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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INTRODUCTION |
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The genomic and antigenomic replication promoters (G/Pr and AG/Pr) of paramyxoviruses are found within the terminal 96 nt or 16 N-subunits of each RNA and are bi-partite in nature (Murphy et al., 1998; Pelet et al., 1996
; Tapparel et al., 1998
). Approximately 30 nt at the 3' end constitute the first element (PrE-I) in which the first 12 nt are conserved between genomes and antigenomes, and across each genus. A second downstream element (PrE-II) is found within the 5'-UTR of the N gene or the 3'-UTR of the L gene (Fig. 1
). For SeV and Human parainfluenza virus 3, PrE-II is a simple but phased hexameric sequence repeat (3' [C1n2n3n4n5n6]3) bound to the fourteenth, fifteenth and sixteenth N protein subunits (Hoffman & Banerjee, 2000
; Tapparel et al., 1998
). For simian virus 5, [n1n2n3n4G5C6]3 is repeated in subunits 13, 14 and 15 (Murphy et al., 1998
). PrE-II, then, is adjacent to PrE-I in the helical nucleocapsid, forming a common or contiguous surface on two turns of the helix. The hexamer (or N-subunit) phase of at least PrE-II is known to be critical for its function (Tapparel et al., 1998
; Murphy et al., 1998
). This phase effect is thought to be due to the different chemical environments of each of the 6 nt bases associated with each N-subunit, as revealed by chemical attack studies of resting viral nucleocapsids (Iseni et al., 2002
). Adenosines in any hexamer phase are largely protected from dimethylsulfate, whereas cytosine reactivity is high only in hexamer positions one and six, precisely the positions of the conserved cytidines in the PrE-II element of G/Pr and AG/Pr.
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METHODS |
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Sequence and plasmids.
All the plasmids harbouring the mini-replicons expressing the green fluorescent protein (GFP) are derived from pSV-DI-H496 described in Vulliémoz & Roux (2001
, 2002)
. In all derivatives used in this study, the 3' end of the mini-genome RNA complementary to the T7 RNA transcript (intermediate replicon for GFP template RNA replication) contains an AG/Pr (see Fig. 2a
of the present work, construct [AGP]). The GFP open reading frame (ORF), flanked by the SeV transcription start and stop signals, was introduced between the CelII and MunI restriction sites (as in Vulliémoz & Roux, 2001
), and the sequence located between MunI and DraIII was deleted to generate [GP]. A cassette containing the adequate promoter(s) between DraIII and BamHI generated constructs [GP-GP], [AGP] and [AGP-GP]. Site-directed substitutions (C58A) and deletions in the genomic promoter of [GP58A-GP], [GP58A], [GP58A-GPd1258A], [GPd12] and [AGP-GPd1258A] were introduced by fusion PCR with adequate oligonucleotides and primers. Constructs [GP58A-GPd12] as well as [AGP-GPd12] and [AGP GPd48] were derived from constructs [GP58A-GP] and [AGP-GP], respectively, with specific substitutions. Plasmids pTM1-N, -P/Cstop and -L were constructed by introducing in the pTM1 vector the SeV-N, -P/Cstop and -L genes as described in Calain et al. (1992)
by using the NcoI site following the Encephalomyocarditis virus IRES.
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Replication of mini-replicons in the presence of support plasmids.
Confluent BSR-T7, seeded as described above the day before, were transfected with a mixture of plasmids including the plasmid harbouring the mini-genome (5 µg), the pTM1-N (1·5 µg), pTM1-P/Cstop (1·5 µg), pTM1-L (0·5 µg), 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 cells were collected as described above. Nine-tenths was 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 5 mM in EDTA and loaded onto linear 2040 % 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 in the pellet were separately collected as described previously (Calain & Roux, 1995
). Poly adenylated RNAs were selected in the non-encapsidated RNA fraction using the Oligotex mRNA mini kit (Qiagen), according to the supplier's instructions.
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. To score non-encapsidated RNAs present in the CsCl gradient pellets, a GFP-specific probe was prepared. The blotted membranes were exposed to Kodak X-Omat films. The autoriadiographs were scanned and the intensity of the replication signal was measured using ONE-Dscan version 1.0 (Scananalytics; CSP).
Primer extensions.
