Proc. Nati. Acad. Sci. USAVol. 77, No. 2, pp. 847-851, February 1980Biochemistry

Two species of full-length cDNA are synthesized in high yield bymelittin-treated avian retrovirus particles

(toxic peptide/envelope permeability/infectious cDNA)

LAWRENCE R. BOONE AND A. SKALKADepartment of Cell Biology, Roche Institute of Molecular Biology, Nutley, New Jersey 07110

Communicated by B. L. Horecker, November 8, 1979

ABSTRACT A method of activating endogenous cDNAsynthesis in avian retroviruses that results in the formation oftwo species of full-length cDNA in high yield is described. Testsof biological activity show infectivity of at least the same orderof magnitude as for full-length cDNA made by other procedures.Melittin, the major component of bee venom, is used as an al-ternative to nonionic detergents to make the viral envelopepermeable and thus activate the endogenous RNA-dependentDNA polymerase. This compound is a toxic peptide known tointeract with phospholipid membranes. It appears to be lessdisruptive to the viral structure than detergents, resulting in amore efficient transcription of the viral genome. Preliminarytests indicate that this method will also prove useful for studyingenzymatic activities associated with other enveloped viruses.

The standard method of synthesizing cDNA by the endogenousreverse transcriptase reaction has been to make the virus en-velope permeable with a nonionic detergent in the presence ofthe appropriate salts, buffers, reducing agent, and deoxynu-cleoside triphosphates. It is clear from several studies that theconcentration of some components is critical for the synthesisof a product that contains full-length transcripts of the viralgenome. The detergent concentration has a very narrow opti-mal range for the synthesis of large cDNA molecules from aviansarcoma virus, and no large transcripts are made when thisconcentration is exceeded (1). The importance of balancing thedivalent cation and deoxynucleoside triphosphate concentra-tions for the synthesis of large transcripts of Moloney murineleukemia virus has also been documented (2, 3). However, highdeoxynucleoside triphosphate concentration and long incu-bation are not required for full-length cDNA synthesis byequine infectious anemia virus (4). Finally, it has been shownthat the addition of ribonucleoside triphosphates can increasethe yield of full-length transcripts in reactions using purifiedcomponents (5) and in the endogenous cDNA reaction (6). Thefull-length in vitro synthesized cDNAs of both murine andavian retroviruses have been shown to be infectious by trans-fection assays (6, 7).

In the present study we examined the use of melittin, themajor component in bee (Apis mellifera) venom as an alter-native to nonionic detergent for disruption of virions for cDNAsynthesis. This cationic peptide of 26 amino acids has an unusualarrangement of largely hydrophobic (positions 1-20) and hy-drophilic (positions 21-26) residues which makes it highlysurface active. Its lytic activity on both natural and artificialmembranes has been well characterized (8-10). We report herethat melittin can be used in place of nonionic detergents for thesynthesis of cDNA that is the length of in vivo proviral DNAand is infectious. Our data suggest that this compound can makethe viral envelope permeable so that substrates for cDNA syn-

thesis can enter, without disruption of structural componentsthat may be required for authentic provirus synthesis. Pre-liminary results indicate that this method will prove to begenerally useful for the study of enzymatic activities associatedwith enveloped viruses.

MATERIALS AND METHODSChemicals. Melittin was purchased from Sigma. Nonidet

P-40 (NP-40; batch 18164) was a gift from Shell.Viruses and Cells. Rous associated virus-2 (RAV-2) and

NY68(B), a recombinant of RAV-2 and a mutant of Schmidt7Ruppin Rous sarcoma virus, temperature sensitive for trans-formation (11), were propagated in our laboratory from stocksoriginally obtained from H. Hanafusa. Chick embryo fibroblastcell culture and virus harvests were as described (12).

