Proc. Ndst Acad. Sci. USAVol. 79, pp. 6142-6146, October 1982Biochemistry

Priming of reovirus transcription by GppppG and formation ofCpG(5')pppp(5')GpC

(mRNA initiation)

MINORU YAMAKAWA, YASUHIRO FURUICHI, AND AARON J. SHATKINRoche Institute of Molecular Biology, Nutley, New Jersey 07110

Contributed by Aaron J. Shatkin, June 28, 1982

ABSTRACT Compounds of general structure N(5')p.(5')Nwere used by the reovirus-associated RNA polymerase as primersfor template-directed synthesis of-virus-specific oligonucleotides-and RNA. Structural requirements for activity included a guano-sine residue and at'least four phosphates-i.e., G(5')pppp(5')N.Gp4G incubated with viral cores in the presence of CTP yieldedGp4GpC and CpGp4GpC. In a complete transcription mixture,Gp4G was also incorporated into the 5' termini of.full-length tran-scripts in the unmethylated forms Gp4GpC and CpGp4GpC, incontrast to viral mRNAs that contain 5'-terminal m7GpppGmpCformed de novo. Gp5G, Gp6G, and Gp4A but not Gp2G, Gp3G,and Ap4A also primed reovirus transcription and inhibited RNAmethylation.

Dinucleoside tetraphosphates ofgeneral structure N(5')pppp(5')Nhave been isolated from prokaryotic and eukaryotic cells. Thefirst of these compounds to be identified was Gp4G, a.majornucleotide constituent and purine storage product in dormantembryos of the brine shrimp Artemia salina (1). Gp4G was later'found in several other lower eukaryotes (2-5), and Ap4A wasdetected in Escherichia coli (6), rat liver (7), and several typesof 'vertebrate tissue culture cells (8). Ap4A is apparently pro-duced by the back reaction of amino acid activation in whichaminoacyl-tRNA synthetase transfers the covalently. boundAMP to ATP instead of to the normal acceptor, cognate amino.acid (9). Recently it was shown that Gp4N compounds accu-mulate during transcription in, vitro by purified insect cyto-plasmic -polyhedrosis virus and vaccinia virus '(10). The viraldinucleoside tetraphosphate products are probably formed bycondensation of enzyme-bound GMP and pppN, a reversiblereaction that closely resembles the 5'-terminal guanylylation-step of eukaryotic mRNA capping. Synthesis of Gp4N duringinitiation of viral. transcription may occur when capping andelongation of transcripts are uncoupled, a situation possiblyanalogous to generation ofAp4A at an early step of protein syn-thesis under conditions of amino acid depletion.The physiological significance of Np4N structures is notwell

established. However, Ap4A has been implicated as a regula-tory element in the initiation of DNA replication on the basisthat it associates with DNA polymerase a (11) and can primethe synthesis of short deoxyoligonucleotides in vitro (12). Wetested Gp4N for priming effects on the initiation of reovirusmRNA synthesis following the suggestion that cap formationmay be a pre-transcriptional event in cytoplasmic polyhedrosisvirus transcription (13). The transcriptase complex in purified.reovirus, unlike that in the polyhedrosis virus, does not syn-thesize or degrade Gp4N. compounds to any detectable extent.However, exogenous preformed Gp4G is incorporated into reo-virus transcription products, including full-length transcripts

and a molar excess of short oligonucleotides that correspond insequence to mRNA 5' termini and probably arise by abortiveinitiation (14). Analyses of the reovirus products made in thepresence of Gp4G- indicate. that phosphodiester bonds areformed on either or both guanosine residues of the preformedtetraphosphate primer, yielding unusual structures of the typeG(5')pppp(5')GpC and CpG(5')pppp(5')GpC.

