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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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The family Paramyxoviridae is segregated into the subfamilies Paramyxovirinae and Pneumovirinae, which differ, among other things, by their obedience to the rule of six and in the organization of their promoters (for a general review on paramyxoviruses, see Lamb & Kolakofsky, 2001
).
The rule of six stipulates that efficient replication of the paramyxovirinae genomes and antigenomes only takes place when the total number of nucleotides is a multiple of six. Originally demonstrated for mini-genome replication (Calain & Roux, 1993), this rule was eventually confirmed for the full-length genome and antigenome replication (Rager et al., 2002; Skiadopoulos et al., 2003). The significance of this rule has to be appreciated with the understanding that some nucleotides are recognized by the vRdRp in the context of the nucleocapsid protein (N), which interacts with exactly 6 nt. Consequently, certain nucleotides, representing signals for the vRdRp, are only found in a particular N-interacting context, a fact that was dubbed the ‘N phase context’ (Kolakofsky et al., 1998). This is the case for the phosphoprotein (P) gene editing signal and for some conserved promoter replication motifs (Tapparel et al., 1998; Murphy & Parks, 1999; Iseni et al., 2002, see below).
Paramyxovirinae promoters are bipartite, with two elements essential for replication, PrE-I and Pr-EII, situated at both ends of a 96 nt sequence stretch (Tapparel et al., 1998; Murphy & Parks, 1999; Hoffman et al., 2006). For G/Pr, in between PrE-I and PrE-II, sits a stretch of 10 nt (nt 56–65) representing the first transcription signal (gs1 for Sendai virus: 3'-UCCCAGUUUC, genome sense). In AG/Pr, at nt 58–66, also sits a signal involved in transcription in the form of a sequence complementary to the last gene end signal (ge, 3'-AAAAAGAAU, antigenome sense). This bipartite promoter organization does not apply to pneumoviruses. For Human respiratory syncytial virus (HRSV), for example, a prototype member of the Pneumovirinae, G/Pr is 52 nt. The 3'-terminal 11 nt constitute a vRdRp-binding site and the first 34 nt contain all of the sequences required for antigenome synthesis (Fearns et al., 2002). At the other end of the promoter, nt 43–52 constitute a gs1, sufficient to signal transcription initiation, whose efficiency depends, however, on the direct upstream U-rich sequence (33–42 nt) (reviewed by Cowton et al., 2006). For vesicular stomatitis virus (VSV), the prototype of the family Rhabdoviridae, G/Pr is 60 nt, with nt 50–60 constituting gs1 at the inner border of the promoter sequence (Pattnaik et al., 1995; Conzelmann et al., 1990; Whelan & Wertz, 1999).
Regardless of the difference in these promoter primary structures, the fact remains that G/Pr promotes replication and transcription and AG/Pr solely promotes replication (Vulliémoz et al., 2005; Cordey & Roux, 2006). This has raised questions about the regulation of replication compared with transcription. The original model proposed that vRdRp initiates mRNA synthesis at gs1 after synthesis of a leader RNA, initiated at the genomic 3' extremity (Kolakofsky & Blumberg, 1982). Release of the non-encapsidated leader RNA engages transcription. In contrast, when leader RNA encapsidation takes place, vRdRp ignores the leader/gs1 junction to produce an encapsidated full-length copy of the genome RNA. In this model, the incoming vRdRp is uncommitted to transcription or replication.
With the advent of reverse genetics, experiments involving insertion of paramyxovirus (Sendai virus) gs1 in AG/Pr, in place of ge, or involving mini-genome constructs with tandem promoters in which gs1 was positioned downstream of AG/Pr, have led to the implementation of a model with the notion of competition by the replication and transcription signals for available vRdRp (Le Mercier et al., 2003; Vulliémoz et al., 2005). However, vRdRp would always enter the template at the genomic 3' end to start synthesis eventually at gs1 (Cordey & Roux, 2006). This model also appears to be coherent with the data available for HRSV (reviewed by Cowton et al., 2006, and, in particular, Cowton & Fearns, 2005). In contrast, data obtained recently in the study of VSV have challenged this model. First, two populations of VSV vRdRp were purified, exhibiting the exclusive ability to replicate or transcribe (Qanungo et al., 2004). Secondly, initiation at VSV gs1 was shown in vivo (but not in vitro) not to be sensitive to UV cross-links between the replication signal and gs1, suggesting that vRdRp can bypass leader RNA synthesis to initiate transcription directly at gs1 (Whelan & Wertz, 2002). For VSV then, it appears that a committed replicase or transcriptase initiates its respective RNA synthesis after engaging the template at its respective initiation signal.
