The RNA Polymerase of Influenza A Virus Is Stabilized by Interaction with Its Viral RNA Promoter

The RNA polymerase of the influenza virus is responsible forthe transcription and replication of the segmented RNA viralgenome during infection of host cells. Polymerase function isknown to be strictly dependent on interaction with its RNA promoter,but no attempts to investigate whether the virion RNA (vRNA)promoter stabilizes the polymerase have been reported previously.Here we tested whether the vRNA promoter protects the polymeraseagainst heat inactivation. We prepared partially purified recombinantinfluenza A virus RNA polymerase, in the absence of influenzavirus vRNA promoter sequences, by transient transfection ofexpression plasmids into human kidney 293T cells. The polymerasewas found to be heat labile at 40°C in the absence of addedvRNA. However, it was protected from heat inactivation if boththe 5' and 3' strands of the vRNA promoter were present. Byusing the ability of vRNA to protect the enzyme against heatinactivation, we established a novel assay, in conjunction witha mutagenic approach, that was used to test the secondary structurerequirement of the vRNA promoter for polymerase binding. Bindingrequired a panhandle structure and the presence of local hairpinloop structures in both the 5' and 3' ends of vRNA, as suggestedby the corkscrew model. The interaction of the vRNA promoterwith the influenza virus RNA polymerase heterotrimeric complexis likely to favor a particular closed conformation of the complex,thereby ensuring the stability of the RNA polymerase withinboth the infected cell and the isolated virus.

INTRODUCTION

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Introduction

Influenza virus is a negative-sense, segmented RNA virus witheight RNA segments coding for 10 genes. The three longest genesegments code for the three subunits PB1, PB2, and PA of theheterotrimeric influenza virus RNA polymerase complex, whichis the enzyme responsible for transcription and replicationof the viral genome in the infected cell. In addition to itsrole as an RNA-dependent RNA polymerase, which copies virionRNA (vRNA) $->$ mRNA in transcription and vRNA $->$ cRNA and cRNA $->$ vRNA inreplication, the complex is also an endonuclease, snatchingcapped RNAs from host cell pre-mRNA for use as primers for thesynthesis of influenza virus-specific mRNA, and a poly(A) polymerase,catalyzing polyadenylation of influenza virus-specific mRNA(for reviews, see references 19, 24, and 31).

There has been significant progress in understanding the sequenceand secondary structure properties of the vRNA promoter, whichconsists of 13 nucleotides (nt) at the 5' end and 12 nt at the3' end of the vRNA, all of which are conserved in each RNA segmentof every influenza A virus. These conserved nucleotides, alongwith an extra one to five segment-specific bases at the 5' and3' ends of vRNA, form a partially double-stranded panhandlestructure. Additional short hairpin loops, consisting of a 2-bpstem and a tetraloop, have been proposed either at the 5' end(5' hairpin loop model) or at both the 5' and 3' ends (corkscrewmodel) of the vRNA (4, 33).

The hairpin loop near the 5' end of the vRNA promoter is probablyrequired for binding to the polymerase complex, since wheneverrequirements for this hairpin loop in, e.g., endonuclease cleavage(21) or polyadenylation (33) have been critically examined,its presence has been found to be absolutely essential. Thehairpin loop near the 3' end of vRNA is also required for endonucleasecleavage (20) but not for ApG-primed transcription (8) or polyadenylation(33).

The influenza virus RNA polymerase binds vRNA and cRNA promotersequences specifically (7-9, 32). The purpose of the presentstudy was to test whether vRNA promoter binding stabilized thepolymerase to heat inactivation, and if so, what the secondarystructure requirements for this stabilization were. To investigatethis problem, we required a source of recombinant influenzavirus RNA polymerase prepared in the absence of influenza virusRNA, since active, nonrecombinant polymerase cannot readilybe isolated from viral cores without contamination by residualinfluenza virus RNA (8). We therefore adapted a plasmid-basedsystem to express influenza virus RNA polymerase transientlyin human kidney 293T cells. We found that the vRNA promoterprotected the isolated influenza virus RNA polymerase from heatinactivation. By utilizing this property of vRNA, we establisheda novel assay, in conjunction with a mutagenic approach, thatwas used to test the secondary structure requirement of thevRNA promoter for polymerase binding. Binding required a panhandlestructure and the presence of local hairpin loop structuresin both the 5' and 3' ends of vRNA, as suggested by the corkscrewmodel.

MATERIALS AND METHODS

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Materials and Methods

Plasmids. Full-length cDNA copies of the PB1, PB2, PA, and NP segmentsof influenza A/WSN/33 virus were cloned into the AgeI site ofpcDNA3A, a variant of pcDNA3 (Invitrogen) modified by the insertionof an AgeI sequence into the HindIII site (further details areavailable from the authors of the present article), giving plasmidspcDNA-PB1 (abbreviated PB1), pcDNA-PB2 (abbreviated PB2), pcDNA-PA(abbreviated PA), and pcDNA-NP (abbreviated NP). These plasmidsare under the control of a cytomegalovirus promoter/enhancer.The plasmid pPOLI-GFP (abbreviated vRNA; sequence availablefrom the authors of the present article) (gift of Peter Palese)encodes the RNA of green fluorescent protein in negative senseflanked by the 5' and 3' ends of the vRNA promoter of segment8 (slightly modified in their 5' and 3' noncoding regions) ofinfluenza A/WSN/33 virus under the control of a truncated humanRNA polymerase I promoter and a ribozyme terminator (5). Thefirst 23 residues of an ApG-primed influenza virus RNA polymerasetranscript of the vRNA promoter derived from pPOLI-GFP havethe sequence AGCAAAAGCAGGGUGACAAAGAC.

