV, suggesting that low levels of the viral component involved in translation inhibition are made even in the presence of cerulenin. These results suggest that cerulenin has no direct inhibitory effect on viral translation,

PEREZ, GUINEA, AND CARRASCO

78

A

-

1

5 -__

h p.i.

0

I

1

RNA+

+

---A. 1

C

5

CRL hp.i.

RNA 1 -+

5 ~ -+

h.p. i. CRL

FIG. 4. Effect of cerulenin on the synthesis of SFV plus- or minus-stranded RNAs. (A, C) Analysis by Northern blot of RNA extracted from HeLa cells infected with SFV at a m.o.i. of 50 PFU per cell. 0.08 mM cerulenin (CRL) was present from the beginning of infection (+). (6, D) Densitometric scans of A and C. respectively.

but rather it blocks a step in the growth of SFV located after virus entry and before late viral translation. Since cerulenin interferes with fatty acid synthesis, it has been reported that this antibiotic inhibits the acylation of the VSV glycoproteins and thus blocks infectious virus production (Schlesinger and Malfer, 1982). Figure 1 I3 shows that the p62 and El proteins are labeled with palmitic acid and the addition of cerulenin does not block this acylation. These results lead to the conclusion that when cerulenin is present once the 26 S mRNA has been made, it has no effect on viral protein synthesis nor on the cleavage or acylation of viral polypeptides. Cerulenin

inhibits the synthesis

of SFV RNA

As a first step toward the analysis of cerulenin action on SFV RNA synthesis, we measured the incorporation of [3H]uridine in the presence of actinomycin D, in order to prevent cellular RNA synthesis. Figure 2

shows the two peaks of [3H]uridine incorporation observed during SFV infection, one at 4 hr and another one at 7-8 hr postinfection. Addition of cerulenin at 0, 2, or 4 hr postinfection (h.p.i.) drasticaly inhibits [3H]uridine incorporation into TCA-precipitable material (Fig. 2). The finding that protein synthesis does not decrease when the antibiotic is added at 2 or 4 h.p.i. in spite of the fact that the synthesis of most viral RNA is inhibited indicates that the amount of late 26 S mRNA used for translation is not influenced by the addition of cerulenin at the 2 or 4 hr postinfection. These results agree well with the suggestion that the inhibition of VSV nucleocapsid formation by cerulenin can be explained by the inhibition of virus RNA synthesis and expand our previous findings that not only the synthesis of poliovirus RNA, but also the synthesis of another RNA-containing animal virus depends on continuous lipid synthesis (Guinea and Carrasco, 1990). In order to analyze the synthesis of the different species of viral RNA, they were labeled with [3’P]-

RNA REPLICATION

at different times postinfection. Figure 5 shows that the infection of HeLa cells by SFV induces a reduction of [3H]glycerol incorporated in lipids after the second hour of infection. On the other hand, cerulenin clearly blocks [3H]glycerol incorporation when added any time postinfection. A more detailed analysis of the lipids synthesized was achieved by chromatography on silica gel plates and quantitation of labeled phospholipids by densitometry. Figure 6 indicates that the incorporation of [3H]glycerol into phospholipids is clearly stimulated during the first four hours of infection, whereas the synthesis of neutral lipids was decreased (Results not shown). Besides, cerulenin clearly reduces phospholipid synthesis both in mock-infected and in SFV-infected cells. Finally, membranes labeled with [3H]glycerol were obtained from uninfected, or SFV-infected HeLa cells and analyzed by sucrose gradient centrifugation. Soon

‘b

012345678 h p.i. FIG. 5. Effect of cerulenin on lipid synthesis. HeLa cells were grown in 24-well plates and infected with SFV (50 PFU/cell). At the times indicated 10 &i/ml [3H]glycerol was added to the medium. Each point was plotted in the middle of the incubation time, i.e. incubation from 0 to 1 hr was plotted at 0.5 hr and so on. Cells were harvested after 1 hr of labeling and the lipidic fraction was extracted as described under Materials and Methods. (Q Control of viral infection. (0) 0.08 mM cerulenin added at the times indicated by the arrows.

