Dept of Genetics and Microbiology, University of Geneva School of Medicine, CMU, 9 Ave de Champel, CH1211 Geneva, Switzerland1
Author for correspondence: Oliver Gubbay. Present address: MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9ET, UK. Fax +44 131 228 5571. e-mail o.gubbay{at}hrsu.mrc.ac.uk
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Abstract |
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Introduction |
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During transcription, vRNAP (or transcriptase) initiates at the genome 3' end, with synthesis of the short leader region. The transcriptase is thought to terminate the leader RNA and reinitiate at the start of the first gene, to synthesize the N mRNA. This transcriptase then continues across the genome, in turn polyadenylating and terminating each mRNA, reinitiating and capping the mRNA of the following gene. Polyadenylation is achieved by the transcriptase reiteratively copying a short run of template uridylates (vRNAP stuttering) before releasing the mRNA with a tail of adenylates. The transcriptase then crosses a short intergenic region, without synthesis, and reinitiates at the beginning of the adjacent downstream gene. During replication, vRNAP (or replicase) initiates synthesis at the 3' end of the genome and antigenome. However, unlike the transcriptase, the replicase ignores all the junctions (and editing signals) to produce an exact complement of the 15·384 kb genome. The fact that full-length antigenomes and genomes, in contrast to the mRNAs, are found only as assembled nucleocapsids has led to the notion that genome/antigenome synthesis and encapsidation are coupled (see below).
Much of our knowledge of mononegavirus RNA synthesis has come from studying vesicular stomatitis virus (VSV), a model rhabdovirus with a particularly robust virion transcriptase activity. Iverson & Rose (1981) studied the kinetics of VSV transcription in vitro by hybridizing short DNA fragments corresponding to specific genome sequences to the VSV virion reaction products. They found that VSV RNAP synthesized mRNA at an average rate of 3·7 nt/s within genes and at 1·4 nt/s over regions that span gene junctions. Assuming that VSV RNAP synthesizes mRNA at a constant rate, the extra time spent in crossing the junctions was presumably due to vRNAP stuttering to form the poly(A) tail and reinitiation of the next mRNA. Even for VSV, however, there is as yet little information on how vRNAP proceeds across the template during genome replication and how this differs from the process of transcription.
In contrast to the VSV virion transcription reaction, that of SeV is much less active and requires high concentrations of polyanions and other special conditions for optimal activity (Vidal & Kolakofsky, 1989 ). A previous study to measure transcriptional read-through of the leader (le)/N junction by SeV vRNAP in in-vitro virion reactions demonstrated that the extent of read-through varies between different SeV strains (510% for strain H and 2040% for strain Z). In SeVZ virion reactions, vRNAP that had initiated at position 1 (the genome 3' end) and read through the le/N junction (position 55/56) did not proceed further than 250300 nt from the genome 3' end. In contrast, >90% of vRNAP that had (re)initiated at position 56 synthesized the complete N mRNA (terminating at position 1737). These experiments suggest that vRNAP initiates RNA synthesis on the template as a relatively non-processive enzyme, but becomes processive (independent of concurrent assembly) upon (re)initiation at position 56 (and possibly also capping of the mRNA). The reinitiated transcriptase also responded to junction signals at very high frequency (close to 100%). Alternatively, vRNAP that has initiated at position 1 may also become processive during genome synthesis, as a replicase. Unlike transcriptases, however, replicases are expected to exhibit a sufficiently high degree of processivity that allows them to ignore junction (and editing) signals. The precise difference between vRNAPs engaged in transcription and replication is unknown; these two forms of vRNAPs have been defined so far essentially by the RNA products they generate.
Besides high concentrations of polyanions, the SeV virion reaction can be stimulated by high concentrations of cytoplasmic extracts (Curran et al., 1992 ). In either case, however, the limited activity of these reactions, and the fact that SeV cores contain about 50 vRNAPs scattered across each nucleocapsid (Portner et al., 1988
), makes them unsuitable for detailed kinetic analysis. This paper reports a reconstituted SeV in-vitro reaction, in which the progress of vRNAP along the templates during transcription, as well as replication, can be followed.
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Methods |
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In-vitro transcription and replication.
RNA synthesis was performed essentially as described by Curran et al. (1994) and Curran (1998)
. Briefly, N:RNA (non-defective) templates were isolated from SeV-infected (strain Z) egg allantoic fluid by banding twice on 2040% CsCl linear gradients. Templates were resuspended at a concentration of about 250 µg/ml in TE (10 mM TrisHCl, pH 7·4, 1 mM EDTA) containing 1 mM DTT and 10% glycerol and stored at -70 °C. A549 cells (in 9 cm diameter Petri dishes) were infected with a recombinant vaccinia virus expressing T7 polymerase (vTF7-3; Fuerst et al., 1986
) and transfected with 2·5 µg pGEM-N, 2·5 µg pGEM-PHA and 1 µg pGEM-L. At 24 h p.i., medium was removed and the cells were incubated for 1 min with 1 ml ice-cold buffer A (150 mM sucrose, 30 mM HEPESNaOH, pH 7·4, 33 mM NH4Cl, 7 mM KCl, 4·5 mM magnesium acetate) containing 250 µg/ml lysolecithin. The cells were then scraped into 300 µl energy mix (100 mM HEPESNaOH, pH 7·4, 150 mM NH4Cl, 4·5 mM magnesium acetate, 1 mM DTT, 1 mM ATP/CTP/UTP, 10 µM GTP, 40 U/ml creatine phosphokinase, 1 mM creatine phosphate) and cell membranes were disrupted by pipetting up and down 20 times. Nuclei and cell debris were removed by centrifugation at 12000 g for 5 min at 4 °C.
