7) 430–436www.elsevier.com/locate/yviro

Virology 363 (200

Differential inhibition of cellular and Sindbis virus translation by brefeldin A

Susana Molina ⁎, Miguel A. Sanz, Vanesa Madan, Iván Ventoso, Alfredo Castelló, Luis Carrasco

Centro de Biología Molecular (CSIC-UAM). Facultad de Ciencias, Universidad Autónoma. Cantoblanco, 28049 Madrid, Spain

Received 14 December 2006; returned to author for revision 4 January 2007; accepted 1 February 2007Available online 23 March 2007

Abstract

Brefeldin A is a macrolide compound that interferes with the secretory pathway and also affects protein synthesis in mammalian cells. As aresult, this antibiotic impedes the maturation of viral glycoproteins of enveloped viruses and viral genome replication in several virus species. Inthe present work, we show that translation of subgenomic mRNA from Sindbis virus, which in contrast to cellular translation is resistant tobrefeldin A after prolonged treatment. The phosphorylation of eIF2α as a result of brefeldin A treatment correlates with the inhibition of cellulartranslation, while late viral protein synthesis is resistant to this phosphorylation. The effect of brefeldin A on Sindbis virus replication was alsoexamined using a Sindbis virus replicon. Although brefeldin A delayed viral RNA synthesis, translation by non-replicative viral RNAs was notaffected, reinforcing the idea that brefeldin A delays viral RNA replication, but does not directly affect Sindbis virus protein synthesis.© 2007 Elsevier Inc. All rights reserved.

Keywords: Brefeldin A; Sindbis virus; Cellular translation; Viral protein synthesis; Sindbis virus replication

Sindbis virus (Mirazimi et al., 1996) is the prototype memberof the Alphavirus genus of the Togaviridae family. The genome,a positive-strand non-segmented RNA of almost 12 kb, consistsof two open reading frames: the first two-thirds encode non-structural proteins (nsPs), while the remaining third encodesstructural proteins (Frolov, 2004; Strauss and Strauss, 1994). Thegenomic 49S RNA also serves as a template for the synthesis ofnegative RNAwhich is used to generatemore genome copies andto transcribe the subgenomic 26S mRNA from an internalpromoter (Strauss and Strauss, 1994). The lytic cycle of SVinfection has two well-defined stages. In the first one, SVgenomic RNA is translated to render the non-structural proteins,nsp1, 2, 3 and 4. These proteins are required for viral replicationand transcription (Kaariainen and Ahola, 2002). At about 2–4 hpost infection (h p.i.), the pattern of protein synthesis drasticallychanges and subgenomic RNA translation increases notably,while genomic RNA is encapsidated in new virus particles. Invertebrate cells, a rapid inhibition of host protein synthesisoccurs at this time of infection (Frolov and Schlesinger, 1994,1996; Strauss and Strauss, 1994; Ventoso et al., 2006). Theability of a virus to inhibit the translation of cellular mRNAsunder conditions in which viral mRNAs are translated has been

⁎ Corresponding author.E-mail address: smolina@cbm.uam.es (S. Molina).

0042-6822/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.virol.2007.02.001

observed in mammalian and other higher eukaryotic cells for avariety of positive- and negative-strand RNA viruses, includingpoliovirus and influenza virus, and for many species of DNAviruses such as adenoviruses, herpesviruses and poxviruses(Bushell and Sarnow, 2002; Schneider and Mohr, 2003;Thompson and Sarnow, 2000). How this blockade is accom-plished remains still poorly understood in many cases, anddifferent viruses may use different mechanisms to achieve thisdifferential inhibition of translation (Bushell and Sarnow, 2002;Schneider and Mohr, 2003; Thompson and Sarnow, 2000).

Brefeldin A (BFA) is a macrolide compound capable ofdisrupting the vesicular system and blocking glycoproteinsecretion in eukaryotic cells (Klausner et al., 1992; Lee andLinstedt, 1999; Nebenfuhr et al., 2002; Pelham, 1991; Sata et al.,1999). The molecular target of BFA is a subset of Sec7-typeGTP-exchange factors (GEFs), which activate a GTP-bindingprotein known as ADP-ribosylation factor 1 (ARF1) (Jacksonand Casanova, 2000). ARF1 recruits the COPI coat and AP-1/clathrin coat protein complexes involved in the formation oftransport vesicles (Scales et al., 2000). BFA inhibits the GDP-GTP-exchange reaction between GEF and ARF1, leading to theinhibition of ARF activation (Jackson and Casanova, 2000;Renault et al., 2003). As a consequence, coat proteins arereleased fromGolgi membranes, provoking the loss of control offusion and budding membranes (Klausner et al., 1992; Kreis

Fig. 1. Effect of BFA on cellular and SV late protein synthesis. Mock-infected(A) or SV-infected BHK cells (multiplicity of infection, 20 PFU/cell) (B) weretreated for 6 h and then labeled with [35S]-Met/Cys during 30 min in thepresence of the same concentrations of BFA. Samples were processed by SDS–PAGE, fluorography and autoradiography as indicated in Materials andmethods. P160, precursor of SV structural proteins; PE2, precursor of E2glycoprotein; E1, mature SV E1 glycoprotein. (C) SV C protein and a cellularprotein, both indicated with arrows, were subjected to densitometric analysis toestimate the percentage of protein synthesis compared to untreated controls.