Preparation of recombinant influenza virus RNA polymerase by transient transfection of 293T cells. Exponentially growing 293T cells (usually 5 x 10⁵ cells) weretransfected with 1 µg of plasmids, as specified in theindividual experiments; e.g., pcDNA-PB1, pcDNA-PB2, and pcDNA-PAwere transfected with Lipofectamine 2000 (Gibco BRL) in suspensionand plated onto 3.5-cm-diameter dishes in antibiotic-free Dulbecco'smodified Eagle's medium containing 10% fetal calf serum. After48 h (typically), cells were harvested, washed in phosphate-bufferedsaline, and lysed for 5 min at 0°C with 1 ml of buffer A(0.1% Nonidet NP-40, 10 mM Tris-HCl [pH 7.8], 10 mM KCl, 1 mMEDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride).Nuclei were isolated by centrifugation at 1,000 x g and lysedfor 10 min at 0°C in 0.1 to 0.2 ml of buffer B (0.5% NonidetNP-40, 50 mM Tris-HCl [pH 7.8], 0.2 M KCl, 0.2 mM EDTA, 1 mMdithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 25% glycerol).After a further centrifugation at 1,000 x g, the nuclear extractwas either used directly or concentrated by precipitation withan equal volume of 3 M ammonium sulfate. The protein pelletwas finally dissolved in 20 to 30 µl of buffer B and storedat -20°C. In some experiments, the transfection was scaledup seven times and plated on 9-cm-diameter dishes.

Preparation of RNA templates. Short 15- or 14-nt synthetic oligonucleotides at the 5' end(5' AGUAGAAACAAGGCC 3') or 3' end (5' GGCCUGCUUUUGCU 3'), respectively,of wild-type influenza A virus vRNA were purchased from Dharmacon,unblocked by following the manufacturer's instructions, andused without further purification. These oligonucleotides containedthe consensus sequence (underlined) plus an additional two residues.Mutant oligonucleotides (15 and 14 nt) corresponding to the5' and 3' ends of vRNA, respectively, were obtained from ourcollection of synthetic RNAs, which had been synthesized andgel purified in 1993 to 1994 and stored at -20°C (8). Purityof the RNA was assessed, essentially as described previously,as >75% (8) by [ ${gamma}$ -³²P]ATP labeling with T4 polynucleotidekinase and analysis on 20% polyacrylamide gel electrophoresis(PAGE) in 7 M urea. The main contaminants were oligonucleotidesone residue shorter than the desired sequence (results not shown).Nucleotides are numbered with prime numbers from the 5' endof the 5' strand of the promoter and without prime numbers fromthe 3' end of the 3' strand of the promoter (see Fig. 5 to 7).

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FIG. 5. Base pairing between the conserved 5' strand and 3' strand of the vRNA promoter is required to protect the RNA polymerase from heat inactivation. (A) The transcriptional activity remaining after heat inactivation of the polymerase in the presence of structures 1 to 11. Mutations are shown in bold; base pairs are shown by vertical lines. For sequences 3, 5, 7, 9, and 11, activity was expressed as a percentage of wild-type (sequence 1) activity (means and standard deviations of three independent experiments), by quantitation of the major bands a and a₁ to a₅; activity values for sequences 2, 4, 6, 8, and 10 were from single measurements. (B) RNA polymerase, obtained by transfection of 293T cells with PB1, PB2, and PA, was heated for 15 min at 40°C in the presence of the indicated sequences (1 µM), transcription was evaluated with the [ ${alpha}$ -³²P]GTP incorporation assay, and the polymerase was fractionated by 20% PAGE in 7 M urea. Lanes 1, 3, 7, 9, 11, 4, and 5 depict the results for the corresponding sequences shown in panel A. Bands b and c depict minor, 13-nt or 12-nt transcripts, shorter than the main 14-nt product, band a; the minor band, x, depicts a longer, 15-nt transcript, possibly formed by the nontemplated addition of an extra base. Bands a₁ to a₅ differ in gel mobility from one another because of their differing G content.

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FIG. 7. Base pairs in the stem of the 3' hairpin loop of the promoter are required to protect the RNA polymerase from heat inactivation. (A) The wild-type vRNA (1) and mutants (17 to 24) tested. Only the 3' hairpin loop (or potential loop) of a 14-nt 3' strand is shown; all experiments used in addition an equimolar amount of the 15-nt wild-type 5' strand of vRNA. Sequences are numbered from their 3' ends. (B and C) RNA polymerase, reconstituted in vivo from PB1, PB2, and PA plasmids, was heated for 15 min at 40°C in the presence of the indicated sequences (1 µM), transcription was evaluated by the [ ${alpha}$ -³²P]GTP incorporation assay with ApG and added wild-type vRNA sequences (2.5 µM), and the polymerase was fractionated by 22% PAGE in 7 M urea. See legend to Fig. 6 for explanation of additional symbols. A duplicate experiment gave similar results.