This VSV model does not account adequately for recent results obtained with Sendai virus or HRSV. Transcription efficiency from a gs1 positioned downstream of an AG/Pr decreased proportionally with the increasing distance separating the two signals (Cordey & Roux, 2006), suggesting that vRdRp traverses the separating distance and therefore does not enter the template directly at gs1. Similar experiments supporting the recruitment of a transcribing HRSV vRdRp at the genomic 3' end have been produced (Cowton & Fearns, 2005).
Another question regarding paramyxovirus gs1 concerns its conserved position relative to the genomic 3' end (nt 56). Extension of this distance from 56 to 146 nt was, however, feasible with no detrimental effect on transcription (Vulliémoz et al., 2005). This raised the possibility that the N phase context of gs1 is important for transcription.
In this paper, we pursued two goals that we thought would shed light on the mechanism by which vRdRp accesses gs1. On the one hand, we undertook characterization of nt 31–55 preceding gs1 with the rationale that direct entry of vRdRp at gs1 would require nucleotides essential for transcription in that region. In addition, we set up experiments to explore the importance of the gs1 phase context for transcription efficiency. The results obtained are discussed in the context of the models presented above.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), were propagated in Dulbecco’s minimal Eagle’s medium (DMEM) supplemented with 5 % fetal calf serum (FCS) in a 5 % CO2 atmosphere. The AGP-55 recombinant Sendai virus (rSeV-AGP55) was constructed and rescued as described previously (Le Mercier et al., 2002). Virus stocks reached titres ranging from 5x108 to 1x109 p.f.u. ml–1.
Plasmids.
All of the plasmids harbouring the mini-genome expressing the hygromycin-resistant gene and/or the green fluorescent protein (GFP) gene were derived from a pGem plasmid harbouring the AGP-GPdel12-GFP-AGPc construct (here called mini-GFP; Vulliémoz et al., 2005, GenBank accession no. DQ902685
[GenBank]
, 1434 nt). An oligonucleotide harbouring three linked copies of the 9 nt Gtx IRES module (Chappell et al., 2000, 2004) was introduced between the MunI (nt 211) and NcoI (nt 244) restrictions sites (this cistron encoding the GFP protein under Gtx IRES control remained silent in the construct). Next, the hygromycin resistance gene, which was PCR amplified from plasmid pEBS-PL-PrI (Bontron et al., 1997), was inserted in the MunI restriction site, just upstream of the Gtx IRES module, creating the AGP-GPdel12-HygRG-IRES-GFP-AGPc mini-genome (here called mini-HygRG; GenBank accession no. DQ914804
[GenBank]
, 2520 nt). Substitutions of the GPdel12 hexamer sequences 37–42, 43–48, 49–54, 52–57 or 56–61 with random nucleotides within mini-HygRG were carried out by insertion of fusion PCR products carrying each randomized hexamer between the XhoI (nt 98) and XbaI (nt 204) sites flanking the GPdel12 sequence. Ninety per cent of the transformed XL1 supercompetent cells (Stratagene) were directly grown in 100 ml L-broth medium. The remaining 10 % were plated on agar to select individual clones for each random hexamer preparation. To change the gs1 phase context, the whole GPdel12 cassette of AGP-GPdel12-GFP-AGPc was displaced by inserting, in between the XhoI and XbaI sites, PCR-amplified fragments containing the adequate addition/deletion sequences (see Fig. 5a). Similar fusion PCR products containing the modifications outlined in Fig. 6(a) were cloned in between the same two sites.
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Replication of mini-genomes in the presence of support plasmids only.
Confluent BSR-T7 cells, seeded the day before as described above, were transfected with a mixture of plasmids including the plasmid harbouring the mini-genome (5 µg), pTM1-N (1.5 µg), pTM1-P/Cstop (1.5 µg), pTM1-L (0.5 µg) and 20 µl Fugene (Roche). At 36 h post-transfection, cells were collected and treated as above for GFP expression analysis by flow cytometry and for Northern blot analysis.
Recovery of encapsidated and non-encapsidated viral RNA.