431S. Molina et al. / Virology 363 (2007) 430–436

et al., 1995; Lippincott-Schwartz et al., 1990, 1991; Orci et al.,1991; Pelham, 1991; Scales et al., 2000; Torii et al., 1995). BFApossesses antiviral activity against enveloped viruses since itimpedes the maturation of viral glycoproteins and the productionof infectious particles which mature on the plasma membrane orwithin the cell (Dasgupta and Wilson, 2001; Irurzun et al., 1993;Madan et al., 2005; Mirazimi et al., 1996; Suikkanen et al.,2003). The replication of viruses such as poliovirus or vesicularstomatitis virus is inhibited by BFA since RNA replication ofthese viruses requires continuous synthesis of lipids to providenew membranes to which viral replication complexes attach.Therefore, this compound acts against some non-envelopedviruses, interfering with genome replication, which requires anintact vesicular system (Cuconati et al., 1998; Gazina et al.,2002; Irurzun et al., 1992, 1993; Maynell et al., 1992). BFA alsodecreases protein synthesis in culture cells. Thus, BFA treatmentof rat GH3 pituitary cells leads to an inhibition of proteinsynthesis at the initiation level (Fishman and Curran, 1992;Mellor et al., 1994). Presumably, this effect is due to the stresssituation of the endoplasmic reticulum caused by the disorga-nization of the membrane system (Liu and Kaufman, 2003;Mellor et al., 1994; Prostko et al., 1993). We have now analyzedthe action of BFA on the translation of BHK-21 cells and SV-infected cells and also tested the effect of BFA on SV mRNAsynthesis. Our results indicate that BFA delays viral translationby retarding viral RNA synthesis.

Results

Effect of BFA on protein synthesis in BHK-21 and SV-infectedcells

To test the effect of BFA on protein synthesis, SV-infected oruninfected BHK-21 cells were treated with different concentra-tions of BFA after viral adsorption. Concentrations of BFAranged from 1 to 20 μg/ml. Cellular (Fig. 1A) and late viralprotein synthesis (Fig. 1B) were analyzed after 6 h of BFAtreatment by metabolic labeling with 35S-Met/Cys. At all theconcentrations analyzed, BFA induced an inhibition of cellularprotein synthesis of around 80%, whereas very little effect wasobserved on viral translation. Notably, SV protein synthesis wasnot inhibited at the highest concentration of BFA whereascellular translation was strongly blocked by only 1 μg/ml of thisantibiotic. The comparative activity of BFA on cellular and viralprotein synthesis is represented in Fig. 1C. There is a cleardifferential inhibition caused by BFA on cellular as compared tosubgenomic SV mRNA translation.

Our next aim was to examine the action of BFA on the earlyand late phases of the viral cycle. This was achieved by addingBFA after virus adsorption or at 2 h p.i. and analyzing thesynthesis of structural viral proteins at 2, 4 and 6 h p.i. byprotein radiolabeling (Fig. 2A). As a control, translation inuntreated cells infected by SV, as well as in non-infected cells(Fig. 2B), was estimated. Addition of BFA after viral adsorptioncaused 91% inhibition of viral translation at 2 h p.i. Thisinhibition decreased throughout the course of infection and wasonly 10% at 6 h p.i. However, when BFAwas added at 2 h p.i.,

the reduction in viral translation was about 39% at the begin-ning of infection, and no differences in protein synthesis withrespect to the untreated cells were observed at 6 h p.i. Therefore,BFA only delays but does not inhibit synthesis of SV structuralproteins.

