Proc. Natl. Acad. Sci. USAVol. 89, pp. 11136-11140, December 1992Biochemistry

Active complete in vitro replication of nodavirus RNArequires glycerophospholipid

(viral RNA replicase/host cell membranes/RNA-depe t RNA polymerase)

SHI-XUAN WU*t, PAUL AHLQUIST*f, AND PAUL KAESBERG*t§*Institute for Molecular Virology and Departments of tBiochemistry and *Plant Pathology, University of Wisconsin, Madison, WI 53706

Contributed by Paul Kaesberg, August 19, 1992

ABSTRACT Flockhouse virus (FHV) is a member of thenodavirus group ofpositive-strand RNA viruses. In the absenceof additional compounds, a template-dependent RNA-dependent RNA polymerase extracted from FHV-infected cellssynthesizes complementary (-)-strand copies of added FHVRNA to yield a double-stranded RNA product. Upon inofglycerophosphoilpid (GPL), this system reproducibly carriesout complete highly active replication of added FHV RNA,producing newly synthesized (+)-strand RNA in predomi-nantly single-stranded RNA form. This accounts for previouslyobserved effects of Lipofectin (a mixture of GPL and cationiclipid) in the system. AlU tested neutral and negatively chargedGPLs except phosphatidic acid support complete FHV RNAreplication in this in vitro system, as do phospholipid extractsfrom uninfected and FHV-infected cells. Neither pigsmy-elin, a membrane phospholipid that is not derived from glyc-erol, nor cholesterol supported FHV RNA replication. Testingofcompounds derived from GPL shows that theablt ofactiveGPL to support FHV (+)-strand RNA synthesis isd ton the struc of both the head group and the acyl chains.Neither the phosphorylated head group nor the diacylgycerollipid moiety alone supports RNA replication. The length andsaturation of acyl chains strongly infiuence the ability of GPLto support RNA replication. Other characteristics of this invitro RNA replication system and the possible role played bymembranes and their components in FHV RNA replication arediscussed.

Viruses encapsidating (+)-strand (i.e., messenger sense)RNA genomes are important pathogens of humans, animals,and plants. Replication of (+)-strand RNA virus genomesoccurs by synthesis of complementary (-)-strand RNAsfrom the parental (+)-strand, followed by synthesis of newprogeny (+)-strands from the (-)-strand RNA intermediates.Despite prolonged efforts with many eukaryotic (+)-strandRNA viruses, this complete RNA-dependent RNA replica-tion cycle has been reproduced in vitro for only a few viruses,frequently due to failure to proceed beyond (-)-strand RNAsynthesis to the production of new (+)-strand RNA (1-3).

Recently, we reported (4) the isolation of a template-dependent RNA replicase extract from Drosophila cellsinfected with Flockhouse virus (FHV), an insect virus in thenodavirus family. This FHV replicase completely replicatesinput FHV RNA in vitro, producing (-)-strands in double-stranded (ds) RNA form and new (+)-strands in mainlysingle-stranded (ss)RNA form. Analogous in vitro replicationof a eukaryotic viral genome has been achieved to date onlyfor cucumber mosaic virus (5). In addition, indirect evidencehas recently been presented for poliovirusRNA replication ina cell-free system (6). The level of RNA replication reported

in these latter systems, however, is significantly lower thanthat obtained for FHV.Complete replication of FHV RNA by the membrane-

associated FHV replicase depended on the absence ofmem-brane-disrupting detergents and the presentation of templateRNA in combination with Lipofectin (BRL), a reagent com-monly used to introduce RNA and DNA into membranecompartments (4, 7). In the presence of detergent or absenceof Lipofectin, added FHV RNA was copied to give (-)-strand RNA, but no new (+)-strands were synthesized.

