JOURNAL OF VIROLOGY, May 1980, p. 550-5590022-538X/80/05-0550/10$02.00/0

Vol. 34, No. 2

Site on the Vesicular Stomatitis Virus Genome SpecifyingPolyadenylation and the End of the L Gene mRNA

MANFRED SCHUBERT,' * JACK D. KEENE,2 RONALD C. HERMAN,' AND ROBERT A.LAZZARINI'

Laboratory ofMolecular Biology, National Institute of Neurological and Communicative Disorders andStroke, Bethesda, Maryland 20205,' and Department ofMicrobiology and Immunology, Duke University

Medical Center, Durham, North Carolina 277102

The 5'-terminal nucleotide sequence from positions 50 to 130 of vesicularstomatitis virus RNA was determined indirectly by using a defective interferingparticle RNA which contains covalently linked genomic minus and antigenomicplus sense RNAs. The last 18 nucleotides of the L gene coding for the viralpolymerase were identified and isolated by specific duplex formation between 5'terminally labeled oligonucleotides from a small single-stranded defective inter-fering particle RNA and L gene mRNA. The L gene ends at position 60 from the5' terminus of the vesicular stomatitis virus genome. The data demonstrated thatthe first seven adenine residues in the polyadenylic acid tail of L gene mRNAmay be coded for in the genome and suggested that the viral transcriptase itselfmay carry out polyadenylation, possibly by chattering at the uridine-rich sequenceat the end of the L gene. Analysis of the 5'-terminal sequence of vesicularstomatitis virus genomic RNA revealed that it might fold into a complex second-ary structure with possibly 62% of the bases paired.

The genome of vesicular stomatitis virus(VSV) consists of a single-stranded minus senseRNA which is approximately 10 kilobases longand codes for the five structural proteins in theorder 5' L-G-M-NS-N 3' (1-3). It serves as atemplate for the replicative intermediate, a full-length plus strand, as well as for five monocis-tronic mRNA's which are modified and carry a5'-terminal cap structure (11, 20, 22) and a 3'-terminal polyadenylic acid [poly(A)] tail (5, 17,28).At present, the details of the transcription of

VSV and the modification of the mRNA's arenot known. The discrete sizes of the messagesmay result from a cleavage of a polycistronicRNA involving specific processing sites or fromspecific start and stop signals from transcription.The available data are compatible with either oftwo general mechanisms for transcription. In thefirst, the transcriptase initiates synthesis at theprecise 3' end and moves down the entire ge-nome in a single synthetic event, without reini-tiating. The individual modified mRNA's aregenerated by cleavage and processing of thenascent transcript (5). In the second model, thetranscriptase enters the template at the precise3' terminus, moves down the template, and ini-tiates and terminates synthesis at the beginningand end, respectively, of each cistron, so that apolycistronic intermediate is never generated. In

both cases, one might expect to find character-istic nucleotide sequences at the beginnings andthe ends of the mRNA's adjacent to the poly(A).The mRNA's of both Indiana serotype and

New Jersey serotype VSV have the same se-quence at their 5' termini, 7-mGpppAACAG (11,20, 22), although the two serotypes share littlesequence homology (7, 18, 19). Little is knownabout the sequences adjacent to the poly(A)tails of the mRNA's except for the one on NmRNA, which was recently determined (15).

In this communication we report the 3'-ter-minal sequence of the message coding for the Lprotein and the location of the complement ofthis sequence on the VSV genomic RNA. Wededuced the sequence of the first 130 nucleotides(23) from the 5' end of VSV genomic RNA andisolated an 18-nucleotide RNase T,-resistant ol-igonucleotide representing the last 18 nucleo-tides of the L gene. We demonstrate that the Lgene ends at position 60 from the 5' terminus ofVSV RNA and that the last seven nucleotidesof this gene consist of uridine residues whichmay code for the first seven adenine residues inthe poly(A) tail. These findings suggest that theviral transcriptase either alone or with othercomponents might be responsible for the gener-ation of poly(A) tails at the ends of messages,possibly by chattering at the uridine-richstretches on the template. A comparison of the

550

VSV POLYADENYLATION SITE 551

sequences adjacent to the poly(A) shows thatthe N (15) and L messages both have the follow-ing sequence: ... UAUGpoly(A).