Reverse Transcriptase Assay. The exogenous template-primer reaction was modified from Kacian et al. (13). The re-action mixture contained 50 mM NaCl, 50 mM Tris-HCl (pH8.0), 5 mM MgCl2, 5 mM dithiothreitol, 50 Ag of (rC).(dG)12_18per ml, 0.1 mM [3H]dGTP, and the indicated concentrationsof melittin or NP-40. Incubation was for 1 hr at 370C and wasterminated by adjustment to 0.5% in NaDodSO4 and 50 mMin EDTA. Each sample was spotted onto GF/B filters (What-man) and -washed three times in cold 5% trichloroaceticacid/1% sodium pyrophosphate and three times in cold 95%ethanol. The filters were dried and assayed for radioactivity byliquid scintillation counting.DNA Synthesis and Purification. The reaction conditions

for synthesizing cDNA were modified from those of Lai et al.(14). The reaction mixture contained 0.1 M Tris HCl (pH 8.3),25 mM NaCI, 3mM magnesium acetate, 30mM dithiothreitol,1 mM dCTP, 1 mM dGTP, 1 mM TTP, 0.2 mM [a-32P]dATP(75 mCi/mmol; 1 Ci = 3.7 X 1010 becquerels), 1 mg of virusprotein [determined by the fluorescamine assay (15)] per ml,and the indicated concentrations of melittin or NP-40. Thereactions were incubated at 41°C for 18 hr and terminated byadjustment .to 0.5% in NaDodSO4 and 10 mM in EDTA. Thesamples were adjusted to 0.1 M in NaCl. Wheat embryo RNAwas added (100 ,g/ml) as carrier and then the mixture wasextracted with an equal volume of phenol/chloroforrm/isoamylalcohol, 25:24:1 (vol/vol), and once with an equal volume ofchloroform/isoamyl alcohol, 24:1. The cDNAs were ethanolprecipitated, chromatographed on Sephadex G-50 to removeunincorporated [a-32P] dATP, and precipitated in ethanol. Theprecipitates were redissolved in 10 mM Tris-HCI, pH 7.5/10mM NaCI/1 mM EDTA. Any exceptions to these proceduresare indicated in the appropriate figure legend.

Electrophoresis and Autoradiography. DNA samples weresubjected to electrophoresis in submerged horizontal slab gels(25 X 19 X 0.5 cm) of 1.0% Agarose (SeaKem). The running

Abbreviations: NP-40, Nonidet P-40; kbp, kilobase pair(s); kb, kilo-base(s).

847

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848 Biochemistry: Boone and Skalka

buffer was 40 mM Tris acetate, pH 7.8/5mM sodiukn 40eat/1mM EDTA for neutral agarose and 30 mM NaOH/2 mMEDTA for alkaline agarose (16). Gels were dried under reducedpressure and exposed to Kodak X R-5 film at -70'C with apreflash (17) or with a chromium tungstate intensifier screen(Ilford) or both.Recombinant DNA Techniques. Procedures were carried

out in compliance with the National Institutes of HealthGuidelines on Recombinant DNA, which requires P2-EK2 orP3-EK1 containment for DNA of avian retroviruses isolatedfrom infected cells.RAV-2 proviral DNA was isolated by Hirt fractionation (18)

from chicken embryo fibroblasts 24 hr after infection. Afterisopycnic centrifugation in CsCl/propidium diiodide (19), thecovalently closed circular DNA [two species, 7.7 and 8.0 kilo-base pairs (kbp), in approximately equal amounts] was digestedwith the restriction endonuclease Sal I which makes a singlecut 1.2 kbp from the 3' end of the linear proviral DNA map(unpublished observations). The fragments were inserted intoSal I-cut X Charon 21A DNA* and packaged in vitro (20) forinfection of Escherichia coli ED8767 (recA-, EK1 host) orDP50 supF (EK2 host). Screening of recombinants was doneby the procedure of Benton and Davis (21) with 32P-labeledRAV-2 RNA as a probe (12). XRAV2-11, the recombinant usedin these studies, has a 7.7-kbp insert which is a complete geneticequivalent of RAV-2 RNA without the terminal redundancypresent in the 8.0-kbp covalently closed circular proviralDNA.DNA Transfection Assay. Purified cDNA was diluted into

transfection buffer containing, per liter, 8 g of NaCl, 0.37 g ofKCI, 0.188 g of Na2HPO4-7H20, 1.0 g of dextrose, 6.34 g ofpiperazine-N,N'-bis(2-ethanesulfonic acid) at pH 6.8, and 25mg of bacteriophage A DNA carrier. The DNA solutions wereadjusted to 135 mM in CaCI2, incubated for 30 min at roomtemperature, and added to duplicate 60mm chicken embryofibroblast cultures (designated A and B) in a final volume of 0.5ml. After 30 min at room temperature, 2.5 ml of medium 199without serum was added and the plates were incubated at37°C for 4 hr, washed once with medium, and then incubatedas usual with medium 199 plus 2% calf serum. Cultures werefed as usual, split into 100-mm dishes at day 6, and split 1:2 onday 12 and again on day 18. Samples for reverse transcriptaseassay were taken at day 21.

