Given the Opportunity, the Sendai Virus RNA-Dependent RNA Polymerase Could as Well Enter Its Template Internally

JOURNAL OF VIROLOGY, Aug. 2002, p. 7987–7995 0022-538X/02/$04.00⫹0 DOI: 10.1128/JVI.76.16.7987–7995.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved. Vol. 76, No. 16 Given the Opportunity, the Sendai Virus RNA-Dependent RNA Polymerase Could as Well Enter Its Template Internally Diane Vulliémoz and Laurent Roux* Department of Genetics and Microbiology, University of Geneva Medical School, 1211 Geneva 4, Switzerland Received 18 January 2002/Accepted 15 May 2002 Viral RNA synthesis on negative-stranded RNA virus infection consists of two distinct processes: genome replication and transcription of the viral mRNAs. Replication initiates at the very 3⬘-OH end of the viral RNA and proceeds via the synthesis of its full complement, the antigenome, which in turn serves as the template to amplify the viral genome. The genomic promoter (GP) and antigenomic promoter (AGP) for replication of the genome and the antigenome, respectively, are positioned at the RNA 3⬘ end of the genome and the antigenome, respectively (for general information, see reference 23). For Sendai virus, a member of the Paramyxoviridae family with a nonsegmented genome of 15,384 nucleotides, the replication promoters are within the first 96 nucleotides (34). While the GP and AGP have the same gross primary structure organization, only the first 12 nucleotides of the promoters are identical. With the possible exception of members of the Pneumovirus genus (e.g., respiratory syncytial virus), the replication promoters of all members of the Paramyxoviridae family are composed of two discontinuous elements (graphically portrayed in reference 36). The first 20 to 30 nucleotides most proximal to the 3⬘ end are fairly conserved among members of the same genus and are likely to contain the signal for RNA synthesis initiation. The second element stretches into the internal extremity of the promoter. A three-times-repeated motif common to the respiro- and morbilliviruses [(CNNNNN)3, nucleotides 79 to 96] and the rubulaviruses [(NNNNGC)3, nucleotides 77 to 94] is absolutely required in number and location for replication to occur (26, 27, 34). By use of a reverse genetic system that recently became available for negative-stranded RNA viruses (1, 4, 8, 11, 13, 16–18, 20, 24, 29, 30, 37), the Sendai virus GP and AGP were found to perform differentially, in that a minireplicon under the control of the AGP replicated ⬃20-fold more efficiently than that controlled by the GP (7, 35). This difference in promoter strength depended on the promoter’s primary sequence, since reciprocal exchanges of the first 30 nucleotides between the two promoters was sufficient to adjust their replication efficiencies (7). Finally, the amplitude of the difference in promoter strength was found to depend on the expression of the accessory C proteins (10), which appear to specifically restrict replication at the GP (5, 33). For Sendai virus, transcription initiates 56 nucleotides away from the 3⬘ end of the viral RNA in a region of about 10 nucleotides found repeated at each mRNA start site. Once initiated, transcription proceeds through the sequential synthesis of capped and polyadenylated messengers via a stop-restart signal recognition mechanism (for general information, see reference 23). It is generally accepted that the transcription promoter overlaps the genomic replication promoter, although it is clear that certain portions of the primary sequence must be devoted to only one process, e.g., the transcription start signal, devoted to transcription, or the first 12 to 30 nucleotides, which are interchangeable between the GP and AGP and required for replication. How the viral RNA-dependent RNA polymer- * Corresponding author. Mailing address: Department of Genetics and Microbiology, University of Geneva Medical School, CMU, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland. Phone: (44 22) 702 56 83. Fax: (44 22) 702 57 02. E-mail: Laurent.Roux@medecine.unige.ch. 7987 Downloaded from http://jvi.asm.org/ on July 24, 2015 by guest The negative-stranded RNA viral genome is an RNA-protein complex of helicoidal symmetry, resistant to nonionic detergent and high salt, in which the RNA is protected from RNase digestion. The 15,384 nucleotides of the Sendai virus genome are bound to 2,564 subunits of the N protein, each interacting with six nucleotides so tightly that the bases are poorly accessible to soluble reagents. With such a uniform structure, the question of template recognition by the viral RNA polymerase has been raised. In a previous study, the N-phase context has been proposed to be crucial for this recognition, a notion referring to the importance of the position in which the nucleotides interact with the N protein. The N-phase context ruled out the role of the template 3ⴕ-OH congruence, a feature resulting from the obedience to the rule of six that implies the precise interaction of the last six 3ⴕ-OH nucleotides with the last N protein. The N-phase context then allows prediction of the recognition by the RNA polymerase of a replication promoter sequence even if internally positioned, a promoter which normally lies at the template extremity. In this study, with template minireplicons bearing tandem replication promoters separated by intervening sequences, we present data that indeed show that initiation of RNA synthesis takes place at the internal promoter. This internal initiation can best be interpreted as the result of the polymerase entering the template at the internal promoter. In this way, the data are consistent with the importance of the N-phase context in template recognition. Moreover, by introducing between the two promoters a stretch of 10 A residues which represent a barrier for RNA synthesis, we found that the ability of the RNA polymerase to cross this barrier depends on the type of replication promoter, strong or weak, that the RNA polymerase starts on, a sign that the RNA polymerase may be somehow imprinted in its activity by the nature of the promoter on which it starts synthesis. 