In culture cells, such as rat GH3 pituitary cells, BFAinduces eIF2α phosphorylation, which could be responsiblefor the inhibition of translation (Mellor et al., 1994). Therefore,phosphorylation of eIF2α during BFA treatment and after SVinfection of BFA-treated or non-treated cells was analyzed(Fig. 3). To avoid the potential inhibition of virus entry byBFA, this compound was added after virus adsorption. Cellswere collected at the times indicated in the figure, andphosphorylated or total eIF2 were detected by Western blottingwith specific antibodies. As a control, eIF2α phosphorylationwas estimated in mock-infected non-BFA-treated cells. InBFA-treated cells, the phosphorylated form is detectable from2 h of treatment, both in mock-infected and SV-infected cells,while phosphorylation of eIF2α in SV-infected non-treatedcells was detected from 4 h p.i., as described previously (Ven-toso et al., 2006). In addition, eIF2 remains phosphorylated at4 and 6 h p.i. in SV-infected BFA-treated cells when SV tran-slation reaches the levels of non-treated controls. This resultsuggests that, although in SV-infected BFA-treated cells theeIF2α phosphorylation was initially induced by BFA treat-ment, it did not affect SV RNA translation. On the other hand,inhibition of cellular protein synthesis can be observed after2 h of treatment with BFA, which coincides with an increase inthe phosphorylated form of eIF2α.

Fig. 2. Kinetics of cellular and SV late protein synthesis: effect of BFA. (A) SV-infected cells were treated at 0 or 2 h p.i. and radiolabeled at 2, 4 and 6 h p.i.during 30 min. (B) Mock-infected cells were treated with BFA and radiolabeledafter 2, 4 or 6 h of treatment. In both cases, BFA was present during labeling.Samples were processed by SDS–PAGE, followed by fluorography andautoradiography. The numbers below each lane represent the percentage ofprotein synthesis with respect to non-treated controls calculated by densitometryof SV C protein and a cellular protein, indicated by the arrows. P160: precursorof SV structural proteins; PE2, precursor of E2 glycoprotein; E1, mature SV E1glycoprotein.

432 S. Molina et al. / Virology 363 (2007) 430–436

To analyze the effect of BFA on translation directed by SVgenomic RNA, a recombinant virus SV-Luc with the luciferasegene inserted into the nsp3 sequence was employed (Bick et al.,2003). BHK cells were treated with 5 μg/ml of BFA at 2 hbefore infection with SV-Luc, after virus adsorption, or at 2 hpost infection. Although in SV-infected cells, the synthesis ofnon-structural proteins ceases at 4 h p.i., SV-luc has a delayedearly phase. For this reason, luciferase activity was measured at2, 4 and 6 h p.i., the time when genomic RNA is being translated(Ventoso et al., 2006). The greatest inhibition of luciferase

Fig. 3. Phosphorylation of eIF2α. SV-infected or mock-infected cells weretreated with BFA, added after virus adsorption. After 2, 4 or 6 h, cells werecollected in sample buffer and subjected to SDS–PAGE andWestern blotting, asdescribed in Materials and methods, using specific antibodies for thephosphorylated (eIF2-P) and the non-phosphorylated form of eIF2. As acontrol, non-treated mock-infected BHK cells were used.

synthesis occurred when cells were treated with BFA beforeinfection (Fig. 4A). When BFAwas present from the beginningof infection or from 2 h p.i., luciferase synthesis was about 70%as compared to the non-treated control at 4 h p.i., and no effectwas observed at 6 h p.i. These results indicate that theexpression of genomic and subgenomic mRNAs of SV isdelayed by BFA treatment. Interestingly, these data also suggestthat the inhibitory effect of BFA would not be correlated with

Fig. 4. Effect of BFA on SV non-structural proteins and RNA synthesis. SV-lucinfected cells were treated with 5 μg/ml of BFA 2 h before infection and at 0 and2 h p.i. Cells were collected at 2, 4 and 6 h p i. to measure luciferase activity (A)or the amount of luciferase, fused to nsP3 or as part of the nsPs polyprotein byWestern blot using a rabbit polyclonal antiserum (B), as described in Materialsand methods. (C) Quantitation of total RNA and genomic RNA (D) molecules inSV-infected cells in the presence or absence of 5 μg/ml of BFA. BFAwas addedat 0 h p.i. and cells were collected at 2, 4 and 6 h p.i. RNA was extracted andquantified by real-time PCR as described in Materials and methods. Data arepresented as percentages of number of molecules of RNA in BFA-treated withrespect to non-treated cells.

433S. Molina et al. / Virology 363 (2007) 430–436

eIF2α phosphorylation since this phosphorylation inhibitstranslation of SV genomic RNA (Ventoso et al., 2006). Toassay if BFA alters the proteolytic processing of early SVproteins, BHK cells, untreated or treated with 5 μg/ml BFA for2 h, were infected with SV-Luc. At 2, 4 and 6 h p.i., cells werecollected and the amount of luciferase fused with the nsP3, aswell as the uncleaved precursors, was detected by Westernblotting (Fig. 4B). The levels of unprocessed polyprotein wereundetectable in BFA-treated cells or proportional to thosedetected in the non-treated control, indicating that thiscompound does not affect the proteolytic cleavage of SV non-structural proteins.