Infected/transfected BSR-T7 cells were collected, pelleted and resuspended in 0.7 ml lysis buffer I [0.6 % NP-40, 50 mM Tris/HCl (pH 8.0), 10 mM NaCl]. EDTA was added to a concentration of 5 mM to post-nuclei supernatants and loaded onto linear 20–40 % (w/w) CsCl gradients (Beckman SW55). After centrifugation (40 000 r.p.m., Beckman SW55, 12 °C, overnight), the nucleocapsid band in the gradient and the non-encapsidated cellular and viral RNAs in the pellet were collected separately, treated with SDS, phenol extracted and ethanol precipitated, as described previously (Calain & Roux, 1995).
RT-PCR.
The mini-genome nucleocapsid RNAs were resuspended in 10 µl TNE [20 mM Tris/HCl (pH 7.5), 50 mM NaCl, 2 mM EDTA] to which 2 pmol primer 5'-GGATAAGTCCAAGACTTCCTCGAG-3' (RT-PCR primer, nt 79–102, see Fig. 2a) of positive polarity complementary to AG/Pr were added. After a 5 min incubation at 70 °C, the mixture was immediately chilled on ice for 5 min. The reverse transcriptase reaction was carried out at 42 °C for 1 h using the Moloney murine leukemia virus (H–) reverse transcriptase (Promega) according to the supplier’s protocol. For the PCR step, 5 µl reverse transcription reaction was used with 30 pmol each of the RT-PCR primer plus a primer of negative polarity complementary to the hygromycin-resistant gene (PCR primer: 5'-CACGAGATTCTTCGCCCTCCGAG-3', nt 323–301, see Fig. 2a). Taq DNA polymerase (2.5 U; Invitrogen) was added together with MgCl2 and dNTPs to final concentrations of 1.5 mM and 200 µM, respectively. Twenty-five cycles of standard PCR amplification yielded a 244 bp DNA fragment (see Fig. 2a). Negative-control reactions with omission of the reverse transcriptase reaction were carried out to ensure that amplification was specific to the RNA.
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Viral RNA analysis.
Primer-extension reactions using CsCl pellet RNAs (mRNAs) as template and Northern blot analysis of the nucleocapsid RNAs (mini-genome RNAs) were carried out exactly as described previously (Cordey & Roux, 2006). For primer extension, a 5'-32P-labelled primer of negative polarity (nt 352–375) leading to an extension product of 228 nt was used.
Cytofluorometry.
Mean GFP fluorescence was measured by flow cytometry, performed on a Becton Dickinson FACScan2, as previously described (Vulliémoz et al., 2005).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, mini-HygRG). Rather than making systematic single nucleotide mutations in the gs1 upstream sequence, we prepared a series of mini-genomes harbouring randomized nucleotides, with the rationale that essential nucleotides, if any, would eventually be selected under the pressure to exhibit hygromycin resistance.
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). The physical separation of the two signals allowed us to study transcription without changing mini-genome replication. Furthermore, the copy-back nature of mini-HygRG (containing an AG/Pr promoter on both the plus and minus sense mini-genomes) induced infected cell survival and establishment of persistent infection (Roux & Holland, 1979), as shown in Fig. 1(b)
. Cell survival was essential to permit hygromycin resistance selection.
BSR-T7 cells were transfected with the plasmids expressing the various mini-HygRGs and then infected with rSeV-AGP55 (see Methods). rSeV-AGP55 is a helper virus in which the trailer region of its antigenomic promoter has been replaced with the leader region, allowing mini-genomes to be amplified more effectively (Le Mercier et al., 2002). After amplification of mini-HyrRG by rSeV-AGP55, cells and media were collected and used to infect fresh BSR-T7 cells. The freshly infected cells were cultured to confluence for 2 days before the addition of hygromycin, which was replaced fresh every day for 4 days. At the end of this time, cells were rinsed with PBS and stained with crystal violet to assess cell viability or collected to isolate the mini-genome nucleocapsid RNAs (see Fig. 1c for a summary of the sequence of events, and Methods for details).
Fig. 1(d) shows that mock-transfected/infected cells, as well as cells transfected/infected with a control plasmid expressing GFP (Fig. 1a, mini-GFP), did not grow under hygromycin treatment above 0.5 mg ml–1, in contrast to cells transfected/infected with mini-HygRG expressing the hygromycin resistance gene. These cells could be passaged further (data not shown) and were shown to establish a persistent infection containing the mini-HygRG genome in the form of nucleocapsids (Fig. 1a, inset), as did cells transfected/infected with mini-GFP and not treated with hygromycin (Fig. 1a, inset), as mini-GFP-1 also exhibited the copy-back defective RNA property to induce cell survival.