7988 VULLIÉMOZ AND ROUX J. VIROL. and Neubert and colleagues (28). Whenever explicit, the viral sequence is written as DNA according to standard convention, 5⬘ to 3⬘, as the positive strand. The plasmids expressing the Sendai virus N protein (pGem-N), P and C proteins (pGem-P/Cwt), P without the C proteins (pGem-P/Cstop), and the L protein (pGem-L) under the control of the T7 RNA polymerase promoter have been described previously (6, 33). The structure of the minireplicon Sendai virus DI-H4-(⫹)⌬DRA/AGP96 (referred to as pSV DIH4-⌬96, construct 1) and its cloning into pSP65 have been described (34). As before (35), the 3⬘ ends of the viral genome and antigenome contain the genomic (GP) and antigenomic (AGP) promoters, respectively. In the case of the H4 RNA, which harbors the AGP on both the minus- and plus-strand RNA, AGPL or AGPR, respectively, denote these two promoters (see Fig. 1A). In all derivatives used in this study, the 3⬘ end of the H4 minus-strand RNA containing AGPL was never modified. Most of the plasmids used here were derived from two different constructs containing two promoters in tandem at the 3⬘ end of the antigenome, Sendai virus DI-H4/AGP-GP and Sendai virus DIH4/AGP-AGP, described previously (36), and are referred to in this study as AGPint-GPext (construct 3) and as AGPint-AGPext (construct 10), respectively. The site-directed base substitution (G91C) was introduced in either promoter of constructs 9 and 13 by fusion PCR with adequate overlapping oligonucleotides at the inner modification sites and external primers containing the MunI site in the minireplicon sequence and the BamHI site at the 3⬘ end of the ribozyme sequence (see Fig. 3A) (36). The opening of an XhoI restriction site introduced between the adjacent promoters by fusion PCR (constructs 4 and 11) (36) allowed the introduction of gradually increasing DNA fragments originating from the Sendai virus M gene to gradually extend the distance between the two promoters. Fragments of 42, 84, 120, 162, and 240 nucleotides were prepared by PCR amplification of portions of the Sendai virus M gene (accession number X53056), comprising nucleotides 595 to 823, with primers flanked by XhoI sites (constructs 5, 6, 7, 8, 9, 12, and 13). The introduction of the stretch of 10 A’s (10A stretch) in construct 9 to generate construct 9A10 was done by fusion PCR. The 240(A10) cassette of 9A10 was then removed by XhoI digestion and introduced in the XhoI site to generate constructs 9A10GP(G91C), 13A10, 13-A10Ex(G91C), and 13-A10In(G91C). The 10A stretch preceding either AGP or GP in constructs 1A10 and 2A10 was introduced by cloning a 10A cassette (5⬘-GTGAAAAAAAAAAGCTCACACTGG-3⬘) flanked with two DraIII sites into the unique DraIII site. The portions of the constructs that had undergone modifications were all extensively sequenced with the Licor DNA 4000 automated sequencing apparatus. Replication system. The intracellular replication system has been extensively and repeatedly described before (6, 35, 36). Its main features are illustrated once more in Fig. 1A and commented on in the first section of the Results. All the replication assays were performed with the P/Cstop plasmid, i.e., in the absence of the C proteins, except for the experiment presented in Fig. 1D, as indicated in the figure legend. RNA analysis. The encapsidated RNA (minireplicon) resulting from replication was analyzed by Northern blotting with a 32P-labeled riboprobe of positive polarity described before (5⬘ex-probe) (25). The in vitro T7 RNA transcripts produced from plasmids 1, 1A10, 2, and 2A10 (Fig. 3) were synthesized in standard conditions. They were probed with a 32P-labeled Sp6 transcript of negative polarity produced from plasmid 1 linearized at the DraIII site downstream of the 10A stretch so that the probe only scored the T7 RNA transcripts that crossed the 10A stretch (see Fig. 3A). Primer extensions, used marginally in this study to verify the precise initiation of the replicated RNA (see Fig. 4C), were performed as previously described (36). Reverse transcription (RT)-PCR amplification was performed after a further purification of the replicated minireplicon RNA through a 5.7 M CsCl cushion to get rid of possible plasmid contamination. Moloney murine leukemia virus reverse transcriptase (Promega) and Taq polymerase (Gibco-BRL Life Technologies) were used in standard conditions with appropriate primers to obtain a 336-nucleotide-long amplified product spanning the 10A stretch region (see Fig. 2D). MATERIALS AND METHODS RESULTS Virus and cells. A549 cells and HeLa cells were grown in regular minimal essential medium (MEM) and in Dulbecco’s modified medium (DMEM) supplemented with 5% fetal calf serum in a 5% CO2 atmosphere, respectively. Recombinant vaccinia virus expressing the T7 RNA polymerase, vTF7-3, a gift from Bernard Moss (National Institute of Health, Bethesda, Md.), has been described by Fuerst and colleagues (14) and used accordingly. vTF7-3 stocks were grown in HeLa cells, with titers around 5 ⫻ 108 PFU/ml. Sequence and plasmids. The complete Sendai virus RNA primary sequence (15,384 nucleotides) was taken from the work of Shioda and colleagues (31, 32) Minireplicon replication system. The