Effects of BFA on SV RNA synthesis

We next determined the effect of BFA on SV RNA synthesis.In this experiment, SV-infected BHK cells were treated withBFA immediately after infection, and at 2, 4 and 6 h p.i. RNAwas extracted and quantitated by real-time PCR, using specificoligonucleotides for measuring total RNA (Fig. 4C) or genomicRNA (Fig. 4D). The amount of total RNA in BFA-treated cellswas lower than in non-treated cells during the initial hours ofinfection. However, at later times a similar amount of total RNAwas detected in BFA-treated and non-treated cells. Thesefindings suggest that SV replication is delayed by BFA, thusaccounting for the decreased synthesis of both structural andnon-structural proteins. Analysis of genomic RNA levelsrevealed that they were lower in BFA-treated cells during theentire treatment period, which suggests that BFA interferes withSV RNA synthesis rather than protein synthesis.

Effect of BFA on viral replication

Since BFA blocks genome replication of some viruses(Gazina et al., 2002; Irurzun et al., 1992, 1993; Maynell et al.,1992), our next aim was to determine whether BFA inhibitedSV replication using the SV replicon repL26S C-luc (Fig. 5A).BHK cells were treated for 4 h with 5 μg/ml BFA and thenelectroporated with repL26S C-luc. BFA was maintained afterelectroporation, and cells were collected at 2, 4 and 6 h p.e.Luciferase activity, which reflects the level of proteinstranslated from subgenomic mRNA, is shown in Fig. 5B. Anacute effect of BFA can be observed within the first hours ofelectroporation, while at 6 h p.e. the luciferase activity issimilar to that found in untreated controls. In addition, theeffect of BFA on translation of the non-replicative RNAs 49S-luc and L26SC-luc was assayed. A schematic representation ofthese RNAs is shown in Fig. 5A. Both RNAs wereelectroporated in BHK cells previously treated with BFA for4 h. BFA was maintained during and after electroporation, and

Fig. 5. Effect of BFA on SV replication. (A) Schematic representation of thedifferent viral RNAs repL26S C-luc, L26SC-luc and 49S-luc. (B) Luciferaseactivity at 2, 4 and 6 h p.e. of BHK cells with the replicative RNA repL26S C-luc,in the presence or absence of BFA. (C) and (D) show the measurement of lucifera-se activity at 2, 4 and 6 h p.e. in BHK cells electroporated with the non-replicativeRNAs L26SC-luc and 49S-luc, respectively, in the presence or absence of BFA.

cells were collected at 2, 4 and 6 h p.e. to measure luciferaseactivity (Figs. 5C and D). In both cases, luciferase activity inBFA-treated cells was higher than in untreated control cells,

434 S. Molina et al. / Virology 363 (2007) 430–436

indicating that BFA had no inhibitory effect on the translationof non-replicative RNAs. The lack of inhibition of SV 49S-derived RNA by BFA is consistent with the results obtainedwith the SV-Luc virus (Fig. 4A).

These findings suggest that BFA interferes with the SVreplicative RNA but does not inhibit the non-replicative forms,supporting the idea that the action of BFA on SV is due to thedelay in RNA synthesis.

Discussion

The fungal metabolite BFA is a well characterized inhibitorof the secretory pathway in mammalian cells (Lippincott-Schwartz et al., 1991; Orci et al., 1991; Pelham, 1991). BFAalso causes a potent inhibition of cellular protein synthesis,presumably due to the unfolded protein response (UPR)triggered in the endoplasmic reticulum as a consequence ofdisorganization of the membrane system (Fishman and Curran,1992; Liu and Kaufman, 2003; Mellor et al., 1994; Prostko etal., 1993). Notably, SV protein synthesis is maintained after aprolonged treatment with this compound, thus reflecting adifferential behaviour between cellular and viral mRNAs.Indeed, BFA strongly inhibits cellular protein synthesis,whereas SV translation is only slightly delayed. Notably, theamount of phosphorylated eIF2α increases after 2 h treatment ofBHK cells with BFA, in agreement with previous resultsreported for GH3 pituitary cells (Mellor et al., 1994). Thisfinding can be interpreted as a consequence of UPR activation(Dinter and Berger, 1998; Harding et al., 1999; Lee andLinstedt, 1999). However, this phosphorylation might not be thesole cause of translation inhibition induced by BFA sincecellular protein synthesis is not completely abrogated. Further-more, the synthesis of luciferase from SV-Luc and the SV-derived genomic RNA in BFA-treated cells is not inhibited, andthe translatability of exogenous viral mRNAs transfected inBFA-treated cells was not affected (data not shown).