RNA synthesis was carried out in 150 µl reactions containing 510 µl of the N:RNA template, 20 µg/ml actinomycin D, 100 µl of a vTF7-3-infected and transfected A549 cell extract and either 30 µCi [-32P]GTP or 1 mM GTP at 30 °C for 3 h. Nucleocapsid and non-nucleocapsid RNAs were separated by centrifugation on 20%, 40%, 5·7 M CsCl step gradients and analysed on agaroseformaldehyde gels. During a kinetic analysis, aliquots were removed at the times indicated in the figure legends and the reaction was stopped by addition of 20 mM EDTA.
RNase protection assay.
Riboprobes, corresponding to sequences at the ends of the M, N and P genes, were prepared as SP6 run-off transcripts from pGEM-M (linearized with NdeI; 227 nt), pGEM-N (linearized with MscI; 253 nt) and pGEM-P (linearized at an MluI site that was introduced at position 1436 on the P gene; 307 nt), respectively. The 142 nt riboprobe corresponding to sequences of the leader and first 87 nt of the N gene was described by Vidal & Kolakofsky (1989) . Riboprobes were purified from 6% acrylamideurea gels and co-precipitated with CsCl-purified RNA from SeV in-vitro reactions and RNase protection analysis was then performed essentially as outlined by Vidal & Kolakofsky (1989)
. After treatment with RNase A, RNA duplexes were recovered by precipitation with an inactivation buffer (Ambion), according to the manufacturers instructions. Protected fragments were resolved on a 6% acrylamideurea gel, visualized by autoradiography and quantified by using a phosphorimager.
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Results |
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The radiolabelled RNA products of an in-vitro reaction were separated by CsCl density gradients into banded and pelleted material on a formaldehydeagarose gel. The banded material contains encapsidated RNA and thus represents the nucleocapsid products of replication. Pelleted material, however, contains unencapsidated RNA and largely represents the products of transcription (see below). The replication (nucleocapsid-assembled) products formed defined bands that migrated progressively more slowly with time of reaction, until the full-length genome of approximately 15·384 kb was completed (Fig. 1A). The relative intensities of bands, when normalized for their G content (by reference to RNA size markers), were roughly the same (data not shown). This reaction is thus highly processive for a significant fraction of the replicases, in that most of these replicases completed their chains. The recovery of genome chains of intermediate length as nucleocapsid-assembled material finally provides direct evidence that genome synthesis and assembly are concurrent events. From these data, we calculated that genome synthesis occurred at approximately 1·22·2 nt/s throughout the time-course of the reaction. Thus, many of the vRNAPs engaged in genome replication began synthesis at the beginning of the reaction, as expected, and continued in a relatively synchronous fashion across the template at an average rate of 1·7 nt/s. In particular, processivity of vRNAPs during replication is emphasized by the fact that replicases showed no evidence of slowing down towards the end of the reaction.
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The kinetics of mRNA synthesis, determined by analysing unencapsidated RNAs in these reactions, differed from those of antigenome synthesis in several respects (Fig. 4B). Firstly, the unencapsidated N and P gene sequences accumulated linearly throughout the entire course of the reaction, without any tendency to level off. Secondly, the N and P mRNAs accumulated at clearly different rates; the P gene sequences accumulated about half as fast as the N gene sequences (as indicated by the relative gradients of each graph). Lastly, the fastest transcriptases (analysed by linear regression analysis) completed the N mRNA at an average of 3·0 nt/s, whereas they appeared to have completed the P mRNA at an average of only 2·1 nt/s. Assuming that vRNAP synthesizes mRNA at a constant rate (of 3·0 nt/s), the extra time spent synthesizing the complete P gene (calculated to be 8·5 min) may be due to the combined effects of the N/P intergenic and P editing sites. However, even though the fastest transcriptases thus traversed the N and P genes of the genome template at least as rapidly as the replicases, this reaction appeared to be very poorly processive. For example, most of the transcriptases that initiated the N mRNA apparently did not finish this chain (Fig. 1B
) and only about half the transcriptases that finished the N mRNA continued to finish the P mRNA (Fig. 4B
). Furthermore, very few (if any) of the transcriptases then continued to finish the M mRNA (Fig. 3
). We suspect that most of these transcriptases have paused heterogeneously along the template and eventually released their chains. vRNAPs without nascent chains would then, presumably, be free to disengage from the template and re-initiate mRNA synthesis at the beginning of the leader or adjacent start sites.