MATERIALS AND METHODS

Virus growth and RNA purification. The VSV(Mudd-Summers strain) used in this study and thedefective interfering (DI) particles DI-T (San Juanstrain) and DI 011 (Mudd-Summers strain) were all ofthe Indiana serotype. The geneology, propagation, andpurification of the virus and the DI particles were

described previously (13, 14).Isolation of I-protein mRNA from infected

cells. BHK-21 cells were grown in suspensions aspreviously described (6). A total of 1.5 x 108 BHK-21cells were collected by centrifugation, suspended in 10ml of medium without serum, and infected with VSVat a multiplicity of 10. After 1 h at 37°C, the suspensionwas diluted with 90 ml of medium containing 5%serum, 100 Mg of actinomycin D, and 100 MCi of [3H]-uridine (specific activity, 20 Ci/mmol) and incubatedat 37°C for 3.5 h. The cells were collected by centrif-ugation, washed twice in cold phosphate-buffered sa-

line, swollen in 3 ml of cold 0.03 M Tris-hydrochloride(pH 7.6)-0.01 M NaCl-1.5 mM MgCl2 and lysed withNonidet P-40 (final concentration, 0.5%). The nucleiwere removed by centrifugation, and the supernatantwas adjusted to 1% sodium dodecyl sulfate (SDS),layered onto two 35-ml 10 to 30% sucrose gradientscontaining 10 mM Tris-hydrochloride (pH7.4), 1 mMEDTA, and 0.5% SDS, and centrifuged for 16 h at22,000 rpm in an SW27 rotor. The 31S RNA wascollected by precipitation, further deproteinized withphenol-SDS, and reprecipitated with ethanol. TheRNA precipitate was washed with 80% ethanol anddissolved in 80% dimethyl sulfoxide-0.01 M Tris-hy-drochloride (pH 7.6)-0.01 M LiCl-0.5 mM EDTA-0.02% SDS. The solution was heated for 15 s at 60°Cand for 3 min at 37°C, diluted with 6 ml of 10 mMTris-hydrochloride (pH 7.6)-0.4 M NaCl-0.5% SDS,and repeatedly passed over a 0.8-ml oligodeoxythy-midylic acid [oligo(dT)] cellulose column (P. L. Bio-chemicals, Milwaukee, Wis.). The column was washedextensively with the same buffer, and poly(A)-contain-ing L mRNA was eluted with 0.01 M Tris-hydrochlo-ride (pH 7.6)-0.05% SDS. After the addition of 50 Mgof carrier tRNA, the RNA was precipitated withethanol and dissolved in water.

In vitro synthesis and purification of L mes-

sage. L message was kindly provided by L. A. Ball. Itwas synthesized in vitro by using the heat-resistantstrain of VSV in the presence of a mouse L-cell S-10cytoplasmic extract, as previously described (4).Poly(A)-containing RNAs were selected by chroma-tography on oligo(dT)-cellulose. The poly(A)-contain-ing RNA was denatured with dimethyl sulfoxide andN,N-dimethylformamide (8) and sedimented in an

aqueous 15 to 30% sucrose gradient in 0.01 M Tris-hydrochloride (pH 7.4)-i mM EDTA-0.5% SDS for 17h at 23,000 rpm in an SW56 rotor. The materialsedimenting at 31S was pooled and ethanol precipi-tated.

Electron microscopy. The 31S RNA was an-

nealed to 42S genomic RNA in a 5-f1 mixture contain-ing 53% (vol/vol) formamide, 2.4 M urea, 18 mMEDTA, and 0.18 M tricine-NaOH (pH 8.3) for 20 minat 37°C. The RNA was spread from a hyperphasecontaining 82% formamide, 3.8 M urea, 8 mM EDTA,and 80 mM tricine-NaOH (pH 8.3) onto a water hy-pophase (12). Grids were prepared and shadowed aspreviously described (29). Tracing of the moleculesand methods of data processing have been describedpreviously (16).

3' and 5' labeling of DI 011 RNA. DI 011 RNAwas labeled at its 3' end by using cytidine 3',5'-bis-[32P]phosphate (*pCp) and RNA ligase and at its 5'end by using [y-32P]ATP and polynucleotide kinase, aspreviously described (10, 13, 23).

Partial digestion conditions. Terminally labeledRNA was subjected to partial digestions with RNasesU2, T,, and Phy 1, as described previously (9, 24, 25).Partial alkali hydrolysis was carried out by using acarbonate buffer (pH 9) for 15 min at 90°C. For thegeneration of small random RNA fragments from 5'terminally labeled RNA, the RNA was dried, dissolvedin 99% formamide, and incubated at 100°C for 1 h.Complete digestion was achieved by using 10 ug ofnuclease P1 per ml in 20 mM sodium citrate (pH 5)-1 mM EDTA at 37°C for 2 h and complete alkalinehydrolysis in 0.3 M NaOH at 37°C overnight.