Lipofectin and its analogs are mixtures of a specific glyc-erophospholipid (GPL) and a cationic lipid. We have nowanalyzed the contribution of each component and found thatonly the GPL is required for complete FHV RNA replicationin vitro. We further show that a number of GPLs supportcomplete FHV RNA replication and that the activity ofeffective GPLs is dependent on the presence and structure ofboth the polar head group and the acyl chains. The amountofGPL required to support (+)-strand synthesis increases asmore FHV replicase extract is added to the reaction. Thisresult, the dispensability of the cationic lipid, and otherobservations suggest that the role of GPL in facilitating(+)-strand RNA synthesis is independent of delivery of theinitial template RNA. Since the RNA replication complexesof most or all (+)-strand RNA viruses are membrane-associated and (+)-strand RNA synthesis has generally beenthe block to complete RNA replication in vitro, the effects ofGPL on FHV RNA replication might have general implica-tions for many viruses.

MATERIALS AND METHODS

Phospbolrps and Related Compounds. The following com-pounds were obtained from Sigma in the highest purity gradesavailable: dimethyldioctadecylammonium bromide (DDAB);L-a-phosphatidic acid (PA, from egg yolk); L-a-phosphati-dylcholine (PC, from egg yolk); L-a-phosphatidylethanol-amine, dioleoyl (PE, synthetic); L-a-phosphatidyl-rac-glycerol, dioleoyl (PG, synthetic); L-a-phosphatidylinositol(PI, from soybean); L-a-phosphatidyl-L-serine (PS, from bo-vine brain); sphingomyelin (SPM, from bovine brain); cho-lesterol; 1,2-dimyristoyl-rac-glycerol [14:0 (i.e., carbons:double bonds in each acyl chain = 14:0)]; 1,2-dioleoyl-rac-glycerol [18:1 (double bond position = cis-9)]; phosphoryl-choline; L-a-glycerophosphate; O-phospho-L-serine; andL-a-glycerophosphorylcholine. Additional synthetic PC spe-cies bearing defined symmetric acyl chaihs ofvarious lengthsand saturation (carbons:double bonds = 3:0; 6:0; 10:0; 12:0;

Abbreviations: FHV, flockhouse virus; dsRNA, double-strandedRNA; ssRNA, single-stranded RNA; GPL, glycerophospholipid;DDAB, dimethyldioctadecylammonium bromide; PA, phosphatidicacid; PC, phosphatidylcholine; PE, diolcoyl phosphatidylethanol-amine; PG, dioleoyl phosphatidylgycerol; PI, phosphatidylinositol;PS, phosphatidylserine; SPM, sphingomyein.§To whom reprint requests should be addressed.

11136

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Proc. Nat. Acad. Sci. USA 89 (1992) 11137

14:0; 16:0; 18:0; 18:1, cis-9; 18:2, cis-9,12; and 18:3, cis-9,12,15) were purchased from Avanti Polar Lipids. Lipidextracts from uninfected and FHV-infected Drosophila cellswere prepared as described for extracting lipids from ratbasophilic leukemia cells (8). Suspensions ofphospholipids inwater were prepared by sonication.Replcase Preparation, RNA Synthesis Amys, and RNA

Product Analysis. Micrococcal nuclease-treated FHV repli-case was prepared as described (see CRCN-stage preparationof ref. 4). Standard FHV RNA synthesis reactions had a totalvolume of 12.5 jul and contained 50 mM Tris acetate (pH 8.2),15 mM MgAc2, 250 mM potassium glutamate, 50 ng ofFHVRNA2, 10 units of placental ribonuclease inhibitor(Promega), 1 mM ATP, 1 mM GTP, 1 mM UTP, 20 ILMunlabeled CTP, 5 p.Ci of [a-32P]CTP (3000 Ci/mmol; 1 Ci =37 GBq), 250 ng of actinomycin D, 5 1ul of micrococcalnuclease-digested FHV replicase extract, and GPL as indi-cated. Reactions were incubated at 300C for 3 hr (see Figs.1-3) or 8 hr (see Figs. 4 and 5). The longer incubation time wasused in later experiments after time-course studies revealedthat nucleotide incorporation remained approximately linearthrough 8 hr of incubation. After phenol extraction andethanol precipitation, the resulting RNA products were elec-trophoresed on a 1.5% agarose gel, which then was dried andautoradiographed.

