5
Proc. Nati. Acad. Sci. USA Vol. 83, pp. 5043-5047, July 1986 Biochemistry In vitro transcription of infectious RNAs from full-length cDNAs of tobacco mosaic virus (viral expression) TETSUO MESHI*, MASAYUKI ISHIKAWA*, FUSAO MOTOYOSHIt, KENTARO SEMBA*f, AND YOSHIMI OKADA* *Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Tokyo 113, Japan; and tNational Institute of Agrobiological Resources, Tsukuba Science City, Ibaraki 305, Japan Communicated by Heinz Fraenkel-Conrat, March 24, 1986 ABSTRACT We have cloned full-length double-stranded cDNAs of tobacco mosaic virus (TMV) (tomato strain L) RNA into a transcription vector, pPM1, which facilitates the correct transcription initiation from the first nucleotide of the inserted double-stranded cDNA, corresponding to the 5' end of TMV RNA. When plasmid DNA is linearized at a unique restriction site (Mlu I) introduced just downstream of the double-stranded cDNA insert and used as a template for in vitro transcription by Escherichia coli RNA polymerase in the presence of m7GpppG, the transcribed RNAs are infectious for tobacco plants. A simple reconstitution procedure increases the infectivity >100 times. Unexpectedly, both the uncapped transcript and the transcript from the uncut plasmid DNA are also infectious, although their infectivities are very low. The progeny viruses multiplying in tobacco plants accurately reflect the cloned sequence. By the same method, we succeeded in the in vitro transcription of infectious RNA of attenuated strain L11A, which is phenotypically distinguishable from wild-type TMV on both tobacco and tomato plants. Tobacco mosaic virus (TMV) is one of the well-characterized plant viruses (1). The genome of TMV is a messenger-sense single-stranded RNA of -6400 nucleotides with a cap struc- ture at the 5' end (1-3). The genomic RNA carries informa- tion of four proteins-130-kDa, 180-kDa (the read-through product of the former), 30-kDa proteins, and the coat protein. The functions of proteins other than the coat protein are not understood at the molecular level, although the involvement of the 130-kDa and/or 180-kDa protein in viral replication and of the 30-kDa protein in viral cell-to-cell movement have been suggested (4-6). A genetic approach is useful for resolving not only the functions of TMV-coding proteins but also the signals on the genomic RNA for the syntheses of plus and minus strands and subgenomic mRNAs. However, proper tools for manipulating the viral RNA-that is, for producing appropriate mutants efficiently and for localizing the muta- tions on the genome-have been lacking. Ahlquist and Janda recently constructed a transcription vector, pPM1, that facilitates the controlled in vitro synthesis of infectious viral RNA molecules from cloned cDNAs (7, 8). pPM1 carries a unique Sma I recognition sequence at the transcription initiation site from the modified Pr promoter of X DNA, designated as the Pm promoter. When double- stranded (ds) cDNA of a given viral RNA is inserted into the Sma I site with correct orientation, transcription starts at the first nucleotide of the inserted ds cDNA. By adding m7G- pppG to the reaction mixture, capped transcripts can be obtained efficiently (9). When a recombinant plasmid linear- ized just downstream of the ds cDNA insert is used as a template, the capped run-off transcript from the Pm promoter shows infectivity, as shown by Ahlquist et al. using brome mosaic virus (BMV), a multicomponent plant virus (8). Such an in vitro transcription system could be very useful for manipulating the TMV RNA in order to understand TMV- coding genetic information. TMV L (wild-type tomato strain) and its attenuated deriv- ative ,11A are well-characterized strains. The complete genomic sequences of both strains have been determined (3, 10) and 10 nucleotide substitutions have been found between the two, 3 of which cause the amino acid substitution (all in the 130/180-kDa protein). TMV L11A is able to multiply in tomato plants without any remarkable symptoms (11) and is used in Japan to protect them from later infection with virulent strains (12). In this study, we cloned full-length cDNAs of TMV L and ,11A RNAs into pPM1 and synthe- sized infectious TMV RNAs from the recombinant plasmids in vitro. MATERIALS AND METHODS Plasmids. The TMV cDNA clones pLT-D27, pL-1-13, and pL11A-A25 carry cDNA copies of residues 1-6215 (3), about 1.6 kilobase pairs (kbp) (13) of the 3' end of TMV L RNA, and the complete sequence of TMV L11A RNA (10), respectively (Fig. 1). pCG9F2 has a cDNA insert of "1.6 kbp derived from the sequence near the 3' end of cucumber green mottle mosaic virus RNA (14) in which the Mlu I recognition sequence exists. These clones have G-C and/or AT tails at least at one end of the cDNA insert. Transcription vector pPM1 (8), generously supplied by P. Ahlquist, was construct- ed by inserting a 0.9-kbp DNA fragment containing the Pm promoter and its repressor gene derived from X DNA be- tween the Pst I and Sma I sites of pUC9 (15). Synthesis of ds cDNA. TMV L genomic RNA was extracted from the purified virus as described (13). The genomic RNA was annealed with an excess amount of the synthetic oligo- nucleotide, pdTGGGCCCCTACCGGGGGT, which is com- plementary to the 3' end of the genomic RNA except for the T residue at the ninth position (13, 17). cDNA was synthe- sized in a 200-1.l reaction mixture containing 55 mM Tris'HCl (pH 8.3), 30 mM KCl, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM each dNTP, 50 pug of annealed RNA per ml, and 250 units of reverse transcriptase (Bio-Rad) per ml at 420C for 90 min. After the genomic RNA had been degraded under alkaline conditions, cDNA was fractionated by 5-20% alka- line sucrose density gradient centrifugation (16) or 2.5% polyacrylamide/8.3 M urea gel electrophoresis (18). The full-length cDNA thus obtained was annealed with pdGTAT- TTTTACAACAATTAC, which corresponds to residues 1-19 of the genomic RNA, in 10 mM Tris HCl, pH 7.5/10 mM MgCl2/50 mM NaCl, with heating at 90'C for 5 min, followed Abbreviations: TMV, tobacco mosaic virus; BMV, brome mosaic virus; ds cDNA, double-stranded cDNA; kbp, kilobase pair(s). =Present address: Institute of Medical Science, University of Tokyo, Tokyo 108, Japan. 5043 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Downloaded by guest on May 20, 2020