Isolation of the 5' terminus ofthe L gene. A 0.4-Mug amount of carrier-free DI-T RNA was digested withRNase T, (20 U/ml) at 37°C for 30 min in 12 Id ofwater. The large (unique) oligonucleotides were sepa-rated by chromatography on a 9-ml Sephadex G-25column equilibrated with 10 mM Tris (pH 7.6)-0.4 MNaCl-0.2% SDS. The materials in the exclude volumewere precipitated with ethanol, washed with 80%ethanol, and dried. These oligonucleotides were la-beled at their 5' termini by using 10 U of polynucleo-tide kinase and [y-32P]ATP (3,000 to 5,200 Ci/mmol).In some experiments the gel filtration step was omit-ted. After labeling, the RNA was diluted with 500,ul of10 mM Tris-hydrochloride (pH 7.5)-i mM EDTA andextracted three times with phenol saturated with thesame buffer. The deproteinized oligonucleotides weremixed with 9 Mug of carrier tRNA, precipitated withethanol, washed with 80% ethanol, and dried. Poly(A)-selected 31S L mRNA derived from 3.8 x 107 VSV-infected BHK-21 cells was mixed with the labeledoligonucleotides in 9 MlI of water, heated for 30 s at100°C, and adjusted to 0.01 M Na2HPO4/NaH2PO4(pH 6.8)-0.4 M NaCl. After 70 min of annealing at37°C, 2 ML of a mixture containing RNase T, (100 U/ml) and RNase A (500 Mig/ml) in annealing buffer wasadded. The digestion was carried out for 15 min at37°C. Then, 1ML of proteinase K (1 mg/mil) was added,and the incubation was continued for 10 min at 37°C.The reaction mixture was diluted into 500 Ml of 10

mM Tris-hydrochloride (pH 7.5)-0.4 M NaCl-0.5%SDS and passaged three times over a 0.4-mi columnof oligo(dT)-cellulose (P. L. Biochemicals) equili-brated with the same buffer, and the bound oligonu-cleotide was eluted with 10 mM Tris-hydrochloride(pH 7.5)-0.05% SDS. The unbound material, as wellas that bound and eluted, was collected by ethanolprecipitation with carrier tRNA and electrophoresedin a 20% polyacrylamide-urea gel.

VOL. 34, 1980

552 SCHUBERT ET AL.

Separation of the RNA fragments. RNA frag-ments resulting from partial digestions were separatedby electrophoresis on 20% polyacrylamide-urea gels aspreviously described (9). Mononucleotides were iden-tified by thin-layer chromatography and electropho-resis on polyethyleneimine sheets. Two-dimensionalpolyacrylamide gel electrophoresis was carried out asreported previously (24).

RESULTSThe 5'-terminal nucleotide sequence of VSV

RNA can be determined indirectly by takingadvantage of a double-stranded hairpin DI par-ticle RNA (DI 011). We demonstrated by an-nealing and RNA sequencing that the 5' termi-nus of DI 011 RNA is identical to the 5' terminiof VSV RNA and other DI particle RNAs forapproximately 1 kilobase (23). In this researchwe also used DI 011 RNA to investigate the 5'-terminal sequence of VSV RNA beyond the 60nucleotides described previously (23), in order

3'

TI OH-U2

.~~~~ A

GAGAA AA G

AAG A

ak GA AAAAAAG

G A

AG

- A AA~

s A ~~~~A~~~~~AibGA- ~~~~AG

AA

130

120

1 10

100

90

80

70

to determine the sequences at the end of the Lgene.Sequence analysis of DI 011 RNA. DI 011

RNA was specifically labeled at its 3' end byusing 32P-labeled pCp and RNA ligase, and the5' terminus was labeled by using [y-32P]ATP andpolynucleotide kinase. Since the two strands ofDI 011 RNA are complementary, the purines onone strand identify the pyrimidines on the otherstrand. The positions of the purines were deter-mined on both strands by using RNases T, andU2 (Fig. 1). The sequence between positions 50and 130 from the 5' end of DI 011 RNA issummarized in Fig. 2. As noted previously, thesequence of the minus strand is identical to thesequences of the 5' ends of VSV and DI particlegenomic RNAs.Electron microscopy of 31S message-ge-

nomic RNA duplexes. To obtain an estimateof the distance between the terminus of the L

5'

U2 Q T

AGG 130G G'

- A G AGA

AA

.i..*w Am GGAA

AG

aw- G

AA

4_

AAA

A 60A

AG

A

AGG

A

* 120

110

100

90

80

A 70

G 60

A 50

FIG. 1. Determination ofpurines on both strands ofDI 011 RNA. 3' and 5' terminally labeled DI 011 RNAswere digested after heating for I min at 100'C and quick cooling with RNases T, and U2; they were thenseparated on a 20%, polyacrylamide gel next to a partial alkaline hydrolysate as described in the text. Thelengths of the RNA fragments are indicated (without the terminal Cp introduced by the ligase reaction). Thepositions of the purines in the 3' terminally labeled RNA are removed by I nucleotide because the RNasescleave on the 3' sides of the purines.

J. VIROL.

A

VSV POLYADENYLATION SITE 553

gene and the 5' end of VSV RNA, we annealedL mRNA from an in vitro transcription reactionto VSV RNA. Figure 3 shows a partial duplexformed between the 42S VSV genome and the31S poly(A) message. The double-stranded re-gion includes approximately 60% of the VSVgenome. This, together with previous electronmicrographic studies, indicates the location ofthe intercistronic boundary between the 17S (G)and 31S (L) messages at about 1.7 ,um, or 40% ofthe way from the 3' end of the VSV genome(12). Short single-stranded regions are observedoccasionally at the end of the duplexes formedbetween the genomic RNA and the 31S message.