ApG-primed transcription, using an [ ${alpha}$ -³²P]GTP incorporation assay, of short model vRNAs. Transcription was performed as described before (8), exceptthat reactions were performed with 2.5 µCi of [ ${alpha}$ -³²P]GTP(>3,000 Ci/mmol; Amersham) in the presence of 1 mM ATP-0.5mM CTP-0.1 µM GTP-0.5 mM UTP-1 mM ApG (Sigma) using upto 1.5 µl of recombinant RNA polymerase and 5' (15 nt)and 3' (14 nt) wild-type or mutant templates (up to 15 pmolof each) in a 3-µl reaction mixture. After 1 h at 30°C,formamide dyes (10 µl) were added and the mixture washeated at 95°C for 2 min. Fractionation was by either 18%,20%, or 22% PAGE in 7 M urea. Gels were autoradiographed usingKodak BioMax Trans-screen HE intensifying screens and BiomaxMS film. Quantitation was by phosphorimage analysis. When productshad a differing G content, the relative molar yields were calculatedby dividing the raw phosphorimage data by the number of G residuesin the transcript. In the case of the base-paired as opposedto the mismatched sequences (Fig. 5A, sequences 1, 3, 5, 7,9, and 11), the [ ${alpha}$ -³²P]GTP incorporation assay was modified bythe omission of wild-type vRNA, since preliminary experimentshad shown that the addition of wild-type vRNA was unnecessary(data not shown). In the case of Fig. 6, the [ ${alpha}$ -³²P]GTP incorporationassay was modified by the addition of a pseudo-wild-type panhandlestructure derived from the specified mutant 15-nt 5' and 14-nt3' vRNA oligonucleotides, which resulted in the formation ofan A $->$ U base pair mutation at positions 11 and 12'.

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FIG. 6. Base pairs in the stem of the 5' hairpin loop of the promoter are required to protect the RNA polymerase from heat inactivation. (A) Pseudo-wild-type (3) (see Fig. 5A, sequence 3, for full structure) and point (12 to 16) mutants tested. Only the first 10 residues of the 15-nt 5' hairpin loop are shown; for the full duplex structure, see Fig. 5A. Sequences are numbered from their 5' ends (1', 2', 3', etc.). (B) The RNA polymerase preparation, reconstituted in vivo from PB1, PB2, and PA plasmids, was heated for 15 min at 40°C in the presence of the indicated sequences (1 µM) and controls. Transcription was evaluated by the [ ${alpha}$ -³²P]GTP incorporation assay with added pseudo-wild-type vRNA sequences (1.25 µM), and the polymerase was fractionated by 22% PAGE in 7 M urea. a, 14-nt transcription product; b, 13-nt product. Control lanes: No RNA, no added RNA; 3', only the 3' strand of the promoter was present; No heat, unheated enzyme. An independent experiment gave essentially similar results.

Heat inactivation of recombinant RNA polymerase. The recombinant enzyme (in buffer B) was heated at 40°Cfor 15 min with a <20% volume of added oligonucleotides ordeionized water and placed on ice. Heated extracts were assayedimmediately for ApG-primed transcription by the [ ${alpha}$ -³²P]GTP incorporationassay (see above).

RESULTS

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Results

Recombinant RNA polymerase isolated from 293T cells is transcriptionally active. We had to prepare a source of recombinant RNA polymerase thatwas active in transcription in the absence of influenza virusvRNA promoter sequences before testing the heat stability ofinfluenza virus RNA polymerase in the presence of the influenzavirus vRNA promoter. To detect polymerase activity with thelimited amounts of RNA polymerase present in transfected cells,we modified an existing transcription assay (8) by reducingthe concentration of GTP to 0.1 µM, thereby improvingits sensitivity. In this assay, a short synthetic 14-nt modelvRNA 3' end sequence, in the presence of a 15-nt 5' end vRNAsequence, was transcribed in the presence of ApG, which resultedin the incorporation of [ ${alpha}$ -³²P]GTP into a major 14-nt transcript,5' AGCAAAAGCAGGCC 3' (Fig. 1, lane 1, band a). A minor, 13-nttranscript (Fig. 1, lane 1, band b) was also present, presumablytranscribed from a 13-nt oligonucleotide (5' GCCUGCUUUUGCU 3')contaminating the 14-nt template (see Materials and Methods).No activity was detected if any of the three polymerase subunitswas omitted from the transient transfection (Fig. 1, comparelanes 1 to 4). This result confirmed that all three polymerasesubunits (PB1, PB2, and PA) were required for transcription.

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FIG. 1. ApG-primed transcription of 14-nt influenza virus vRNA model templates requires a RNA polymerase complex containing PB1, PB2, and PA. The results of in vitro transcription of a model 14-nt vRNA 3' sequence in the presence of an equimolar concentration (2.5 µM) of a model 15-nt 5' end sequence, evaluated using the [ ${alpha}$ -³²P]GTP incorporation assay with different preparations of RNA polymerase prepared in parallel from the indicated plasmids (see Materials and Methods) and fractionated by 20% PAGE in 7 M urea, are shown. a, 14-nt product; b, 13-nt product (see text).

Recombinant RNA polymerase requires both the 5' and 3' strands of the vRNA promoter for ApG-dependent transcriptional activity. Evidence that both the 5' and 3' ends of the influenza virusvRNA promoter are required for transcription by influenza virusRNA polymerase has been derived mainly from early in vitro studieswith RNA polymerase isolated from virus (7, 8). More recentstudies of recombinant RNA polymerase isolated from baculovirus(13) confirmed these findings. Nevertheless, before studyingheat inactivation of influenza virus RNA polymerase isolatedfrom nuclear extracts of transiently transfected 293T cells,it was necessary to confirm the vRNA promoter requirements forthe ApG-dependent [ ${alpha}$ -³²P]GTP incorporation assay that we proposedto use to monitor heat inactivation. Figure 2 shows the effectof adding the 15 nt from the 5' end, the 14 nt from the 3' end,or oligonucleotides from both the 5' and 3' ends of the vRNApromoter (see Materials and Methods) to an influenza virus RNApolymerase (prepared in the presence of NP) with or withoutendogenous influenza virus-like vRNA derived from the plasmidpPOLI-GFP. In the case of nuclear extracts prepared from 293Tcells cotransfected with pPOLI-GFP, in addition to transfectionwith expression plasmids for PB1, PB2, PA, and NP, we foundthat the RNA polymerase transcribed the endogenous vRNA withoutneeding any added vRNA 5' or 3' end nucleotides (Fig. 2, lane1). This result confirmed that the 5' and 3' ends of the influenzavirus vRNA promoter were supplied by the endogenous RNA transcribedby the host Pol I from the transfected plasmid, pPOLI-GFP.