Polarity analysis of RNA products was performed withribonuclease T1 (4). As controls for ribonuclease T1 analysis(see Fig. 3), [a-32P]CTP-labeled (+)- and (-)-strand tran-scripts were synthesized by in vitro transcription of FHVRNA2 cDNA clones. (+)-Strand RNA2 transcripts weresynthesized by bacteriophage 17 RNA polymerase transcrip-tion ofXba I-linearized p2B1OMJ, which was created (RanjitDasgupta, personal communication) by first excising a com-plete biologically active cDNA copy of FHV RNA2 fromplasmid p2B1OSP (9) through sequential Pst I, T4 DNApolymerase, and Xba I treatments and then ligating thiscDNA between the Stu I and Xba I sites of transcriptionplasmid pMJ5 (10). (-)-Strand RNA2 transcripts were syn-thesized by bacteriophage SP6 RNA polymerase transcrip-tion of Pst I-linearized pKSP65B2(-), which was created bysubcloning the complete Pst I-Xba I-bounded FHV RNA2cDNA copy from p2B1OSP into the Pst I and Xba I sites ofpSP65 (ref. 1; note from ref. 11 that the earlier nodavirusstrain designation BBV-W17 is synonymous with FHV).

RESULTSGPL Alone Stmultes sRNA Synthesis by FHV RNA-

DependentRNA Polymerame. We have previously shown that,in unsupplemented reactions, a membrane-associated tem-plate-dependent RNA-dependent RNA polymerase extractfrom FHV-infected cells synthesizes (-)-strand RNA copiesof added FHV RNA, yielding dsRNA product (4). However,when the initial FHV template RNA is introduced in thepresence of Lipofectin, a reagent used to deliver RNA andDNA to membrane compartments, this RNA-dependentRNA polymerase extract acts as a true RNA replicase andadditionally synthesizes new (+)-strand RNA, which accu-mulates in both dsRNA and ssRNA forms (4). Thus, the mostobvious effect of Lipofectin addition to the system is theinduction of ssRNA synthesis.

Lipofectin is a 1:1 mixture of a neutral GPL, i.e., PE, anda patented cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (7). To compare theeffects of these components, we sought to identify a Lipo-fectin analog whose individual components were both readilyavailable. Fig. 1, lanes 1-6, shows that ssRNA synthesis bythe FHV polymerase extract is also induced by a mixture ofPE and another cationic lipid, dimethyldioctadecylammo-nium bromide (DDAB). This same combination is used as a

- DDAB+PE DDAB PEmI F

4g DDAB: 0 .02.06 .2 .6 2 .02 .06 .2 .6 2g PE: 0 .1 .3 1 3 10 .1 .3 1 3 10

dsRNA-i.-

.9__WssRNA-_-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

FIG. 1. Autoradiograph of a 1.5% agarose gel showing thecombined and individual effects of DDAB and PE on in vitro RNAsynthesis in FHV replicase reactions initiated by addition of purifiedFHV RNA2, the smaller of the two FHV genomic RNAs, andincubated for 3 hr at 300C. Lane 1 contains the products ofa controlreaction without DDAB or PE. The amount (jug) of DDAB, PE, orboth in each of the remaining 12.5-;ul reaction mixtures is shownabove the corresponding lanes. The positions of the dsRNA2 andssRNA2 product bands are shown at the left.

Lipofectin analog in the commercial transfection reagentTransfectACE (BRL). When using a 5:1 (wt/wt) PE/DDABmixture, maximal ssRNA synthesis was obtained at 3.6 ,tgper 12.5 ;LI of reaction mixture (lane 5), with no increase inssRNA synthesis and reduced dsRNA synthesis at 12 jyg perreaction mixture (lane 6). Lipofectin produces similar dose-dependent effects on ssRNA synthesis, giving optimal ss-RNA synthesis at 2-3 Mg of Lipofectin per reaction mixture(results not shown).We compared the individual effects of PE and DDAB to