We suspect that these single-stranded tails arethe poly(A) of the L message since this portionof the duplex structures anneals to polybromo-deoxyuridine-tailed simian virus 40 DNA (datanot shown). The fact that only one and not twotails could be detected suggests that transcrip-tion of L mRNA terminates within approxi-mately 100 nucleotides from the 5' end of VSVRNA, the limit of resolution by electron micros-copy. The sequencing data presented above spanthis region and therefore should contain thesequences specifying the 5' end of the L gene.

Isolation of the 5' terminus of the L gene.The 5'-terminal sequence of the VSV genome

50 60 70 80(-)5'...AGGAUCAAAGUUUUUUUCAUACUUAAAGUUUGGAGUCUCC(+)3'...UCCUAGUUUCAAAAAAAGUAUGAAUUUCAAACCUCAGAGG

90 100 110 120 130UCAUGAUUUUUUAAUCUCUCCAAGAGUUUUCCUCGUGUAGG...3'AGUACUAAAAAAUUAGAGAGGUUCUCAAAAGGAGCACAUCC...5'

FIG. 2. Nucleotide sequence of DI 011 RNA from positions 50 to 130. The positions of the purines weredetermined by using 3' and 5' terminally labeled DI 011 RNA (Fig. 1). The positions of the pyrimidines werededuced from the partial purine sequences of the complementary strands.

;*¢@'...'''..';, , ''

*~~~~~~~~~.~~~

IL ~ ~ 4 .

N~~~~~~~~~~9i'4. . . .L '**. ' ~ . '

