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Department of Microbiology and Molecular Medicine, University of Geneva Medical School, CMU, 1 rue Michel-Servet, CH-1211 Geneva 4, Switzerland
Correspondence
Laurent Roux
laurent.roux{at}medecine.unige.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Their genomic and antigenomic replication promoters (G/Pr and AG/Pr) are organized in a bipartite fashion (Fig. 1a), with essential promoter elements (PrE-I and -II) located not only at the genome/antigenome 3' ends (nt 1–30), but within the 3' untranslated region (UTR) of the first gene (G/Pr) or the 5' UTR of the last gene (AG/Pr) (Murphy et al., 1998; Tapparel et al., 1998; Hoffman & Banerjee, 2000; Walpita, 2004; Marcos et al., 2005; Vulliémoz et al., 2005). The conserved decanucleotide mRNA initiation (or gene start, gs) site of the first gene (gs1) is invariably found at nt 56–65, and N mRNAs always start opposite genomic U56, i.e. the second position of the tenth genome hexamer.
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) (Pattnaik et al., 1995; Li & Pattnaik, 1997, 1999; Fearns et al., 2000, 2002; McGivern et al., 2005).
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; Vulliémoz et al., 2005). How this competition between gs1 and PrE-I/-II for the two subunits (P and L) of the vRdRp occurs is unclear, but it suggests that this vRdRp is not yet committed to its specialized task. An attractive mechanism that would explain this competition is that it occurs ‘in cis’, e.g. during vRdRp scanning of the genome 3' end for a new RNA start site, after vRdRp initiation at the genome 3' end and release of leader RNA (Le Mercier et al., 2003).
There are different views of how NNV RdRp acts as a transcriptase that begins the process of transcription at gs1. For the rhabdovirus VSV, vRdRp is thought to be committed to its mutually exclusive task of replicase or transcriptase before it engages the template. Committed transcriptase interacts directly with and initiates at gs1 (Whelan & Wertz, 2002), whereas replicase does so at the genome/antigenome 3' end (Fig. 2a
). VSV RdRp complexes committed to transcription or replication can be prepared from recombinantly expressed VSV proteins (Qanungo et al., 2004) and, during infection, mRNA expression from gs1 is not affected by UV cross-links that impede leader RNA synthesis (Whelan & Wertz, 2002). This view is also supported by the properties of the VSV defective interfering (DI) genome DI-LT2. VSV DI-LT2 contains all of the cis-acting sequences of the wild-type VSV genome (Fig. 2a), as well as four intact genes (missing the L gene). It also contains an additional 70 nt at the genome 3' end derived from AG/Pr, which displaces G/Pr (and gs1) 70 nt from the genome 3' end (Fig. 2b; Semler et al., 1978; Johnson et al., 1979). This naturally occurring DI-LT2 replicates extremely well during coinfection with wild-type virus, but fails to transcribe mRNAs in vitro and in vivo, presumably because only replicase initiates at AG/Pr (Fig. 2b) and therefore cannot go on to initiate at gs1. Alternatively, VSV transcriptase cannot initiate directly at gs1 because the precise location of gs1 relative to the genome 3' end is also important for the initiation of transcription from gs1.
In striking contrast to VSV DI-LT2, a corresponding Sendai virus (SeV) DI, produced artificially, in which gs1 is displaced 90 nt from its natural position in the genome, expresses mRNAs from gs1 [Vulliémoz et al., 2005; Fig. 1b (AGP-6-GPd12)]. Thus, either SeV RdRp, which initiates at the genome 3' end, can go on to initiate at gs1, meaning that this vRdRp is not a committed replicase, or the SeV RdRp, which initiates at gs1, is a committed transcriptase, which does so, in apparent contrast to VSV, independently of its precise location relative to the genome 3' end. Such a transcriptase, then, should not be affected by the distance separating gs1 and the genome 3' end. We therefore investigated the effect of progressive further displacement of gs1 from the genome 3' end.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5 % fetal calf serum (FCS) in a 5 % CO2 atmosphere. The AGP-55 recombinant Sendai virus (rSeV-AGP-55) was constructed and rescued as described by Le Mercier et al. (2002). Virus stocks, prepared in 9-day-old embryonated chicken eggs from three times plaque-purified virus, reached titres ranging between 5x108 and 109 p.f.u. ml–1.
Sequence and plasmids.
All of the plasmids harbouring the minigenome expressing the green fluorescent protein (GFP) are derived from pSV-DI-H4
96, described previously (Vulliémoz & Roux, 2001, 2002). The constructs used here were produced in a manner similar to that described previously (Vulliémoz et al., 2005) and they all represent derivatives from published constructs. The intervening sequence originates from the SeV M gene sequence.
Rescue of minigenomes in the presence of the helper rSeV-AGP-55.
