Proc. Natl. Acad. Sci. USAVol. 93, pp. 4370-4373, April 1996Genetics

Amplification of the full-length hepatitis A virus genome by longreverse transcription-PCR and transcription of infectious RNAdirectly from the ampliconRAYMOND TELLIER, JENS BUKH, SUZANNE U. EMERSON, AND ROBERT H. PURCELL*Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health,Bethesda, MD 20892

Contributed by Robert H. Purcell, January 31, 1996

ABSTRACT The genetic study of RNA viruses is greatlyfacilitated by the availability of infectious cDNA clones. How-ever, their construction has often been difficult. While ex-ploring ways to simplify the construction of infectious clones,we have successfully modified and applied the newly describedtechnique of "long PCR" to the synthesis of a full-length DNAamplicon from the RNA of a cytopathogenic mutant (HM175/24a) of the hepatitis A virus (HAV). Primers were syn-thesized to match the two extremities of the HAV genome. Theantisense primer, homologous to the 3' end, was used in boththe reverse transcription (RT) and the PCR steps. With theseprimers we reproducibly obtained a full-length amplicon of

7.5 kb. Further, since we engineered a T7 promoter in thesense primer, RNA could be transcribed directly from theamplicon with T7 RNA polymerase. Following transfection ofcultured fetal rhesus kidney cells with the transcriptionmixture containing both the HAV cDNA and the transcribedRNA, replicating HAV was detected by immunofluorescencemicroscopy and, following passage to other cell cultures, byfocus formation. The recovered virus displayed the cytopathiceffect and large plaque phenotype typical of the original virus;this result highlights the fidelity of the modified long reversetranscription-PCR procedure and demonstrates the potentialof this method for providing cDNAs of viral genomes andsimplifying the construction of infectious clones.

The genetic study of many RNA viruses has benefited from theavailability of cDNA clones of the entire viral genome, whichcan serve as templates for the generation of infectious RNAs(for review, see ref. 1). However, their construction has oftenbeen difficult, involving the cloning and ligation of manysubgenomic cDNA fragments. In spite of recent improvementsafforded by the PCR, the process is still laborious and manydifficulties remain, including the deleterious effect of addi-tional nucleotides at either end of the viral genome and theinstability of some viral sequences in the bacterial host (1-4).Furthermore, the "quasi-species" nature of many viruses (5)challenges the concept of a clonal population as an accurate orcomplete description of the natural infection.

In principle, the application of the recently described tech-nique of "long PCR" (6) should simplify the construction ofinfectious clones and diminish some of the problems notedabove. We have used as a model for this approach a cyto-pathogenic mutant of the hepatitis A virus (HAV). In thisstudy, we report the synthesis of a full-length cDNA of thisHAV mutant in a single round of long PCR, following thereverse transcription (RT) of the viral RNA. We furtherdemonstrate that infectious RNAs can be generated directlyfrom the amplicon by in vitro transcription. The recoveredvirus preserved the characteristic cytopathic effect and large

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plaque phenotype of the mutant, demonstrating the highfidelity of this process.

MATERIALS AND METHODSVirus Stocks.A cytopathogenic mutant strain (HM175/24a)

of HAV (7) was passaged in the fetal rhesus kidney cell line11-1; cell lysates were prepared as described (8) and stored in100 ,tl aliquots at -80°C. The viral titer of the stock wasestablished by radioimmunofocus assay as described (8) andexpressed as radioimmunofocus-forming units per ml.RNA Purification. RNA from 100 u.l of viral stock was

extracted with the TRIzol system (GIBCO/BRL), accordingto the manufacturer's recommendation, except that all mixingsteps were performed by repeated inversion or tapping of thetube to prevent RNA shearing. The RNA pellet was resus-pended in 10 ,ul of 10 mM dithiothreitol containing 5%(vol/vol) RNasin (20-40 units/,ul) (Promega) and frozen at-80°C.Primers. The sense primer CLT7HALA (5'-CCATCGAT-