In transcription RNAs full-length cDNAsof tobacco · Full-Length cDNACloning. (i) Construction ofpUCG91. Tointroduce the MluI site into the polylinker sequence of pUC9(15), 0.21 kbpofthe

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Page 1: In transcription RNAs full-length cDNAsof tobacco · Full-Length cDNACloning. (i) Construction ofpUCG91. Tointroduce the MluI site into the polylinker sequence of pUC9(15), 0.21 kbpofthe

Proc. Nati. Acad. Sci. USAVol. 83, pp. 5043-5047, July 1986Biochemistry

In vitro transcription of infectious RNAs from full-length cDNAs oftobacco mosaic virus

(viral expression)

TETSUO MESHI*, MASAYUKI ISHIKAWA*, FUSAO MOTOYOSHIt, KENTARO SEMBA*f, AND YOSHIMI OKADA**Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Tokyo 113, Japan; and tNational Institute of AgrobiologicalResources, Tsukuba Science City, Ibaraki 305, Japan

Communicated by Heinz Fraenkel-Conrat, March 24, 1986

ABSTRACT We have cloned full-length double-strandedcDNAs of tobacco mosaic virus (TMV) (tomato strain L) RNAinto a transcription vector, pPM1, which facilitates the correcttranscription initiation from the first nucleotide of the inserteddouble-stranded cDNA, corresponding to the 5' end of TMVRNA. When plasmid DNA is linearized at a unique restrictionsite (Mlu I) introduced just downstream of the double-strandedcDNA insert and used as a template for in vitro transcription byEscherichia coli RNA polymerase in the presence of m7GpppG,the transcribed RNAs are infectious for tobacco plants. Asimple reconstitution procedure increases the infectivity >100times. Unexpectedly, both the uncapped transcript and thetranscript from the uncut plasmid DNA are also infectious,although their infectivities are very low. The progeny virusesmultiplying in tobacco plants accurately reflect the clonedsequence. By the same method, we succeeded in the in vitrotranscription of infectious RNA of attenuated strain L11A,which is phenotypically distinguishable from wild-type TMVon both tobacco and tomato plants.