replication system used in this study has been used successfully previously (6, 7, 33–36). It roughly corresponds to the method classically developed to replicate minireplicons of the other negative-stranded RNA viruses (for reviews, see references 3, 9, and 15) and is similar to that which allowed the recovery of infectious Sendai virus from cDNA (16). This method is based on a multiplasmid Downloaded from http://jvi.asm.org/ on July 24, 2015 by guest ase (RdRp) is committed to replication or transcription is still a matter of debate and research. The active genome of negative-stranded RNA viruses is not the naked RNA but the nucleocapsid (NC), i.e., an RNAnucleocapsid protein (N) complex of helicoidal symmetry. Although the structure at atomic resolution of this nucleocapsid has yet to be determined, the tightness of the RNA-N protein complex varies among the virus families. The resistance to high-salt conditions and/or to equilibrium density centrifugation (23), as well as the accessibility of the bases to watersoluble reagents and their ability to base pair (2, 19, 21a), are criteria that allow this differentiation. Compared to the orthomyxoviruses (e.g., influenza virus) and the rhabdoviruses (e.g., vesicular stomatitis virus), the paramyxoviruses (e.g., Sendai virus) appear to exhibit the highest binding of the RNA and N (19a). The interaction of the N protein with six nucleotides possibly relates to the fact that the rule of six applies to this family (12). This rule stipulates that the total number of nucleotides in the genome must be a multiple of six for efficient genome replication (6). Importantly, the rule implies that the nucleotides are seen by the viral RdRp in the context of the nucleocapsid (N) protein (22). Recently, the importance of the N protein phase context received experimental support with the demonstration that recognition by the viral RdRp of the replication promoters depended on the position of the nucleotides vis-à-vis the six N protein interaction sites (36). This result was obtained with minigenomes carrying two promoters in tandem separated by six nucleotides of intervening sequence that allowed changing the N-phase context of the inner promoter while keeping the original N-phase context of the external promoter. Despite efficient replication from the external promoter, the internal promoter was not used under these conditions. This contrasted with the use of both promoters when the inner promoter’s original N-phase context was conserved as well. More than supporting the importance of the N-phase context for promoter recognition, these experiments raised questions about the mechanism of template recognition by the viral RdRp. For instance, when replication initiation took place at the internal promoter, was the viral RdRp entering the template at the internal promoter location or was it initiating internal RNA synthesis by scanning from the template extremity? In this study, we address this question and we provide data that support entry at the internal promoter as well. Moreover, by confronting the viral RdRp with what appears to be an elongation barrier, we provide evidence for an imprinting of the viral RdRp by the type of promoter (GP or AGP) on which it first starts. The importance of these results to our understanding of the mechanisms of viral RNA synthesis is discussed. VOL. 76, 2002 TEMPLATE ENTRY OF PARAMYXOVIRUS RNA POLYMERASE 7989 transfection of cells (Fig. 1A) previously infected with a vaccinia virus recombinant expressing the T7 RNA polymerase (vTF7-3). The minireplicons used here were derived from a naturally amplified copyback-defective interfering RNA which originally harbored the antigenomic replication promoter (AGP) at the 3⬘ end of both the positive and the negative strands. The template plasmids are designed so that the T7 RNA transcript produced is of positive polarity and contains the exact viral 5⬘ and 3⬘ natural extremities due to the adequate positioning of the T7 RNA promoter and the presence of the hepatitis virus delta ribozyme (Fig. 1A, diagram b) (6). The T7 transcript is then encapsidated by the N protein (Fig. 1A, diagram c), and as such becomes a template for the Sendai virus RdRp. The viral RdRp now copies this template and generates an encapsidated RNA of negative polarity that becomes the hallmark of replication (Fig. 1A, diagram d). At the end of the reaction (generally 42 h posttransfection or postinfection), the encapsidated RNA is purified and analyzed by Northern blotting or primer extension, with scoring only for the RNA of negative polarity (Fig. 1A, diagram e) (for details, see Materials and Methods) (36). In the present study, the promoter manipulations were performed exclusively at the 3⬘ end of the positive-strand RNA (AGPR, Fig. 1A, diagram c), leaving the 3⬘ end of the negativestrand RNA (AGPL) unaltered. In consequence, the replication potential of the minigenomes was only differentially affected by the promoter (or the double promoters) of the positive-strand RNAs. Multiple rounds of replication with the minus-strand minireplicon (and AGPL) as the template were in principle nondiscriminative, since all the minus-strand minireplicons harbored identical AGPs. Internal initiation is independent of the length of intervening sequence between the two promoters. In our previous study (36), the two promoters in tandem, in the configuration AGPint-GPex (Fig. 1B, construct 3), were adjacent or separated by at most six nucleotides (construct 4). To verify whether the use of the internal AGP (AGPint) was dependent on the proximity of the external GP (GPext), the intervening distance between the two promoters was sequentially increased from 6 to Downloaded from http://jvi.asm.org/ on July 24, 2015 by guest FIG. 1. Replication of the AGPint-GPext constructs. (A) Schematic representation of the replication system. For a detailed description, see Results. NoP and PE refer, respectively, to the Northern blot riboprobe and to the primer extension oligonucleotide, both of positive polarity, complementary to the replicated RNA, depicted in diagram e. (B) Schematic representation of the AGPint-GPext series constructs. 