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FIG. 2. Both 5' and 3' vRNA promoter arms are needed to initiate transcription by RNA polymerase prepared without endogenous influenza virus-like RNA. ApG-primed in vitro transcription using the [ ${alpha}$ -²P] GTP incorporation assay was performed in the presence of short oligonucleotides (0.2 µM) corresponding to the 5' end (15 nt), 3' end (14 nt), or both ends of wild-type vRNA as shown, and the polymerase was fractionated by 20% PAGE in 7 M urea. Lanes 1 to 4, RNA polymerase prepared from PB1, PB2, PA, NP, and pPOLI-GFP; lanes 5 to 8, RNA polymerase prepared from PB1, PB2, PA, and NP. a', 14-nt product AGCAAAAGCAGGGU, b', 13-nt product AGCAAAAGCAGGG derived from endogenous pPOLI-GFP; z, longer transcription products, estimated as 17 to 19 nt in length, derived from endogenous pPOLI-GFP; a, 14-nt major end product derived from the added 14-nt 3' end of wild-type vRNA; b, 13-nt products derived from added vRNA template (see text); c, 12-nt product AGCAAAAGCAGG, present in transcripts derived from both pPOLI-GFP and the added 14-nt 3' end of wild-type vRNA.

A major 14-nt partial transcription product, AGCAAAAGCAGGGU(Fig. 2, lane 1, band a'), was present in good yield under ourreaction conditions. We deduced the sequence of this product,firstly because it was present at the 14-nt position on thegel and migrated only slightly slower than a previously characterized(7) 14-nt product (Fig. 2, lane 1, band a), AGCAAAAGCAGGCC,derived from a known added 14-nt vRNA template (see Materialsand Methods and Fig. 2, lane 8). Secondly, the 14-nt partialtranscription product has the expected sequence derived fromthe plasmid pPOLI-GFP (see Materials and Methods). Thirdly,the 14-nt partial transcription product migrates marginallymore slowly than band a, despite its similar length, becauseof its higher G content (five versus four G residues), whichis consistent with the greater retardation effect of the presenceof G residues in dense denaturing polyacrylamide gels (35).Fourthly, it accumulates in high yields, because the next, or15th, residue to be incorporated is a G residue, after whicha G is not incorporated until residue 21 (see Materials andMethods for the sequence derived from the vRNA promoter of pPOLI-GFP).The reaction conditions, in which GTP is present at low concentrations(0.1 µM), should, therefore, favor its accumulation. However,other unknown factors may be responsible for the high yieldof this reaction product.

The addition of the 14-nt 3' end of the influenza virus vRNApromoter catalyzed the synthesis of a 14-nt transcription product(a) (Fig. 2, lane 3) that migrated slightly faster than the14-nt product (a') derived from pPOLI-GFP (see above). No significantimprovement in the yield of band a was observed if the 5' endof vRNA was present in addition (Fig. 2, lane 4). This confirmedthat the synthesis of the 14-nt product a in lanes 3 and 4 hadoccurred through the copying of the added 3' end in trans bythe RNA polymerase, which was bound to the endogenous vRNA promoter,as suggested before (30).

In contrast, influenza virus RNA polymerase prepared from nuclearextracts of 293T cells transfected with expression plasmidsfor PB1, PB2, PA, and NP but in the absence of pPOLI-GFP transcribedan added 3' end vRNA template only in the presence of an added5' end of vRNA (Fig. 2, lane 8). Neither the 5' nor the 3' endalone of vRNA stimulated transcription (Fig. 2, lanes 6 and7). However, when used at a 10-fold-higher concentration (2.5µM), weak (<10%) activity was reproducibly detectedwith the 3' end of the vRNA promoter alone (data not shown;see Discussion).

Overall, we conclude that both the 5' and 3' ends of the vRNApromoter are required for significant transcriptional activityin an ApG-dependent assay using a mammalian recombinant influenzavirus RNA polymerase prepared in the absence of endogenous influenzavirus-like vRNA promoter sequences.

Influenza virus vRNA promoter sequences protect the RNA polymerase from heat inactivation. Preliminary experiments with an RNA polymerase that expressedendogenous influenza virus-like vRNA, prepared by transfectionof 293T cells with the PB1, PB2, and PA expression plasmidsplus an NP-expressing plasmid and pPOLI-GFP, showed that transcriptionactivity was stable for 15 min at 40°C but became unstableat 45°C and higher temperatures (results not shown). Wethen tested if the heat stability of the RNA polymerase wasa function of the presence of NP alone or of NP and influenzavirus-like vRNA together. We prepared nuclear extracts of transientlytransfected 293T cells expressing PB1, PB2, and PA and eitherNP alone or NP with influenza virus-like vRNA derived from theplasmid pPOLI-GFP. This plasmid contains the promoter and 5'and 3' noncoding regions of influenza A/WSN/33 virus segment8, controlling the expression of a negative-sense green fluorescentprotein. After 15 min of heat treatment at 40°C, the activityof the polymerase was evaluated with the [ ${alpha}$ -³²P]GTP incorporationassay in the presence of added 15- and 14-nt oligonucleotidesderived from the 5' and 3' ends, respectively, of the influenzavirus vRNA promoter (see Materials and Methods). Only the RNApolymerase reconstituted in the presence of influenza virus-likevRNA derived from the plasmid pPOLI-GFP was heat stable (Fig.3; compare lanes 7 and 8), thus giving rise to the major 14-nttranscript (band a) and minor transcripts b and c (see legendto Fig. 3). In addition, the previously detected 14-nt partialproduct, band a' (5' AGCAAAAGCAGGGT 3'), derived from pPOL1-GFP(see Fig. 2), accumulated preferentially. RNA polymerase, derivedeither from PB1, PB2, and PA or from PB1, PB2, PA, and NP, washeat labile (Fig. 3, lanes 4 and 6). Unheated controls (Fig.3, lanes 3, 5, and 7) showed the expected major 14-nt transcriptionproduct. Thus, it was likely that the vRNA promoter was criticalin protecting the polymerase against heat inactivation, althoughthe influence of other nucleotides within the 5' and 3' noncodingregions, and the possibility that the NP cooperated with vRNA,could not be excluded at this stage.