On the other hand, SV protein synthesis is reduced in thepresence of BFA only at early times after infection. Viraltranslation recovers throughout the time of treatment, so that thelevels of SV protein synthesis reach those observed in untreatedinfected cells. The inhibitory effect of BFA is lower when addedlater during infection, suggesting that BFA has an indirect effecton SV protein synthesis. Previous studies that analyzed theaction of BFA on togavirus protein synthesis added thiscompound or analyzed protein synthesis at later times ofinfection, thus, no effect of BFAwas observed on genomic norsubgenomic RNA translation (Carleton and Brown, 1996; DaCosta and Rebello, 1999; Madan et al., 2005). Interestingly, theamount of genomic RNA is lower in the presence of BFA ascompared to untreated cells, indicating that BFA interferes withviral RNA replication. This possibility is reinforced by theobservation that BFA only reduced the expression of the SV-derived replicon, whereas non-replicative mRNAs were trans-lated under these conditions. The finding that the 49S non-replicative RNA is not inhibited by BFA treatment agrees withthe idea that the action of BFA on SV is exerted on replication.The resistance of 26S mRNA to BFA, which increases eIF2α

phosphorylation, could be due to its hairpin structures that mayprovide translational resistance to the conditions induced by thiscompound (Ventoso et al., 2006).

The action of BFA on the vesicular system impairs thematuration of viral glycoproteins, suppressing the formation ofviral particles of enveloped viruses (Dasgupta andWilson, 2001;Irurzun et al., 1993; Madan et al., 2005; Mirazimi et al., 1996;Suikkanen et al., 2003). In addition, BFA also has an inhibitoryeffect on replication of many non-enveloped viruses withoutglycoproteins, such as enteroviruses and rhinoviruses. In thesecases, BFA abrogates viral replication since this process isassociated with the formation of vesicular structures (Cuconatiet al., 1998; Gazina et al., 2002; Irurzun et al., 1992; Maynell etal., 1992). BFA is known to affect the control of fusion andbudding of membranes, and so also influences the process ofvacuole formation (Klausner et al., 1992; Kreis et al., 1995;Lippincott-Schwartz et al., 1990, 1991; Orci et al., 1991;Pelham, 1991; Scales et al., 2000; Torii et al., 1995). Replicationof SV alters intracellular membranes, creating new vesicleswhich are linked to viral RNA synthesis (Kujala et al., 2001;Peranen et al., 1995; Salonen et al., 2005). Most probably, BFAinterferes with the formation of the new vesicles by decreasingthe replication efficiency, and hence affecting viral proteinsynthesis (Cuconati et al., 1998; Irurzun et al., 1992; Maynellet al., 1992). This hypothesis also explains the differential effectobserved when BFA is added at different times after infection,with the greatest effect when BFAwas present before infection.When SV infection takes place in cells previously treated withBFA, the vesicular system is disorganized before the replicativecomplexes are formed. In this case, replication may be moreaffected than when BFA is added after virus adsorption or at 2 hp.i. The formation of the vesicles needed for viral replicationbefore BFA addition may result in a lower inhibition of replica-tion. However, this inhibition is partial even when BFA is addedbefore infection. Thus, viral replication is not completelyabolished by the presence of BFA since RNA and proteinsynthesis increases throughout infection. Therefore, BFA maydisturb and delay, but not completely block, the formation ofviral replication complexes. In such conditions, more time isneeded to reach adequate levels of SV non-structural proteins inBFA-treated cells. Thus, the amount of viral RNA and proteinsynthesis finally recovered in the presence of BFA and at 6 h p.i.is comparable to non-treated controls.

Taken together, these results provide further insight into theeffect of the macrolide compound BFA on alphavirus infection.

Materials and methods

Cell culture and viruses

Baby hamster kidney (BHK-21) cells were grown at 37 °Cin Dulbecco's Modified Eagle's medium (DMEM) supplemen-ted with 5% fetal calf serum (FCS) and non-essential aminoacids. Wild-type (wt) SV and SV-Luc stocks were obtainedfrom the cDNA clones pT7SVwt (Hahn et al., 1992; Sanz andCarrasco, 2001) and pToto1101/Luc, respectively (generouslyprovided by Charles M. Rice, Rockefeller University, NY)

435S. Molina et al. / Virology 363 (2007) 430–436

(Bick et al., 2003). They were propagated and titered in BHK-21 cultures.

Plasmids

The replicon repL26S C-luc was obtained by in vitrotranscription from the plasmid pT7 repL26S C-luc. This plasmidis derived from pT7SVwt and contains the luciferase genebetween the SV C protein sequence and the SV 3′ non-codingregion with deletion of the sequence encoding for E3, E2, 6Kand E1 proteins. The first three codons encoding for E3 aremaintained to facilitate the autoproteolytic cleavage of C protein.