0, Partial scheme of the pSP65 plasmid harboring the basic copyback minireplicon pSV-DI-H4⌬96 flanked by the T7 RNA polymerase promoter (T7p) and the hepatitis delta virus ribozyme (Rbz). 1, T7 RNA transcript produced from 0, carrying at its 5⬘ and 3⬘ ends the complementary sequence of the antigenomic promoter (AGPLC) and the antigenomic promoter sequence (AGPR), respectively. 2, Minireplicon T7 transcript harboring at its 3⬘ end the genomic promoter (GP) instead of the AGP. 3, Minireplicon T7 transcript harboring at its 3⬘ end an internal AGP sequence adjacent to an external GP. 4 to 9, Series of minireplicon T7 transcripts harboring at their 3⬘ ends a double promoter, as in construct 3, but separated by increasing intervening sequences as indicated (nt, nucleotides). Note that all the constructs harbor the same 5⬘ end sequence (AGPLC). (C) Northern blot analysis (see Materials and Methods) of the encapsidated minireplicon RNAs of negative polarity produced upon transfection of A549 cells with the various plasmids described for panel B in replication assays made in the absence of the C proteins (P/Cstop conditions; see Materials and Methods). Open and solid arrowheads, external and internal initiation, respectively. (D) As for panel C, but the replication assays were performed in HeLa cells in the presence of the C proteins (Cwt conditions; see Materials and Methods). 7990 VULLIÉMOZ AND ROUX FIG. 2. Effect of the 10A stretch on minireplicon replication. (A) Schematic representation of the single-promoter minireplicons used. 0, as described in the legend to Fig. 1B, with further features related to the present experiment: the restriction sites [DraIII, BamHI (BH1), and MunI], the sequencing primer (SE), and the Sp6 promoter. 1 and 2, as for Fig. 1B. 1A10 and 2A10, Derivatives of 1 and 2, respectively, containing a 10A stretch inserted at the DraIII site. (B) Northern blot analysis of the in vivo-replicated minireplicons. Lanes ⫺L, replication assays performed in the absence of the L-expressing plasmid. (C) Northern blot analysis of in vitro T7 RNA polymerase transcriptions of pSp65 plasmids carrying the indicated minireplicons. The plasmids were linearized at the BamHI (BH1) site (see panel A). The probe of negative polarity resulting from an Sp6 RNA polymerase transcription of plasmid 1 or 2 linearized at the DraIII site (indicated as probe ⫺ in panel A) was used. Open arrowhead, plasmids. Solid arrowhead, T7 transcripts. ⫺T7p, in vitro transcription without T7 RNA polymerase. (D) The plasmid-carrying construct 1A10 and the RT-PCR product amplified (see text) from the in vivo-replicated minireplicon 1A10 were sequenced with a primer situated as indicated in panel A (SE). Only the relevant portions of the sequence are shown. ⬎⬎ indicates further extension of A’s past the 10A stretch, ⴱ indicates longer replication products, and ⬍⬍⬍ indicates the large accumulation of heterogeneous shorter products. can it have the same effect on the T7 RNA polymerase, since the level of the T7 RNA transcript obtained in vitro from the corresponding template plasmid was not affected (Fig. 2C, compare lanes 1 and 2 with 1A10 and 2A10). In Fig. 3B (left panel), comparison of lanes 2 and 2A10 again shows the blocking effect of the 10A stretch on the single-GP construct. When positioned between GP and AGP, the 10A stretch had a similar inhibitory effect on GPext (lane 9A10). AGPint, however, remained perfectly functional (compare with lane 9), showing that replication from the internal promoter did not depend on replication from the external promoter. The function of the external promoter was also obliterated by a G91C point mutation shown to prevent replication (34, 36). In this case, internal initiation was observed in the total Downloaded from http://jvi.asm.org/ on July 24, 2015 by guest 240 nucleotides (Fig. 1B, constructs 4 to 9). Figure 1C presents a Northern blot of the replication products where the use of AGPint corresponds to the signal of construct 1 (single-AGP construct) (Fig. 1B) or 2 (single-GP construct), and the use of GPext is reflected by the upper signals whose sizes increase sequentially with the increasing intervening sequence (lanes 5 to 9). The constant presence of the lower band was evidence that AGPint was used regardless of its distance from GPext (the initiation at both promoters for constructs 3 and 4 was verified by primer extension; data not shown) (36). In construct 9, the 240 nucleotides of intervening sequence represented more than 3 helix turns of distance between the two promoters. As shown previously (7), the replication of the minigenome carrying a single GP (construct 2) was less efficient than that harboring an AGP (construct 1) (difference of about 10-fold in the experiments presented). For the promoters in tandem, this was still the case, but the difference was statistically reduced to 1.5- to 2.0-fold (not shown). The replication assay in Fig. 1C was performed in the absence of the C proteins (P/Cstop conditions; see Materials and Methods). Since the presence of the C proteins was shown to represent more stringent conditions for the viral RdRp function (35), it was of interest that initiation