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FIG. 3. Heat stability of the influenza virus RNA polymerase. The results of assays of the heat stability of the RNA polymerase are shown. RNA polymerase, isolated from 293T cells transfected with the plasmids as indicated, was preincubated for 15 min at 40°C (even-numbered lanes) or not preincubated (odd-numbered lanes), evaluated by the [ ${alpha}$ -³²P]GTP incorporation assay in the presence of added 5' and 3' ends (each at 5 µM) of the vRNA promoter, and fractionated by 20% PAGE in 7 M urea. a', 14-nt partial product AGCAAAAGCAGGGT, derived from the RNA of plasmid pPOLI-GFP; a, 14-nt product, derived from the added 14-nt 3' end of wild-type vRNA; b, 13-nt minor transcript, probably derived from a contaminating 13-nt vRNA template; c, 12-nt product (see legend to Fig. 2). The nuclear extracts (see Materials and Methods) used in lanes 1 and 2 were prepared from mock-transfected (no added plasmids) 293T cells.

Next we tested whether the addition of the 5' and 3' ends ofthe vRNA promoter to the RNA polymerase prior to heat treatmentwould give the same protective effect in vitro. Figure 4 showsthat when the 15- and 14-nt oligonucleotides (see Materialsand Methods), corresponding to the 5' and 3' ends, respectively,of the vRNA promoter, were added to the RNA polymerase (reconstitutedwithout influenza virus-like vRNA or NP) prior to heat treatment,the enzyme was protected against heat inactivation when evaluatedin the [ ${alpha}$ -³²P]GTP incorporation assay. Significant protectionrequired the presence of both oligonucleotides (Fig. 4, lane4), although a very minor level of protection was reproduciblyobserved with the 3' end alone (see lane 3 and Discussion).No protection was seen with the 5' end alone (Fig. 4, lane 2).Since the 15- and 14-nt vRNA promoter oligonucleotides containonly the consensus promoter sequences plus two additional basepairs, this suggests that the segment 8-specific 5' and 3' noncodingregions present in pPOLI-GFP and the NP were unlikely to havecontributed to the heat stability of the polymerase observedin vivo (Fig. 3, lane 8). Overall, we conclude that both the5' and 3' ends of vRNA are required to protect the influenzavirus RNA polymerase against heat inactivation.

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FIG. 4. Both the 5' and 3' ends of the vRNA promoter are needed to obtain significant protection of the RNA polymerase from heat inactivation. RNA polymerase, obtained by transfection with PB1, PB2, and PA, was heated for 15 min at 40°C in the presence of the indicated oligonucleotides (5 µM), and transcription was subsequently evaluated with the [ ${alpha}$ -³²P]GTP incorporation assay with ApG in the presence of both 5' and 3' vRNA oligonucleotides (2.5 µM). Lane 1, no added RNA; lane 2, 5' vRNA strand (15 nt); lane 3, 3' vRNA strand (14 nt); lane 4, 5' plus 3' strand; lane 5, unheated control; a, 14-nt product; b, 13-nt minor product; c, 12-nt minor product.

Base pairing between the 5' and 3' strands of the vRNA promoter is needed to protect the RNA polymerase from heat inactivation. By introducing single or multiple mutations between residues10 and 14 of the 3' end of the vRNA promoter, thereby disruptingone to three base pairs of the panhandle, we tested whetherboth the 5' and 3' ends of the vRNA promoter were needed toprotect the influenza virus RNA polymerase from heat inactivation.Then, using a previously established methodology (8), we complementedthe mismatched base(s) by mutating the appropriate base(s) ofthe 5' end of the vRNA promoter to reform alternative base pairs.Figure 5A shows the wild type and 10 mutant panhandle structurestested. Overall, the activity levels of the mismatched constructsremaining after heat inactivation, as evaluated by the [ ${alpha}$ -³²P]GTPincorporation assay, ranged from 1 to 9% of the wild-type activitylevel (Fig. 5A). A potential complication is the need to ensurethat the ApG-dependent transcription assay can effectively measurethe polymerase activity of mutants, which are known from previouswork to be inactive or only minimally active in transcription(7, 8). Thus, we performed control experiments in which we showedthat, prior to testing by the [ ${alpha}$ -³²P]GTP incorporation assay,wild-type vRNA promoter sequences efficiently displaced thesemismatched constructs from the polymerase. These results (datanot shown) revealed that the wild-type promoter used in theApG transcription assay can displace any potential binding ofthese mismatched promoter sequences to the polymerase. Thisconfirms that the low activities observed after heat inactivationin the presence of the mismatched panhandle constructs (Fig.5A, sequences 2, 4, 6, 8, and 10) were valid results and werenot an artifact of the testing procedure. Significantly, inthe case of mutants 3, 5, 7, 9, and 11, for which we complementedmismatched base pairs with either single, double, or triplebase pairs, we were able to recover activity levels rangingfrom 47 to 104% (Fig. 5A) of the wild-type activity level. Figure5B shows a typical result for those mutants (lanes 1, 3, 5,7, 9, and 11). It may be noted (Fig. 5B) that because of theirdiffering sequences, the mutant transcripts (bands a₁ to a₅),although all were 14 nt in length, differed slightly from oneanother and from the wild-type transcript (band a) in theirgel mobility, as expected from previous work (7, 8). Furthercontrol experiments (data not shown) confirmed that in the caseof the base pair mutants which provided significant protectionagainst heat inactivation (Fig. 5B, mutants 3, 5, 7, 9, and11), the results obtained were independent of the addition ofwild-type vRNA promoter sequences. This confirmed that the vRNApromoters for these particular mutants remained bound to thepolymerase and, under our experimental conditions, were notsignificantly displaced by the inclusion of wild-type vRNA promotersequences in the ApG-dependent transcription assay which wasused as a readout of polymerase activity.