phosphate and the RNA separated in a nondenaturing agarose gel. A major band corresponding to replication complexes appears in these gels and only a faint band of 26 S mRNA is visible (Fig. 3). Once again, the presence of cerulenin during the labelling time drastically decreases the phosphate incorporated in RNA. Finally, to examine the appearance of plus- or minus-stranded SFV RNAs in the absence or in the presence of cerulenin, total RNA from SFV-infected cells was extracted, separated by agarose gels and hybridized with specific riboprobes. Figure 4 indicates that both minus- and plus-stranded RNA synthesis are hampered if cerulenin is present from the third hour of infection. In conclusion these results indicate that the antibiotic cerulenin is a potent inhibitor of viral RNA synthesis. Lipid synthesis and membrane formation SFV infection: Action of cerulenin

79

REQUIRES LIPID SYNTHESIS

A

PE

PC

-+

-+

----

O-2

-+-+

O-2

2-4

Hela

2

4-6

6-8

I:RL h.p.i

SFV

during

Virus infection usually induces profound modifications in phospholipid synthesis (Carrasco et al., 1989). However, little is known about the effects of SFV or SIN virus infection on lipids. Conflicting results were reported on the effects of SFV on the synthesis of phosphatidyl choline in BHK cells (Vance and Burke, 1974; Whitehead et a/., 1981). Therefore, we first examined the incorporation of glycerol into lipid material in SFVinfected cells and the action of cerulenin when added

0-2

Hela

o-2

2-4

4-6

6-6

h p.i.

SFV

FIG. 6. Analysis of phospholipids in SFV-infected HeLa cells by thin-layer chromatography. HeLa cells grown in 60.mm plates were infected with SFV at 50 PFU/cell. Lipids were labeled with 10 &i/ml of [3H]glycerol during 2 hr. 0.08 mMcerulenin was present (+) or not (-) during the labeling period. The phospholipid fraction was extracted, and analyzed by thin-layer chromatography as described under Materials and Methods.

80

PEREZ, GUINEA, AND CARRASCO mock-infected

cells

SFV 3-4 hp.i.

SFV 1-2 hp.i. 3ooo I--

o0 fradon

number

10 fraction

20 number

0

10 fraction

number

FIG. 7. Action of cerulenin on SFV-infected HeLa cells membranes. Analysis by discontinuous sucrose gradients. Mock-infected or SFV-infected HeLa cells were labeled with 20 &i/ml [3H]glycerol at the times indicated. Cell cultures were harvested, the cytoplasmic extract was prepared and fractionated as described under Materials and Methods. Each fraction was extracted by chloroform:methanol, and the radioactiviv determined. The top of the gradient is on the left. (lXl) Mock-infected, or SFV-infected cells. (0) Treatment with 0.08 mM cerulenin was from time 0 hr.

after infection, the pattern of membranes made is modified (Fig. 7). The peak migrating in the middle fractions, corresponding to smooth membranes, increases with respect to the membranes present in the rough endoplasmic reticulum, that migrate at the bottom fractions of the gradient. Addition of cerulenin, by blocking the synthesis of phospholipids, has consequences for the formation of labeled membranes. Thus, the amount of label in almost all the fractions was reduced when cerulenin was present, although it seems that the synthesis of some membranes, corresponding to the SER, is perhaps more affected. These results lead us to suggest that SFV infection increases the synthesis of phospholipids during the first hours of infection and that cerulenin powerfully blocks the synthesis of lipids, thus reducing the formation of new membranes. DlSCUSSlON A number of modifications in phospholipid metabolism and membrane traffic occur when animal cells are infected by viruses (Carrasco et a/., 1989). Thus, alphavirus-infected cells induce the formation of cytopathic vacuoles that have been subdivided in two morphologically distinct groups (Grimley et al., 1968; Griffiths et al., 1989). Type I cytopathic vacuoles (CPV-I) are seen early in the infectious cycle and contain a series of membrane buds attached to the interior surface of vacuoles. Viral RNA synthesis takes place in close association with CPV-I (Grimley et a/., 1968). The CPV-II are structurally different and are more numerous at late times of infection. The viral RNA polymerase has been localized in complex ribonucleoprotein structures associated with the cytoplasmic surface of the