GGTAATACGACTCACTATAGTTCAAGAGGGG-TCTCCGGGA-3') consisted of a Cla I site followed by thecore sequence of the T7 promoter (9-11), a single guanosinenucleotide, and the first 20 nt of the wild-type HAV HM175(12). The antisense primer CLOTHALA (5'-CCATCGAT-GGTTTTTTTTTTTTTTTATTTACTGATAAAA-GAAATAAAC-3') consisted of a Cla I site followed by astretch of 15 thymidine nucleotides and 23 nt corresponding tothe sequence of the wild-type HAV HM175 (12) immediatelybefore the poly(A) tail. The antisense primer KPOTHALA(5'-GGGGTACCCCTTTTTTTTTTTTTTTATT-TACTGATAAAAGAAATAAAC-3') was essentially identi-cal to CLOTHALA, except for the flanking sequence, whichnow contained a Kpn I site.The primers were used in two pairs: the first pair consisted

of the primers CLT7HALA and CLOTHALA, synthesized inour laboratory using an Applied Biosystems model 392 DNA/RNA synthesizer, without size purification after synthesis; thesecond pair consisted of the primers CLT7HALA and KPO-THALA, which were synthesized and size purified by anoutside contractor. In the experiments described below theantisense primer of a given pair was used for both the RT andthe PCR.RT. The RNA aliquot was thawed on ice, incubated 2 min

at 65°C, and chilled on ice. From a master mix, 0.5 ,/l of RNasin(20-40 units/ml) (Promega), 1 p.l of 100 mM dithiothreitol(Promega), 1 pul of 10 mM stock solution of dNTP (Pharma-cia), 2.5 pl of 10 puM primer stock solution, 1 ,pl (200 units) ofSuperscript II reverse transcriptase (GIBCO/BRL), and 4 u.lof 5x 1st Strand Synthesis buffer (GIBCO/BRL) was addedto the RNA. The reaction was incubated for 1 h at 42°C; 1 p.lof RNase H (1-4 units/pul) (GIBCO/BRL) and 1 pul of RNase

Abbreviations: HAV, hepatitis A virus; RT, reverse transcription.*To whom reprint requests should be addressed.

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T1 (900-3000 units/til) (GIBCO/BRL) were then added andthe reaction incubated for 20 min at 37°C.

In one series of experiments, the cDNA was then purifiedwith the DNA Clean Sweep kit (Amresco, Solon, OH), usedessentially according to the manufacturer's recommendationexcept that 20 ttg of glycogen (Boehringer Mannheim) wasused as a carrier instead of the supplied yeast tRNA and mixingwas performed as above. The cDNA pellet was resuspended in10 ,l of double distilled H20, incubated 3 min at 95°C, andkept on ice until added to the long PCR reaction mix. Inanother series of experiments, after incubation and treatmentwith RNases, 2 Atl of the unpurified RT reaction mixture wasadded directly to a long PCR reaction mix (13) (R.T., unpub-lished data). In these experiments, we considered the contri-bution of antisense primer, buffer, and dNTP from the 2 ,l ofRT reaction to the PCR reaction to be negligible.Long PCR with the KlenTaq LA-16 Polymerase Mix. The

reactions were performed in a total volume of 50 ,l with finalconcentrations of 20 mM of Tris-HCl (pH 8.55 at 25°C), bovineserum albumin at 150 ,xg/ml, 16 mM of (NH4)2804, 3.5 mMof MgC12 ("PC2" buffer; ref. 6), and 250 ,M of each dNTP.Each reaction contained 20 pmol of each primer and 0.4 ,ul ofthe "KlenTaq LA-16" enzyme mix (6). This enzyme mix wasprepared in a proportion of 15 ,tl of KlenTaql (25 units/,ul)(Ab Peptides, St. Louis) to 1 /xl of cloned Pfu (2.5 units/Al;Stratagene). The mix used in these experiments synthesized anamplicon of 11 kb from as little as 10-4 ng of A phage DNA(R.T., unpublished data).

Reactions were performed in thin-wall PCR tubes (Strat-agene). Each reaction was overlaid with exactly 40 ,ul ofmineral oil (Sigma). The PCR was performed using a Robo-cycler thermal cycler (Stratagene) with the following cyclingparameters: denaturation at 99°C for 35 sec, annealing at 67°Cfor 30 sec, and elongation at 68°C for 9 min 45 sec during thefirst 15 cycles, 11 min during the next 10 cycles, and 13 minduring the last 10 cycles.Long PCR with the Advantage KlenTaq Polymerase Mix.

The reactions were performed essentially as above, except thatthe buffer supplied with the kit (Clontech) was used instead ofthe PC2 buffer, and 1 ,ul per reaction of the AdvantageKlenTaq polymerase mix (a mix of KlenTaql, Deep Vent, andTaqStart anti-Taq antibody) was used instead of the KlenTaqLA-16 mix. The Advantage KlenTaq polymerase mix synthe-sized an amplicon of 11 kb from as little as 10-5 ng of A phageDNA (R.T., unpublished data).