Primer extensions were done as described previously (Vulliémoz & Roux, 2001). An infrared dye-labelled oligonucleotide (IRD 800, 5'-CAGCTTGCCGTAGGTGGCATCGCCC-3') of negative polarity positioned in the GFP ORF was used. Due to built in properties of the automated sequencer (LI-COR DNA 4000; MWG-Biotech), the results can only be interpreted semi-quantitatively and to define the position of RNA synthesis initiation.
Analysis of the GFP expression.
Infected/transfected cells were collected in PBS. Flow cytometry was performed on a Becton-Dickinson FACSCan2. R1 and M1 parameters were adjusted on BSR-T7 cells infected with rSeV-AGP55 and transfected with the plasmid harbouring [GPd12]. Data analysis was performed with a Becton Dickinson software.
GFP relative expression.
Estimation of GFP gene transcription by measures of mean GFP fluorescence depended on the amount of the template available responsible for GFP expression. However, due to differences in replication ability of the different templates, the fraction of the cells harbouring these templates varied. The flow cytometry measures, however, allow one to estimate, for each template, its distribution which corresponds to the percentage of gated cells (%gated). Replication was then corrected for the variable distribution among the different templates as follows: Replication/%gatedx100=Corrected Replication. In the end GFP fluorescence was expressed as: Mean fluorescence/Corrected Replication = GFP fluorescence. 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 legend, as is the number of independent experiments (three or four) performed, and the mean of these values (see also text of Results).
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RESULTS |
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Transcription downstream of AG/Pr
To create mini-replicons in which GFP mRNAs do not initiate from within the elements used for replication, mini-replicons with tandem 96 nt promoters were used, in which the external promoter was AG/Pr preceding a G/Pr, harbouring the active gs1 (Fig. 2). The series of GFP mini-replicons used to characterize this general configuration is shown in Fig. 2(a)
. [AGP] is a single promoter construct with AG/Pr directly upstream of the GFP ORF. These double promoter constructs initiate replication only from the external AG/Pr (AG/Prext) (Vulliémoz & Roux, 2001
, 2002
). Whether they are transcription competent was open to question, either when the internal GP is integer [AGP-GP] or when it carries a 12 nt deletion in PrE-I. Construct [AGP-GPd1258A], further carries a C58A mutation on the internal G/Pr (G/Print) that strongly decreases transcription from gs1 (Le Mercier et al., 2002
). It was introduced here to verify whether transcription from [AGP-GPd12] can be suppressed. Finally, [GPd12] was designed to test the effect of the 12 nt deletion on [GP] transcription.
Fig. 2(b) shows that all the constructs were replication competent, within the same range, with the exception of [GPd12] for which the 12 nt deletion totally suppresses replication. As indicated by the difference in migration between the [AGP] and constructs 2, 3 and 4, replication of the double promoter constructs initiated only at AG/Prext as expected (see also Fig. 6
).
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Internal gs1 still functions without the two replication promoter elements
The 12 nt deletion at the 3' end of PrE-I of G/Print allows transcription from gs1146 (see Fig. 2); this part of PrE-I that is essential for replication is then dispensable for transcription. When the 12 nt deletion was extended to 48 nt ([AGP-GPd48], Fig. 3
), transcription was decreased ca. fivefold, indicating that nucleotides 1248 play a role in transcription efficiency. Nevertheless, transcription can take place with a completely truncated PrE-I. We then examined the requirement for PrE-II, the other element essential for replication. [AGP-GPd48dBB], which also lacks PrE-II, showed no further penalty in transcription than [AGP-GPd48] (Fig. 3c
). In conclusion, significant transcription takes place from gs1 that lies downstream of AG/Prext and which is removed from the totality of the G/Pr sequences known to be essential for replication. Assuming that PrE-I and PrE-II together directly recruit vRdRp to initiate at the genome 3' end, it appears unlikely that a G/Pr devoid of these two elements can directly recruit vRdRp to initiate at gs1. The most likely explanation for GFP expression from the ectopic gs1 is that vRdRp is recruited to the template by AGPext before it can initiate at the downstream gs1. Presumably, trailer RNA synthesis would occur first, and vRdRp could then scan the template for the downstream gs1.