CPV-I (Froshauer et a/., 1988). The suggestion that translation, transcription, and assembly of viral nucleocapsids occurs within a single large CPV has been advanced (Froshauer et a/., 1988). A clear similarity between these vacuoles and the cytoplasmic membrane structures induced by picornaviruses is evident (Dales et a/., 1965; Acheson and Tamm, 1967; Caliguiri and Tamm, 1970). The translation of poliovirus mRNA, its replication and even virus assembly occur in close connection with the virus-induced cytoplasmic vesicles (Koch and Koch, 1985). Picornaviruses stimulate the synthesis of phospholipids by a still unidentified mechanism and at least part of these newly made phospholipids participate in the formation of the membranes that form the cytoplasmic vacuoles, where RNA replication complexes attach (Butterworth eta/., 1976; Semler et a/., 1988; Carrasco et al., 1989). Our findings indicate that SFV is also able to stimulate the synthesis of phospholipids. It was previously reported that the infection of BHK cells by SFV led to a drastic inhibition of phosphatidyl choline synthesis as measured bycholine incorporation (Vance and Burke, 1974). In addition, a decrease in the activity of the enzyme CDP-choline: 1,2-diglyceride choline phosphotransferase present in microsomes was found in BHK cells infected by SFV (Vance and Burke, 1974). Further analyses suggested that the rate of phosphatidylcholine biosynthesis was not inhibited in the infected cells, but was even stimulated (Whitehead et al., 1981). The use of glycerol, or glucose (results not shown) clearly indicates that SFV, like poliovirus, (Guinea and Carrasco, 1990) enhances the synthesis of phospholipids after infection.

RNA REPLICATION

REQUIRES LIPID SYNTHESIS

The biological implications of phospholipid synthesis in the replication cycle of animal viruses remain largely unknown. Recently, we found that the inhibitor of phospholipid synthesis cerulenin, decreased both lipid synthesis and poliovirus RNA replication in infected HeLa cells (Guinea and Carrasco, 1990). Our present results point to the idea that the synthesis of new phospholipids is required for membrane formation. In turn, the newly made membranes are necessaryfor SFV RNA synthesis, but are not required for the translation of the 26 S mRNA. In agreement with our findings is the recent report that Sindbis virus does not replicate in a CHO cell mutant unable to synthesize phospholipids (Kuge et al., 1989). Although Sindbis virus can be internalized in the mutant cells, no viral RNA is synthesized. However, Kuge er al., (1989) had no explanation for their findings and did not correlate the inhibition of phospholipids with the blockade of viral RNA replication. We suggest that the effect described is akin to the action of cerulenin on SFV RNA synthesis that we report here. In fact, a previous report on the action of cerulenin on VSV-infected chicken embryo fibroblasts showed a decreased synthesis of viral nucleocapsids (Schlesinger and Malfer, 1982). The authors point out that this inhibition “could be explained by inhibition of viral RNA synthesis,” although they indicate that acylation of viral glycoproteins does not occur in the presence of cerulenin and this effect may be responsible for the inhibition of virus assembly and budding (Schlesinger and Malfer, 1982). A plausible explanation for the lack of inhibition by cerulenin of fatty acid acylation of SFV proteins in our system is that labelled palmitic acid is provided to the cultured cells. Thus, the cells do not have to synthesize this fatty acid and this step would not be inhibited by cerulenin. Nevertheless, even if this antibiotic affects late steps of the virus life cycle, such as assembly and budding, our results indicate that cerulenin has a potent effect on an earlier step of virus replication, i.e., viral RNA synthesis. Therefore, some of the previous findings reported with cerulenin in another virus-cell systems need to be reconsidered in the light of the potential inhibition that the antibiotic has on viral RNA synthesis. ACKNOWLEDGMENTS The expert technical assistance of Ms. M. Chorro and Mr. M. A. Sanz is acknowledged. L.P. and R.G. are holders of a Comunidad Autonoma de Madrid and a Ministerio de Education y Ciencia fellowships respectively. CICM (Project 91088-0233) and Fundacibn Ram6n Areces are acknowledged for financial support.