MATERIALS AND METHODSMethods. Purified reovirus type 3 was converted to cores by

chymotrypsin digestion as described (14). Transcription reac-tion mixtures were 70 mM Tris-HCl, pH 8/0.2 mM ATP/0.2mM CTP/0.2 mM UTP/0.4 mM GTP/8 mM Mg(OAc)2/0.1mM S-adenosylmethionine or 3 ,M S-adenosyl[methyl-3H]me-thionine (specific activity, 72 Ci/mmol; 1 Ci = 3.7 X 101Obecquerels)/1 mM phosphoenolpyruvate containing 1 to 2 unitsof pyruvate kinase and 6 pug of reovirus cores in a total, volume,of50 1.l. a-32P-Labeled CTP or UTP was used at a final specificactivity of 0.5 Ci/mmol unless otherwise specified.

For product analysis, incubation mixtures were extractedwith an equal volume of H20-saturated.phenol, and newly syn-thesized mRNAs and oligonucleotides in the ether-washedaqueous layer were. digested in 0.1 M Tris-HCl (pH 8) for 1 hrat 370C with calf intestine alkaline phosphatase at 20 units/ml.The phosphatase-treated products were separated by electro-phoresis in 20% polyacrylamide slab gels (0.1 x 28 x 37 cm)containing 7 M urea as described (14). 'Individual oligonucleo-tides located by autoradiography were extracted from gels andcharacterized further by DEAE-cellulose column chromatog-raphy for determination of net negative charge and by enzy-matic digestion and high-voltage paper electrophoresis for basecomposition (14).

Materials. a-32P-Labeled NTPs (specific activity, 410 Ci/mmol) were from Amersham, and S-adenosyl[methyl-3H]methionine was from. New England Nuclear. Marker com-pounds including GpC, Ap4A, and Gp2G, Gp3G, Gp4G, Gp5G,and Gp6G were from P-L Biochemicals. Nuclease P1, calf in-testine alkaline phosphatase, and RNase T2 were from YamasaShoyu (Tokyo), Boehringer Mannheim, and Calbiochem, re-spectively. Tobacco pyrophosphatase was kindly provided byM. Miwa and T. Sugimura, National Cancer Center ResearchInstitute (Tokyo, Japan). Gp4A was synthesized and kindly pro-vided by M. Manami and A. Kuninaka, Yamasa Shoyu ResearchInstitute.

RESULTSAlteration of- Reovirus Transcription Products by Gp4G.

Reovirus cores contain several enzymes that function together.to produce viral mRNAs. Messengers made in the presence ofS-adenosylmethionine contain 5' termini that are almost exclu-sively' blocked and methylated by a cap structure, m7G(5')-

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The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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B234567 2 3 4 5 6

ORIGIN-

XC-

rG-c-uncapped.4

G-C-U-LG-C-U-A

BPS-

capped -C-U=

-E GP2G Gp4G Gp6G IECON Gp;3G Gp5G

_ w.*1!F_ _ -G-C

w G-C-U uncapped

-G-C-UJ-A

24

2

.CO A. 1 - _CON Gp4G GP4A Ap4A, [Gp4G Gp4A Ap4A

+4 rNTP CTP ONLY

FIG. 1. Effects of Np.N compounds on oligonucleotide synthesis by reovirus cores. (A) Complete transcription reaction mixtures (50 I-l) con-

taining 5 ,Ci of [a-32P]CTP and the indicated Gp.G compounds (1 mM) were incubated for 3 hr at 420C, and the products were analyzed by gelelectrophoresis. -E, no cores; CON, control reaction without Gp.G. (B) Comparative effects of Gp4G, Gp4A, and Ap4A on synthesis of mRNA andoligonucleotides in complete reaction mixtures as inA and in partial reaction mixtures containing 0.2 mM [a-32P]CTP as the only ribonucleosidetriphosphate (rNTP). XC, xylene cyanol; BPB, bromphenol blue.

ppp(5')GmpC (15). In addition to full-length transcripts, a seriesofvirus-specific di-, tri-, and tetranucleotides are also producedin large molar excess. They apparently result from a process ofreiterative initiation by the core-associated RNA polymerase.The oligonucleotides correspond to the 5'-terminal sequencecommon to reovirus mRNAs but, in contrast to mRNA, they aremainly (>90%) uncapped structures like ppGpC (14, 16). Thecombined observations that most reovirus initiation events are

abortive and yield uncapped 5'-terminal oligomers while es-

sentially all of the full-length products are capped suggests thatnascent chains with guanylylated 5' termini are more effectivelyelongated than uncapped transcripts.