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Discussion |
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Within the family Paramyxoviridae, respiroviruses such as SeV and human parainfluenza virus type 3 apparently require only host cell components to promote vRNAP processivity during mRNA synthesis. However, pneumoviruses such as respiratory syncytial virus (RSV) encode a protein (M2-1) for this purpose, which is required in addition to possible host cell components. In the absence of the M2-1 protein, RSV mRNA synthesis rarely continues beyond the first two (very short) NS1 and NS2 genes, yet the absence of this protein has no effect on genome replication (Fearns & Collins, 1999 ). Moreover, M2-1 promotes vRNAP read-through of gene junctions to form dicistronic mRNAs (Hardy et al., 1999
; Hardy & Wertz, 1998
). Both these effects of M2-1 could thus be due to its ability to prevent RSV RNAP pausing during transcription. However, none of these cellular and viral factors required for vRNAP processivity during paramyxovirus mRNA synthesis is required for vRNAP processivity during genome synthesis (Fearns & Collins, 1999
). Thus, for at least two genera of this virus family, replicases acquire processivity independent of the cellular and viral factors required for RNAP processivity during mRNA synthesis.
The effect of nascent chain assembly on RNAP processivity during replication
In these in-vitro reactions, genome, antigenome and mRNA synthesis all take place simultaneously using the same pool of P and L proteins (as transfected cell extract). Nevertheless, vRNAPs engaged in replication are highly processive, whereas those engaged in transcription are not. The main difference between these two modes of vRNA synthesis is that RNA synthesis and assembly are coupled during replication. As a consequence of this coupling, replicases are envisaged to interact with PN assembly complexes involved in nascent chain assembly (Curran, 1998 ; Horikami et al., 1992
). We cannot rule out that the presence of N (and P) protein may lead to a conformational change in vRNAP that itself promotes processivity (Mooney & Landick, 1999
). However, we argue that the coupling of assembly and synthesis promotes processivity simply by preventing vRNAP pausing, independent of possible changes in vRNAP conformation (see below).
Bacterial and eukaryotic DNA-dependent RNAPs synthesize RNA at 30100 nt/s in vitro, which is nevertheless insufficient to account for the rates observed in vivo (Reines et al., 1996 ; Uptain et al., 1997
; Shilatifard, 1998
). In addition to their slower overall rate in vitro, transcription elongation by these cellular RNAPs is disrupted by frequent pausing. Pausing is thought to occur at specific sites where NTP addition is disfavoured, and RNAP has a propensity to backtrack on the template and nascent RNA, removing the nascent RNA 3' end from the RNAP active site (Reeder & Hawley, 1996
; Guajardo & Sousa, 1997
; Komissarova & Kashlev, 1997a
, b
; Nudler et al., 1997
). During the pause, RNAP is envisaged as oscillating between the backtracked inactive state and the active (forward) state. SeV vRNAP backtracking is thought to be essential for the vRNAP stuttering that forms the mRNA poly(A) tail (as well as the G additions for P mRNA editing); this is envisaged to be achieved by nascent RNAtemplate hybrid realignment within the elongating vRNAP (Hausmann et al., 1999a
, b
).
Although we have provided direct evidence that genome synthesis and assembly take place concurrently, how closely the assembly of the nascent genome/antigenome chain follows its synthesis is unclear. It is possible that assembly occurs as soon as the nascent RNA emerges from the vRNAP exit channel (Mooney & Landick, 1999 ) and is available for interaction with the PN assembly complex, since replicases traverse their templates relatively slowly (1·8 nt/s). In this case, the nascent chain assembled with N subunits would hinder vRNAP backtracking sterically, as backtracking requires the most recently exposed RNA chain to be drawn back within the exit channel (Reeder & Hawley, 1996
; Komissarova & Kashlev, 1997a
, b
; Mooney & Landick, 1999
); this model is summarized in Fig. 5
. The close coupling of genome synthesis and assembly would thus favour vRNAP read-through of the gene junctions and other transcriptional pause sites and promote vRNAP processivity throughout genome synthesis.
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Following on from the model described above, the uncoupling of assembly from genome synthesis would permit vRNAP backtracking at poly(A)/termination sites and produce truncated genomes. A rare polyadenylated transcript representing the leader and N gene sequences (as read-through) is found in measles virus-infected cells, but only as a nucleocapsid, whereas the N mRNA is found only in a non-assembled form (Castaneda & Wong, 1990 ). This rare measles virus fusion transcript was presumably made by vRNAP that began the synthesis of an antigenome nucleocapsid but nevertheless polyadenylated and terminated the chain at the second junction. However, both these examples of uncoupling are rare events. Some mechanism must therefore exist to ensure that genome synthesis and assembly are tightly coupled, especially as replicases approach poly(A) signals. For example, replicase progression along the template could be driven by its interaction with the PN assembly complex once genome synthesis and assembly have become coupled. The idea that the interaction between elongating replicases and PN assembly complexes would not only promote but also maintain vRNAP processivity is therefore a corollary of the above model.
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Acknowledgments |
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References |
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Received 29 May 2001;
accepted 10 August 2001.