Tobacco mosaic virus (TMV) is one of the well-characterizedplant viruses (1). The genome ofTMV is a messenger-sensesingle-stranded RNA of -6400 nucleotides with a cap struc-ture at the 5' end (1-3). The genomic RNA carries informa-tion of four proteins-130-kDa, 180-kDa (the read-throughproduct ofthe former), 30-kDa proteins, and the coat protein.The functions of proteins other than the coat protein are notunderstood at the molecular level, although the involvementofthe 130-kDa and/or 180-kDa protein in viral replication andofthe 30-kDa protein in viral cell-to-cell movement have beensuggested (4-6). A genetic approach is useful for resolvingnot only the functions of TMV-coding proteins but also thesignals on the genomic RNA for the syntheses of plus andminus strands and subgenomic mRNAs. However, propertools for manipulating the viral RNA-that is, for producingappropriate mutants efficiently and for localizing the muta-tions on the genome-have been lacking.

Ahlquist and Janda recently constructed a transcriptionvector, pPM1, that facilitates the controlled in vitro synthesisof infectious viral RNA molecules from cloned cDNAs (7, 8).pPM1 carries a unique Sma I recognition sequence at thetranscription initiation site from the modified Pr promoter ofX DNA, designated as the Pm promoter. When double-stranded (ds) cDNA of a given viral RNA is inserted into theSma I site with correct orientation, transcription starts at thefirst nucleotide of the inserted ds cDNA. By adding m7G-pppG to the reaction mixture, capped transcripts can beobtained efficiently (9). When a recombinant plasmid linear-ized just downstream of the ds cDNA insert is used as atemplate, the capped run-offtranscript from the Pm promotershows infectivity, as shown by Ahlquist et al. using brome

mosaic virus (BMV), a multicomponent plant virus (8). Suchan in vitro transcription system could be very useful formanipulating the TMV RNA in order to understand TMV-coding genetic information.TMV L (wild-type tomato strain) and its attenuated deriv-

ative ,11A are well-characterized strains. The completegenomic sequences of both strains have been determined (3,10) and 10 nucleotide substitutions have been found betweenthe two, 3 of which cause the amino acid substitution (all inthe 130/180-kDa protein). TMV L11A is able to multiply intomato plants without any remarkable symptoms (11) and isused in Japan to protect them from later infection withvirulent strains (12). In this study, we cloned full-lengthcDNAs of TMV L and ,11A RNAs into pPM1 and synthe-sized infectious TMV RNAs from the recombinant plasmidsin vitro.

MATERIALS AND METHODSPlasmids. The TMV cDNA clones pLT-D27, pL-1-13, and

pL11A-A25 carry cDNA copies of residues 1-6215 (3), about1.6 kilobase pairs (kbp) (13) ofthe 3' end ofTMV L RNA, andthe complete sequence ofTMV L11A RNA (10), respectively(Fig. 1). pCG9F2 has a cDNA insert of "1.6 kbp derived fromthe sequence near the 3' end of cucumber green mottlemosaic virus RNA (14) in which the Mlu I recognitionsequence exists. These clones have G-C and/or AT tails atleast at one end of the cDNA insert. Transcription vectorpPM1 (8), generously supplied by P. Ahlquist, was construct-ed by inserting a 0.9-kbp DNA fragment containing the Pmpromoter and its repressor gene derived from X DNA be-tween the Pst I and Sma I sites of pUC9 (15).