Checking the nucleotide sequence requirement upstream and in the first 6 nt of gs1
A series of mini-HygRG constructs was produced containing a randomized sequence of 6 nt organized in the hexameric pattern described in Fig. 2(a). Randomization separately covered four hexamers preceding gs1 (nt 31–54), the hexamer representing the first 6 nt of gs1 (nt 56–61) and the hexamer encompassing 4 nt preceding gs1 and the first 2 nt of gs1 (nt 52–57). Surviving cultures were obtained for each construct mixture run through the transfection/infection protocol and submitted to hygromycin treatment (not shown). Nucleocapsid RNAs were purified from the cultures and RT-PCR amplifications were performed to derive a 244 nt DNA fragment encompassing G/Pr (see Fig. 2c for two examples).
Sequences of the fragments derived from the gs1 upstream hexamers exhibited a randomized pattern, supporting a lack of nucleotide selection following the hygromycin treatment (Fig. 3a). This contrasted with the sequence pattern of the hexamer encompassing the first 6 nt of gs1 (Fig. 3b). In this case, the wild-type sequence 3'-TCCCAG-5' emerged above the background of a randomized sequence. This result validated the experimental approach. To explain this background, it should be noted that the mini-genomes will replicate efficiently and propagate regardless, as long as the cells survive the hygromycin treatment. In the case of hexamers 31–36 and 49–54, resistance could then derive from the presence of more than one combination of sequences leading to enough hygromycin resistance gene expression to allow survival. This explanation appears equally pertinent for the results of Fig. 3(b), where on top of the selection of the wild-type sequence leading to cell survival, transcription-incompetent mini-genomes would simultaneously propagate.
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Based on this explanation supporting a lack of essential nucleotide selection, we postulated that any non-wild-type sequence in the pre-gs1 hexamers would be allowed. As a corollary, we expected this assertion not to be valid for the gs1 hexamer. Individual clones were then derived from the randomized plasmid preparations (see Methods) and used to produce mini-HygRGs with determined non-wild-type sequences.
Fig. 4(a) shows the sequences obtained for five of these clones for the indicated hexamers. When passed through the hygromycin resistance protocol, the mutated sequences of the pre-gs1 hexamers all led to cell culture survival (Fig. 4b, clones 43–48 and 49–54), in contrast to the five mutated gs1 hexamers (Fig. 4b, clones 56–61). In these latter cases, however, the cells survived to the transfection/infection protocol in the absence of hygromycin (not shown). This demonstrated that the mutated mini-HygRGs replicated efficiently as they protected the cells from the lytic effect of the helper virus. However, they did not express the hygromycin resistance gene.
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). Surprisingly, selection of nt 56, which in hexamers 56–61 was weak but present (Fig. 3b), was now clearly absent (arrow, Fig. 3c). On the other hand, nt 57 stood out above the background of degenerated sequences (arrowhead, Fig. 3c). In summary, this analysis suggested that no particular nucleotides upstream of gs1 are required exclusively for transcription. This contrasted with the situation in gs1, where at least part of the wild-type sequence 3'-UCCCAG-5' appeared to be required.
Analysis of the phase context requirement for gs1 function
Starting with a 3'-AG/Pr-G/Prdel12 tandem promoter mini-genome expressing GFP (mini-GFP, Fig. 1a), we derived a series of constructs in which the first nucleotide of gs1 was adjusted in all of the six phase contexts (Fig. 5a, mini-GFP-1). A transfection/infection (with rSeV-AGP55) protocol was then applied to the six mini-GFP-1s (called phases 1 to 6). Mini-genome amplification was monitored by Northern blotting and GFP gene expression was estimated by flow cytometry to follow GFP protein expression and by primer extension to follow GFP mRNA levels (Fig. 5). As expected, the six derivatives replicated at a similar level (Fig. 5b). The amounts of GFP mRNA were monitored by primer extension assays (Fig. 5c). In parallel, a fraction of the transfected/infected cells were analysed for mean GFP fluorescence content. Primer extension and mean fluorescence values were plotted on the same graph (Fig. 5d) relative to the phase 2 construct (the wild-type configuration), which was taken as 100 %. The data from the two assays coincided and showed that the wild-type configuration was the most efficient. The flanking phase 1 led to a moderate but reproducible loss in transcription. This loss was increased in phase 3 and reached levels of reduction of five- to tenfold for the remaining phase contexts.