those ofthe PE/DDAB mixture. DDAB alone did not supportssRNA synthesis and, when added at or above 0.2 Mug perreaction mixture, DDAB inhibited dsRNA synthesis (Fig. 1,lanes 7-11). In contrast, PE alone strongly stimulated ssRNAsynthesis (Fig. 1, lanes 12-16). In the absence of DDAB, noinhibition of either ssRNA or dsRNA synthesis was seeneven at 10 Mug of PE per reaction mixture. Rather, there wasa direct increase in stimulation of ssRNA synthesis throughthe entire range of PE concentrations tested, up to andincluding 10 Mg per reaction mixture. The levels of ssRNAproduced in the presence of higher amounts of PE alone(lanes 15 and 16) considerably exceeded ssRNA levels seenwhen an equivalent amount of PE was introduced in thepresence ofDDAB (lanes 5 and 6). Thus, cationic lipid is notonly unnecessary for stimulation but also has an inhibitoryeffect on both ssRNA and dsRNA synthesis by the FHVRNA-dependent RNA polymerase.

Effect of Different GPLs onFHV RNA Replcation. Next wetested the effect of other common GPLs on FHV RNAsynthesis in vitro. PE, PC, PG, PI, and PS showed someability to stimulate ssRNA synthesis (Fig. 2). The thresholdconcentration required to stimulate ssRNA synthesis variedamong GPLs and was lowest for PG. Fig. 2 and otherexperiments revealed that, for PG, optimal stimulation ofssRNA synthesis was obtained at 0.5-2.5 Mg of PG perreaction mixture. Above 2.5 ug of PG per reaction mixture,synthesis of both ssRNA and dsRNA was reduced (Fig. 2,lane 13). PC, PE, PI, and PS gave less stimulation of ssRNAsynthesis than PG when used at or below 1 ,ug per reactionmixture. However, the level ofssRNA synthesis continued toincrease as the level of these GPLs was increased as high as25 Mg per reaction mixture (Fig. 2 and results not shown). Ofthese latter four GPLs, PC gave the highest stimulation ofssRNA synthesis. Total lipid extracts from either uninfestedor FHV-infected Drosophila cells also stimulated ssRNAsynthesis in the FHV in vitro system (Fig. 2, lanes 23-28).

Biochemistry: Wu et al.

Proc. Natl. Acad. Sci. USA 89 (1992)

Drosophila GPLGPL: - PA PC PE PG Pi PS SPM Uninf. FHV-inf.

: 0 .1 1 10 .1 1 10 1 1 10 1 1 10 .1 1 1O .1 1 10 .1 1 10 .1 1 10 .1I1-0

ds-_ m- "*wto do; VW*** S

i.** *.m'.b a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

0._

ClD

um

EUU

z

PA

0 0.1 1 10GPL. lug per reaction mixture

FiG. 2. Effect of various phospholipids on in vitro FHV RNA synthesis assays performed as in Fig. 1. (Left) Lane 1 contains the productsof a control reaction without any added GPL. The type and level (ug per 12.S-pl reaction mixture) of phospholipid added to each of the otherreaction mixtures are shown above the corresponding lanes. Lanes 2-22 show the efcts of PA, PC, PE, PG, PI, PS, and SPM. Lanes 22-28show the effects of total lipid extracts from uninfected (Uninf.) and FHV-infected (FHV-inf.) Drosophila cells, as indicated. (Right) ssRNAaccumulation vs. pig ofGPL added per reaction mixture for each ofthe defined GPL species shown in the autoradiograph. ssRNA accumulationvalues were determined by laser densitometry of the autoradiograph and were normalized to set the highest measurement to 100 on an arbitraryscale. Note that the horizontal axis is a logarithmic scale.

The simplest GPL, PA, did not demonstrably stimulatessRNA accumulation above a minor basal level sometimesseen in the absence of any 'added GPL (Fig. 2, lanes 1-4).Unlike the other tested GPLs, PA lacks an additional alcoholmoiety esterified to the head group phosphate. While PA isan intermediate in the biosynthesis of other GPLs, little PAis present in' most biological membranes. We also testedSPM, the only common membrane phospholipid not derivedfrom glycerol. Like PA, SPM was not able to detectablystimulate FHV ssRNA synthesis (Fig. 2, lanes 20-22). An-other important membrane lipid, cholesterol, was also unableto support FHV ssRNA synthesis (results not shown).