Synthesis of ds cDNA. TMV L genomic RNA was extractedfrom the purified virus as described (13). The genomic RNAwas annealed with an excess amount of the synthetic oligo-nucleotide, pdTGGGCCCCTACCGGGGGT, which is com-plementary to the 3' end of the genomic RNA except for theT residue at the ninth position (13, 17). cDNA was synthe-sized in a 200-1.l reaction mixture containing 55 mM Tris'HCl(pH 8.3), 30 mM KCl, 10 mM MgCl2, 10 mM dithiothreitol,1 mM each dNTP, 50 pug of annealed RNA per ml, and 250units of reverse transcriptase (Bio-Rad) per ml at 420C for 90min. After the genomic RNA had been degraded underalkaline conditions, cDNA was fractionated by 5-20% alka-line sucrose density gradient centrifugation (16) or 2.5%polyacrylamide/8.3 M urea gel electrophoresis (18). Thefull-length cDNA thus obtained was annealed with pdGTAT-TTTTACAACAATTAC, which corresponds to residues1-19 of the genomic RNA, in 10mM Tris HCl, pH 7.5/10mMMgCl2/50 mM NaCl, with heating at 90'C for 5 min, followed

Abbreviations: TMV, tobacco mosaic virus; BMV, brome mosaicvirus; ds cDNA, double-stranded cDNA; kbp, kilobase pair(s).=Present address: Institute ofMedical Science, University ofTokyo,Tokyo 108, Japan.

5043

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

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Proc. Natl. Acad. Sci. USA 83 (1986)

130 kDa

E P A BII

I E

30 kDa0180 kDa CP

B N

3 4 5 6(

P

pLM 51

p N

N

N

__l~

*Z)u'Zuln,s's.''C

-

E

-EI

E A

-.9e ds-cDNA(kbp)

}- pLT-D27AI- pL..A-A25AM- pL-1-13

pLM31

z- pLFW1

pLFW3

pLFW4A MZf- pLFAl

FIG. 1. Schematic representation of the genomic organization ofTMV and ds cDNA cloned in each plasmid. The cistrons of the fourproteins coded are shown above the restriction map for ds cDNA ofTMV L (wild type). The derivation ofthe cDNA sequence ofeach pLFplasmid is shown by the same shading. Only the cleavage sites used toconstruct pLF plasmids are shown on the plasmid structures. Opencircles in pLM31, pLFW1, and pLFW4 indicate the artificially intro-duced point mutation near the 3' end. Open and closed triangles inpLFA1 show the locations of base substitutions found in the L11Agenomic sequence (10). Closed triangles are the ones causing amino acidchanges. Pm designates Pm promoter of pPM1. CP denotes the coatprotein. E, EcoRI; P, Pst I; A, Apa I; B, Bgl II; N, Nco I; M, Mlu I.

by slow cooling to 20'C. The second strand was synthesizedin 10 mM Tris HCI, pH 7.5/10 mM MgCl2/25 mM NaCl/5mM dithiothreitol/0.2 mM each dNTP/250 units of Esche-richia coli DNA polymerase I large fragment (Toyobo,Osaka, Japan) per ml at 21TC for 3 hr. The full-length dscDNA was purified from 0.8% low melting point agarose gel(19).

Full-Length cDNA Cloning. (i) Construction ofpUCG91.To introduce the Mlu I site into the polylinker sequence ofpUC9 (15), 0.21 kbp of the HindIII/filled-in Mlu I fragmentof pCG9F2 (14) was inserted into the HindIII/filled-in Sal Isites of pUC9 to create pUCG91 (Fig. 2A).

(it) Construction of pLM51. Full-length ds cDNA wasdigested with Bgl II, and the 2.62-kbp fragment derived fromthe 5' portion of the genomic RNA was purified and ligatedwith 3.03 kbp of the Aat II/Sma I fragment of pPM1containing the Pm promoter and 0.47 kbp of the AatII/BamHI fragment of pUC9. The ligation products weretransformed into E. coli MC1061 (20) with high transforma-tion efficiency. After the selection of pLM51 by colonyhybridization (21) and restriction mapping, it was trans-formed into E. coli HB101 with the RecA- background (22).The junction between the Pm promoter and ds cDNA wasconfirmed by sequencing (18).

(ifi) Construction of pLM3L. Full-length ds cDNA wascleaved by Pst I, and the 4.54-kbp fragment derived from the3' portion was isolated and ligated with the 2.18-kbp AatII/Pst I fragment of pUC9 and the 0.49-kbp Aat II/fled-inMlu I fragment of pUCG91. Transformation and selectionwere performed as described above.