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AG/Pr and G/Pr are not equivalent in promoting GFP expression
To examine whether the nature of the external replication promoter influenced the efficiency of GFP expression from an ectopic gs1, we directly compared two series of mini-replicons which contain either AG/Prext or G/Pr -C58Aext (Fig. 5a). The three AGP constructs were amplified (2050-fold) better than their GP counterparts, as expected, as AG/Pr is known to be the stronger of the two replication promoters (Fig. 5b
). Despite their lower level of replication, the G/Prext mini-replicons constantly exhibited a higher mean GFP fluorescence (data not shown). After normalization to template levels, the G/Prext constructs expressed 2050-fold more GFP than the AG/Prext counterparts (Fig. 5c
).
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Primer extensions were also performed to precisely determine the position of the RNA 5' ends. For instance, the strong decrease in GFP expression due to the C58A mutation introduced in the internal G/Pr (Fig. 2, [GP58A-GPd1258A]; Fig. 3
, [AGP-GPd1258A]) suggests that the internal gs1 is the site of mRNA initiation. If so, this will be undoubtedly demonstrated by primer extension. Fig. 6(a)
shows the results of extending a primer of negative polarity situated in the GFP ORF on the various CsCl pellet RNAs. A double band (presumably due to the presence of a 5' cap group) is present only at the transcription start site of the internal gs1. When primer extensions were performed with the encapsidated (CsCl band) RNAs, the 5' ends of the mini-antigenomes were found to be displaced according to the nucleotide deletion in G/Print (Fig. 6b
). Identical results were obtained with the three constructs of the GP58A series (data not shown). These results confirm that GFP expression is due to mRNAs that have initiated at the ectopic gs1.
GFP expression in the absence of the helper virus
The above mini-replicon system uses a helper virus to provide the replication and transcription functions. As the various mini-replicons compete more or less effectively with the helper genome, the amounts of replication substrates available could vary and this could in some way affect the relative use of vRdRp as a transcriptase or replicase. It was therefore of interest to also examine the two series of mini-replicons of Fig. 5 when they were amplified and expressed by the N, P and L proteins derived from plasmids (Fig. 7
). vRdRp availability here only depends on the extent of plasmid transfection. In contrast to the helper virus mediated mini-genome amplification (Fig. 5
), there was only a two- to fivefold difference between the levels of AGPext and GPext mini-replicons amplified by plasmid-expressed N, P, and L (Fig. 7b
). The absence of competition with the helper genomes is probably responsible for this difference, and vRdRp provided by plasmids may be less limiting as well. More importantly, the mean fluorescence of the GP58A-GP constructs was again higher than that of the AGP-GP constructs (Fig. 7a
). After correction for template levels, G/Prext constructs expressed GFP four-to 40-fold more efficiently than corresponding AG/Prext constructs (Fig. 7c
).
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DISCUSSION |
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There are two views of how non-segmented negative-stranded vRdRp gains access to gs1. vRdRp can either directly interact with gs1 without having first initiated leader RNA, as recently proposed for vesicular stomatitis virus (VSV) (Whelan & Wertz, 2002). Alternatively, vRdRp can reach gs1 after having entered the nucleocapsid at its 3' end. Our results suggest that this latter mechanism applies for SeV. Although in most experiments gs1 is part of an internal replication-incompetent G/Pr (due to deleting the conserved first 12 nt), it is possible to delete up to the first 48 nt of G/Print and its PrE-II element as well without eliminating GFP expression (Fig. 3
). In these latter cases, it is unlikely that there are sufficient cis-acting sequences remaining for SeV RdRp to enter the template directly at gs1, given that RNA initiation at the genome 3' end requires at least two essential sequence elements spread over 96 nt of G/Pr. If, on the other hand, vRdRp arrives at gs1 after having entered the nucleocapsid at the 3' end, gs1 of the highly deleted G/Print would still function.