The non-replicative RNA 49S-luc was obtained by in vitrotranscription from pToto1101/Luc digested with BssHII, whicheliminates the 3′ non-coding region. The resulting RNA waspolyadenylated post-transcriptionally with PolyA polymerase(Invitrogen).

The non-replicative RNA L26SC-luc was obtained by invitro transcription from pT7 C+ Luc. This plasmid contains thesubgenomic sequence from repL26S C-luc after the T7 promotersequence, which permits the in vitro production of this mRNA.

Viral infections

BHK-21 cells were infected with wild-type SVor SV-Luc ata multiplicity of infection of 10 PFU/cell. After 30 min ofadsorption, the medium was removed and culture plates wereincubated with fresh DMEM supplemented with 5% FCS.

Effects of different compounds on SV and cellular translation

BFA (5 μg/ml) (Sigma) was added to the SV-infected culturesat different h p.i. At the indicated times, proteins were labeled for30 min with 200 μl methionine/cysteine-free DMEM supple-mented with 1 μl Trans label [35S]-Met/Cys (15 mCi/ml,Amersham Biosciences) per well in the presence or absence ofthe inhibitor. Cells were collected in sample buffer, boiled for4 min and analyzed by SDS–PAGE and fluorography.

Western blot analysis

The phosphorylation state of the translation initiation factoreIF2 was determined by Western blotting (Ventoso et al., 2006),as well as the proteolytic cleavage of the nsPs. BHK-21 cellswere infected, treated with BFA or infected and treated withBFA after virus adsorption. At different h p.i., cells werecollected in sample buffer and proteins were fractionated bySDS–PAGE in 15% polyacrylamide gels and transferred tonitrocellulose membranes by wet transfer. Membranes wereblocked with PBS containing 5% low-fat dry milk. Anti-eIF2(Santa Cruz), anti-phosphorylated-eIF2 (Biosource) or anti-Luciferase (Promega) antibodies were then added, and themembranes were washed with PBS containing 0.2% Tween 20.Goat anti-rabbit horseradish peroxidase-conjugated antibodies(Pierce) and the ECL kit (Amersham Biosciences) were used todetect bound antibodies. Chemiluminescence was detected byexposure to Agfa X-ray film.

Measurement of luciferase activity

BHK-21 cells were infected with SV-Luc or electroporatedwith 20 μg of RNA. At different hours post-electroporation (h p.e.), cells were lysed in a buffer containing 0.5% Triton X-100,25 mM glycylglycine (pH 7.8) and 1 mM dithiothreitol.Luciferase activity was determined using a Monolight 2010apparatus (Analytical Luminescence Laboratory), as describedpreviously (Ventoso and Carrasco, 1995).

Analysis of mRNA by real-time PCR

SV RNA levels in infected cells were determined by real-time quantitative reverse transcription (RT)-PCR as previouslydescribed (Alvarez et al., 2003; Castello et al., 2006). Briefly,total RNAwas extracted from 25×10 cells at the times indicatedin each figure, using the RNeasy commercial kit (Qiagen)following the manufacturer's recommendations. The isolatedRNAwas resuspended in 30 μl of nuclease-free water, and 3 μlwas subjected to analysis. Real-time quantitative RT-PCR wasperformed with the LightCycler thermal cycler system (RocheDiagnostics) using the RNA Master SYBR Green I kit (RocheDiagnostics) as described (Alvarez et al., 2003; Castello et al.,2006). The primers C-forward (5′-GAA CGA GGA CG GAGATGT CAT CG-3′) and C-reverse (5′-CAG CGC CAC CGAGGA CTATCG C-3′) were employed to quantify the total SVRNA. Data analysis was done using the Roche MolecularBiochemicals LightCycler software, version 3.3. The specificityof the amplification reactions was confirmed by analyzing thecorresponding melting curves.

Acknowledgments

This study was supported by Grant (BFU2006-02182/BMC)from DGICYT and an Institutional Grant awarded to the Centrode Biología Molecular “Severo Ochoa” by the FundaciónRamón Areces.

References

Alvarez, E., Menendez-Arias, L., Carrasco, L., 2003. The eukaryotic translationinitiation factor 4GI is cleaved by different retroviral proteases. J. Virol. 77(23), 12392–12400.

Bick, M.J., Carroll, J.W., Gao, G., Goff, S.P., Rice, C.M., MacDonald, M.R.,2003. Expression of the zinc-finger antiviral protein inhibits alphavirusreplication. J. Virol. 77 (21), 11555–11562.

Bushell, M., Sarnow, P., 2002. Hijacking the translation apparatus by RNAviruses. J. Cell Biol. 158 (3), 395–399.

Carleton, M., Brown, D.T., 1996. Events in the endoplasmic reticulum abrogatethe temperature sensitivity of Sindbis virus mutant ts23. J. Virol. 70 (2),952–959.