RESULTSDemonstration of Capacity of Melittin to Activate De-

oxynucleotide Polymerization by RAV-2 Virions. Fig. 1Ashows the results of a standard reverse transcriptase assay whichcompared the effects of melittin and the nonionic detergentNP-40. A wide range of 1:2 dilutions of both the toxin and thedetergent was used and the reaction was terminated after 1 hr.Melittin was effective in stimulating the synthesis of poly[3H]dGwith (rC'dG)1218 as template-primer. A striking contrast be-tween these two compounds was the inhibition of polymeraseactivity at high melittin concentrations. In this assay, themaximal polymerase activity of melittin-treated virus wasreproducibly higher than that of NP-40-treated virus. Theabsence of activity in the virus-free control demonstrates thatthe preparation of bee toxin peptide itself contains no poly-merase activity.

Fig. 1B shows the results of an experiment designed to testthe ability of melittin to facilitate the synthesis of cDNA from

X 20-E0.

io-

16-B

12x

E~~~~~~~~~~~4' 0

4

0.001 0.01 0.1 1.0Melittin, mg/ml

NP-40, %

FIG. 1. Reverse transcription by RAV-2 virions activated bymelittin and NP-40. (A) Exogenous template-primer. Twenty-four-hour culture fluids were harvested from RAV-2-infected chickenembryo cells and clarified at 2500 rpm for 10 min; virus was pelletedby centrifugation at 35,000 rpm for 90 min at 4°C in a Beckman 70Ti rotor. Duplicate 100-Al reaction mixtures were set up containingresuspended virus (protein concentration was not determined) and1:2 dilutions of melittin or NP-40. Duplicates were averaged andcontrols without virus were done in parallel. 0, Melittin'alone; 0,melittin with RAV-2; *, NP-40 alone; o, NP-40 with RAV-2. (B)Endogenous template-primer. Culture fluids were harvested, clarifiedas in A, and pelleted through a cushion of.20% sucrose in 50 mMTris-HCl, pH 7.5/1 mM EDTA at 25,000 rpm for 90 min at 4°C in aBeckman SW 27 rotor. Duplicate 100-Al reaction mixtures were setup containing resuspended virus and used to synthesize cDNA asdescribed in Materials and Methods except that the precursor con-centrations were decreased to 0.1 mM for dATP, dCTP, and dGTP,10 ,uM [3H]TTP (1 Ci/mmol) was present, and virus was at 0.1 mg/ml.Incubation, precipitation, and symbols as in A.

the endogenous RNA template. Melittin was effective in thisreaction also, and the increase in incorporation of labeled nu-cleotide compared to that with NP-40 was even greater thanwith exogenously supplied template. As with exogenouslysupplied template-primer, the reaction was inhibited at highmelittin concentrations. Other experiments (not shown) indi-cated that, at the inhibitory concentrations of melittin, synthesisterminated rapidly; thus, the incorporation values at thoseconcentrations reflect yields rather than rates of synthesis.

Characterization of Products. We used reaction conditionssimilar to those described for detergent-activated cDNA syn-thesis (14) with both melittin- and NP-40-treated RAV-2 virions.Several concentrations of each compound were tested to de-termine optimal activity. The reaction products were extractedand samples normalized to equivalent radioactivity weresubjected to electrophoresis in an alkaline agarose gel. NP-40-activated cDNA synthesis yielded cDNA migrating at theposition expected for a complete transcript of the RAV-2 ge-nome [7.7 kilobases (kb)] and a broad range of lower molecularweight cDNAs (Fig. 2). The melittin-activated reaction yieldsthe same types of products. However, in addition, two speciesof full-length cDNA were resolved, the larger of which wasundetected in any of the NP-40-activated reactions. Thismolecule migrated at the position expected for a moleculeapproximately 300 bases longer than full-length cDNA, cor-responding to the length estimated for the terminal redundancypresent in avian proviral DNA (22, 23).

* Blattner, F. R., Kiefer, D. O., Moore, D. D., de Wet, J. R. & Williams,B. G. (1978) Application for EK2 Certification of a Host-VectorSystem for DNA Cloning, Suppl. 9: Data on Charon 21A.