Purification of Amplicons. The PCR reaction was electro-phoresed on a 0.7% agarose gel. DNA bands were visualizedwith a UV transilluminator and the desired bands excised fromthe gel. (Shielding from UV was provided by illuminating thegel through two trays of Plexiglas to minimize photo-nicking.)The DNA was purified from the agarose by the "glassmilk"method (Geneclean II kit, Bio 101). The final elution was inDNase-free, RNase-free double distilled H20.

Transfection. The in vitro transcription using T7 RNApolymerase, the transfection of fetal rhesus kidney cells andthe immunofluorescence microscopy for HAV were per-formed as described (14). Plaque assays were performedessentially as described (8) except that acetone-fixed cells werestained with crystal violet.

RESULTSWe have used an HAV mutant to evaluate a simplified andrapid method for quickly generating infectious cDNAs ofRNAviruses. A cell lysate containing the cytopathogenic mutantHAV HM175/24a (7) at a titer of 4.4 x 107 radioimmunofo-cus-forming units per ml was the source of viral RNA. Thetotal RNA extracted from a 100-tLI aliquot of this viral stockwas used in each RT reaction.

In a first series of experiments, we performed the RT withthe primer CLOTHALA, purified the cDNA with the DNAClean Sweep kit, and performed long PCR with the primerpair CLT7HALA-CLOTHALA and the KlenTaq LA-16 mix.From this relatively modest amount of RNA, we obtained anamplicon of 7.5 kb, which is the expected length for a completecDNA of HAV. After purification, in vitro transcription withT7 RNA polymerase and transfection of cultured fetal rhesuskidney cells we obtained, in each of two experiments, repli-cation-competent HAV that spread throughout the culture, asdemonstrated by immunofluorescence microscopy.We also tested a recently introduced commercial mix, the

KlenTaq Advantage polymerase mix (Clontech) and at thesame time we simplified the RT-PCR procedure by eliminatingthe cDNA purification step and transferring a small amount ofthe unpurified RT reaction mixture to the PCR reaction. Withthis procedure, either pair of primers (CLT7HALA-CLOTHALA or CLT7HALA-KPOTHALA) consistentlyproduced a 7.5-kb amplicon (Fig. 1A). As is usually the casewith long PCR, extra bands were also observed. In our hands,the KlenTaq Advantage polymerase mix proved to be moresensitive and consistent than the KlenTaq LA-16 mix.

After gel purification and transcription with T7 RNA poly-merase, amplicons synthesized with one or the other pair ofprimers produced RNA that comigrated with the HAVgenomic RNA produced from an infectious clone of HAV,pT7-HAV1 (15), that was derived from pHAV/7 (16) (Fig.1B). The amount of RNA produced appeared to be equivalentwith the two pairs of primers. However, the infectivity of theRNAs produced from the two amplicons was quite different.Amplicons produced with the primer pair CLT7HALA-

CLOTHALA yielded replicating viruses in each of threeexperiments. We also obtained viruses in an additional exper-iment in which 50 ,ul of PCR reaction mixture was treated with

A

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B

kb23.1-9.4-6.6-4.4-2.3-2.0-1.4-1.1-0.9-0.6-

9 q A

FIG. 1. (A) Amplification of the HAV genome by long RT-PCR.The PCR was performed with the primer pair CLT7HALA-CLOTHALA and the Advantage KlenTaq polymerase mix. Reactionmixture (5 /LI) was analyzed on a 0.7% agarose gel. Lane 1, molecularweight markers; lane 2, PCR reaction mixture. The major band (7.5 kb)is of the size expected for the full-length HAV cDNA. (B) Ampliconsand their transcripts. Purified amplicons were transcribed in a 50-,jlreaction with T7 RNA polymerase. Reaction mixture (5 ,ul) wasprecipitated with ethanol and electrophoresed on a 1% agarose gel.Lane 1, molecular weight markers; lane 2, reaction mixture with theamplicon produced with the primer pair CLT7HALA-CLOTHALA;lane 3, reaction mixture with the amplicon produced with the primerpair CLT7HALA-KPOTHALA; lane 4, reaction mixture with thelinearized plasmid pT7-HAV1 (15).

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2.5 ,ul of RNase H in order to destroy any carry-over RNAbefore gel purification of the cDNA. Therefore, with eithermethod of long PCR, this pair of primers generated anamplicon that produced infectious RNAs. In all six instances,the resulting infected cells displayed the characteristic HAVimmunofluorescence pattern of punctate cytoplasmic staining;a representative experiment is illustrated in Fig. 2A. In all ofthe experiments plaque assays of the recovered viruses dem-onstrated a large plaque, cytopathic phenotype characteristicof the mutant HM175/24a. Fig. 2B illustrates a representativeexperiment.