Pulse-chase experiments, nuclease S1 analysis, denatur-ation/renaturation, and other studies show that the minorhigh molecular weight RNA bands near the top of lanes 6, 7,

10, etc., in Fig. 2 and some lanes in later figures containreplicative intermediate structures that can be chased intodsRNA and also contain deadend products that may includeself-primed multimeric dsRNA. Recent experiments alsoshow that visible accumulation of these higher molecularweight RNA products is dependent on reaction conditionsand can be suppressed by a combination of changes (higherpH, higher rCTP, and lower Mg2+ concentration) that simul-taneously improve the yield ofssRNA (unpublished results).

Polarity of ssRNA and dsRNA Synthesized in the Presencemnd Absence of GPL. Subsequent experiments utilized PC as

a model GPL. Ribonuclease T1 analysis (4) was used todetermine the polarity of the radiolabeled ssRNA synthe-sized in the presence ofPC and the proportion ofradiolabeled(+)-' and (-)-strand RNA in dsRNA synthesized in thepresence and absence of PC. In the absence of PC or anyother GPL, the dsRNA product produced was exclusivelylabeled in the (-)-strand (Fig. 3, lane 3). In the presence ofPC, however, the dsRNA product contained radiolabeled(+)- and (-)-strand RNA in approximately equal proportions(lane 2) and the radiolabeled ssRNA was exclusively (+)-strand RNA (lane 1). The equivalent labeling of (+)- and(-)-strand RNA in dsRNA synthesized in the presence ofPCimplies that nearly all (-)-strands were used as templates forat least one round of subsequent (+)-strand RNA synthesis.It also implies that these (-)-strands remained preferentiallyassociated with newly synthesized product (+)-strands.The sequence ofthe FHV RNA2 template (11) reveals that,

for (+)- and (-)-strands, the ribonuclease Ti RNA fragmentsvisualized in Fig. 3 include representatives from near bothends of the 1400-base RNA2. The similar intensity of bandsin lanes 1 and 2 shows that the [a-32P]CTP label is distributedthroughout the length of newly synthesized RNA strands of

both (+)- and (-)-polarity. Thus, GPL alone, as reported forLipofectin (4), is able to support the filfl replication ofFHVRNA in this in vitro system.

Inability of GPL Substituents to Support FHV (+)-StrandRNA Syntbess. We tested whether FHV ssRNA synthesiscould be stimulated by either the diacylglycerol lipid portionor the polar head group (phosphoryl alcohol) portion of theactive GPLs, rather than the intact GPL molecule. Twodiacylglycerols were tested, 1,2-dimyristoylglycerol 14:0 and1,2-dioleoyl-glycerol 18:1, double bond position = cis-9.Both of these diacylglycerols are components of GPLs thatare active in stimulating ssRNA synthesis by the FHVreplicase (see PI 18:1 in Fig. 2 and PC 18:1 and PC 14:0 in Fig.

+PC -PC trRNA2_-ds

ss ds ds - +

(+)

inAl -o*28b

24b

s. Al_

19b-p-

18b '. X

4 6~ :

_ 21 b

I7b

1 2 3 4 5

FIG. 3. Ribonuclease T1 analysis of the polarity of FHV RNAreplicase products. FHV RNA replicase was primed with RNA2template in the presence and absence ofPC (20pg per 100-gl reactionmixture; other reaction conditions were as in Fig. 1). The [32PICTP-labeled ssRNA2 and dsRNA2 product bands were separated byelectrophoresis in a low-melting-point agarose gel, eluted, digestedwith ribonuclease T1, electrophoresed on the indicated lanes of a