These findings raised the possibility that reovirus transcrip-tion, like influenza virus mRNA synthesis (17), can be primed,for example by incorporation of G(5')ppp(5')G or other similarcompounds into transcript 5' termini. To test the effects ofpre-formed Np0N structures on transcription, we analyzed theproducts made by reovirus cores in reaction mixtures that in-cluded Gp3G or related compounds and [a-32P]CTP as the la-beled precursor. Polyacrylamide gel electrophoresis resolvedthe labeled viral mRNAs and a series offast-migrating uncappedand capped oligonucleotides that have been shown (14) to cor-

respond in sequence to mRNA 5' termini (Fig. 1A, lane 2).Addition to the incubation mixtures of 1 mM Gp2G or Gp3G hadlittle effect on the transcription pattern (lanes 3 and 4). How-ever, in the presence of diguanosine tetra-, penta-, or hexa-phosphate, mRNA synthesis was partially inhibited, and therewas a dramatic change in oligonucleotides (lanes 5-7). Theyields of both the capped and the uncapped oligomers de-creased, and several new radiolabeled bands were evident.Another pattern ofoligonucleotides was obtained in the pres-

ence of Gp4A as compared with Gp4G (Fig. 1B, lanes 3 and 4).By contrast, Ap4A appeared to have no effect on the profile of32P-labeled control products that included mRNA, uncappedoligomers, and a low level of capped oligonucleotides (lanes 2and 5). Synthesis of at least some and probably all of the newoligomers clearly involved incorporation of exogenous Gp4Nbecause they were not produced in similar reaction mixturesthat lacked Gp4N. For example, incubation of cores with [a-32P]CTP and Gp4G produced only bands 1 and 2 (lane 6), and

Gp4A yielded a different band 3 (lane 7), which was identifiedas CGGp4A (data not shown). Consistent with its lack of effecton the complete transcription reactions (lane 5), Ap4A also wasnot utilized by the core enzymes in partial reactions, and noradiolabeled bands were observed after incubation ofAp4A with[a-32P]CTP (lane 8). Other analyses ofthe oligonucleotides pro-duced in the presence of Gp4G and various combinations ofrNTP precursors indicate that incorporation of the preformedtetraphosphate is template directed and virus specific. For ex-ample, cores incubated with Gp4G, ATP, GTP, and [a-32P]UTP

1 2 3 4 5 6 7

ORIGIN-

XC-

uncapped._.._,G-C

BPB-

copped,--2copped *_.

-E GppppG(mM) O.

0.00.2 0.4 0.8 16

FIG. 2. Inhibition of m7GpppG'pC synthesis and formation of oli-gonucleotides 1 and 2 in the presence of Gp4G. Partial incubation mix-tures (50 ,ul) contained 0.4 mM GTP, 0.2 mM [a-32P]CTP, 6 ,g of reo-virus cores, 0.1 mM S-adenosylmethionine, and Gp4G as indicated.After 1 hr at 42°C, the samples were analyzed as in Fig. 1.