~\.** *.. **'3%.* *~ Ta'I0IV.3.1Al~~~~~4

? .. * ~~~~ b.e \ '~~~~b*4 ~~* . k.4vb .~

*9 a ,..' ,~~~~ ~e #4. f

~~~~~~~~~~~ ~ ~ ~ ~~~~~ 1a.~~~~~~~~~~~~~~~~~~~-0

FIG. 3. Electron micrograph of a 31S message-genomic RNA duplex. 31S message and 42S genomic RNAwere annealed and prepared for electron microscopy as described in the text. Bar = 0.2 ,tm.

VOL. 34, 1980

554 SCHUBERT ET AL.

contains stretches of seven and six uridine resi-dues at positions 60 to 66 and 96 to 101. Wepostulated that one of these uridine stretchesmight be positioned exactly at the end of the Lgene and might code for the first few adeninesof the poly(A) tail of L mRNA. The sequencealso shows that these uridine stretches are partof 18- and 19-base oligonucleotides that could beformed by complete digestion of VSV RNA withRNase T1. If this assumption' is correct, one ofthese oligonucleotides which are of minus senseshould be able to enter duplexes with L mRNA,and the resultant duplex should contain the firstsix or seven adenine residues of the poly(A) tailof the mRNA. These duplexes retain the poly(A)tail even after digestion with a mixture of pan-creatic and T1 RNases, since no pyrimidine orguanosine residues intervene between the du-plex and the poly(A) tail. Furthermore, this spe-cific duplex should be recoverable from the di-gest by adsorption to oligo(dT)-cellulose. Sincethe two stretches of uridines abut either a gua-

DI-T RNA (-)

Complete RNase T, digestion

(X)nGp

Polynucleotide kinase

y_3'2P-ATP

Phenol extraction

*p(X) GP

J. VIROL.

nine or a guanine-adenine at their 5' sides, wewere able to design an experiment which allowedus to label and isolate whichever oligo-nucleotide formed a stable duplex with the se-quences at the L mRNA-poly(A) junction. Toavoid other termination sites in the VSV genomeand also to increase the specific activity of la-beling, we chose a small single-stranded DI par-ticle RNA (DI-T RNA), which is approximately2 kilobases long, is homologous to VSV RNA forat least 1 kilobase from the 5' terminus (23), andlacks sequences from the 3' one-half of the VSVgenome (27). The isolation procedures are de-scribed in detail above. The principle of themethod is summarized in Fig. 4.DI-T RNA was completely digested by using

RNase T1 to generate the oligonucleotides men-tioned above. In some experiments the oligonu-cleotides were preselected by gel filtration overSephadex G-25 to limit the number of oligonu-cleotides to those larger than 10 nucleotides andto increase the labeling of oligonucleotides big

Poly A selectedL messenger RNA

Annealing

IAAAAAAAAA (A) n

G * G * G - G

I Complete RNases Ti and A digestion

Proteinase K digestion

G - G - G -

IOligo dT cellulose

IFlow through

AAAAAAAAA(A) nG - r

Eluate

- AAAAAAAAAA(A)nG -G -*

FIG. 4. Procedure for isolation of the 5' terminus of the L gene. For details, see text.

VOL. 34, 1980

enough to form stable duplexes. The 5' ends ofthe oligonucleotides were labeled by using [y-32P]ATP and polynucleotide kinase, and the en-

zymes were removed by phenol extraction. Thelabeled oligonucleotides were annealed in thepresence of 0.4 M NaCl to poly(A)-selected 31Sin vivo L mRNA. After digestion with an excess

of RNase T, and RNase A and inactivation ofthe RNases with proteinase K, the reaction mix-ture was passed three times over a column ofoligo(dT)-cellulose. The column was washed ex-

tensively, and bound oligonucleotides were

eluted by decreasing the salt concentration. Theadsorbed and the unadsorbed materials were

precipitated with ethanol, dissolved, denaturedin 8 M urea, and directly applied onto a 20%polyacrylamide gel (Fig. 5). Although numerousRNA bands were found in the flow-through frac-tion, only a single band, migrating as an approx-

imately 18- or 19-nucleotide oligonucleotide, wasdetected in the eluate. Since the complete flow-through and eluate fractions were applied ontothe gel, it can be seen that the oligonucleotide inthe eluate was quantitatively removed from theflow-through fraction. Thus, the duplex formedwith this oligonucleotide must be directly linkedto a poly(A) tail without any intervening single-stranded regions containing pyrimidines or gua-nine.Characterization of the oligonucleotide.

The oligonucleotide was eluted from the gel, anda portion of it was completely digested withnuclease P1. The terminally labeled nucleotidewas identified by thin-layer chromatographyand electrophoresis as 5'-UMP (data not shown).The nucleotide sequence of this oligonucleotidewas determined by using formamide (25) forrandom cleavage and RNases T1, U2, and Phy 1for partial enzymatic digestions. The fragmentswere separated on a 20% polyacrylamide gel(Fig. 6). Partial hydrolysis with formamide re-

vealed that the oligonucleotide was 18 nucleo-tides long; the terminal mononucleotide pUpmigrated very rapidly and ran off the gel underthe conditions used in the experiment shown inFig. 6. As expected from the isolation scheme,the oligonucleotide was RNase T, resistant evenat the high RNase concentration used in thisexperiment. It contains a guanine residue inposition 18 of the 3' terminus of the fragment, asindicated by a large space between bands 17 and18 of the formamide ladder. The pyrimidineresidues, which were not as easy to read becauseof the more complex specificity of RNase Phy 1,were confirmed by using a two-dimensional pol-yacrylamide gel system (Fig. 7), as well as a