The rescue of the minigenomes by the helper rSeV-AGP-55 virus was performed exactly as described by Vulliémoz et al. (2005), except that, at the end of the procedure, the infected cells collected in PBS were divided into fractions used for GFP expression by flow cytometry and into fractions used to characterize the extent of replication or transcription by Northern blotting, primer extension or RNase protection, respectively.
Replication of minigenomes in the presence of support plasmids.
Confluent BSR-T7/5 cells, seeded as described above the day before, were transfected with a mixture of plasmids, including the plasmid harbouring the minigenome (5 µg), pTM1-N (1·5 µg), pTM1-P/Cstop (1·5 µg) and pTM1-L (0·5 µg), and 20 µl Fugene (Roche). Thirty-six hours post-transfection, the cells were collected and treated as above for GFP expression analysis by flow cytometry and for Northern blot analysis.
Recovery of the encapsidated and non-encapsidated viral RNAs.
Infected/transfected BSR-T7/5 cells were collected as described above. Ninety per cent were pelleted and resuspended in 1 ml lysis buffer [0·6 % NP40, 50 mM Tris/HCl (pH 8·0), 10 mM NaCl; Mottet & Roux, 1989]. Post-nuclei supernatants were made in 5 mM EDTA and loaded onto linear 20–40 % (w/w) CsCl gradients (Beckman SW60). After centrifugation (40 000 r.p.m., 12 °C, overnight), the nucleocapsids banding in the CsCl gradient and the non-encapsidated cellular and viral RNAs sent to the pellet were collected as described previously (Calain & Roux, 1995).
Northern blot analysis.
Northern blots were performed as described previously (Calain & Roux, 1995). To score replication, the 32P-labelled riboprobe contained promoter- and GFP-specific sequences, which allow detection of the helper virus genome. The blotted membranes were quantified by using a PhosphoImager (ImageQuant version 5.0; Molecular Dynamics).
Primer extension.
The primer for primer extension, shown in Fig. 3(a) (PE), was end-labelled with [
-32P]ATP by T4 polynucleotide kinase (Promega) and annealed with 20 µg (1x) or 60 µg (3x) infected-cell CsCl gradient-pelleted RNAs. Reverse transcription was carried out at 42 °C according to the supplier's protocol by using M-MLV reverse transcriptase (Promega). The reverse transcription product was analysed by electrophoresis on a 6 % polyacrylamide gel. The gel was fixed by acetic acid/ethanol, dried and quantified by using a PhosphoImager (ImageQuant version 5.0; Molecular Dynamics).
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, RNaseP probe, was synthesized from the appropriate plasmid by the T7 RNA polymerase using [
-32P]U. The probe was then treated with DNase I (Qiagen; 1 h, 37 °C) and purified by 5 % polyacrylamide gel electrophoresis in 8 M urea. The RNase-protection assay was carried out with an RPA III kit (Ambion) using 10 µg (1x) or 50 µg (5x) total RNA to provide evidence of quantitative analysis. The gel was fixed by acetic acid/ethanol, dried and quantified by using a PhosphoImager (ImageQuant version 5.0; Molecular Dynamics).
Analysis of GFP expression.
Infected/transfected cells were collected in PBS. Flow cytometry was performed on a Becton-Dickinson FACSCan2. R1 and M1 were adjusted on BSR-T7/5 cells infected with rSeV-AGP-55 only and transfected with the plasmid harbouring AGP-GPd12. Data analysis was performed with Becton Dickinson software.
GFP relative expression.
The method has been described previously in detail (Vulliémoz et al., 2005). In brief, GFP gene transcription, by measures of mean GFP fluorescence, was standardized to the amount of the template available for GFP mRNA synthesis. Replication itself was corrected for the fraction of cells carrying the minigenomes, taken as the percentage of cells positively gated in the flow-cytometry analysis. In the end, GFP fluorescence was expressed as the mean fluorescence divided by the corrected replication value. To be able to integrate the data of more than one experiment, the results were expressed as a percentage relative to one template taken as the reference for the series; this reference is indicated in the figure legends [see Figs 3(f), 4(b) and 5(c)], as is the number of independent experiments (three or four) performed and the average of the mean.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). It contains, at the DI genome 3' end, 96 nt of AG/Pr, 6 nt of spacer and 84 nt of G/Pr in which the first 12 nt have been deleted (Fig. 3a, construct 1). This construct generates minigenomes that replicate via the external AG/Pr and express GFP from gs1 of the internal G/Pr. AG/Pr was chosen as the genome 3'-end replication promoter because of the absence of gs1 and because no transcripts other than the trailer RNA and antigenomes are known to be initiated from AG/Pr. The deletion of the first 12 nt of the internal G/Pr ensures that it cannot function as a replication promoter (Vulliémoz et al., 2005). Further minigenome constructs were generated containing intervening sequences of increasing size (240, 474 and 720 nt) between the tandem promoters (Fig. 3a, constructs 2, 3 and 4). gs1 was thus moved progressively downstream, from position 146 to positions 384, 618 and 864 relative to the minigenome 3' end. It is noteworthy that, in all of the minigenomes, the phase context of gs1 is identical and corresponds to that found on the natural genome (second position of the hexamer). The minigenomes of these constructs were first rescued from plasmids by using helper SeV and were found to replicate to similar levels (Fig. 3b, c). This is expected, as all of these minigenomes have identical replication promoters. In contrast, measurement of GFP fluorescence by flow cytometry indicated that GFP expression decreased steadily with increasing distance between the replication and transcription signals (Fig. 3f, FLUO bars).