7 8

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6144 Biochemistry: Yamakawa et al.

pU-

2~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

2 .*0

pC- _GpC-

Ongin-Spot 2 2 2 2 2

a1 2 3 4 6 78

pU-'

pA-

pC-

origin-

FIG. 3. Analysis of oligonucleotides 1 and 2. (A) 32P-Labeled oli-gomers 1 and 2 were extracted from a polyacrylamide gel identical toFig. 1B (lane 6), and samples treated as indicated were analyzed byhigh-voltage paper electrophoresis atpH 3.5. Lanes: 1 and 2, untreatedsamples; 3 and 4, spots 2 and 1, respectively, were digested with tobaccopyrophosphatase (0.06 units/ml, 1 hr, 3700) and then with calf intes-tine alkaline phosphatase; 5 and 6, spots 2 and 1, respectively, weretreated with HCl (0.1 M, 1000C, 3 min) and then with alkaline phos-phatase; 7 and 8, spots 2 and 1, respectively, were digested with RNaseT2 (50 units/ml, 1 hr, 3700); 9 and 10, spots 2 and 1, respectively, weredigested with nuclease P1 (0.125 mg/ml, 1 hr, 37°C). Samples in lanes3 and 4 were extracted with phenol and ether between enzymatic diges-tions. (B) Oligonucleotide 2 (10,000 cpm in each 50-1,u sample) wasdigested at 370C with nuclease P1 in 20 mM NaOAc buffer (pH 6.0).At the indicated times, samples were heated at 100°C for 1 min to stopthe digestion, and products were separated by paper electrophoresisat pH 3.5. Lanes: 1 and 2, untreated samples; 3, spot 2 was digestedfor 2 min with nuclease P1 at 1.25 ,ug/ml; 4-8, spot 2 was digested withnuclease P1 at 12.5 ng/ml for 0, 2, 4, 8, and 16 min, respectively.

but without CTP synthesized no labeled oligonucleotide prod-ucts, in agreement with the viral RNA 5'-terminal sequence,m7GpppGmpCpUpA (18).Gp4G was effectively utilized as a preformed "pseudocap"

for reovirus core-catalyzed oligonucleotide formation with [a-32P]CTP (Fig. 2). Addition of increasingly high concentrationsofGp4G abolished the formation ofm7GpppG(m)pC ("capped G-C") but had little effect on the synthesis of (p)ppGpC ("un-capped G-C"). At Gp4G/GTP ratios (mol/mol) greater than 2,capped G-C synthesis was essentially completely replaced bythe formation of oligonucleotides 1 and 2 (lanes 6 and 7). Asshown below, compounds 1 and 2 correspond to Gp4GpC andCpGp4GpC, respectively.

Characterization of Oligonucleotides 1 and 2. Radiolabeledoligonucleotides 1 and 2 extracted from a gel identical to thatshown in Fig. lB (lane 6) were characterized by high-voltagepaper electrophoresis after various treatments. Spot 1 yielded32P-labeled GpC exclusively on digestion with tobacco pyro-

phosphatase and alkaline phosphatase (Fig. 3A, lane 4), con-sistent with the pyrophosphate-containing structure Gp4G*C.By contrast, spot 2, which migrated more slowly than spot 1(lanes 1 and 2), was resistant to the two phosphatases (lane 3).Brief exposure of spots 1 and 2 to HCl under conditions thatpartially hydrolyze polyphosphate linkages followed by alkalinephosphatase treatment yielded G6C and residual undigestedstarting material (lanes 5 and 6). Cleavage with RNase T2 mark-edly increased the electrophoretic mobility of both compounds,but enzyme-treated oligomer 2 (lane 7) migrated considerablymore rapidly than oligomer 1 (lane 8). These results are in goodagreement with the relative net negative charges determinedby DEAE-cellulose column chromatography; for oligomers 1and 2, the values were -4.5 and -5.5, which increased to-5.5 and -7.5, respectively, following RNase T2 digestion

Table 1. Differential inhibition of mRNA methylation by Gp4Gand related compounds

Addition Methylation, RNA synthesis, Methylation/(1 mM) % % RNA synthesis ratioNone 100 100 1.00Gp2G 84 85 0.99Gp3G 84 97 0.87Gp4G 8 52 0.15Gp5G 7 52 0.13Gp6G 12 47 0.26Gp4A 54 116 0.47Ap4A 122 141 0.87

Values of 100% for methylation and RNA synthesis correspond to0.51 and 0.28 pmol of C3H3 and [32P]pC incorporated into acid-precip-itable products in 1 hr at 420C from radiolabeled S-adenosylmethio-nine and CTP, respectively.