wandering spot analysis, using cellulose acetatestrips at pH 3.5 in the first dimension and ho-mochromatography on DEAE-cellulose thin-layer plates in the second dimension (data not

VSV POLYADENYLATION SITE 555

FT E

- Origin

- xc

U.

BPBU.

FIG. 5. Isolation of the 5' terminus of the L gene.5' terminally labeled RNase T,-resistant oligonucle-otides of DI-T RNA were annealed to 31S poly(A) invivo L mRNA. After complete digestion of the single-stranded RNAs with RNases T, and A, the mixturewas applied to oligo(dT)-cellulose. The flow-throughfraction (lane FT), as well as the material eluted ata low salt concentration (lane E), was precipitatedand subjected to electrophoresis on a 20% polyacryl-amide gel. Dyes: XC, xylene cyanol FF; BPB, bro-mophenol blue.

shown). The determined sequence was5' *pUUUUUUUCAUACUUAAAGp 3'.This sequence corresponds exactly to that in

positions 60 to 77 from the 5' terminus of VSV

556 SCHUBERT ET AL.

U U, FA Phyl PhylT2 4'

I-

J. VIROL.

DISCUSSIONIn this report we describe the positions of

adenine and guanine residues on the comple-G 18 mentary 5'- and 3'-terminal regions of double-

stranded DI 011 RNA. From the purines on oneA strand, the pyrimidines on the other were de-A < - duced. Since this DI RNA is single stranded inA1 the viral nucleocapsid, the complementary re-

U gions are not simultaneously replicated (14).U Thus, both regions can also mutate independ-C ently, resulting in mismatches between the twoA strands of the hairpin RNA. Ifmismatches occur

in DI 011 RNA, those containing a purine on theU 1o 3' strand would introduce an error in the derived

5'-terminal sequence reported here and viceversa. We do not believe that the sequence con-tains this type of error for the following reasons.Under appropriately severe conditions, single-

U strand-specific RNases can recognize single basemismatches other than possibly guanine-uridinepairs and cleave the duplex at those points (J.Perrault, personal communication). The fact

UD

U

u

BPB0U ?

FIG. 6. Sequence analysis of the oligonucleotide.The RNA band shown in Fig. 5, lane E, was elutedfrom the gel as previously described (24), partiallydigested with RNases U2 and Phy 1, and separatedon a 20%o polyacrylamide gel next to a partial hy-drolysate of the same RNA generated in 99o form-amide for 60 min at 100°C. The oligonucleotide wasalso incubated for 15 min at 50°C in the presence of60 x 10-3 U ofRNase T, per ,ug of tRNA.

RNA (Fig. 2) and is different from the oligonu-cleotide in positions 95 to 113.

In Fig. 8 we summarize the data and indicatethe cleavage sites of RNase T1 on minus senseDI-T RNA, as well as the RNase A cleavage siteon the L mRNA. We conclude from this seriesof experiments that the polymerase gene ends inposition 60 from the 5' terminus of VSV RNAand that the first seven adenine residues in thepoly(A) tail of L mRNA may be coded by thegenome.

3

Iu

Cp~~~4

I U

TuA U

-1A 10

C;"rSA.u

FIG. 7. Two-dimensional polyacrylamide gel elec-trophoresis of the oligonucleotide. The 18-nucleotideRNA band shown in Fig. 5 was partially hydrolyzedby using formamide and was separated in a two-dimensional polyacrylamide gel as described previ-ously (24). BPB, Bromophenol blue dye marker.

VSV POLYADENYLATION SITE 557

i 140 50 60 70 80 90

5'...GAGGGUCUUAAGGAUCAAAGUUUUUUUCAUACUUAAAGUUUGGAGUCUCC U....3 (-) VSVAA A A A A A A G U A U G A A U U U C A A A C C U C A G A G G A ...5 (+) L MESSAGE

AA

AA

A3 (A),

FIG. 8. L gene ends in position 60 from the 5' terminus of VSV RNA. The VSV sequence, as well as the LmRNA sequence, was deduced from the sequencing data on DI 011 RNA, as presented in the text andpreviously (23). The arrows mark the cleavage sites ofRNase T, on VSVRNA and ofRNaseA on the message.The sequences between the arrows correspond to the poly(A)-containing duplex that bound to oligo(dT)-cellulose. This duplex was labeled with 32P at the uridine residue in position 60.

that DI 011 RNA retains its overall size, asmeasured by gel electrophoresis, after digestionwith single-strand-specific RNases (23) suggeststhat if mismatches other than guanine-uridinepairs are present, they must be near the junctureof the plus and minus strands. Furthermore, wehave not found a single instance of a purine onone strand positioned opposite a purine on theother.By annealing 3' terminally labeled and nicked

DI 011 RNA, we demonstrated previously thatthe 5'-terminal sequence of DI 011 RNA is iden-tical to the 5'-terminal sequences of VSV RNAand other DI RNAs for 1 kilobase (23). Theelectron microscopic study showed that the Lgene ends within approximately 100 nucleotidesfrom the 5' terminus of VSV RNA, a regioncovered by the RNA sequencing. Based on thesequence, we made the assumption that thepoly(A) in the L message might be partly en-coded in the genome by a stretch of either six orseven uridine residues. We designed a methodwhich, if this were the case, would allow us toselect the specific oligonucleotide representingthe terminal nucleotides of the L gene out of amixture of RNase Ti-resistant oligonucleotides.This was done by generating 5' terminally la-beled RNase Ti-resistant oligonucleotides froma short single-stranded DI particle RNA whichhas the 5' end of VSV RNA conserved in it (23).The labeled oligonucleotides were annealed to Lmessage, and duplexes resistant to RNases T1and A were selected over oligo(dT)-cellulose.Only a single oligonucleotide was detected in theeluate of the column. This oligonucleotide wasresistant to RNases T1 and A when annealed toL message and also bound to oligo(dT)-cellulose,indicating that it contained a poly(A) tail de-rived from the L message which was linked to aterminal adenine residue in the duplex. Anyother nucleotides between the poly(A) tail andthe duplex would have led to a separation of thepoly(A) tail during subsequent digestions withRNases T, and A. The comparison of the se-quences of the oligonucleotide and the 5' termi-nus of VSV RNA revealed that the oligonucle-