The fact that disruption of even one base pair of the panhandleinterfered with its ability to protect the polymerase in theheat inactivation assay, coupled with the fact that alternativebase pairs partially or completely restored protection, arguesstrongly in favor of a role for the panhandle in polymeraseprotection.

Hairpin loops in both the 5' and 3' strands of the vRNA promoter are needed to protect the RNA polymerase from heat inactivation. As the panhandle structure protected the RNA polymerase fromheat inactivation, it was of interest to establish whether protectionrequired a panhandle with both a 5' and a 3' hairpin loop, asin the corkscrew model, or whether only a 5' hairpin loop wasneeded, as in the 5' hairpin loop model (33).

To address this question, we initially used mutant 15-nt-longoligonucleotides of the 5' strand of vRNA with point mutationswithin the 5' hairpin loop. These were tested in the presenceof 14-nt 3'-strand oligonucleotides with wild-type 3' hairpinloop sequences. Four mutants were designed for disruption ofbase pairs in the stem of the 5' hairpin loop (Fig. 6 mutants12, 13, 15, and 16 at positions 2', 3', 8', and 9', respectively).However, one mutant, mutant 14, had a G $->$ U mutation at position5' of the hairpin loop. All 5'-strand mutants had an additional12' G $->$ U panhandle mutation, because they had been originallydesigned for other experiments. However, this additional 12'G $->$ U mutation in the panhandle did not interfere in the experimentalapproach, because the 5' mutants were complemented with a 14-ntmutant 3' strand containing an A $->$ U base pair mutation at position11. This mutant 3' strand restored base pairing with the 5'mutant strand in the panhandle, giving rise to an 11, 12' A $->$ Ubase pair that was known to be 47% as effective as wild-typevRNA (see Fig. 5A, sequence 3) in protecting the RNA polymeraseagainst heat inactivation. We used the heat inactivation assay,in the presence of added pseudo-wild-type vRNA promoter (seeMaterials and Methods) to provide an active vRNA template formutants that were known to be inactive in transcription, fortesting the remaining polymerase activity in the [ ${alpha}$ -³²P]GTP incorporationassay (7). Figure 6 shows that only mutant 14 significantlyprotected against heat inactivation. All of the other mutants,i.e., mutants 12, 13, 15, and 16, failed to give a convincinglevel of protection above that of a control with the 3' strandof the vRNA promoter alone (Fig. 6, lane labeled 3'). Theseresults suggest that base pairs in the stem of the 5' hairpinloop are important in protection against heat inactivation ofthe influenza virus RNA polymerase.

To establish if the hairpin loop in the 3' end of the vRNA promoteris required to protect against heat inactivation, we testedpoint and multiple mutations of the 14-nt 3' end of vRNA (Fig.7) in the presence of the 15-nt wild-type 5' strand of vRNAto evaluate activity in the subsequent ApG-primed [ ${alpha}$ -³²P]GTPincorporation assay, again in the presence of wild-type vRNApromoter sequences (see above). Initially we selected mutants18 and 19 (see Fig. 7A) because these mutants, with multiplesequence changes from wild-type vRNA, are known to inhibit ApG-primedtranscription (8). Mutant 17, with a triple mutation in thehairpin loop, was selected as a potential positive control,since it is known to retain full transcriptional activity (8).The results (Fig. 7B) suggest that the 3' hairpin loop was requiredto protect against heat inactivation. If both base pairs inthe stem of the loop were disrupted (as for mutant 18), theactivity level was no higher than that of a control (Fig. 7B,3') in which inactivation was carried out in the presence ofthe 3' strand of the promoter alone. If only one base pair wasretained (as for mutant 19), slight residual activity was apparent(Fig. 7B). However, when both base pairs of the stem were present(as for mutant 17), significant protection was observed.

To confirm the hypothesis that the 3' hairpin loop was requiredto protect against heat inactivation, five further point andmultiple mutants were selected. These differed from the mutantsselected above in that they were all known to be as active,or nearly as active, as the wild type in ApG-primed transcription(8). The results (Fig. 7C) suggest that none of the mutantsefficiently protected against heat inactivation, except mutants21 and 22, which showed partial protection. Interestingly, thesetwo mutants retained one of the two base pairs of the 3' hairpinloop, possibly suggesting that some secondary structure resemblingthe 3' hairpin loop was forming. Mutant 20 (9, G $->$ C) (Fig. 7C),however, showed no evidence of significant protection.

Overall, we conclude that both the 5' and 3' hairpin loops ofvRNA appear to be important if vRNA is to efficiently protectthe influenza virus RNA polymerase against heat inactivation.