Castello, A., Sanz, M.A., Molina, S., Carrasco, L., 2006. Translation of Sindbisvirus 26S mRNA does not require intact eukaryotic initiation factor 4G.J. Mol. Biol. 355 (5), 942–956.

Cuconati, A., Molla, A., Wimmer, E., 1998. Brefeldin A inhibits cell-free,de novo synthesis of poliovirus. J. Virol. 72 (8), 6456–6464.

Da Costa, L.J., Rebello, M.A., 1999. Effect of brefeldin A on Mayaro virusreplication in Aedes albopictus and Vero cells. Acta Virol. 43 (6),357–360.

Dasgupta, A., Wilson, D.W., 2001. Evaluation of the primary effect of brefeldin

436 S. Molina et al. / Virology 363 (2007) 430–436

A treatment upon herpes simplex virus assembly. J. Gen. Virol. 82 (Pt 7),1561–1567.

Dinter, A., Berger, E.G., 1998. Golgi-disturbing agents. Histochem. Cell Biol.109 (5–6), 571–590.

Fishman, P.H., Curran, P.K., 1992. Brefeldin A inhibits protein synthesis incultured cells. FEBS Lett. 314 (3), 371–374.

Frolov, I., 2004. Persistent infection and suppression of host response byalphaviruses. Arch. Virol. Suppl. (18), 139–147.

Frolov, I., Schlesinger, S., 1994. Translation of Sindbis virus mRNA: effects ofsequences downstream of the initiating codon. J. Virol. 68 (12), 8111–8117.

Frolov, I., Schlesinger, S., 1996. Translation of Sindbis virus mRNA: analysis ofsequences downstream of the initiating AUG codon that enhance translation.J. Virol. 70 (2), 1182–1190.

Gazina, E.V., Mackenzie, J.M., Gorrell, R.J., Anderson, D.A., 2002. Differentialrequirements for COPI coats in formation of replication complexes amongthree genera of Picornaviridae. J. Virol. 76 (21), 11113–11122.

Hahn, C.S., Hahn, Y.S., Braciale, T.J., Rice, C.M., 1992. Infectious Sindbisvirus transient expression vectors for studying antigen processing andpresentation. Proc. Natl. Acad. Sci. U.S.A. 89 (7), 2679–2683.

Harding, H.P., Zhang, Y., Ron, D., 1999. Protein translation and folding arecoupled by an endoplasmic-reticulum-resident kinase. Nature 397 (6716),271–274.

Irurzun, A., Perez, L., Carrasco, L., 1992. Involvement of membrane traffic inthe replication of poliovirus genomes: effects of brefeldin A. Virology 191(1), 166–175.

Irurzun, A., Perez, L., Carrasco, L., 1993. Brefeldin A blocks proteinglycosylation and RNA replication of vesicular stomatitis virus. FEBSLett. 336 (3), 496–500.

Jackson, C.L., Casanova, J.E., 2000. Turning on ARF: the Sec7 family ofguanine-nucleotide-exchange factors. Trends Cell Biol. 10 (2), 60–67.

Kaariainen, L., Ahola, T., 2002. Functions of alphavirus nonstructural proteinsin RNA replication. Prog. Nucleic Acid Res. Mol. Biol. 71, 187–222.

Klausner, R.D., Donaldson, J.G., Lippincott-Schwartz, J., 1992. Brefeldin A:insights into the control of membrane traffic and organelle structure. J. CellBiol. 116 (5), 1071–1080.

Kreis, T.E., Lowe, M., Pepperkok, R., 1995. COPs regulating membrane traffic.Annu. Rev. Cell Dev. Biol. 11, 677–706.

Kujala, P., Ikaheimonen, A., Ehsani, N., Vihinen, H., Auvinen, P., Kaariainen,L., 2001. Biogenesis of the Semliki Forest virus RNA replication complex.J. Virol. 75 (8), 3873–3884.

Lee, T.H., Linstedt, A.D., 1999. Osmotically induced cell volume changes alteranterograde and retrograde transport, Golgi structure, and COPI dissocia-tion. Mol. Biol. Cell 10 (5), 1445–1462.

Lippincott-Schwartz, J., Donaldson, J.G., Schweizer, A., Berger, E.G., Hauri,H.P., Yuan, L.C., Klausner, R.D., 1990. Microtubule-dependent retrogradetransport of proteins into the ER in the presence of brefeldin A suggests anER recycling pathway. Cell 60 (5), 821–836.

Lippincott-Schwartz, J., Yuan, L., Tipper, C., Amherdt, M., Orci, L., Klausner,R.D., 1991. Brefeldin A's effects on endosomes, lysosomes, and the TGNsuggest a general mechanism for regulating organelle structure andmembrane traffic. Cell 67 (3), 601–616.