REFERENCES ACHESON, N. H., and TAMM, I. (1967). Replication of Semliki forest virus: An electron microscopic study. Virology 32, 128-l 43.

81

BIENZ, K., EGGER,D., TROXLER,M., and PASAMONTES,L. (1990). Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region. 1. Viral. 64, 1156-l 163. BUIXRWORTH, 9. E., SHIMSHICK, E. J., and YIN, F. H. (1976). Association of the poliovirus RNA polymerase complex with phosphoiipid membranes. J. Viral. 19, 457-466. CALIGUIRI, L. A., and TAMM, I. (1970). The role of cy-toplasmic membranes in poliovirus biosynthesis. Virology 42, 100-l 1 1. CARRASCO,L., OTERO, M. J., and CASTRILLO,J. L. (1989). Modification of membrane permeability by animal viruses. Pharmacol. Ther. 40, 171-212. CORNATZER,W. E., SANDSTROM, W., and FIERS, R. G. (1961). The effect of poliomyelitis virus type 1 (Mahoney strain) on the phospholipid metabolism of the HeLa cell. Biochim. Biophys. Acfa 49,414415. DALES, S., EGGERS,H. J., TAMM, I., and PALADE, G. E. (1965). Electron microscopic study of the formation of poliovirus. Virology 26,379389. FROSHAUER,S., KARTENBECK,J., and HELENIUS.A. (1988). Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes. 1. Cell Ho/. 107, 2075-2086. GAEDIGK-NITSCHO,K., DING, M., LEVY, M. A., and SCHLESINGER,M. J. (1990). Site-directed mutations in the Sindbis virus 6K protein reveal sites for fatty acylation and the underacylated protein affects virus release and virion structure. Virology 175, 282-291. GAROFF, H., FRISCHAUF,A. M., SIMONS, K., LEHRACH, H., and DELIUS, H. (1980a). Nucleotide sequence of cDNA coding for Semliki forest virus membrane glycoproteins. Nature 288, 236-241. GAROFF, H., FRISCHAUF,A. M., SIMONS, K., LEHRACH, H., and DELIUS, H. (1980b). The capsid protein of Semliki forest virus has clusters of basic amino acids and prolines in its amino-terminal region. Proc. Natl. Acad. Sci. USA 77, 6376-6380. GIRARD, M.. BALTIMORE, D., and DARNELL. J. E. (1967). The poliovirus replication complex: Site for synthesis of poliovirus RNA. J. MO/. Biol. 24, 59-74. GRIFFITH& G., FULLER,S. D., BACK, R., HOLLINSHEAD,M., PFEIFFER,S., and SIMONS, K. (1989). The dynamic nature of the Golgi complex. J. Cell Biol. 108, 277-297. GUINEA, R., and CARRASCO,L. (1990). Phospholipid biosynthesis and poliovirus genome replication, two coupled phenomena. EMBO J. 9, 201 l-2016. GRIMLEY, P. M., BEREZESKY,I. K., and FRIEDMAN, R. M. (1968). Cytoplasmic structures associated with an arbovirus infection: loci of viral ribonucleic acid synthesis. J. Viral. 2, 1326-l 338. HARFORD, C. G., HAMLIN, A., and RIDERS, E. (1966). Electron microscopic autoradiography of DNA synthesis in cells infected with vaccinia virus. Exp. Cell Res. 42, 50-57. IKUTA, K., and LUFTIG, R. 9. (1986). Inhibition of cleavage of Moloney murine virus gag and env coded precursor polyproteins by cerulenin. Virology 154, 195-206. KOCH, F.. and KOCH, G. (1985). “The Molecular Biology of Poliovirus.” Springer-Verlag, New York. KOTWAL,G. J., and GHOSH, H. P. (1984). Role of fatty acid acylation of membrane glycoproteins. J. Biol. Chem. 259, 4699-4701. KUGE, O., AKAMATSU, Y., and NISHIJIMA, M. (1989). Abortive infection with Sindbis virus of a Chinese hamster ovary cell mutant defective in phosphatidylserine and phosphatidylethanolamine biosynthesis. Biochim. Biophys. Acra 986, 6 l-69. MOSSER. A. G., CALIGUIRI.L. A., and TAMM, I. (1972). Incorporation of lipid precursors into cytoplasmic membranes of poliovirus-infected HeLa cells.Virology 47, 39-47. OMURA. S. (1970). The antibiotic perulenin, a novel tool for biochemis-