otide represents the Ti oligonucleotide frompositions 60 to 77. The data demonstrate thatthe L gene ends in position 60 from the 5' ter-minus of VSV RNA with a stretch of sevenuridine residues and also shows that the firstseven adenine residues in the poly(A) tail maybe encoded in the genome. The method used inthis study depends on the following assumptions:(i) the poly(A) is partly encoded in the genome,and (ii) the end of the gene is part of an oligo-nucleotide large enough to form a stable duplexwith the message. These stringent prerequisites,together with the introduction of RNase A afterduplex formation, resulted in a highly specificisolation procedure. It should be emphasizedthat because of its specificity, this method onlygives a positive signal with L mRNA's that ter-minate at one of the oligouridylic acid stretches.Other conjectural or potential termination sitesare not tested by this method. At present wecannot rule out the possibility that there areseveral sites at which the L mRNA can termi-nate.The fact that the L gene terminates with a

stretch of seven uridine residues indicates thatthe viral transcriptase might be involved in thepolyadenylation of L message, possibly by chat-tering at the seven uridine residues. Electronmicroscopic studies (12) which showed VSVmRNA's linked by intervening poly(A)'s alsolead to the speculation that the poly(A) mightbe added by the viral transcriptase. Anotherpossibility is that the seven encoded adenineresidues at the beginning of the poly(A) tailserve as a primer for an as-yet-unidentified en-zyme which completes the polyadenylation.The last 70 nucleotides adjacent to the

poly(A) tail on the L and N messages (15) donot show any obvious similarities, except for 4nucleotides next to the poly(A) tail. Both mes-sages share the following sequence: ... UAUG-poly(A). The complement of this sequence, to-gether with the seven uridines, might be part ofa common polyadenylation signal (D. T. Mc-Geoch, personal communication).The 5'-terminal sequences of the eight RNA

VOL. 34, 1980

A

558 SCHUBERT ET AL.

segments of influenza virus (21, 26) bear similar-ities to VSV RNA in that they are all rich inadenine. Each of the influenza virus RNA seg-ments also carries stretches of five to sevenuridine residues starting in position 17 from the5' terminus. It is not clear at present whetherthese uridine-rich sequences are signals for ter-mination and polyadenylation of the influenzamRNA's. However, it should be noted that theresults of Skehel and Hay (26) suggest that thetranscriptions of the mRNA's terminate nearthe stretches of uridine.

Analysis of the 5'-terminal sequence of VSVRNA revealed a polarity in the distribution ofadenine and uridine residues. The first 60 nu-cleotides are rich (53%) in adenine residues, andthe next 60 positions contain 48% uridine resi-dues. This distribution suggests that the 5' ter-minus might be able to form an extensive sec-ondary structure. Figure 9 shows a potentialsecondary structure of the 5' terminus involvingpositions 1 to 125. The 3' portions of intracellular42S plus strand RNA and DI 011 RNA also canbe drawn in such a configuration. We have in-dicated the end of the L gene in position 60 andthe sequence GGUCUU in positions 43 to 48,which represents a polymerase recognition siteinvolved in the generation of DI particle RNAs(23). The secondary structure was designed byfirst pairing adjacent complementary regions,considering small hairpin formations duringtheir syntheses, and then folding the RNA backon itself. In this configuration 78 of 125 bases arepaired (62%), including 12 of 18 guanine residues.The extensiveness of this self-complementarityclearly exceeds that of tRNA's, in which approx-imately 55% of the bases are paired. At present,we do not know whether this hypothetical struc-

ture is stable in naked RNA. It seems likely thatseveral of the short stems that involve princi-pally adenine-uridine pairs may not be stable (C.R. Cantor, personal communication). It shouldalso be noted that the 5' terminus of VSV RNApresumably is covered by the viral nucleocapsidN protein and might not be able to enter intointramolecular secondary structures. The extentof the self-complementarity is very surprisingand might be important early during replicationof the minus strand, adding protection to thenascent naked RNA before encapsidation withN protein. Further studies will be necessary toestablish its significance.

ACKNOWLEDGMENTSWe thank L. A. Ball for providing in vitro synthesized L

message and C. R. Cantor for his helpful comments on thepotential secondary structure at the 5' termini of VSV and DIRNAs.

LITERATURE CITED