GFP mRNA levels were also measured directly by primer extension and RNase protection. For primer extension (Fig. 3d), we used a [32P]5'-phosphorylated primer of negative polarity that extends 228 nt to the 5' end of the GFP mRNA (see Fig. 3a, bottom line). Lanes 1 and 3 of Fig. 3(d) show duplicate samples that contained different amounts (3x and 1x) of CsCl gradient-pelleted RNA to demonstrate the quantitative nature of the analysis. For RNase protection (Fig. 3e), we used a negative-polarity probe of 142 nt, of which 112 nt are complementary to the minigenome sequence and of which 62 nt are complementary to the 3' end of the GFP mRNA (Fig. 3a, bottom line). Duplicate samples of constructs were also used to verify the quantitative aspect of the analysis (lanes 1 and 3). The extension and the RNase-protected products were estimated by using a PhosphoImager (Molecular Dynamics) and are expressed relative to that of construct 1 (AGP-6-GPd12) (Fig. 3f, FLUO, PE and RNaseP bars). The correlation of the data obtained by all three methods is striking. The measure of the mean GFP fluorescence in these experiments thus appears to accurately reflect the synthesis of GFP mRNAs.
Rescue of the minigenomes in the absence of helper virus
The use of a helper virus to rescue minigenomes in transmissible form leads to competition between the helper virus and minigenome promoters for available vRdRp during intracellular replication. To examine whether the nature of the rescue system affected our results, we repeated the experiment in BSR-T7/5 cells in which vRdRp (P and L) and N protein, as well as the minigenomes, were produced ‘constitutively’ from plasmids via T7 RNA polymerase, in the absence of helper virus (Fig. 4). Under these conditions, once more, GFP expression levels as measured by flow cytometry showed a steady decrease proportional to the increasing distance between the replication and the transcription start sites (Fig. 4c). Thus, SeV RdRp that initiates at gs1 is not independent of its precise location relative to the genome 3' end. It is rather affected by the distance between the genome 3' end and gs1, as it progressively initiates at gs1 less frequently as the distance increases.
Removing PrE-I and PrE-II
The cis-acting PrE-I and PrE-II sequences of G/Pr and AG/Pr that are important for replication are spread over 96 nt of the genome and antigenome 3' ends (see Fig. 1a). However, in tandem promoter constructs such as AGP-GPd12, the PrE-I and PrE-II domains, essential for replication, can be removed completely from the internal G/Pr, leaving only 30 nt (49–78) containing gs1 in its bona fide hexamer phase, yet transcription continues (Vulliémoz et al., 2005). To determine whether PrE-I/II of G/Pr are nevertheless important for how Sendai virus RdRp that initiates at gs1 is affected by the distance between the genome 3' end and gs1, we progressively increased the distance between the genome 3' end and this minimal gs1 (nt 49–78). Fig. 5(a) depicts the minigenome constructs (7, 8 and 9) that were analysed in parallel with the corresponding minigenomes carrying PrE-I/II (from Fig. 3a). As expected, the further deletions did not affect the levels of minigenome replication (Fig. 5b). Fig. 5(c) shows that there is a similar, steady decrease in GFP expression with increasing distance of gs1 from the genome 3' end when the GFP mRNA initiates from the minimal gs1 [constructs 7–9, Fig. 5c(ii)], as when it initiates from the efficient gs1 of G/Prd12 [Fig. 5c(i)]. Note the difference in scale of the two graphs of Fig. 5(c), reflecting a general decreased GFP expression due to the PrE-I/II deletions. This was expected, as sequences upstream of nt 49 in G/Pr also contribute to gs1 expression, as described previously (Vulliémoz et al., 2005). Thus, in the end, we were unable to find any evidence that PrE-I/II of G/Pr are important for how the SeV RdRp that initiates at gs1 is affected by the intervening distance between gs1 and the genome 3' end. Our data support the conclusion that SeV RdRp does not initiate directly at gs1, but rather first interacts with the genome 3' end and then reaches gs1 in a manner that senses the distance separating gs1 from the genome 3' end.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) with one subunit bound to the genome 3' end and the other to gs1. In this view, initiation at gs1 by the downstream vRdRp depends on the upstream vRdRp interaction with the genome 3' end. Increasing the intervening distance, then, will decrease the likelihood of interaction of gs1 and the genome 3' end (via the two vRdRp subunits) that is required for the downstream vRdRp to initiate at gs1. Second, all SeV RdRps, both those that go on to make antigenomes and those that go on to make mRNAs, initially initiate RNA synthesis at the genome 3' end. The latter initiate at gs1 after having presumably released the nascent transcript and having scanned the template linearly for gs1. NNV RdRps are unique in that they apparently do not disengage from the template upon release of the mature mRNA, but scan the template linearly for a new start site, even when this gs is upstream of the gene-end site (Stillman & Whitt, 1998; Fearns & Collins, 1999). If similar events occur upon release of the nascent RNA (trailer RNA in the present case?), the increasing distance between gs1 and the genome 3' end will lower the number of scanning RdRps that reach gs1.