In contrast, contemporaneous experiments with ampliconsproduced by the primer pair CLT7HALA-KPOTHALAfailed to produce replicating viruses each of four times, eventhough the quantity and quality of RNA produced in thetranscription reactions were equivalent to that obtained fromamplicons produced with the primer pair CLT7HALA-CLOTHALA.

DISCUSSIONIn this study we used the recently described technique of longPCR (6) to generate in one round of RT-PCR a completecDNA copy of an RNA virus, from which infectious RNAswere transcribed. We have transcribed infectious RNAs fromamplicons produced with either the KlenTaq LA-16 mix orAdvantage KlenTaq polymerase mix, starting with approxi-mately 4 x 106 radioimmunofocus-forming units of HAV.One of the problems encountered with the construction of

infectious clones by traditional recombinant techniques is thereduction of infectivity caused by additional nucleotides in-corporated at the 5' end of the viral genome during transcrip-tion of the enzyme restriction sites used for cloning (1). Anadvantage of the procedure described here is that it permitscomplete control of the apposition of the T7 promoter so thata minimum number of extra nucleotides are incorporated.Since transcription is much more efficient if the first baseimmediately after the core sequence of the T7 promoter is aguanosine nucleotide (9, 10) we added a single guanosinenucleotide between the T7 core sequence and the 5' end of theHAV genome, which begins with a thymidine nucleotide (12).Although successful RNA transcriptions directly from ampli-cons containing a standard T7 promoter have been demon-strated previously with T7 RNA polymerase (17-19), it ap-pears that a 9-20 bp leader sequence before the promoterincreases the yields (19). This requirement was convenientlyfulfilled by adding an upstream restriction site that could beused in future cloning experiments. As designed, the

CLT7HALA primer performed quite well for both PCR andRNA transcription.

Additional sequences at the 3' end of the viral genome canalso cause problems even though the tolerance appears greaterand some sequences may even be beneficial (1); we weretherefore somewhat surprised to see that the 3' addition of a10-nt stretch containing a Kpn I site eliminated virus produc-tion. We speculate that a secondary structure generated by thissequence may inhibit the genome replication or packaging.An important issue with this experiment was to eliminate the

possibility that infectious viruses originated from viral RNAcarried over from the initial cell lysate. The most compellingargument against this was the consistent failure to recoverviruses when experiments were performed with the primerpair CLT7HALA-KPOTHALA. The RT-PCR was per-formed with both primer pairs in parallel from equal aliquotsof the same viral stock. Thus, if the recovery of virusesconsistently observed with the primer pair CLT7HALA-CLOTHALA was attributable solely to a carry over of viralRNA, we should have observed it also when the second primerpair was used. Of course the experimental design made sucha carry over extremely unlikely. After the completion of theRT, the reaction was digested with both RNase H and RNaseT1. The initial motivation for this was to prevent RNAmolecules from interfering with the PCR by binding to theDNA, but it had the advantage of destroying single-strandedviral RNA. Of greater concern was the fact that picornavirusinfections also generate double-stranded RNA replicativeforms known to be infectious and to constitute as much as 10%of the viral RNA at the end of replication (20). Double-stranded RNA is resistant to digestion by RNases. However,because of the accumulation of DNA during PCR, putativeresidual RNAs would end up complexed in an RNA-DNAduplex. These are not known to be infectious and there areexamples of DNA oligomers complexed with the viral RNA ofsome picornaviruses that resulted in inhibition of translation(21) or infection (22). Certainly such a duplex would besensitive to cellular RNase H. Also, an RNase H treatedamplicon obtained with the primer pair CLT7HALA-CLOTHALA still produced infectious RNA. Finally, anyresidual RNA in the PCR reaction mix would have had towithstand hydrolysis catalyzed by divalent metal ions, a processaccelerated by high temperature (23-25). For all of thesereasons, we conclude that the recovered virus was encoded bythe RNAs transcribed from the amplicon.

It is noteworthy that multiple mutations were required forthe large focus (S.U.E., unpublished data) and cytopathic (7)phenotypes of the HAV mutant used in this study. That the

BA

HM175/24a (stock) Recovered Viruses

FIG. 2. Production of infectious viruses from amplicons. (A) Cells transfected with transcribed amplicons produced with the primer pairCLT7HALA-CLOTHALA were examined by immunofluorescence microscopy. The typical punctate staining seen with HAV is apparent in thisphotograph. (B) Plaque assays performed with viruses harvested from transfected cells demonstrated a large plaque cytopathic phenotype identicalto that of the original virus.