Proc. Natl. Acad. Sci. USA 77 (1980)

Proc. Natl. Acad. Sci. USA 77 (1980) 849

Basesx 10

23-

NP-40

1 2 3 4 5

Melittin

1 2 3

-23

-9.89.8-

6.6 - -6.6

A.:

FIG. 2. Alkaline agarose gel electrophoresis of melittin- andNP-40-stimulated RAV-2 cDNA products. Virus prepared as in Fig.1B was used to synthesize [32P]cDNA. Approximately 3000 cpm ofeach purified product was denatured in 0.25 M NaOH and subjectedto electrophoresis at 40 V and 110 mA for 40 hr in a 1.0% alkalineagarose gel. The gel was dried and autoradiographed. Lanes 1-5:NP-40 at 0.04% (lane 1), 0.02% (lane 2), 0.01% (lane 3), 0.005% (lane4), and 0.0025% (lane 5). Lanes 1-3: melittin at 100 gg/ml (lane 1), 25,g/ml (lane 2), and 12.5,ug/ml (lane 3). The size markers are HindIllfragments of X [14C]DNA.

In order to determine the relative amounts of 7.7-kb or (7.7+ 8.0)-kb cDNA synthesized under each of the conditionstested, an equal fraction of each reaction mixture was subjectedto electrophoresis in an alkaline agarose gel and material in theappropriate region was quantitated by densitometry of theautoradiograph and liquid scintillation assay of bands cut fromthe gel. The highest yield obtained in the presence of melittinwas as much as 3.5-fold higher than the highest yield obtainedwith NP-40 (Table 1). The concentrations of NP-40 tested werewithin the range that allows high molecular weight cDNAsynthesis (1), and our yields of full-length cDNA are similar tothe value, 250 ng/mg of virus, reported by Lai et al. (14). Al-though the yields were lower at increased concentrations ofmelittin, the ratio of full-length cDNA to low molecular weightproducts was higher.When we varied a number of the standard reaction condi-

tions, we found that melittin gave a high yield of full-lengthcDNA under all conditions investigated. Using an isotope in-corporation assay and long incubation periods with melittin-treated virus, we found no significant effect on the yield ofcDNA synthesized when Mg2+ concentration was varied be-tween 5 and 40 mM (total nucleoside triphosphate concentra-tion was 4 mM), although incorporation was decreased by more

than 80% at 2.5 mM. The addition of 1 mM NaPP1 or 1 mMATP had no measurable effect on the yield of total cDNA. In

Table 1. Yields of full-length cDNA synthesized with variousconcentrations of melittin or NP-40

7.7-kb orReaction (7.7 + 8.0)-kb cDNA,conditions ng/mg virus protein

Melittin100,g/ml 270-33025 Mg/ml 330-47012.5 jg/ml 430-590

NP-400.04% 90-1300.02% 130-1700.01% 90-1300.005% 30-50

The cDNA products synthesized by equal amounts of virus, withvarying concentrations of melittin or NP-40, were denatured in 0.25MNaOH and subjected to electrophoresis at 40 V and 66 mA for 16 hrin a 1.0% alkaline agarose gel. The amount of 7.7-kb or (7.7 + 8.0)-kbcDNA was quantitated by densitometry of an autoradiograph andliquid scintillation assay of bands cut from the gel. The range indicatesthe discrepancy between the values obtained by the two procedures.

addition, analysis of the products synthesized with and without1 mM ATP showed no effect on the yield of full-length cDNA.Using melittin with the reaction components described byClayman et al. (6), we have found that full-length cDNA wassynthesized. The yields were decreased compared with ourstandard conditions; however, the size distribution of subge-nomic cDNA included larger molecules than those seen withour standard procedure. Finally, actinomycin D (100 gg/ml)inhibited the melittin reaction to the same degree as it did theNP-40 reaction, and the gel pattern of strong-stop and minorbands was identical with either compound under these condi--tions.