at both AGPint and GPext was taking place as well in the presence of the C proteins (P/Cwt conditions) (Fig. 1D). It is finally noteworthy that the two replicated products accumulated with similar kinetics (not shown), which rules out a possible precursor-product relationship between the two bands and minimizes a possible competition between the two minireplicons for amplification. Does internal initiation depend on the activity of the external promoter? The results presented above were consistent with the independent activity of each promoter and could therefore speak for entry of the viral RdRp at each promoter. To be consistent with viral RdRp internal entry, replication from the internal promoter should ideally take place under conditions in which entry at GPext is prevented. Since no direct method to prevent or even to assess entry at the promoter is available, methods that attempted to prevent the viral RdRp activity from the external promoter were developed. A stretch of 10 adenosines (template strand) was inserted just after the inner border of GPext after it was found by serendipity that such a configuration could significantly lower (or even abolish) replication initiated at a perfectly competent promoter (compare replication of constructs 1 and 2 with that of 1A10 and 2A10 in Fig. 2B). This decrease or block in replication likely results from a stuttering of the viral RdRp at the 10A sequence. Indeed, when the RNA from the 1A10 construct replicated (Fig. 2B), the sequencing of its RT-PCR product clearly showed sequence degeneration with further extension of A’s past the 10A stretch (Fig. 2D, RT-PCR sequence). This should lead to longer replication products, as observed, while the large accumulation of heterogeneous shorter products could rather speak for a backward stuttering (Fig. 2D). Following stuttering, the 10A stretch may eventually cause the dissociation of the viral RdRp from its template. Concomitantly, stuttering on the 10A stretch could also result from the production of replicating molecules out of the rule of six, which would subsequently be amplified much less efficiently. Whatever the mechanism, the 10A stretch remains an obstacle for the viral RdRp. In no way J. VIROL. VOL. 76, 2002 TEMPLATE ENTRY OF PARAMYXOVIRUS RNA POLYMERASE absence of initiation at GPext [Fig. 3B, lanes 9GP(G91C) and 9A10GP(G91C)]. If anything, in the presence of 10A, internal initiation could be slightly more efficient [compare lanes 9 and 9GP(G91C) with lanes 9A10 and 9A10GP(G91C), respectively]. Inactivating the internal promoter with the same mutation, however, led to exclusive replication from the external promoter [Fig. 3B, right panel, lane 9AGP(G91C)]. Note that, in this configuration, the presence of the 10A stretch prevented any replication by inhibiting replication from the external promoter [lane 9A10AGP(G91C)]. In general, in the whole series, the activity of AGPint corresponded to that of the single-GP construct (Fig. 3, construct 2), which itself is about 5- to 10-fold lower than that of the single-AGP construct (Fig. 1C, constructs 1 and 2). This was verified even when GPext was mutated [Fig. 3, construct 9 GP(G91C)]. This may indicate that internal RNA synthesis is somehow less efficient than that starting at the 3⬘ end of the template. Despite this observation, this first series of experiments were supportive of independence of function of the internal and external promoters and as such were compatible with independent entry of the viral RdRp at each promoter. Does internal initiation depend on the nature of the external promoter? A second series of minigenomes carrying double promoters was prepared, this time with two AGPs, two strong FIG. 4. Replication of the AGPint-AGPext constructs. (A) Schematic representation of AGPint-AGPext series constructs. Construct 1, as described in the legend to Fig. 1. Constructs 10 to 13, minireplicons harboring two AGP promoters, adjacent (construct 10) or separated by increasing intervening sequence (nucleotides [nt]) as indicated. (B) Northern blot analysis (see the encapsidated minireplicon RNAs produced from the plasmids described for panel A). The open rectangle is meant to help show the different migration of the bands. Note the presence of a single band regardless. (C) Primer extension (PE) analysis (see Materials and Methods) on construct 10 to better demonstrate the preponderant presence of the replicated product initiating at AGPext. The end position of the product elongated on the RNA is given by comparison with the sequence performed on the respective plasmid with the same primer. 3⬘-end sequence (Seq) of AGP, 5⬘. . .TTGTCTGGT-3⬘. Adjacent ribozyme sequence, 5⬘. . .CCGGC CGA. . .3⬘. Only portions of the sequencing gel relevant for the 3⬘ ends of the replicated RNAs are shown. promoters, separated by increasing intervening sequences (Fig. 4A). As shown best by the product obtained with construct 13 (AGP-240-AGP), only one replication signal was observed that corresponded to the use of the external promoter only (Fig. 4B, lane 13). For the three remaining constructs (construct 10, 11, and 12), the difference in size between the putative products made from the internal or external promoter was too small to be informative. Therefore, primer extension analysis of the replicated products was performed (see legend to Fig. 4C and Materials and Methods). For construct 10, the almost exclusive product observed extended to the end of AGPext (Fig. 4C, PE 10), a result verified for the other two constructs (not shown). In conclusion, in contrast to what had been observed in the context of GPext (Fig. 3), AGPint was not used in the context of AGPext. A possible trivial explanation for this discrepancy could be the higher strength of the external promoter in the series AGPintAGPext. If any components were limiting, then the strong external promoter could outcompete the activity of the internal Downloaded from http://jvi.asm.org/ on July 24, 2015 by guest FIG. 3. Replication of the AGPint-GPext constructs harboring a 10A stretch in the intervening sequence. (A) Schematic representation of the minireplicons used. 2 and 9 are as described in the legend to Fig. 1. 