Using a helper virus to sustain amplification and transcription is very efficient. It may, however, introduce an element of competition between the helper virus and mini-genome promoters for available viral RNA polymerase that could skew the results in the case of limiting availability of the helper functions. We therefore repeated the experiment using a mini-genome rescue protocol based on helper functions constitutively produced from transfected plasmids under the control of the T7 RNA polymerase. Another difference in this protocol lies in the absence of the C proteins expressed from the C gene (using a P/Cstop plasmid). The absence of the C proteins has been shown to relieve stringency in the recognition of promoter motifs by the viral RNA polymerase (Cadd et al., 1996; Tapparel et al., 1997). The phase context effect was similarly observed with the same pattern (phase 2>phase 1>phases 3–6, Fig. 5e), although discrimination between the wild-type phase context and phases 4–6 was less prominent.
In the series of constructs presented in Fig. 5, the N-phase context of the whole transcription promoter (G/Prdel12) was changed. Thus, it was possible that the shift of the whole sequence might have amplified the effect, or, alternatively, that the change in phase context of nucleotides other than gs1 might have been involved. A second series of derivatives (mini-GFP-2) was therefore prepared in which two major differences were introduced (Fig. 6). Firstly, the sequence shift was limited to gs1 as far as possible (Fig. 6a). Secondly, the replication promoter was changed from AG/Pr to G/Pr58A, a genomic promoter carrying a C/U transition (genome sense) at nt 58. This mutation has been shown to impede transcription drastically, but to have little effect on replication (Le Mercier et al., 2003; Vulliémoz et al., 2005). G/Pr58A was therefore introduced here as a replication-only promoter to check for possible effects of the upstream promoter on the N phase effect. As previously shown (Vulliémoz et al., 2005), the use of G/Pr58A as a replication promoter in place of AG/Pr leads to a lower level of replication of the constructs, but allows better transcription from the downstream G/Prdel12 (comparison not shown). The GFP mean fluorescence of mini-GFP-2 (wt) (Fig. 6a) was around 2500 units compared with 600 units for the corresponding mini-GFP-1 (wt) (Fig. 5a). Apart from this expected difference, the results obtained with the mini-GFP-2 series paralleled those of the mini-GFP-1 series (Fig. 6c).
In summary, our observations allowed us to conclude that transcription was optimized by setting the first gs1 nucleotide at position 56 in the N phase context 2. It is noteworthy that this optimization was observed under conditions where gs1 did not sit in its natural position relative to the template 3' extremity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) or their replacement by corresponding nucleotides originating from AG/Pr (Calain & Roux, 1995) has been found to affect transcription negatively. This means that providing randomized sequences (as in the present experiments) is not equivalent to providing an unnatural fixed sequence. This has been verified previously. When a similar selection approach was used to define the PrE-II repeated motif, (3'-CNNNNN-5')3, only the C in position 1 of the hexamers stood out (Tapparel et al., 1998). However, some of the nucleotides in positions 2–6 could also influence replication when mutated individually in the context of an otherwise wild-type sequence (Walpita, 2004). Thus, the negative effect of a mutation can be corrected by compensatory changes, when available. The converse rationale could apply. When, in a stretch of sequence, the requirement for critical nucleotides is fulfilled, the flexibility of a nearby position can be increased. This could explain why position 56 in the randomized 52–57 hexamer did not stand out when nucleotides downstream of position 58 corresponded to the wild-type sequence (Fig. 3c, arrow). However, upon randomization of this downstream sequence, as in hexamer 56–61, better selection of nt 56 (Fig. 3b, arrowhead) was observed. Position U56 thus appears to be flexible, in contrast to the next three Cs at nt 57–59, which stood out as the best selected in the randomized 56–61 hexamer. This corroborates the critical role that C58 has previously been shown to play in gs1 function. Indeed, C58U transition was the only gs1 nucleotide substitution that was reproducibly selected to eliminate the transcription activity of a G/Pr replacing AG/Pr in an ambisense recombinant Sendai virus (Le Mercier et al., 2002). It should be noted that tolerance for non-optimal sequences in the mini-genome population, resulting in high background, set up conditions likely to identify only stringently required positions. Thus, these conditions were not optimal to detect patterns such as pyrimidine-rich sequences.