We found that the frequency with which gs1146 initiated mRNA synthesis appeared to depend, in an inverse fashion, on the strength of the upstream replication promoter. When a minimal (10 nt) gs1 was introduced into AG/Pr at position 56, this diminished the use of this 3' end promoter for replication (Le Mercier et al., 2003). In those experiments, there was an inverse relationship between the presence of gs1 and the relative strength of the replication promoter. In the present study, there was an inverse relationship between the relative efficiency of gs1 positioned downstream of the replication promoter and the relative strength of that promoter. Thus, when transcription and replication start sites were in close proximity, each form of viral RNA synthesis negatively affected the other. There are two relatively straightforward, but very different, interpretations for these observations. The first is that mRNA start sites and 3' end replication promoters compete for a common pool of vRdRp (Le Mercier et al., 2003
). The second is that, given the proximity of these two RNA start sites, the interaction of a transcriptase with gs1 interferes with a replicase starting RNA synthesis from the genome 3' end. In this case, the apparent competition would be due to steric interference of the vRdRp that initiates mRNA synthesis with that which synthesizes RNA from the genome 3' end, and vice-versa.
In mini-replicons, in which gs1 was displaced from position 56 to 68, the gene start site was equally effective in reducing replication (Le Mercier et al., 2003). This limited displacement, which maintains gs1 between PrE-I and PrE-II of G/Pr, however, is probably insufficient to alter possible steric interference. In the present experiments the transcription start site of GP58A-GPd12 had been displaced 90 nt downstream to position 146 relative to the genome 3' end. Nevertheless, the apparent competition between gs1 and the 3' end replication promoter could still be observed. It is then less likely that this competition operates via some form of steric interference. In this case, it would appear that the two viral RNA start sites within G/Pr negatively influence each other by competing for a common pool of vRdRp.
Remarkably, the competition between transcription and replication promoters occurred even when vRdRp and N protein were provided by plasmids in the absence of helper virus (Fig. 7). This competition thus appears to be direct, and not due to secondary effects affecting the provision of replication substrates that could occur during helper-virus mediated mini-genome expression. There is good evidence that the negative-stranded vRdRp can scan the nucleocapsid template for new gene start sites once they have released the mature mRNA (Stillman & Whitt, 1998
; Fearns & Collins, 1999
). These vRdRp may also be able to scan the template for gs1. If so, the competition we envision would occur in large part during this template scanning: the presence of a gs near the 3' end replication promoter would divert vRdRp from scanning back to the genome 3' end, and the presence of a strong 3' end promoter would disfavour vRdRp scanning to gs1. It will be of interest to displace a minimal gs1 progressively away from the genome 3' end, and to determine whether the intervening distance affects the efficiency of mRNA synthesis from gs1.
One complication in interpreting our data is that we do not know how the C58A mutation inhibits GFP mRNA synthesis from gs1. It is possible that this mutation simply inhibits mRNA initiation, or it inhibits productive mRNA synthesis. Indeed, in a study of the transcription start signal of VSV, mutations were found that would allow initiation of mRNA synthesis without proper capping (Stillman & Whitt, 1999). In this case, the uncapped transcripts were prematurely terminated and degraded, a finding that could only be made in in vitro studies, and thus more difficult with SeV. Even in this case, however, SeV RdRp would, in effect, simply recapitulate leader RNA synthesis, i.e. synthesize a short, uncapped transcript without being committed to transcription. It is difficult to see how synthesis of an abortive transcript from gs156 would so strongly enhance productive mRNA synthesis from a downstream gs1. Moreover, when [GP58A-GPd12] and [GP-GP] were compared (Fig. 4
), there was no evidence that the C58A mutation had enhanced expression from gs1146. Finally, we recently produced [GPgsm-GPd12] constructs in which nt 5665 of GPext were all substituted with the corresponding nucleotides of AGP. These replicated two- to threefold better than their [GP58A-GPd12] counterparts, but generated similar high mean GFP fluorescence (data not shown). In the end the more likely possibility is that vRdRp initiates at gs1146 only after releasing the short leader RNAs. It must then scan the template for a new RNA synthesis start site, and it is during this process that the various promoters compete with each other for scanning vRdRp.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Buchholz, U. J., Finke, S. & Conzelmann, K.-K. (1999). Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. J Virol 73, 251259.
Calain, P. & Roux, L. (1993). The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA. J Virol 67, 48224830.[Abstract]
Calain, P. & Roux, L. (1995). Functional characterisation of the genomic and antigenomic promoter of Sendai virus. Virology 212, 163173.[CrossRef][Medline]
Calain, P., Curran, J., Kolakofsky, D. & Roux, L. (1992). Molecular cloning of natural paramyxovirus copy-back defective interfering RNAs and their expression from DNA. Virology 191, 6271.[CrossRef][Medline]
Egelman, E. H., Wu, S.-S., Amrein, M., Portner, A. & Murti, G. (1989). The Sendai virus nucleocapsid exists in at least four different helical states. J Virol 63, 22332243.[Medline]
Fearns, R. & Collins, P. L. (1999). Model for polymerase access to the overlapped L gene of respiratory syncytial virus. J Virol 73, 388397.