DISCUSSION

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Discussion

The RNA polymerase of influenza virus is tightly bound to theviral RNA promoter and packaged with NP into eight ribonucleoproteincomplexes. In the infected cell, however, the RNA polymeraseis present both as free or partially assembled polymerase, intransit from its site of synthesis in the cytosol to the nucleus,and as bound polymerase, complexed to the vRNA or the cRNA promoterin the nucleus. However, most of the time the polymerase wouldbe expected to be bound to either the influenza virus vRNA orcRNA promoter. The purpose of this study was to test whetherthe vRNA promoter was involved in stabilizing the enzyme, andif so, what sequence and secondary structure features of thispromoter were required.

To address these questions, it was first necessary to isolaterecombinant RNA polymerase in the absence of influenza viruspromoter sequences. A plasmid-based, transient expression systemin human kidney 293T cells was devised. This system was a modificationof the 12-plasmid transfection procedure devised to rescue novelinfluenza viruses (5, 27). Although other influenza virus RNApolymerase expression systems are described in the literature,they all had significant disadvantages for our purposes. Baculovirus(13) or Pichia (16) expression vectors were not used, becauseinsect or yeast cells may not express an RNA polymerase withthe correct posttranslational modifications (e.g., phosphorylation)present in naturally infected avian or mammalian cells. Suchmodifications may be required for polymerase binding to hostfactors such as RNA polymerase II or hCLE, which might regulateinfluenza virus replication (6, 15). Vaccinia virus expressionin HeLa cells was not attempted, because RNA polymerase isolatedby this approach supported only limited transcription (12).However, a recently described affinity method for partiallypurifying vaccinia virus-expressed influenza virus RNA polymerasefrom HeLa cells looks more promising (22). Finally, transfectionof plasmids into vaccinia virus T7 polymerase-infected COS-1cells was inappropriate, because active RNA polymerase couldnot be isolated in the absence of endogenous influenza virus-likevRNA (28).

Characterization of the recombinant RNA polymerase. We initially demonstrated that the isolated recombinant RNApolymerase is transcriptionally active by using ApG as a primerin the presence of two short synthetic oligonucleotides containingthe 5' and 3' ends of the vRNA promoter (Fig. 1). Transcriptionalactivity was dependent on the presence of all three subunitsof the RNA polymerase, which is a result consistent with previousplasmid reconstitution experiments (29) but inconsistent withother studies which suggested that all three subunits are notrequired (18, 25, 26). Perhaps all three subunits are neededfor efficient synthesis but residual activity, below our levelof detection, can occur with short model vRNA templates in theabsence of one or more subunits. Although NP is essential forin vivo replication of influenza virus RNA (2, 14), the presenceof NP was not required for the synthesis of short 14-nt transcripts(Fig. 1), confirming previous studies with short RNA templates(12, 13, 16, 22). Recombinant RNA polymerase prepared by ourmethod also required, as expected (7, 8, 13), both the 5' and3' ends of the vRNA promoter for significant transcriptionalactivity (Fig. 2). Moreover, the recombinant RNA polymerasehad the other expected properties of an authentic RNA polymerase,e.g., priming with capped (globin mRNA) primers, endonucleaseactivity, and poly(A) polymerase activity (data not shown).Interestingly, we were able to directly demonstrate (Fig. 2)that the RNA polymerase, which bound to an endogenous vRNA promoter,copied an added 3' end of the vRNA promoter in trans, thus confirmingearlier suggestions that this could occur (30).

Influenza virus vRNA promoter in a corkscrew configuration is needed to protect the RNA polymerase from heat inactivation. Having isolated recombinant RNA polymerase, we investigatedits stability with respect to heat. The RNA polymerase was essentiallystable during 15 min of heat treatment at 40°C in the presenceof endogenously synthesized influenza virus vRNA sequences andNP. In the absence of such influenza virus-like vRNA sequences,the polymerase was completely inactivated (Fig. 3). Protectionagainst heat inactivation was restored when the vRNA promoterwas added prior to heat treatment. Protection by the vRNA promoterrequired both the 5' and 3' ends of the vRNA promoter, althougha minor level of protection reproducibly occurred in the presenceof the 3' end of the vRNA promoter alone (Fig. 4). In high concentrations,the presence of the 3' end of the vRNA promoter alone also stimulatedthe synthesis of low yields of transcripts in the ApG-dependenttranscription assay. Because the RNA polymerase enzyme preparationwas impure, we could not determine whether this low-level activityof the 3' end of the vRNA promoter is an intrinsic propertyof the RNA polymerase itself or whether it results from thepresence of cryptic influenza virus 5' vRNA or influenza virus-mimickingvRNA species, e.g., small nuclear RNAs, that might contaminatethe RNA polymerase preparation. Contamination with 5' vRNA promotersequences would seem to be the more likely scenario, however,since all presently available evidence suggests that both the5' and 3' ends of the vRNA promoter are needed for efficientpromoter activity, although the presence of the 5' end of thevRNA promoter alone is sufficient for cap binding (1). Contaminationwith 5' vRNA promoter sequences is certainly possible, becausethe 3' noncoding regions of the mRNA for PB1, PB2, and PA containantisense 5' vRNA promoter sequences derived from their pcDNA3expression plasmids (see Materials and Methods). If these expressionplasmids were transcribed by the host RNA polymerase from acryptic promoter in antisense orientation, albeit inefficiently,this transcription would result in the generation of 5' vRNAinfluenza virus promoter sequences, which could explain whywe observed low-level transcription in the presence of an added3' end of vRNA alone.