A more trivial explanation for the lack of nucleotide selection upstream of gs1 could follow from such a low requirement for the hygromycin resistance protein that even expression from incompetent transcription signals would suffice. However, selection was effective for hexamer 56–61 and, conversely, fixed mutated sequences in this hexamer lead to cell death following hygromycin treatment. These two observations then serve to validate the method using the conditions of our experiments. We thus concluded that no nucleotides upstream of gs1 are critical for transcription.
When we changed the gs1 N phase context, we could observe an effect on transcription and this was proportional to the extent of the change. This represents the first clear demonstration that, among the Paramyxovirinae, the absolutely conserved position 56 of the first gs1 nucleotide is related to its relative point of interaction with the N protein. Our observation was made with a tandem promoter mini-genome in which gs1 had been moved further away from the genome 3' end from position 56 to 146. Therefore, the phase context effect could be observed independently of the distance from the template 3' end. This strongly suggests that N phase context preservation, rather than measuring distance from the genomic 3' end, represents the explanation for the conservation of position 56 in the natural genome. Ultimately, the genomic 3' extremity appears not to be important for gs1 recognition, a conclusion that corroborates the lack of importance of the genomic 3' extremity in replication signal recognition, a feature that was claimed to follow from the application of the rule of six (Vulliémoz & Roux, 2002).
Recently, Hoffman et al. (2006), in a study of human parainfluenza virus 3 G/Pr, arrived at the opposite conclusion that there is no position dependency for gs1 function. However, in contrast to their assertion, Fig. 5(b) of their paper clearly shows that transcription efficiency was affected by the change in gs1 N phase context. We feel then that there is no discrepancy between the two studies. Thus, gs1 becomes another signal, along with the PrE-II replication signal (Tapparel et al., 1997; Murphy & Parks, 1999) and the editing signal, during transcription of the P gene (Iseni et al., 2002), where the N phase context plays a role in recognition. Thus, the vRdRp is sensitive to the N phase context, whether in replication or transcription mode.
Our observation that N phase contexts 2 and 1 are optimal for transcription is remarkable in that these positions correspond to those naturally found for the respiroviruses (including Sendai virus). For the Paramyxovirinae, gs1 is only seen in N phase context 2 (Kolakofsky et al., 1998). This stresses the fact that nucleotides positioned at the ‘3'-edge’ of the N protein (as are the three Cs of the PrE-II replication motif; Tapparel et al., 1998) are part of a motif signalling to the vRdRp. These nucleotides are in fact the first made available to a vRdRp displacing the N protein in its progression on the template. Although we have little insight into the mechanism of action of the vRdRp, this could indicate the presence of an interaction of the leading edge of the vRdRp with the next N protein to be removed to allow polymerase progression.
The conclusions drawn from our two sets of experiments have reinforced our views on how paramyxovirus transcription initiation takes place. First, the absence of critical nucleotides immediately upstream of gs1 weakens the possibility that a landing site for the vRdRp exists in this region. Secondly, the finding of the importance of the gs1 N phase context provides an explanation for the absolute conservation of the first nucleotide positioning of gs1. This weakens the argument that this positioning reflects the measure of distance from the genomic 3' end, a feature that would participate in the correct positioning of a vRdRp entering directly at gs1.
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ACKNOWLEDGEMENTS |
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Received 10 November 2006;
accepted 23 January 2007.
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, Full-length genomic RNA; *, mini-genome RNAs. (b) BSR-T7 cells were infected with rSeV-AGP55 (i) or transfected with mini-HygRG and infected with rSeV-AGP55 (ii). At 48 h post-infection, cells were observed by light microscopy and photographed. (c) Schematic representation of the experimental protocol used to select surviving cells resistant to hygromycin (see Methods). (d) Hygromycin treatment of BSR-T7 cells infected with mock-transfected/infected cells and supernatants (BSR-T7 mock), with supernatants and cells transfected/infected with the mini-GFP and rSeV-AGP55 (BSR-T7+mini-GFP), or with supernatants and cells transfected/infected with mini-HygRG and rSeV-AGP55 (BSR-T7+mini-HygRG), according to the protocol in (c). At the end of the experiment, freshly infected BSR-T7 cells were stained with crystal violet.