20o polyacrylamide/8.3 M urea sequencing gel, and autoradio-graphed. As controls, lanes 4 and 5 contain ribonuclease T1 digestsof (-)strand and (+)-strand RNA2 transcripts (tr RNA2) synthe-sized by bacteriophage T7 and SP6 RNA polymerase in vitrotranscription of full-length RNA2 cDNA clones. b, Bases.

ss-

11138 Biochemistry: Wu et al.

Proc. Natl. Acad. Sci. USA 89 (1992) 11139

PC: - 12:0 14:0 16:0 18:0 18:1 18:2rg-0.21-5.215.215.25I.r1-5fig : 0 .2 1 5 .2 1 5 .2 1 5.2 1 5 '.2 1 5 '.2 1 5

_smm*_M 4d

1 2 3 4 5 6 7 8 9 10 11 12 13141516 1718 19

FIG. 4. Effects of acyl chain length and saturation on FHV RNAsynthesis. Lane 1 contains the products ofa control reaction withoutany added GPL. The remaining lanes show the effect of addingsynthetic PC species with defined symmetric acyl chains. The acylchain length and number of double bonds are shown above each setof lanes in the form carbons:double bonds, and the amount (jg per12.5-p.l reaction mixture) of PC added to each reaction mixture isshown above the corresponding lanes (see text for double bondpositions). Reaction conditions were the same as for Figs. 1-3,except that 20 ng ofRNA2 template per reaction mixture and 100 AMunlabeled CTP were used, and the reactions were incubated for 8 hr.ds, dsRNA; ss, ssRNA.

4). We also tested phosphorylcholine, L-a-glycerophosphate,and O-phospho-L-serine, which are the polar head groups ofPC, PG, and PS, respectively. Over the concentration rangeat which the complete GPLs were active (0.1-10 gg perreaction mixture), none of these compounds supported FHVssRNA synthesis (results not shown). These results suggestthat stimulation of FHV ssRNA synthesis in this in vitrosystem requires intact GPL.Acyl Chain Length and Saturation Influence PC Stimulation

of FHV RNA Synthesis. The PC preparation used in theexperiments in Figs. 2 and 3 was purified from egg yolk andcontains PC species bearing a variety of fatty acid chains. Tofurther explore the significance ofthe lipid portion ofGPL foractivation of FHV (+)-strand RNA synthesis, synthetic PCvariants bearing symmetric acyl chains ofdefined lengths andsaturation were examined. First, we tested PC species withsaturated acyl chains of0, 3, 6, 10, 12, 14, 16, and 18 carbons.PC with saturated acyl chains of 14, 16, or 18 carbonssupported ssRNA synthesis, whereas those with acyl chainsof 12 or fewer carbons did not (Fig. 4 and additional exper-iments). PC bearing 14-carbon acyl chains stimulated ssRNAsynthesis at a lower concentration than PC with 16- or18-carbon chains and, similar to PG (Fig. 2), gave optimalssRNA stimulation at <5 pug per reaction mixture (Fig. 4,lanes 5-7).The effects of double bonds were tested by comparing PC

species with 18-carbon acyl chains containing 0, 1, or 2double bonds (i.e., acyl chains of the form 18:0; 18:1, cis-9;

and 18:2, cis-9,12). The 18:1 acyl chains stimulated ssRNAsynthesis as well as or better than 18:0, while 18:2 gave betterstimulation than either 18:0 or 18:1 (Fig. 4, lanes 11-19). Ina preliminary experiment, freshly resuspended PC with 18:3,cis-9,12,15 acyl chains stimulated ssRNA synthesis with thesame efficiency as 18:2 chains (results not shown). However,the PC 18:3 preparation completely lost its stimulating ac-tivity in less than a week, possibly due to oxidation.In Vitro FHV RNA Replication Depends on the PC/Repli-

case Ratio. Next we tested whether the amount of PCrequired to stimulate FHV RNA replication was influencedby the amount ofFHV replicase preparation in the reaction.In the experiment shown in Fig. 5, five sets of reactions wereassembled. Within each set, the level of replicase was con-stant (at 0.5, 1, 2.5, 5, or 10 p.l ofreplicase per 12.5-pul reactionmixture) while the level ofPC was varied at 0.5, 1, 2.5, 5, and10 uig per reaction mixture. Not surprisingly, the level ofdsRNA synthesis and the maximal level of PC-stimulatedssRNA synthesis both increased as more FHV replicase wasadded to the reaction.More interestingly, the threshold concentration of PC