1. Abraham, G., and A. K. Banerjee. 1976. Sequentialtranscription of the genes of vesicular stomatitis virus.Proc. Natl. Acad. Sci. U.S.A. 73:1504-1508.

2. Ball, L. A. 1977. Transcriptional mapping of vesicularstomatitis virus in vivo. J. Virol. 21:411-414.

3. Ball, L. A., and C. N. White. 1976. Order of transcriptionof genes of vesicular stomatitis virus. Proc. Natl. Acad.Sci. U.S.A. 73:442-446.

4. Ball, L. A., and C. N. White. 1978. Coupled transcriptionand translation in mammalian and avian cell-free sys-tems. Virology 84:479-495.

5. Banerjee, A. K., G. Abraham, and R. J. Colonno.1977. Vesicular stomatitis virus: mode of transcription.J. Gen. Virol. 34:1-8.

6. Banerjee, A. K., and D. P. Rhodes. 1973. In vitrosynthesis ofRNA that contains polyadenylate by virionassociated RNA polymerase of vesicular stomatitis vi-rus. Proc. Natl. Acad. Sci. U.S.A. 70:3566-3570.

7. Cartwright, B., and F. Brown. 1972. Serological rela-tionships between different strains of vesicular stoma-titis virus. J. Gen. Virol. 16:391-398.

8. Derman, E., and J. E. Darnell. 1974. Relationship of

40,G-A

G

A-C; .G\C\. )u'C 50

c \~~~~~~A\'U\ A,A-GG

C A 10 A; U

A C ~~C A\ 20 \A 60 u-u.

p pp A-C-G-A-A-G A-A-A-A-C A-G-A-U-A-A-A-A-A-A-U A-A-G-U-U-U U

-G-G-A-U-GU-G-UC-U-C-C-U-U-U-UG U-C- U-A U-U-U-U-U-U-A U-U-C-A-U-AU U

130 120 A C A 00 G 1A 70

/~~~~~~~~~~\

G U 80.'.\CU U A

\A C ~A 90 G A

110 CG\ AU \

FIG. 9. Potential secondary structure of the 5' termini of VSV and DI RNAs. The arrows mark the 5'terminus of the L gene and the sequence GGUCUU, which is involved in the generation of DI particles (23).This potential structure shows 78 of 125 bases paired, including 12 of 18 guanine residues.

J. VIROL.

VSV POLYADENYLATION SITE 559

chain transcription to poly(A) addition and processingof mRNA in HeLa cells. Cell 3:255-264.

9. Donis-Keller, H., A. M. Maxam, and W. Gilbert. 1977.Mapping adenines, guanines and pyrimidines in RNA.Nucleic Acids Res. 4:2527-2538.

10. England, T. E., and 0. C. Uhlenbeck. 1978. 3'-Terminallabelling ofRNA with T4 RNA ligase. Nature (London)275:560-561.

11. Franze-Fernandez, M. T., and A. K. Banerjee. 1978.In vitro RNA transcription by the New Jersey serotypeof vesicular stomatitis virus. I. Characterization of themRNA species. J. Virol. 26:179-187.

12. Herman, R. C., S. Adler, R. A. Lazzarini, R. J. Co-lonno, A. K. Banerjee, and H. Westphal. 1978. In-tervening polyadenylate sequences in RNA transcriptsof vesicular stomatitis virus. Cell 15:587-596.

13. Keene, J. D., M. Schubert, R. A. Lazzarini, and M.Rosenberg. 1978. Nucleotide sequence homology atthe 3' termini of RNA from vesicular stomatitis virusand its defective interfering particles. Proc. Natl. Acad.Sci. U.S.A. 75:3225-3229.

14. Lazzarini, R. A., G. H. Weber, L. D. Johnson, and G.M. Stamminger. 1975. Covalently linked message andanti-message (genomic) RNA from a defective vesicularstomatitis virus particle. J. Mol. Biol. 97:289-307.

15. McGeoch, D. J., and N. T. Turnbull. 1978. Analysis ofthe 3' terminal nucleotide sequence of vesicular stoma-titis virus N protein mRNA. Nucleic Acids Res. 5:4007-4024.

16. Meyer, J., P. D. Neuwald, S.-P. Lai, J. V. Maizel, Jr.,and H. Westphal. 1977. Electron microscopy of lateadenovirus type 2 mRNA hybridized to double-strandedviral DNA. J. Virol. 21:1010-1018.

17. Murphy, M. F., and R. A. Lazzarini. 1974. Synthesis ofviral mRNA and polyadenylate by a ribonucleoproteincomplex from extracts of VSV-infected cells. Cell 3:77-84.

18. Reichmann, M. E., W. M. Schnitzlein, D. H. L. Bishop,R. A. Lazzarini, S. T. Beatrice, and R. R. Wagner.

1978. Classification of the New Jersey serotype of vesic-ular stomatitis virus into two subtypes. J. Virol. 25:446-449.

19. Repik, P., A. Flammond, H. F. Clark, J. F. Obijeski,P. Roy, and D. H. L. Bishop. 1974. Detection ofhomologous RNA sequences among six rhabdovirusgenomes. J. Virol. 13:250-252.

20. Rhodes, D. P., and A. K. Banerjee. 1976. 5'-Terminalsequence of vesicular stomatitis virus mRNA's synthe-sized in vitro. J. Virol. 17:33-42.

21. Robertson, J. S. 1979. Nucleotide sequences from theterminal regions of fowl plague virus genome RNA.Nucleic Acids Res. 6:3745-3757.

22. Rose, J. K. 1978. Complete sequences of the ribosomerecognition sites in vesicular stomatitis virus mRNAs:recognition of the 40S and 80S complexes. Cell 14:345-353.

23. Schubert, M., J. D. Keene, and R. A. Lazzarini. 1979.A specific internal RNA polymerase recognition site ofVSV RNA is involved in the generation of DI particles.Cell 18:749-757.

24. Schubert, M., J. D. Keene, R. A. Lazzarini, and S. U.Emerson. 1978. The complete sequence of a uniqueRNA species synthesized by a DI particle of VSV. Cell15:103-112.

25. Simonesits, A., G. G. Brownlee, R. S. Brown, J. R.Rubin, and H. Guilley. 1977. New rapid gel sequencingmethod for RNA. Nature (London) 269:833-836.

26. Skehel, J. J., and A. J. Hay. 1978. Nucleotide sequencesat the 5' termini of influenza virus RNAs and theirtranscripts. Nucleic Acids Res. 5:1207-1219.

27. Stamminger, G., and R. A. Lazzarini. 1974. Analysis ofthe RNA of defective VSV particles. Cell 3:85-93.

28. Villarreal, L. P., and J. J. Holland. 1973. Synthesis ofpoly(A) in vitro by purified virions of vesicular stoma-titis virus. Nature (London) New Biol. 246:17-19.

29. Westphal, H., and S.-P. Lai. 1977. Quantitative electronmicroscopy of early adenovirus RNA. J. Mol. Biol. 116:525-548.

VOL. 34, 1980