Both models account for the reduction of gs1 efficiency with its increasing displacement from the genome 3' end, by invoking a required interaction of vRdRp with the genome 3' end for subsequent vRdRp initiation at gs1. However, in the first case, this interaction serves to position a second vRdRp on the template that initiates at gs1, without prior RNA synthesis (de novo initiation). In the second case, this interaction serves to initiate RNA synthesis, thereby stably attaching vRdRp to the N–RNA template, and it is this stable attachment that permits subsequent vRdRp scanning of the N–RNA for gs1 after nascent RNA release (vRdRp reinitiation; Kolakofsky et al., 2004). There are two reasons why we favour the second model.
First, the interaction of the genome 3' end and gs1 via SeV RdRp, independent of RNA synthesis and independent of the precise location of gs1 downstream, i.e. vRdRp searching in space for gs1 by looping out the intervening sequence, would require a remarkable flexibility in the N–RNA template. This flexibility is inconsistent with this structure, as revealed by electron-microscopy image reconstruction of the SeV and measles virus nucleocapsids (Egelman et al., 1989; Bhella et al., 2004; Schoehn et al., 2004) and the general inaccessibility of the RNA bases of this N–RNA assembly to the solvent (Iseni et al., 2002). Second, we were unable to provide evidence that the PrE-I/II sequences, which are essential for the initiation of genome replication, are also important in determining how the vRdRp initiates at gs1. In case of a de novo initiation at gs1, the PrE-I/II sequence would be expected to affect this initiation. This negative result is more consistent with a model in which vRdRp scans the template linearly for gs1, where the essential task of attaching vRdRp stably to the template (so that linear scanning for gs1 can occur) takes place, in our case during trailer RNA synthesis. Reinitiation, rather than de novo initiation at gs1, can presumably take place on a more limited cis-acting sequence (nt 49–78, containing gs1 in its bona fide hexamer phase). In the end, in the same way that the lack of VSV DI-LT2 transcription supports the existence of VSV RdRp committed to replication or transcription before it engages the template, the transcription of SeV (AGP-6-GPd12) and its derivatives supports the existence of an uncommitted pool of SeV RdRp that engages the template at the genome 3' end and becomes committed to transcription after release of the nascent RNA.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 15 July 2005;
accepted 22 November 2005.
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). G/Pr active gene start (grey arrow) allows the transcription of a GFP gene down to a gene-end signal (shown above the gene). The plus-strand 3' end also contains AG/Pr. The distance between AG/Pr and the deleted GP is indicated. Construct 5 harbours only AG/Pr at its 3' end. Below construct 5, schematic representation of the GFP transcription unit with the negative-sense primer of 25 nt used for the primer-extension reaction (PE) and the extended product (228 nt) and with the negative-sense probe of 142 nt used in theRNase-protection assay (RNaseP), of which 62 and 112 nt are complementary, respectively, to the GFP message and to the plus-strand minigenome (see Methods). (b) Northern blot analysis scoring the replication of the constructs depicted in (a). A riboprobe of positive polarity was used to evaluate the template available for transcription. (c) The bands obtained in (b) were quantified and the replication mean values (three experiments) were expressed relative to construct 1 and plotted with SD from the mean. (d) Primer-extension analysis (see Methods). Lane numbers represent minigenome numbers as in (a); No indicates helper virus infection only; 3x and 1x indicate relative fraction of total RNA used. Two independent experiments were carried out. (e) RNase-protection assay (see Methods) carried out using 10 µg (1x) or 50 µg (5x) total RNA. Lane numbers as in (d). (f) GFP transcription levels estimated by GFP mean fluorescence (FLUO, see Methods), primer extension (PE) [mean of two separate experiments (mean and SD)] and RNase protection (RNaseP) are plotted as values relative to those obtained for AGP-GPd12. Black bars, PE; dark-shaded bars, FLUO; light-shaded bars, RNaseP.