2A10, 9A10, 9A10GP(G91C), and 9AGPA10(G91C) harbor a 10A stretch (positive polarity sequence) adjacent to the internal border of GP (see Materials and Methods). 9GP(G91C), 9A10GP(G91C), 9AGP(G91C), and 9A10AGP(G91C) harbor a G-to-C substitution at position 91 of GP or AGP. (B) Northern blot analysis of the replicated minireplicons. When two bands are visible, the upper and lower one correspond to initiation at GP and AGP, respectively. Open and solid arrowheads, as for Fig. 1. 7991 7992 VULLIÉMOZ AND ROUX one. A series of replication assays were therefore performed in which the template level was progressively reduced, keeping the other parameters unchanged. No matter how reduced the replication from the external promoter was, following the reduced delivery of the template plasmid, a higher replication from the internal promoter was never observed (construct 13) (Fig. 5B). This argues against a limiting factor that would prevent activity of the internal promoter. In Fig. 6, the effect of introducing the 10A stretch between the two AGPs was analyzed. As found previously, the inhibitory effect of the 10A was less pronounced when it was downstream of a single AGP (Fig. 6B, compare constructs 1 and 1A10). Consequently, replication from the external AGP in the double promoter was still observed (construct 13A10), although it was partially decreased compared to that of construct 13. Interestingly, for construct 13A10, evidence of internal initiation was present (Fig. 6B, see arrow). The replicated product from the internal promoter was very reproducibly observed (for another example, see Fig. 5, 13A10) and could represent 15 to 20% of the total (statistical analysis not shown). Decreasing the template in otherwise unaltered replication conditions did not change this fraction (Fig. 5, 13A10). Internal initiation was, in contrast, never observed when the activity of the internal promoter was inactivated by the G91C mutation [Fig. 6B, 13In(G91C) and 13-A10In(G91C)]. Note that the internal promoter was active in the doubleAGP construct, since upon inactivation of AGPext by the G91C mutation, replication took place exclusively internally [Fig. 6B, 13-Ex(G91C) and 13-A10Ex(G91C)]. In general, the replication product issued from the double-AGP constructs, either from AGPext (Fig. 6, construct 13) or from AGPint [Fig. 6, 13-Ex(g91C)], was consistently lower than that produced by the FIG. 6. Replication of the AGPint-AGPext constructs harboring a 10A stretch in the intervening sequence. (A) Schematic representation of the constructs. Constructs 1, 13, and 13A10, as described in the legend to Fig. 5. 13-Ex(G91C) and 13-In(G91C), as for construct 13 but harboring the G91C mutation in the external or the internal AGP, respectively. 13-A10Ex(G91C) and 13-A10In(G91C), as for 13Ex(G91C) and 13-In(G91C) but with the 10A stretch in the intervening sequence. (B) Northern blot analysis of the replicated products. Construct 9 (Fig. 3A) was used a size marker for the replication products initiating at both promoters. Open and solid arrowheads, as for Fig. 1. single-AGP construct (Fig. 6, construct 1), alluding to the fact that, as for the series AGPint-GPext, there is some price to pay for the presence of the double promoter on the original template. In summary, this AGPint-AGPext series of constructs appeared to demonstrate that the activity of the external promoter was somehow limiting the activity of the internal one, a notion that contrasted with the conclusion drawn from the results obtained with the AGPint-GPext series. Does internal initiation depend on GP’s being only the external promoter? The difference between the first and second series of constructs lies in the nature of the external promoter. In the end, only GPext was associated with internal initiation. Two new constructs harboring two GP promoters separated by an intervening sequence of 240 nucleotides were therefore analyzed, so that, as in the first series, GP was again the external promoter (Fig. 7). This GPext (construct 14), (Fig. 7B) was used with the same efficiency as a single-GP construct (compare with construct 2) and with an efficiency that could even exceed that exhibited by GPext of the AGPint-GPext construct (construct 9), where the internal promoter is AGP. Despite this high activity, replication from GPint was not apparent (construct 14, solid arrowhead position). The insertion of the 10A stretch between the two GPs (14A10) reduced the replication from GPext, as expected (compare with construct 2A10). Downloaded from http://jvi.asm.org/ on July 24, 2015 by guest FIG. 5. Gradually diminishing the template availability. (A) Schematic representation of the minireplicons used. Constructs 1 and 13, as described in the legend to Fig. 4. 