needed to induce FHV RNA replication also increased asmore replicase was added. With 0.5-1 pul of replicase perreaction mixture, maximal ssRNA synthesis was achievedwith 0.5 1Lg of PC, the lowest PC concentration tested. Incontrast, with 5 and 10 gl of replicase per reaction mixture,little or no ssRNA synthesis was induced until the level ofPCwas raised to 2.5 and 5 ,ug per reaction mixture, respectively.Thus, the ability of PC to stimulate in vitro FHV RNAreplication is not simply a function of the absolute concen-tration of PC but of the relative amounts of PC and replicasepreparation in the reaction.

DISCUSSIONWe have demonstrated here that GPL is required for com-plete high-level replication of FHV RNA in an in vitroextract. The effect ofGPL is to stimulate the (+)-strand RNAsynthesis step of the replication cycle. Subdivision of GPLinto its diacylglycerol and phosphoryl alcohol portionsshowed that GPL integrity is essential to this activation. Theimportance of both the polar head group and the lipid portionwas underscored by the lack of detectable stimulation by PA,which lacks an additional alcohol esterified to the head groupphosphate, and by the dramatic effects of acyl chain lengthand, to a lesser degree, saturation on PC activity. Some othercommon membrane lipids, SPM and cholesterol, were unableto stimulate (+)-strand synthesis.Membrane Association of (+)-Strand RNA Synthesis by

FIIV and Other Viruses. GPLs are major components of mostbiological membranes, and results from other studies suggestthat the influence ofGPLs on FHV (+)-strand RNA synthesis

pI Replicase: 0.5 1 2.5 5 10ig PC: .5 1 2.5 5 10 .5 1 2.5 5 10 .5 1 2.5 5 10 .5 1 2.5 5 10 .5 1 2.5 5 10

ds -- -"I-..-.. sin" W% NM ..'a...

e*0*so .. SA.Am

1 2 3 4 5 6 7 8 9 10 11 121314 IT 16 17 18 19 20 21 22 23 24 25

FIG. 5. Influence of the PC/replicase ratio on in vitro RNA synthesis in FHV replicase reactions primed with FHV RNA2 template. Thevolume (Al) ofreplicase preparation and amount (pg) ofPC (from egg yolk) used per 12.5-pl reaction mixture are shown above each lane. Reactionconditions and abbreviations were as in Fig. 4.

Biochemistry: Wu et aL

*Nb w4,,. "O *A.. as AWL"VW"W,

Proc. Natl. Acad. Sci. USA 89 (1992)

in vitro may reflect a dependent association of (+)-strandRNA synthesis with membranes. For example, we haveshown (4) that the FHV RNA replicase used here is mem-brane-associated. Moreover, when the membrane is dis-rupted with detergent, the extract retains the ability tosynthesize (-)-strand RNA but loses the ability to synthesizenew (+)-strand RNA from that (-)-strand when stimulatedwith GPL (ref. 4 and unpublished results).The association of viral RNA synthesis with membranes is

not limited to FHV. Infection by most (+)-strand RNAviruses induces proliferation of cytoplasmic membranes, andthe RNA replication complexes of most or all (+)-strandRNA viruses are initially extracted from cells in membrane-bound form (2, 3, 5, 12). For poliovirus, among other cases,a variety of additional evidence shows that membrane vesi-cles are involved in RNA replication (13) and may be par-ticularly involved in (+)-strand RNA synthesis. In poliovi-rus-infected cells, the lipid biosynthesis inhibitor ceruleninblocks phospholipid synthesis, membrane proliferation, andviral but not cellular RNA synthesis, preferentially reducing(+)- over (-)-strand RNA synthesis (14). Brefeldin A, whichinhibits formation of certain classes of intracellular vesicles,also potently inhibits poliovirus RNA replication (15). Amutation in poliovirus membrane protein 3AB induces aspecific defect in (+)-strand RNA synthesis (16). Poliovirus(-)-strand RNA synthesis can be achieved in vitro in theabsence of membranes, while (+)-strand RNA synthesisfrom endogenous templates has only been observed in crudemembrane-containing fractions (3). Membranes have alsobeen suggested to play a role in a cell-free poliovirus assem-bly system in which de novo (+)-strand synthesis is believedto occur (6).