In order to compare the in vitro synthesized cDNA withproviral DNA of known length and to determine whether bothstrands are present in the full-length transcripts, the followingexperiment was performed. A bacteriophage X clone containinga 7.7-kbp RAV-2 proviral DNA insert was prepared by re-combinant DNA techniques using a restriction endonucleasethat cleaved the proviral DNA circle at one location. DenaturedcDNA synthesized by melittin- or NP-40-treated virus anddenatured 7.7-kbp proviral DNA, excised from the X vectorwith the one-cut restriction endonuclease, were subjected toelectrophoresis in a neutral agarose gel and transferred to ni-trocellulose. The 7.7- and 8.0-kb cDNA species synthesized inthe melittin-activated reaction were not resolved under theseconditions (Fig. 3). However, this gel did clearly resolve the (+)and (-) strands of the denatured RAV-2 insert. The leadingedge of the cDNA synthesized in the presence of either melittinor NP-40 migrated to the same position as the slower of theseparated 7.7-kb DNA strands which has been identified as the(-) strand. No cDNA at the faster migrating position of the7.7-kb (+) strand was detected. The minor bands which appearto be subgenomic in length have not yet been characterized;however, they are present in both melittin- and NP-40-stimu-lated cDNA products.The infectivity of melittin-stimulated cDNA was assayed by

the transfection procedure of Graham and Van der Eb (25). Wetested the total cDNA product synthesized by melittin-activatedNY68(B) and monitored cell morphology and reverse tran-scriptase activity after several passages. At the highest dosetested (50 ng/60-mm plate), both cultures became fullytransformed and produced reverse transcriptase-containingparticles (Table 2). Even with the assumption of only 1 focus-

Biochemistry: Boone and Skalka

850 Biochemistry: Boone and Skalka

i; RAV-2-11 Melittin NP-40

(-)

(+)

FIG. 3. Comparison of full-length cDNA with cloned 7.7-kbproviral DNA by strand separation in a neutral agarose gel. cDNA(4.5-hr synthesis) activated by melittin (100 ,g/ml) or NP-40 (0.1%)and Sal I-cleaved XRAV2-11 were denatured in 0.25 M NaOH andsubjected to electrophoresis at 100 V and 55 mA for 16 hr in a 1.0%neutral agarose gel. DNA was transferred to a nitrocellulose filter bythe procedure of Southern (24) and hybridized with RAV-2 [32P]RNA,as described (12). This step (not shown) identified the slower mi-grating strand as the (-) strand of the RAV-2 cloned insert. The[32P]RNA probe was subsequently melted off and the filter was

rehybridized with RAV-2 [32PlcDNA [endogenous synthesis activatedwith melittin at 50 ,ug/ml as described in Materials and Methods withthe exception that [a-32P]dATP (42 Ci/mmol) was at 2 Jm] to detectboth strands, as shown.

forming unit per 50 ng, the specific infectivity is of a magnitudecomparable to that reported by others for cDNA (6, 7). Of thelower doses, none showed conclusive signs of transformationand none had detectable levels of reverse transcriptase.

DISCUSSIONOur results show that melittin, a toxic peptide from bee venom,can substitute for nonionic detergents in facilitating endogenouscDNA synthesis by avian retroviruses. The product of this re-

action contains two species of high molecular weight cDNAwhich appear to be (-) strands and correspond in size to linearprovirus with and without the large terminal redundancy (22,23). The yield of full-length cDNA with a wide range of mel-ittin concentrations is higher than with any of the NP-40 con-

centrations tested. The most striking difference, however, is the

Table 2. DNA transfection assay of melittin activated cDNA

cDNA, Reverse transcriptaseng/60-mm Cell morphology* activityt

dish A B A B

50 T (18) T (12) 51,751 18,77525 N N 79 10612.5 N N 102 870 N N 57 68

NY68(B) was prepared as in Fig. 1B and used to synthesize cDNAwith melittin (100 ,ug/ml) as described in Materials and Methodsexcept that all nucleoside triphosphates were at 1 mM. The label was[a-32P]dGTP (25 mC/mmol) and incubation was for 16 hr.* Cultures were determined to be transformed (T) if they exhibitedthe transformed morphology characteristic of NY68(B)-infectedchicken embryo cells at the permissive (370C) temperature and theflattened appearance typical at the nonpermissive (410C) temper-ature. The number in parentheses indicates the first day on whichtransformation was observed. Normal (N) morphology is that ofuntreated chicken embryo cells passaged in parallel.

t Reverse transcriptase was assayed in 5 ml of culture supernatantas described in Fig. 1A and Materials and Methods, using 0.2%NP-40. Samples were precipitated with 10% trichloroacetic acid/1%NaPPj with 100 ,g of bovine serum albumin as carrier; precipitateswere collected on GF/C filters (Whatman) with suction. Filters werewashed with 5% trichloroacetic acid/1% NaPPi and then with eth-anol and were dried for scintillation counting. A background of 56cpm, from a control reaction mixture, has been subtracted from eachvalue.