(iv) Construction ofpLFWJ, pLFW3, pLFW4, andpLFAI(Fig. 1). pLFW1 was constructed by inserting the Pst Ifragment of pLM51 containing the Pm promoter and the 5'portion of ds cDNA into the unique Pst I site of pLM31.pLFW4 and pLFA1 were constructed by replacing theEcoRI/Nco I and EcoRI/Apa I fragments ofpLFW1 with thecorresponding fragments of pLT-D27 and pL61A-A25, re-spectively. pLFW3 had the same structure as pLFW4 exceptthat its Nco I/Apa I sequence was derived from pL-1-13.

In Vitro Transcription and Reconstitution. In vitro tran-scription using E. coli RNA polymerase was carried out bythe reported method (8). The transcribed RNA was purifiedby phenol extraction and ethanol precipitation. Reconstitu-tion was performed by using the coat protein of commonstrain OM (4 mg/ml) purified by the acetic acid method (23)in 0.1 M Na phosphate (pH 7.0) at 20'C for 16 hr. TemplateDNA did not inhibit the reconstitution reaction and so wasnot removed.

Infectivity Test and Analysis of Progeny Viruses. Nicotianatabacum L. cv. Xanthi nc and Samsun were used as the locallesion and systemic hosts, respectively. Inocula were pre-pared by diluting the reconstitution mixture five times withH20 or by suspending viruses or nucleic acids in 20 mM Naphosphate (pH 7.0). Fifty to 60 FLd of the inocula was used perhalf leaf. Viruses were propagated on the systemic tobaccovariety directly inoculated with the transcripts or with theirprogeny after a single lesion isolation and then purified (24).The RNA sequence at the 3' end was determined by thechemical method (25). Sequencing at the 5' end was carriedout by the dideoxy-chain termination method (17). Theprimer used was an end-labeled single-stranded DNA gener-ated from the cDNA clone by restriction digestion, comple-mentary to residues 144-178 of the genomic RNA. Thephenotype of TMV L11A as an attenuated strain was testedon Licopercicon esculentum Mill cv. Fukuju no. 2.

RESULTSCloning of Full-Length cDNA of TMV L into pPMl. The

rationale of in vitro expression of infectious viral RNAthrough run-off transcription is correct transcription initia-tion at the 5' terminus of the genomic RNA and terminationat or near the 3' end (8). It is possible to clone the 5' end ofTMV RNA at the proper distance from the Pribnow box byligating Sma I-cut pPM1 and the 5' end of the ds cDNA (7).On the other hand, to stop the transcription it is necessary tointroduce a unique restriction site just downstream of the dscDNA insert. We chose Mlu I as the enzyme for linearizationof the full-length cDNA plasmids from among enzymes thatdo not cut either the ds cDNA of TMV L or pPM1, and weintroduced the Mlu I recognition sequence into the polylinkersite of pUC9 (Fig. 2A). By ligating the filled-in Mlu I site andthe 3' end of ds cDNA, the Mlu I recognition sequence(ACGCGT) was recreated (Fig. 2B).cDNA synthesis was primed by using a synthetic oligonu-

cleotide complementary to the 3' end of the genomic RNA ofTMV L except for the T residue at the ninth position (Fig.2B). This substitution makes it possible to discriminate theprogeny virus derived from the in vitro-synthesized RNAfrom standard TMV L. This mismatching did not influencethe priming efficiency. The mutation of this position on theTMV L genomic sequence was conceived not to causedefectiveness, because the mutated position is not base-paired on the conserved tertiary structure (26) and thechanged sequence becomes the same as that of commonstrain TMV (13), closely related to the tomato strain.