(data not shown). Finally, treatment of oligomers 1 and 2 withnuclease P1 yielded fC as the only radiolabeled product (lanes9 and 10). The combined findings indicate that oligonucleotides1 and 2 synthesized in the presence of Gp4G and [a-32P]CTPare Gp4G*C and C6Gp4G*C, respectively. Consistent withthese structures, both radiolabeled compounds yielded all the32P radioactivity as 6C after complete digestion with nucleaseP1, and partial digestion converted oligomer 2 to oligomer 1with release of pC (Fig. 3B). The extent of cleavage was de-pendent on both incubation time and enzyme concentration andapparently proceeded stepwise:

C*Gp4G6C (spot 2) P1. Gp4G*C (spot 1) + pC P1 Gp4G + pC

Incorporation of Gp4G into mRNA. Addition of Gp3G totranscription incubation mixtures containing [a-32P]CTP andS-adenosyl[methyl-3H]methionine was without striking effecton mRNA synthesis or methylation as measured by 32P and 3Hincorporation into acid-precipitable products (Table 1). How-ever, as observed for oligonucleotide synthesis (Figs. 1 and 2),the presence of 1 mM Gp4G markedly altered RNA formation.32P-Labeled RNA yields were decreased partially while product

'o.03-

a)0.

cm0O

20FRACTION

2

0

a.

rtO

FIG. 4. Sedimentation analysis of RNA products. Transcriptionreaction mixtures containing [a-32P]CTP and S-adenosyl[methyl-3H]methionine were incubated for 2 hr at 420C, and the products wereextracted with phenol, washed with ether, and purified by SephadexG-50 gel filtration (0.9 x 28 cm column) in 20 mM Tris-HCl (pH 8.0).The peak fractions of excluded radiolabeled material were pooled, andsamples of the mRNAs recovered by ethanol-precipitation were ana-lyzed by sedimentation (SW-40, 18 hr, 26,000 rpm) in linear 5-30%glycerol gradients (14). e, 32p; o, 3H. m and s, medium and small reo-virus species, respectively.

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methylation was more significantly inhibited (Table 1). The re-sults are consistent with incorporation ofpreformed Gp4G intotranscript 5' termini that are not subsequently methylated, incontrast to the usual GpppGpC 5' ends that are converted tom7GpppGmpG. Other polyphosphate compounds, includingGp5G, Gp6G, and Gp4A, that altered the pattern of oligonu-cleotide products (Fig. 1) also differentially inhibited transcriptmethylation (Table 1).To demonstrate directly that exogenous Gp4G was incorpo-

rated into transcripts as unmethylated "pseudocaps," viralRNAs were synthesized in the presence of [a-32P]CTP and S-adenosyl[nmthyl-3H]methionine in the absence and presenceof 1 mM Gp4G. Sedimentation analyses indicated that bothRNA preparations consisted mainly of small and medium reo-virus RNA species (Fig. 4). While the products were similar asjudged from the 32P profiles, the relative incorporation ofC3H3was decreased by 83% in the presence of Gp4G.