13A10 harbors a 10A stretch (positive polarity sequence) adjacent to the internal border of external AGP. (B) Northern blot analysis of the replicated products. ␮g, micrograms of the template plasmid added. Note the presence of a faint signal below the main band for lanes 13A10, 5 and 0.5 ␮g (see text). Open and solid arrowheads, as for Fig. 1. J. VIROL. VOL. 76, 2002 TEMPLATE ENTRY OF PARAMYXOVIRUS RNA POLYMERASE 7993 Interestingly, replication from GPint was now visible. The replicated product from GPint was, as expected, lower than that obtained when AGP was the internal promoter (construct 9, solid arrowhead). However, it reproducibly exceeded by twoor threefold the product issued from GPext. In summary, the presence of GPext was not sufficient to permit visible internal initiation. DISCUSSION FIG. 8. Schematic summary of the data. The explanations of the symbols are presented at the right of the figure. The two promoters are shown, in their wild-type and mutated configurations, as well as the intervening sequence and the 10A stretch. Below, the curved arrows portray external or internal initiations of RNA synthesis, as well as interdicted initiations. The sizes of the arrows reflect the inherent activity of the promoter, AGP⬎⬎GP. This activity is further modulated by the relative use of the promoter, as indicated by the width of the straight arrows (in the left part of the figure) depicting the importance of the replicated products. In the left part of the figure, only the two promoters at the 3⬘ end of the encapsidated T7 RNA transcripts are shown. (A) AGPint-GPext series. (B) AGPint-GPext series. (C) GPint-GPext series. Downloaded from http://jvi.asm.org/ on July 24, 2015 by guest FIG. 7. Replication of the GPint-GPext constructs. (A) Schematic representation of GPint-GPext series constructs. Construct 2, as described in the legend to Fig. 1. Construct 2A10, as for Fig. 2. Constructs 9 and 9A10, as for Fig. 3. Constructs 14 and 14A10, minireplicons harboring two GP promoters, adjacent or separated by 240 nucleotides, respectively. (B) Northern blot analysis of the replicated constructs. 2, 2A10, 9, and 9A10 are shown for comparison and as size markers. 14 and 14A10 constitute the newly analyzed constructs GPintGPext. Open and solid arrowheads, as for Fig. 1. ⴱ, residual non-fully denatured RNA, occasionally seen upon poor solubilization. Note its position close to the main band. The ability of the viral RdRp to initiate replication at a promoter not positioned at the 3⬘ end of the template is evident in the three series of experiments presented above. This took place efficiently when the internal promoter was inherently stronger than the external one, as in the series AGPintGPext. In the AGPint-AGPext and GPint-GPext series, where the two promoters were of equal strength, internal initiation was seen only when the activity of the external promoter was impaired (10A stretch) or obliterated (G91C mutation) (see schematic summary of the results in Fig. 8). The mechanism by which this internal initiation took place is open to question. The following observations have to be taken into account in order to try to derive such a mechanism. First, and this is not new, replication from AGP was more efficient than that from GP. Second, the strength of the promoter appeared to be adjusted by its relative position. Replication from the external position appeared to be favored relative to that from the internal position. In consequence, when the two promoters were identical, replication from the external position alone was observed. Replication from the internal promoter occurred only when the inherent strength of the promoter compensated for the internal positioning, as in the first series 7994 VULLIÉMOZ AND ROUX way “imprint” the viral RdRp with different properties vis-à-vis the internal promoter recognition. This interpretation, however, as appealing as it appears, does not agree with the results of the GPint-GPext series, for which it predicts an internal initiation at GPint, a result that was not observed (Fig. 7, construct 14). More generally, these models, although impossible to absolutely disprove, are difficult to reconcile with the results obtained when RNA synthesis is impaired by the G91C mutations or by the 10A stretch insertion. For example, scanning would have to take place without apparent RNA synthesis initiation at the external promoter. Finally, the minireplicons carrying only the internal promoter could have been generated during the replication of the opposite strand by the polymerase terminating at the internal promoter. Again, this possibility cannot be formerly excluded. It is, however, not consistent with the lack of replication resulting from internal initiation in the AGPint-AGPext or GPint-GPext. In conclusion, although the data presented do not provide direct and unquestionable evidence for internal entry of the viral RdRp, they can still be best interpreted in that way. Furthermore, the possibility of internal entry agrees with and confirms our previous proposal that the proper N-phase context is sufficient for the viral RdRp to recognize its template, this at the expense of the template 3⬘-end congruence that was found not to be critical (36). This implies that the viral RdRp must detect or interact with the nucleotides of the promoter region despite the tight interactions between the RNA and the N protein subunits. The three cytidines of the threetimes-repeated motif interacting in position 1 with the N subunits might represent the detected signal. Present in the second helix turn, directly superimposed over the three most proximal hexamers of the promoter, they exist in a configuration that is not found in any of the 13 other hexamers constituting the promoter (34). Alternatively, some nucleotides (again the three-times-repeated motif?) of the promoter region might induce the nucleocapsid to adopt a local structure sufficiently different from the rest of the nucleocapsid to represent this signal. The two hypotheses may not be exclusive. Until the high-resolution structure of the