Since the ligation efficiency is very low when both ends ofDNA are blunt, we cut full-length ds cDNA with a restrictionenzyme (Bgl II or Pst I) to produce cohesive ends and clonedthe 5' and 3' portions separately to create pLM51 and pLM31(Fig. 1). Next, the two plasmids were recombined and thefull-length cDNA clone pLFW1 was obtained (Fig. 1). Sincethe transcript from pLFW1 did not show infectivity (seebelow), the TMV sequence of pLFW1 was replaced by thecorresponding ones derived from the already sequencedcDNA clones, pLT-D27 (3) and pL-1-13 (13). pLFW3 andpLFW4 were constructed by changing the EcoRI/Apa I(residues 274-6382) and EcoRI/Nco I (residues 274-5462)fragments of pLFW1, respectively (Fig. 1). The artificially

---

I~~~~~~~~~ ~ ~ of

5044 Biochemistry: Meshi et al.

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Proc. Natl. Acad. Sci. USA 83 (1986) 5045

A

B

pCG9F2 -a '- pUC9----CCCTATTACGCG TCGACGGAT---- pUCG91

Miul Soil C

ds-cDNA ^- - . PtlU----CCGGAACCCCCGGTAGGGGCCCA CGCGTC5ACGGA

Apal Miul

-T--

C ----CCGGAACCCCCGGTAGGGGCCCA3----GGCCTTGGGGGCCATCCCCGGGTGCGC5.

+ 6384

----CCGGAACCCCCGGUAGGGGCCCACGC3'

U

ICG91AT---- pLFW1, pLFW4

------ pLFW3, pLFAl

Mlul-cut Template

Transcribed RNA

FIG. 2. (A) Sequence surrounding the introduced Mlu I site ofpUCG91. (B) Sequence surrounding thejunction between fiull-lengthds cDNA and pUCG91 ofpLF plasmids. The sequence complemen-tary to the synthetic oligonucleotide used for the priming of cDNAsynthesis is underlined. Arrow ( t ) indicates the introduced thymine-to-adenine transversion. (C) Mlu I-cut template DNA and tran-scribed RNA from the Mlu I-cut template. Bold arrow meanstranscription. Three additional bases derived from the Mlu I recog-nition sequence are designated by small capitals. Arrow (t) indi-cates the resultant uracil-to-adenine transversion.

introduced point mutation (adenine residue) near the 3' endhad reverted to the wild-type sequence (thymine residue) inpLFW3 (Fig. 2B). At the same time, we constructed pLFA1by replacing the sequence of EcoRI/Apa I by a pLl1A-A25-derived fragment, which included all of the substitutionsfound between the L RNA and that of its attenuated strain,L11A (Fig. 1). The biological activity of the transcribed RNAfrom pLFA1 should be phenotypically distinguishable.

Infectivity Test for in Vitr Transcripts. Mlu I-cut fulfl-lengthcDNA clones, pLF plasmids, were used as templates for invitro transcription under the conditions described by Ahl-quist et al. (8). The 5'-terminal sequence of the transcriptswas determined by the dideoxy method (17). Both capped

A

GCC

A

(i

Transcript Virion

A C A CG v v u G v v U

G U GU

and uncapped genome-sized transcripts had the same 5'-terminal sequence as the viral RNA (not shown), indicatingthat the transcription from Pm promoter started from theguanine residue corresponding to residue 1 of the genomicRNA. The efficiency of the incorporation of m7GpppG intothe transcripts was analyzed by DEAE-cellulose paper elec-trophoresis (27) of the complete RNase T2 digests of thegel-purified transcripts synthesized in the presence of m7G-pppG. The fraction of the transcripts carrying the cap structurewas >60%. The 3'-terminal sequence of the genome-sizedtranscripts was determined by the chemical method (25). Wefound that most of the genome-sized transcripts had threeadditional nucleotides (CGC) at the 3' end, derived from theMlu I recognition sequence (Figs. 2C and 3A), consistent withthe previous report (7). In most experiments, 100-130 ng (inthe presence of m7GpppG) and 250-340 ng (in the absence ofm7GpppG) of genome-sized transcripts were obtained from 1pg of linearized template DNA.The infectivity of transcripts was assayed on both local

lesion and systemic hosts. The typical results with the locallesion host are summarized in Table 1. The results wereconsistent with those of the systemic host. The plasmid DNAof either a linear or circular form was not detectably infec-tious (Table 1, nos. 1 and 2). Infectivity could only be seenafter transcription and it was RNase-sensitive and DNase-resistant (nos. 3-6). The infectivity could be increased >100times by a simple reconstitution procedure (nos. 3 and 4) asin the case of the authentic TMV RNA (nos. 13 and 14), sowe added the reconstitution step before the infectivity assayin most experiments.The transcript from pLFW1 was not infectious (Table 1,