Liu, C.Y., Kaufman, R.J., 2003. The unfolded protein response. J. Cell Sci. 116(Pt 10), 1861–1862.

Madan, V., Sanz, M.A., Carrasco, L., 2005. Requirement of the vesicular systemfor membrane permeabilization by Sindbis virus. Virology 332 (1),307–315.

Maynell, L.A., Kirkegaard, K., Klymkowsky, M.W., 1992. Inhibition ofpoliovirus RNA synthesis by brefeldin A. J. Virol. 66 (4), 1985–1994.

Mellor, H., Kimball, S.R., Jefferson, L.S., 1994. Brefeldin A inhibits proteinsynthesis through the phosphorylation of the alpha-subunit of eukaryoticinitiation factor-2. FEBS Lett. 350 (1), 143–146.

Mirazimi, A., von Bonsdorff, C.H., Svensson, L., 1996. Effect of brefeldin A onrotavirus assembly and oligosaccharide processing. Virology 217 (2),554–563.

Nebenfuhr, A., Ritzenthaler, C., Robinson, D.G., 2002. Brefeldin A: decipheringan enigmatic inhibitor of secretion. Plant Physiol. 130 (3), 1102–1108.

Orci, L., Tagaya, M., Amherdt, M., Perrelet, A., Donaldson, J.G., Lippincott-Schwartz, J., Klausner, R.D., Rothman, J.E., 1991. Brefeldin A, a drug thatblocks secretion, prevents the assembly of non-clathrin-coated buds onGolgi cisternae. Cell 64 (6), 1183–1195.

Pelham, H.R., 1991. Multiple targets for brefeldin A. Cell 67 (3), 449–451.Peranen, J., Laakkonen, P., Hyvonen, M., Kaariainen, L., 1995. The alphavirus

replicase protein nsP1 is membrane-associated and has affinity to endocyticorganelles. Virology 208 (2), 610–620.

Prostko, C.R., Brostrom, M.A., Brostrom, C.O., 1993. Reversible phosphoryla-tion of eukaryotic initiation factor 2 alpha in response to endoplasmicreticular signaling. Mol. Cell Biochem. 127–128, 255–265.

Renault, L., Guibert, B., Cherfils, J., 2003. Structural snapshots of themechanism and inhibition of a guanine nucleotide exchange factor. Nature426 (6966), 525–530.

Salonen, A., Ahola, T., Kaariainen, L., 2005. Viral RNA replication inassociation with cellular membranes. Curr. Top Microbiol. Immunol. 285,139–173.

Sanz, M.A., Carrasco, L., 2001. Sindbis virus variant with a deletion in the 6Kgene shows defects in glycoprotein processing and trafficking: lack ofcomplementation by a wild-type 6K gene in trans. J. Virol. 75 (16),7778–7784.

Sata, M., Moss, J., Vaughan, M., 1999. Structural basis for the inhibitory effectof brefeldin A on guanine nucleotide-exchange proteins for ADP-ribosylation factors. Proc. Natl. Acad. Sci. U.S.A. 96 (6), 2752–2757.

Scales, S.J., Gomez, M., Kreis, T.E., 2000. Coat proteins regulating membranetraffic. Int. Rev. Cytol. 195, 67–144.

Schneider, R.J., Mohr, I., 2003. Translation initiation and viral tricks. TrendsBiochem. Sci. 28 (3), 130–136.

Strauss, J.H., Strauss, E.G., 1994. The alphaviruses: gene expression,replication, and evolution. Microbiol. Rev. 58 (3), 491–562.

Suikkanen, S., Antila, M., Jaatinen, A., Vihinen-Ranta, M., Vuento, M., 2003.Release of canine parvovirus from endocytic vesicles. Virology 316 (2),267–280.

Thompson, S.R., Sarnow, P., 2000. Regulation of host cell translation by virusesand effects on cell function. Curr. Opin. Microbiol. 3 (4), 366–370.

Torii, S., Banno, T., Watanabe, T., Ikehara, Y., Murakami, K., Nakayama, K.,1995. Cytotoxicity of brefeldin A correlates with its inhibitory effect onmembrane binding of COP coat proteins. J. Biol. Chem. 270 (19),11574–11580.

Ventoso, I., Carrasco, L., 1995. A poliovirus 2A (pro) mutant unable to cleave3CD shows inefficient viral protein synthesis and transactivation defects.J. Virol. 69 (10), 6280–6288.

Ventoso, I., Sanz, M.A., Molina, S., Berlanga, J.J., Carrasco, L., Esteban, M.,2006. Translational resistance of late alphavirus mRNA to eIF2alphaphosphorylation: a strategy to overcome the antiviral effect of protein kinasePKR. Genes Dev. 20 (1), 87–100.