nucleocapsid is available, it will be difficult to tackle this type of question. If imprinting vis-à vis the internal promoter recognition, as discussed above, is not supported by our data, the difference with which the 10A stretch impairs replication starting at the AGP or GP could well be interpreted as the difference with which the viral RdRp negotiates the barrier to RNA synthesis elongation. Starting at AGP appears to make the viral RdRp less prone to terminate synthesis when crossing the 10A stretch, as if the AGP RdRp would have inherently more processivity than the GP RdRp. Considering that RNA synthesis initiation at AGP leads naturally to a replication-only process, while initiation at GP can proceed to replication or transcription, it would not be surprising if the two promoters were to imprint the viral RdRp with a different sensitivity for recognition of downstream signals. In fact, as artificial as the double-promoter constructs presented here can be—in nature the replication promoters are normally found at the extremity of the templates—an old report describing a vesicular stomatitis virus defective interfering (DI) RNA relates to the double promoter and the imprint- Downloaded from http://jvi.asm.org/ on July 24, 2015 by guest (AGPint-GPext). Finally, whenever replication from the external promoter was impaired, replication from the internal promoter took place regardless of the promoter configuration. These observations are consistent with the ability of the viral RdRp to initiate replication at either promoter, provided that the promoter is accessible. While accessibility to the external promoter in its wild-type configuration is permanent, that of the internal promoter appears to be regulated by the use of the external promoter. Taking these observations into consideration, we propose the following mechanism to best account for the results obtained. Internal initiation reflects internal entry, an entry regulated by the availability of the internal promoter, a promoter that can be occluded by the use of the external promoter. In the light of this model, the higher strength of AGP over GP allows replication from the internal promoter in the AGPint-GPext series (Fig. 8A) by minimizing promoter occlusion resulting from GPext activity. Promoter occlusion is furthermore decreased by addition of the 10A stretch, seen here mainly by the decrease in replication from GPext, and not so much by an increased replication from AGPint, which must reach the upper limit of the system. Replication from AGPint takes place a fortiori when the activity of GPext is abolished by the G91C mutation (see Fig. 3). For the AGPint-AGPext series (Fig. 8B), both promoters are of equal strength, so that replication from AGPint cannot overcome the occlusion resulting from AGPext. The 10A stretch, by decreasing the ability of the viral RdRp to elongate, results in a slight decrease in promoter occlusion, allowing minimal replication from AGPint. Note that the release of promoter occlusion by the 10A stretch, evidenced by the importance of replication from AGPint, is less important than that seen in the AGPint-GPext series, a feature that correlates with a lower effect of the 10A stretch in blocking replication from AGP than from GP. In contrast, the G91C mutation, by inhibiting replication from AGPext, fully releases promoter occlusion, so that replication from AGPint becomes maximal (see Fig. 6). Finally, in the GPint-GPext series, the equal strength of the internal and external promoters prevents, as in the AGPintAGPext series, the release of promoter occlusion, and only replication from GPext is observed. As in the AGPint-GPext series, the 10A stretch almost completely abolishes replication from GPext and in this way releases promoter occlusion, with concurrent appearance of replication from GPint. Although weak, replication from GPint may in fact correspond to the highest possible replication from a weak promoter positioned internally. It is indeed higher than that initiating at GPext (see Fig. 7). Although this model appears to account most fully for the results obtained, other possibilities can be envisaged. These include external entry followed by template scanning to reach the internal promoter or by reinitiation at the internal promoter after release of the nascent chain initiated at the external promoter. These models could integrate the effect that initiation at the external promoter could have on the ability of the viral RdRp to recognize the internal promoter. Considering the AGPint-GPext and AGPint-AGPext series, one could conclude that a viral RdRp starting replication at GPext would still be in a position to recognize AGPint, while an initiation at AGPext would render the viral RdRp insensitive to an internal promoter. Entry and initiation at GPext or AGPext would in a J. VIROL. VOL. 76, 2002 TEMPLATE ENTRY OF PARAMYXOVIRUS RNA POLYMERASE ing concepts (21). An internal DI deletion (DI-LT2) carrying, before the genomic 3⬘ end, 70 nucleotides complementary to the 5⬘ end of the vesicular stomatitis virus genome (in other words, carrying an AGP sequence in front of the normal GP sequence) was described. 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Whelan, S. P. J., L. A. Ball, J. N. Barr, and G. W. Wertz. 1995. Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones. Proc. Natl. Acad. Sci. USA 92:8388–8392. Downloaded from http://jvi.asm.org/ on July 24, 2015 by guest We, particularly D.V., thank Dominique Garcin for exciting discussions and Daniel Kolakofsky for critical reading of the manuscript. This work was supported by a grant from the Swiss National Foundation for Scientific Research. 7995