no. 9). In vitro translation experiments with the transcriptfrom pLFW1 showed that pLFW1 had a mutation at least inthe 130-kDa coding region (data not shown), Therefore, wereplaced the cDNA sequence in pLFW1 by the correspond-ing ones derived from already sequenced cDNA clones andconstructed pLFW3 and pLFW4 (Fig. 1). The transcriptsfrom pLFW3 and pLFW4 were both infectious (Table 1, nos.4 and 10). pLFW4 has an artificially introduced point muta-tion near the 3' end (Fig. 28). It was revealed that this basechange (thymine to adenine) did not affect the infectivity.

B_pLFW4 L

' -'-- - 1A C A C

G v v U G V v uG u G u

GG

:.

.:' GcCC...C

o. C

G

UGv-- A

GG

G

c

Jeff...g~

G

UU.

ci

GG

G

G

ci

A3C

FIG. 3. (A) Autoradiogram showing the sequences of the 3' end of the genome-sized transcript from Mlu I-cut pLFW4 and of the RNAextracted from the pLFW4-derived progeny virus. RNA was labeled at the 3' end with [32P]pCp (14), gel-purified, and sequenced by the chemicalmethod (25). Three additional bases at the 3' end of the transcript are circled. (B) Comparison of the sequences around the artificially introducedpoint mutation. Genomic RNA was purified from systemic tobacco leaves inoculated with the transcript from pLFW4, or with the authenticTMV L, and then sequenced (25). The point mutation at the ninth position from the 3' end is indicated by an arrowhead.

Biochemistry: Meshi et aL

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Proc. Natl. Acad. Sci. USA 83 (1986)

Table 1. Infectivity assay of in vitro transcribed TMV RNA

Concentration,No. Inocula jig/ml Infectivity

X pLFW4 (circular plasmid DNA) 180 0/64 0/552 pLFW4/MIu I 180 0/72 0/463 pLFW4/Mlu I/Tr (cap') 180 23/46 80/864 pLFW4/MIu I/Tr (cap')/Rec 4 416/138 471/1885 pLFW4/Mlu I/Tr (cap')/DNase/Rec 4.6 154/48 280/786 pLFW4/Mlu I/Tr (cap')/RNase/Rec 13 0/47 0/1187 pLFW4/Mlu I/Tr (cap-)/Rec 13 3/161 0/768 pLFW4/Tr (cap')/Rec 4 8/60 14/1059 pLFW1/Mlu I/Tr (cap')/Rec 4 0/39 0/4010 pLFW3/MIu I/Tr (cap')/Rec 4 308/49 255/6711 pLFA1/MIu I/Tr (cap')/Rec 4 268/49 75/4312 pLFW4/MIu I/Rec 13 0/34 0/10813 TMV L RNA 10* 106/97 92/4314 TMV L RNA/Rec 0.027t 91/83 126/3315 MockW 0 0/79 0/115Concentration is expressed as that of template DNA in the inoculum or that of template DNA from

which the transcripts or reconstituted RNAs are derived. Infectivity is expressed as the ratio of thenumber of local lesions produced by each inoculum on one-half of a leaf of N. tabacum L. cv. Xanthinc to that produced by the standard TMV L virion (0.1 ,ug/ml) on the other half ofa leaf. /Mlu I denotesthe linearization of plasmid DNA with Mlu I. /Tr denotes transcription. (cap') and (cap-) denote thepresence and absence of m'GpppG in the transcription mixture, respectively. /Rec denotes in vitroreconstitution. DNase treatment was performed with 0.83 unit of DNase I (DPRF, Worthington) per,tg of template DNA (28). RNase treatment was carried out in a mixture of 10 mM Tris HCl, pH 7.5/1mM EDTA/1 Ag of RNase A (type 1-A, Sigma) per ml/0.12 ttg of template DNA and its derivedtranscripts per ml, at 37°C for 30 min.*Concentration of TMV L genomic RNA.tConcentration of the reconstituted genomic RNA.tBuffer inoculation.