82

PEREZ, GUINEA, AND CARRASCO try as an inhibitor of fatty acid synthesis.

Bacterial. Rev. 40, 68 I-

697. PAL, R., GALLO, R. C., and SARNGADHARAN,M. G. (1988). Processing of the structural proteins of human immunodefiencyvirus type 1 in the presence of monensin and cerulenin. Proc. Nat/. Acad. Sci. USA 85, 9283-9286. PENMAN, S. (1965). Stimulation of the incorporation of choline in poliovirus-infected cells. L/iro/ogy 25, 148-l 52. SCHLESINGER,M. J., and MALFER, C. (1982). Cerulenin blocks fatty acid acylation of glycoproteins and inhibits vesicular stomatitis and Sindbis virus particle formation. J. Biol. Chem. 257,988-9890. SCHLESINGER,S., and SCHLESINGER,M. (1990). Replication of Togaviridae and Flaviviridae. ln “Virology” (B. N. Fields et a/., Eds.), 2nd ed., pp, 697-7 11. Raven, New York. SEMLER, B. L., KUHN, R. J., and WIMMER, E. (1988). Replication of the poliovirus genome. ln “RNA Genetics” (E. Domingo, J. J. Holland, and P. Ahlquist, Eds.). CRC, Boca Raton, FL. STRAUSS,1. H., and STRAUSS,E. G. (1988). Replication of the RNAs of

Alphaviruses and Flaviviruses. In “RNA Genetics” pp. 72-90. (E. Domingo, J. J. Holland, P. Ahlquist, Eds.). CRC, Boca Raton, FL. TAKKINEN,K. (1986). Complete nucleotide sequence of the nonstructural protein genes of Semliki forest virus. Nucleic Acid Res. 14, 5667-5682. VANCE, D. E., and BURKE, D. C. (1974). Inhibition of 3-sn-phosphatidylcholine biosynthesis in baby hamster kidney 21 cells infected with Semliki forest virus. Eur. J. Biochem. 43, 327-336. VANCE, D. E., TRIP, E. M., and PADDON, H. B. (1980). Poliovirus increases phosphatidylcholine metabolism in HeLa cells by stimulation of the rate-limiting reaction catalyzed by CTP: Phosphocholine cytidylyltransferase. /. Biol. Chem. 255, 1064-l 069. WELCH, W., and SEFTON, B. M. (1980). Characterization of a small, nonstructural viral polypeptide present late during infection of BHK cells by Semliki forest virus. 1. Viral. 33, 230-237. WHITEHEAD, F. W., TRIP, E., and VANCE, D. E. (1981). Semliki forest virus does not inhibit phosphatidylcholine biosynthesis in BHK-21 cells. Can. J. Biochem. 59, 38-46.

Synthesis of Semliki Forest virus RNA requires continuous lipid synthesis.

The involvement of lipid biosynthesis in the replication of Semliki Forest virus (SFV) in HeLa cells has been analyzed by the use of cerulenin, an inh...
3MB Sizes 0 Downloads 0 Views