fact that two species of full-length cDNA can be resolved in themelittin- but not in the NP-40-activated reaction products. Twospecies of large cDNA, the larger one containing a 600-baserepeat, have been observed in detergent-disrupted Moloneymurine leukemia virus (7, 26). This difference may signify thatwe have somehow missed a critical NP-40 concentration neededto activate 8.0-kb cDNA synthesis. Alternatively, it may reflectsubtle differences between murine and avian retrovirus struc-tures. Differences between these two groups have been ob-served by others in studies of in vitro cDNA products (27). Inany case, our results indicate that such a critical concentrationrequirement is not necessary with melittin and suggest thatmelittin may prove useful for activating reverse transcriptionin particles obtained from various sources such as the blood ofhuman leukemia patients (28) or released from certain humantumor cell lines (29).Our melittin-stimulated full-length cDNA was assumed to

be primarily (-) strand as shown by others (1, 3, 4, 6, 14, 30) andwas demonstrated to be so by comparison of its electrophoreticmobility with the mobilities of separated strands of the 7.7-kbRAV-2 provirus insert (Fig. 3). Although the 7.7- and 8-kbspecies were not resolved in this neutral gel, the leading edgeof the full-length cDNA product migrated coincident with the7.7-kb (-) strand, and no discrete band was detected at theposition corresponding to the 7.7-kb (+) strand. We assume,therefore, that most of the cDNA molecules consisted of anintact (-) strand and a segmented (+) strand as does the bulkof proviral DNA synthesized in vivo (31-33; unpublisheddata).The high efficiency of full-length cDNA synthesis and the

presence of extended (8 kb) cDNA suggests that melittin-acti-vated synthesis is more comparable to in vivo provirus synthesisthan is that activated by detergent. This can be explained bythe mechanism of interaction of melittin with membranes.Although melittin resembles a detergent in its amphipathicstructure, it does not lyse membranes by solubilizing theircomponents as detergents do (34). Melittin has a high affinityfor phospholipid and it has been postulated that its insertion

Proc. Natl. Acad. Sci. USA 77 (1980)

Proc. Natl. Acad. Sci. USA 77 (1980) 851

disrupts membranes by producing a wedge-like effect (10).Studies in which membrane-damaging capacity of severaltoxins, including melittin, and the nonionic detergent TritonX-100 were compared show that the membrane lesions causedby melittin are initially smaller than those caused by TritonX-100, although the functional size increases with high con-centration or prolonged exposure (35, 36).The difference between melittin action and detergent action

is readily apparent when these two compounds are added toconcentrated virus suspensions. NP-40 causes a rapid loss inturbidity whereas melittin causes no visible loss. In fact, at highconcentrations, melittin causes aggregation of particles, whichmay explain the inhibition of cDNA synthesis under theseconditions.Many proteins, including reverse transcriptase, retain their

activity in the presence of nonionic detergents. Although viralnucleoprotein cores remain intact, as viewed by electron mi-croscopy and buoyant density centrifugation (37), nonionicdetergents may cause subtle changes within the core. Novaket al. (38) have reported that, at higher-than-optimal detergentconcentrations, the tRNA-primed cDNAioo of avian sarcomavirus endogenous reverse transcripts is not properly elongatedand additional sequences are copied at random from the viralgenome. More recently this has been correlated with the de-creased ability of the reverse transcriptase-associated RNaseH activity to degrade the RNA strand of the initial RNA-DNAhybrid in the presence of high detergent concentrations (39).Our data suggest that the reaction that generates the extended(8 kb) (-) strand may be even more sensitive to detergent andthat these adverse effects can be circumvented by using mel-ittin. This may be important for investigation of the role of corestructure and cellular factors in provirus synthesis.

Finally, preliminary tests indicate that activation withmelittin will also prove useful for the study of in vitro tran-scription with other enveloped viruses. We have observed thatin vitro synthesis of cRNA by influenza virus is 2-3 times moreefficient in reactions in which melittin is substituted for non-ionic detergents; however, the effective concentration rangeis much narrower than with the avian retroviruses (unpublisheddata). Vesicular stomatitis virus cRNA synthesis is also activatedby melittin treatment (unpublished data).

We thank P. De Bona for generous supplies of RAV-2, L. Baer forplaque-purified NY68(B), and G. Ju for analysis of recombinant DNAclones.

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Biochemistry: Boone and Skalka