Role ofGPL in FHV (+)-Strand RNA Synthesis in Vito. Theoriginal motivation to add Lipofectin to FHV RNA polymer-ase reaction mixtures (4) was an attempt to enhance orredirect the delivery of RNA templates to the membrane-associated FHV polymerase. However, the crucial Lipofec-tin component for mediating nucleic acid delivery into mem-brane compartments, cationic lipid (7), not only is unneces-sary but also is inhibitory to FHV (+)-strand RNA synthesis(Fig. 1). Moreover, the GPLs active in supporting FHV RNAreplication included those whose net charge was either neu-tral (PE and PC) or negative (PG, PI, and PS). In contrast,cationic lipid-mediated delivery of nucleic acids to cellstolerates or is assisted by PE but is inhibited by PC andabolished by negatively charged GPLs (7). Thus, the effect ofLipofectin and, more importantly, GPL on FHV (+)-strandRNA synthesis is likely to be independent of template RNAdelivery.The dependence of in vitro FHV RNA replication on the

GPL/replicase ratio (Fig. 5) suggests that GPL interactsdirectly with one or more components of the replicase extractand that some saturation threshold must be exceeded tofacilitate (+)-strand RNA synthesis. How an interaction withexogenous GPL would activate (+)-strand synthesis is un-clear. Conceivably, a direct GPL-replicase interaction mightactivate some enzyme function specifically that is requiredfor initiation or elongation of (+)-strand RNA synthesis.Precedents for such stimulation include activation of theEscherichia coli DNA replication initiation protein DnaA bydiphosphatidylglycerol (17) and the activation of proteinkinase C by its highly cooperative interaction with PS anddiacylglycerol (18). However, while these enzyme activa-tions show exquisite specificity for particular GPLs, FHV(+)-strand RNA synthesis was activated by GPL with any ofa variety of distinct head groups.

Alternatively, the above-mentioned (+)-strand-specific ef-fects of the lipid biosynthesis inhibitor cerulenin on poliovi-rus RNA synthesis (14) and additional effects on Semliki

Forest virus RNA synthesis (19) have been interpreted toindicate that continuous lipid synthesis and/or membraneformation are required for viral RNA replication in vivo.While potential complexities of cerulenin action in vivo (20)and in vitro (21) show that additional work will be required totest these suggestions, a similarly dynamic membrane assem-bly or modification process might be required for FHV(+)-strand RNA synthesis in vitro. This possibility mightexplain why exogenous GPL is required for FHV (+)-strandRNA synthesis, even though the FHV replicase activity inthe extracts used here is associated with cellular membranesthat presumably contain substantial amounts ofGPL (4). Thishypothesis also appears consistent with the ability of anumber of different GPLs to support (+)-strand synthesis andwith the sensitivity of (+)-strand RNA synthesis to PC fattyacid structure (Fig. 4), which should influence the ability ofadded PC to fuse with preexisting membranes associated withthe replicase. In this regard it is interesting to note that thecontrast between the ability of PC with saturated acyl chainsof 14 or more carbons and the inability ofPC with acyl chainsof 12 or fewer carbons to stimulate (+)-strand RNA synthesis(Fig. 4) appears to correlate closely with the threshold acylchain length required for PC to form bilayer or multibilayervesicles rather than spherical micelles in water (22).

We thank G.-Q. Zhang for excellent technical assistance and R.Quadt, D. Sawicki, and S. Sawicki for helpful comments on themanuscript. This research was supported by National Institutes ofHealth Grants A123742 and GM35072.

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