Unexpectedly, the uncapped transcript and the transcriptfrom uncut plasmid DNA were infectious (Table 1, nos. 7 and8). The infectivities of these transcripts were very low (lessby a factor of i0-104for the former and less by a factor of=50 for the latter) but reproducibly observed.Angysis of Progeny Viruses. One week after inoculation on

the systemic host, progeny viruses were purified by thestandard procedure (24). Genomic RNAs were isolated andtheir 5'- and 3'-terminal sequences were determined. Thesequences ofthe 5' ends ofthe progeny viruses were the sameas those of both the in vitro transcripts and the parent virusused for the cloning (not shown). The 3'-terminal sequenceswere determined by the chemical method (25). As shown inFig. 3B, the introduced thymine-to-adenine transversion wasconserved in the genomic sequences of the progeny viruses.These observations indicated that the infectious RNAs re-sulted from the expression of the cloned cDNA. The sametransversions were also found in the progeny viruses derivedfrom uncapped' transcript and transcript from the uncutplasmid DNA. The three additional nucleotides at the 3' endof the genome-sized transcripts were not found in theprogeny viral RNAs (Fig. 3A).Phenotypes of both the capped transcript from linearized

pLFA1 and some progeny viruses isolated from a singlelesion on Xanthi nc tobacco were examined on both tomatoand systemic tobacco plants. Nineteen days after inocula-tion, the viruses were propagating but the plants showed no

symptoms, consistent with'the phenotype of L11A (11), whileplants inoculated with pLFW4-derived transcripts or theirprogeny showed mosaic symptoms. These observations in-dicated the faithful in vitro transcription of the clonedsequence and in vivo replication of in vitro transcribed TMVRNA.

DISCUSSIONWe have described the construction of full-length cDNAclones of TMV RNAs and the in vitro transcription of

infectious RNAs from the cDNA clones by E. coli RNApolymerase. Previously, Ahlquist et al. showed that BMVinfection can be derived from cloned viral cDNA insertedinto pPM1 by transcription (8). Compared with the BMV-barley system, the TMV-tobacco system we used is knownto give higher specific infectivity. Moreover, we could obtainefficient expression ofTMV, especially by adding an in vitroreconstitution step, which increased the infectivity >100times. As a result, it has become possible to manipulate theTMV genome in vitro. This will lead to a better understandingof the TMV genomic structure and its function.The 5' cap structure has been shown to be important for

mRNA stability and translatability (29, 30). Ohno et al.reported the significance of the cap structure for TMVinfectivity by using enzymatically decapped TMV RNA (31).However, it could not be determined whether the lowinfectivity of their preparation was due to the decapped RNAitself or to the low amount of capped RNA still present afterthe decapping treatment. In this work, we prepared com-pletely uncapped RNA and it showed infectivity, although itwas very low, indicating that the cap structure is not essentialfor the TMV infection but drastically increases the infectiv-ity. The cap structure may affect the translation efficiencyand the stability of the TMV RNA in the inoculated cells.

In addition, we detected the infectivity ofthe reconstitutedtranscript even from uncut plasmid DNA. The multiplyingprogeny viruses accurately reflected the cloned sequence andhad the same genomic length as the authentic virus. Althoughwe cannot explain the process by which the additionalnucleotides at the 3' end of transcripts are eliminated, thisobservation indicates that the laborious step of the introduc-tion of a restriction enzyme recognition sequence down-stream of a cDNA insert can be omitted, at least in the caseofTMV expression. This will lead to easier application of thistranscription system to the production of recombinant virus-es or viruses carrying unrelated sequences, if they carryrecognition sequences of the enzyme used to linearize theplasmid.

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Proc. Natl. Acad. Sci. USA 83 (1986) 5047

We thank Dr. P. Ahlquist and Agrogenetics Research Associatesfor the use ofpPM1. E. coli MC1061 was a generous gift from Dr. M.Casadaban. We also thank Dr. K. Ohno for the preparation ofoligonucleotides. This work was supported in part by Grants-in-Aidfrom the Ministry of Education, Science and Culture, and from theMinistry of Agriculture, Forestry and Fisheries, Japan.

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