Gubbay, O., Curran, J. & Kolakofsky, D. (2001). Sendai virus genome synthesis and assembly are coupled: a possible mechanism to promote viral RNA polymerase processivity. J Gen Virol 82, 28952903.
Hoffman, M. A. & Banerjee, A. K. (2000). Precise mapping of the replication and transcription promoters of human parainfluenza virus type 3. Virology 269, 201211.[CrossRef][Medline]
Iseni, F., Baudin, F., Garcin, D., Marq, J. B., Ruigrok, R. W. H. & Kolakofsky, D. (2002). Chemical modification of nucleotide bases and mRNA editing depend on hexamer or nucleoprotein phase in Sendai virus nucleocapsids. RNA 8, 10561067.
Lamb, R. A. & Kolakofsky, D. (2001). Paramyxoviridae: the viruses and their replication. In Fields Virology, 4th edn, pp. 13051340. Edited by D. M. Knipe & P. M. Howley. Lippincott, Williams & Wilkins.
Le Mercier, P., Garcin, D., Hausmann, S. & Kolakofsky, D. (2002). Ambisense Sendai viruses are inherently unstable but are useful to study viral RNA synthesis. J Virol 76, 54925502.
Le Mercier, P., Garcin, D., Garcia, E. & Kolakofsky, D. (2003). Competition between the Sendai virus N mRNA start site and the genome 3'-end promoter for viral RNA polymerase. J Virol 77, 91479155.
Mottet, G. & Roux, L. (1989). Budding efficiency of Sendai virus nucleocapsids: influence of size and ends of the RNA. Virus Res 14, 175187.[CrossRef][Medline]
Murphy, S. K., Ito, Y. & Parks, G. D. (1998). A functional antigenomic promoter for the Paramyxovirus Simian virus 5 requires proper spacing between an essential internal segment and the 3' terminus. J Virol 72, 1019.
Pelet, T., Delenda, C., Gubbay, O., Garcin, D. & Kolakofsky, D. (1996). Partial characterization of a Sendai virus replication promoter and the rule of six. Virology 224, 405414.[CrossRef][Medline]
Rager, M., Vongpunsawad, S., Duprex, W. P. & Cattaneo, R. (2002). Polyploid measles virus with hexameric genome length. EMBO J 21, 23642372.
Skiadopoulos, M. H., Vogel, L., Riggs, J. M., Surman, S. R., Collins, P. L. & Murphy, B. R. (2003). The genome length of human parainfluenza virus type 2 follows the rule of six, and recombinant viruses recovered from non-polyhexameric-length antigenomic cDNAs contain a biased distribution of correcting mutations. J Virol 77, 270279.[CrossRef][Medline]
Stillman, E. A. & Whitt, M. A. (1998). The length and sequence composition of vesicular stomatitis virus intergenic regions affect mRNA levels and the site of transcript initiation. J Virol 72, 55655572.
Stillman, E. A. & Whitt, M. A. (1999). Transcript initiation and 5'-end modifications are separable events during vesicular stomatitis virus transcription. J Virol 73, 71997209.
Tapparel, C., Maurice, D. & Roux, L. (1998). The activity of Sendai virus genomic and antigenomic promoters requires a second element past the leader template regions: a motif (GNNNNN)3 is essential for replication. J Virol 72, 31173128.
Vulliémoz, D. & Roux, L. (2001). Rule of Six: how does the Sendai virus RNA polymerase keep count? J Virol 75, 45064518.
Vulliémoz, D. & Roux, L. (2002). Given the opportunity, the Sendai virus RNA-dependent RNA polymerase could as well enter its template internally. J Virol 76, 79877995.
Whelan, S. P. J. & Wertz, G. W. (2002). Transcription and replication initiate at separate sites on the vesicular stomatitis virus genome. Proc Natl Acad Sci U S A 99, 91789183.
Received 9 July 2004;
accepted 14 October 2004.