Radiolabeled RNA products were digested with RNase T2and analyzed by DEAE-cellulose column chromatography. Allof the 3H radioactivity and 0.21% of the 32P-labeled material inthe digested control sample eluted with a net negative chargebetween -4 and -5-i.e., the position ofauthentic methylatedm7GpppGmpCp caps (Fig. 5A). This structure was confirmedby nuclease P1 digestion and paper electrophoresis of the re-sulting 3H-labeled m7GpppGm and 32P-labeled pC products(data not shown). The RNA products made in the presence ofGp4G after RNase T2 digestion included a reduced amount of

A CONTROL 2 3 -4(no GppppG)

'.5-

1.0

0.5-

'o0

0 B +GppppG - 3 4

t T F T

double-labeled m7GpppGmpCp (0.11% of total 32P) and twoadditional 32P-labeled peaks ofnet charge about - 5.5 and - 7.5(Fig. 5B). These net charge values are the same as determinedfor Gp4Gp and pGp4Gp, the RNase T2 digestion products ofoligonucleotides 1 and 2, respectively. The distribution of total32P radioactivity in peaks 1 and 2 was 0.07% and 0.19%, re-spectively, consistent with their derivation by RNase T2 diges-tion of sequences in the RNA products corresponding toGp4GpC and CGGp4G*C. Peaks 1 and 2 were desalted and theirstructures were confirmed by further analysis. As shown in Fig.6, the 32P-labeled oligonucleotides derived from mRNA byRNase T2 digestion correspond in electrophoretic mobility toRNase T2-digested oligonucleotides 1 and 2. The results indi-cate that Gp4G is incorporated into reovirus transcripts instructures of the type G(5')pppp(5')GpC... and CpG(5')pppp(5')GpC.

DISCUSSIONEukaryotic mRNAs ofdiverse origin are guanylylated and meth-ylated at the 5' end (19). The modification reactions in variousviral and cellular systems are initiation-related mechanisticallysimilar events. Cap sites apparently correspond to RNA poly-merase initiation positions on RNA and DNA templates (20, 21).In the case of reovirus transcription by viral cores, nascent(p)ppGpC is converted to GpppGpC, which is subsequentlymethylated to form m7GpppGmpC (22). This cap structure ispresent on the 5' ends of full-length reovirus mRNAs (15).

-5 -6 -74 4 4

i7GpppG'pCp

3

2 NMlo

a.

-6 -7 -8 -J

I

FRACTION

FIG. 5. Chromatographic analysis of RNA 5' termini. RNAs were synthesized as in Fig. 4 but by a 4-hr incubation of 100-ilI reaction mixturesthat contained 24 pg of reovirus cores, 35 ,Ci of S-adenosyl[methyl-3H]methionine, [a-32P]CTP (specific activity, 12.5 Ci/mmol) and, for B, 1 mMGp4G. RNAs purified by Sephadex G-50 gel filtration were ethanol-precipitated with 100 pg of rRNA as carrier. Gel filtration and ethanol-pre-cipitation were repeated to remove any residual [a-32P]CTP, and the purified mRNAs were digested with RNase T2 (100 units in 0.5 ml, 37°C, 4hW). Digests were applied to DEAE-cellulose (0.9 x 42 cm column) and eluted with a linear gradient of 0.05-0.4 M NaCl in 20 mM Tris-HCl, pH8/7M urea (112 ml of each). One-milliliter fractions were collected and assayed directly for 32P by Cerenkov counting. Aliquots of 50 ,ld were assayedfor 3H by counting in Bray's solution. o, 3H; el 32p.

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6146 Biochemistry: Yamakawa et al.

1 2

pGP4Gp-

pA-

PA-p-.:...

FIG. 6. Identification of oligonucleotides 1 and 2 in RNase T2 di-

gest of mRNA made in the presence of Gp4G. Purified mRNA made

with 1mM Gp4G and [ar-32P]CTP in the transcription reaction mixture

was digested with RNase T2, and the digestion products were sepa-

rated by DEAE-cellulose column chromatography as described in Fig.