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recovered viruses still displayed both phenotypes furthercorroborates the already observed greater fidelity of longPCR mixes compared with the Taq polymerase (6) andsuggests a potential for this method in amplifying viralgenomes or long RNA molecules for other purposes such asfor determination of sequence.

Since a high level of sequence homology is required onlyin the region of the two primers used for PCR, these methodsare well-suited for obtaining complete cDNAs of interestingmutants or of new genotypes. Although the HAV mutantstudied here has been almost completely sequenced (7) theprecise sequence of the termini of the genome has not beenestablished, and we designed our primers based on thewild-type HAV sequence (12). Given that we recoveredcytopathogenic viruses at the end of the process, it appearsthat either the termini are identical to those of wild-typeHAV or any mutations present in the regions correspondingto the primers do not contribute to the cytopathogenicphenotype. We expect that these primers and the amplifi-cation parameters described here will be useful for a broadrange of HAV mutants.

In principle, purified and diluted amplicons could be am-plified again with the same primers. For viruses with sequencesthat are unstable or toxic in bacterial hosts when cloned in aplasmid (1-4), this could provide an alternative way of prop-agating DNA. A potentially interesting consequence is thatwhen amplifying a virus with a quasi-species nature, such ashuman immunodeficiency virus (26) or hepatitis C virus (27),one would obtain a representative population of cDNAs fromwhich infectious RNAs could be transcribed.

In summary, we have described a simple and rapid longRT-PCR protocol to obtain a full-length amplicon fromwhich infectious RNAs can be directly transcribed. Hayesand Buck (28) have reported the construction of infectiousclone for cucumber mosaic virus RNAs by cloning full-lengthamplicons of the genomic RNAs. However, the longest ofthese RNAs was 3.1-kb long and Hayes and Buck started withlarge amounts ofRNA that would not be readily available formany viruses. Other authors have used PCR to amplifyoverlapping fragments of many plant RNA viruses andassembled infectious clones by standard recombinant DNAtechnology (reviewed in ref. 1); this process has also beenused for HAV (29). More recently, Gritsun and Gould (30),in a process taking 10 days, have used a long RT-PCRprotocol to obtain two overlapping cDNA fragments (5.2 kband 5.7 kb) covering the whole genome of a tick borneencephalitis virus, and have generated by fusion PCR afull-length amplicon from which infectious RNAs could betranscribed. In contrast, we have developed a simpler pro-cedure that requires but a single round of RT-PCR toamplify an RNA genome of 7.5 kb. Consequently, we cangenerate an infectious cDNA in useful amounts in only 2days. In principle this method could be used with many otherviruses and should prove most helpful.

We thank Tatiana S. Tsareva for primer synthesis, Ying K. Huangfor technical assistance, and Carmean J. Hutton for assistance with themanuscript. R.T. is the recipient of a Fellowship from the MedicalResearch Council of Canada.

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Biochemistry. In the article ‘‘Characterization of soluble formsof nonchimeric type V adenylyl cyclases,’’ by Klaus Scholich,Ann J. Barbier, Jason B. Mullenix, and Tarun B. Patel, whichappeared in number 7, April 1, 1997, of Proc. Natl. Acad. Sci.USA (94, 2915–2920), the authors request that the followingchanges be noted in the amino acid linker sequence on page2915. The last sentence of the first paragraph in the ‘‘Methods’’section should read: [sequence, AAAGGMPPAAAGGM]instead of [sequence, AAAGGM(PPAAAGGM)2].

Biochemistry. In the article ‘‘Mechanism of protein remodel-ing by ClpA chaperone,’’ by Marie Pak and Sue Wickner, whichappeared in number 10, May 13, 1997, of Proc. Natl. Acad. Sci.USA (94, 4901–4906), the authors wish to correct a printer’serror. On page 4903, in Table 1, line 4 with ‘‘RepA 1 ClpA 1ATP,’’ 46% of the RepA was retained in the condition of thefirst column with ‘‘No NaCl’’ and at ‘‘0°C’’ instead of 10% asstated.

Genetics. In the article ‘‘Amplification of the full-lengthhepatitis A virus genome by long reverse transcription-PCRand transcription of infectious RNA directly from the ampli-con,’’ by Raymond Tellier, Jens Bukh, Suzanne U. Emerson,and Robert H. Purcell, which appeared in number 9, April 30,1996, of Proc. Natl. Acad. Sci. USA (93, 4370–4373), theauthors request that the following correction be noted. Onpage 4371, column 1, line 22 stating ‘‘Each reaction contained20 pmol of each primer . . .’’ should read ‘‘Each reactioncontained 10 pmol of each primer . . . .’’

Corrections Proc. Natl. Acad. Sci. USA 94 (1997) 10485