Moreover, 5'- and 3'-end promoter mutations that disrupted basepairs between the 5' and 3' ends of the vRNA promoter, thuspreventing or interfering in the stability of the panhandle,abolished protection (Fig. 5). However, complementary mutations,which reformed alternative base pairs in the panhandle, restoredprotection partially, or in some cases completely, even althoughdifferent base pairs were now present (Fig. 5). Overall, itwas clear that significant protection against heat inactivationrequired both the 5' and 3' ends of the vRNA promoter, heldtogether in a panhandle configuration. Other results (data notshown) suggested that influenza virus cRNA promoter sequencesalso protected the RNA polymerase against heat inactivation.Again, both the 5' and 3' strands of the cRNA promoter wererequired for significant protection.

Next, we investigated whether the local hairpin loops in the5' and 3' arms of the vRNA promoter had to be present in vRNAto protect the RNA polymerase against heat inactivation. Asexpected, given the importance of the 5' hairpin loop for binding,endonuclease cleavage, transcription, and polyadenylation (7-9,17, 21, 30, 34), mutations which disrupted either of the basepairs in the stem of the 5' hairpin loop failed to protect theRNA polymerase, whereas a mutation in the hairpin loop itselfstill protected against heat inactivation (Fig. 6).

The role of the 3' hairpin loop has been more controversialthan that of the 5' hairpin loop, but recent evidence has confirmedits importance for endonuclease cleavage (20), despite earlierindications that it was not required for ApG-primed transcriptionand polyadenylation (8, 33). Here we found that mutations ofthe stem of the 3' hairpin loop of the promoter convincinglyshowed that this secondary structure feature was required forefficient protection of the polymerase against heat inactivation.All mutants which disrupted both base pairs of the stem failedto confer protection. By contrast, a triple mutation at positions5, 6, and 7 within the hairpin loop itself was tolerated (Fig.7). However, a detailed study of three position 9 mutants showedthat partial protection occurred in two of the mutants whenonly one of the base pairs of the stem was disrupted. This resultsuggests that the role of the 3' hairpin loop may be somewhatless critical than that of the 5' hairpin loop. Some degreeof relaxation of secondary structure is still compatible withpartial protection against heat inactivation of the polymerase.Our heat inactivation data are thus consistent with the hypothesisthat the 3' hairpin loop is a transient structure which hasto melt before transcription can be initiated (20). Thus, the3' hairpin loop may aid binding to the polymerase but its presenceis not as critical as that of the 5' hairpin loop, which mustremain bound to the template for correct polyadenylation (7,8, 30, 33, 34, 37).

It would be interesting to extend the present studies on heatinactivation of the influenza virus RNA polymerase by investigatingwhether the vRNA secondary structure requirements for conferringprotection against heat inactivation would differ if an activityof the polymerase other than ApG-dependent transcription wereused as a readout assay. Thus, an endonuclease cleavage assay(21) or mRNA poly(A) assay (34) might give different resultsfrom those of the ApG-dependent transcription assay used inthis study, e.g., for mutations which disrupt the stem of the3' hairpin loop of vRNA, as discussed above. However, to investigatethis fully would require a detailed study that is beyond thescope of this paper.

General implications. It is well known that the vRNA promoter binds strongly to thepolymerase. Indeed, progress has been made in localizing theamino acid residues in PB1 involved in the various interactions(10, 11, 23). However, to our knowledge, there have been noprevious experimental data showing that the vRNA promoter stabilizesthe RNA polymerase and protects it against heat inactivation.In the absence of the vRNA promoter, we envisage a polymerasestructure that is open and labile to heat inactivation. In thepresence of the promoter, the polymerase structure might beclosed and more stable. Since the RNA polymerase has to surviveinside the infected cell throughout the infection, a polymerasecomplex with the maximum possible half-life could have evolvedto give the virus a selective advantage. It would be interestingto test if avian influenza viruses, which replicate at the temperaturesfound in birds (i.e., higher than those found in mammals), haveRNA polymerases which are more heat stable. It would also beinstructive to test whether pathogenic human influenza viruseshave polymerases that exhibit greater heat stability than lesspathogenic strains. Such a property of human viruses could allowreplication at the higher temperatures found in the lung, thusfacilitating the development of pneumonia, whereas the lesspathogenic influenza viruses, because of their heat lability,might be confined to the lower temperatures of the upper respiratorytract.

We have not studied the mechanism of heat inactivation of theRNA polymerase. One possibility, however, is that the PA subunitis bound more weakly than the other subunits to the polymerasecomplex and dissociates when subjected to heating, leaving aninactive, dimeric complex. This hypothesis is supported by previousobservations that PA is less stably associated with PB1 andPB2 in a recombinant polymerase complex, formed in the absenceof influenza virus vRNA, than in a complex isolated from influenzavirus-infected cells (3, 36).

In conclusion, we have found that the presence of both the 5'and 3' hairpin loops of vRNA in the corkscrew model (4) appearsto be important if vRNA is to efficiently protect the influenzavirus RNA polymerase against heat inactivation. This resultis further evidence of the importance of the panhandle and the5' and 3' hairpin loops of the vRNA promoter in providing thenecessary secondary structure to ensure tight binding of thepromoter to the polymerase as a prelude to endonuclease cleavage,transcription, and polyadenylation.

ACKNOWLEDGMENTS

G.G.B. and J.L.S. were supported by MRC program grant G9523972to G.G.B.

We thank Peter Palese for the pPOLI-GFP, Ervin Fodor and MandyCrow for helpful discussions, and Alice Taylor for DNA sequencing.

FOOTNOTES

* Corresponding author. Mailing address: Chemical Pathology Unit, Sir William Dunn School of Pathology, University of Oxford, S. Parks Rd., Oxford OX1 3RE, United Kingdom. Phone: 44 1865 275559. Fax: 44 1865 275556 E-mail: George.Brownlee{at}path.ox.ac.uk.

REFERENCES

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