5. The peaks of radioactivity eluting with net charges of -5.5 (oligo-

mer 1) and -7.5 (oligomer 2) were pooled and desalted by binding to

DEAE-Sephadex A-25 and elution in NH4HCO3. Samples eluted in 1

M NH4HCO3 (>90% of the radioactivity) were lyophilized and ana-

lyzed by paper electrophoresis at pH 3.5. Marker compounds Gp4Gp

(lane 1) and pGp4Gp (lane 2) were obtained by RNase T2 digestion of

oligonucleotides 1 and 2 isolated from a gel identical to that in Fig. lB

(lane 6). Lanes: 3 and 5, oligomer 2 isolated from two different RNA

preparations; 4, oligomer 1 obtained from the same RNA digest ana-

lyzed in lane 5.

However, most reovirus RNA polymerase starts are abortive

and yield unmodified 5'-terminal oligonucleotides, mainly(p)ppGpC (14, 16).

The correlation between 5'-terminal guanylylation and na-

scent chain completion suggested that the presence of pre-

formed G(5')ppp(5')G might promote reovirus RNA formation.Although diguanosine triphosphate was not utilized by reovirus

cores, similar 5'-5' linked dinucleoside polyphosphate com-

pounds were incorporated into virus-specific oligonucleotides,and Gp4G was obtained in messenger-size transcripts. A single

guanosine residue and four phosphates were minimum require-

ments for utilization of the GpnN compounds. Presumably, the

similar net negative charge (2i-4) and spatial resemblance of

Gp4N to GTP allowed binding to the transcriptase in place of

the authentic nucleotide, followed by incorporation into RNA

products. Ap4A was not a substrate for the reovirus polymerase

and only the guanosine residue in Cp4A was utilized for 3'-5'

phosphodiester bond formation. This occurred exclusively by

addition of CMP from CTP, consistent with template-directed

transcription that, in the absence of Gp4N primers, begins by

synthesis of the sequence (p)ppGpC (22).

Surprisingly, Gp4G was extended from both guanosine res-

idues, and Gp4GpC was apparently a more effective primer

than Gp4G since CeGp4GhC was made fromGp4GuC in the

presence of a i04 molar excess of Gp4G (estimated from Fig.

2, lane 3). The limited number of oligonucleotide products syn-

thesized in complete incubation mixtures (Fig. 1B, lane 3) sug-

gests that template-directed abortive initiation also occurs dur-

ing Gp4G-primed reovirus RNA formation. In addition toGp4GpC and CpGp4GpC, analyses of [a-32P]UTP-labeledproducts indicate that Gp4GpCpU, CpGp4GpCpU, and Up-

CpGp4GpCpU may also be produced (data not shown).

Transcripts made in the presence of Gp4G sedimented like

full-length mRNAs and yielded Gp4Gp and pGp4Gp on RNaseT2 digestion. The results show that RNA synthesis can beprimed by Gp4G, but it is not yet known whether polynucleo-tide chains are made on only one or on both sides of the poly-phosphate linkage. cDNA clones of individual reovirus genomesegments, which have recently become available (unpublishedresults), should be useful as probes to determine whetherGp4G-primed RNAs are chimeric. For example, it will be ofinterest to test whether the RNA that cosediments with the mclass of viral mRNA includes Gp4G-joined transcripts of twodifferent small genome segments. If transcript chimeras areformed, it may be possible to use this approach to decipher thein situ organization of the reovirus genome template RNAs.

The formation of Gp4N compounds by cytoplasmic polyhe-drosis virus and vaccinia virus (10) and their incorporation intoRNA products by reovirus-associated polymerase may occur byreactions that are restricted to these viral in vitro transcriptionsystems. On the other hand, enzymes that cleave Gp4G andother dinucleoside tetraphosphate compounds have been iso-lated from normal rat and mouse livers (23, 24). In addition, thepresence in HeLa nuclear poly(A)+RNA of a RNase T2-resistantconstituent having some of the properties of pNpnNp has beenreported recently (25). The possible physiological significanceof dinucleoside 5'-5' polyphosphate compounds for initiationof RNA synthesis in vivo remains to be explored.We thank Maureen A. Morgan and Alba J. LaFiandra for excellent

assistance.

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