Journal of General Virology (2005), 86, 171–180
DOI 10.1099/vir.0.80435-0
Nature of a paramyxovirus replication promoter
influences a nearby transcription signal
Diane Vulliémoz, Samuel Cordey, Geneviève Mottet-Osman
and Laurent Roux
Correspondence
Laurent Roux
Department of Microbiology and Molecular Medicine, University of Geneva Medical School,
CMU, 1 rue Michel-Servet, CH-1211 Geneva 4, Switzerland
Laurent.Roux@medecine.
unige.ch
Received 9 July 2004
Accepted 14 October 2004
The genomic and antigenomic 39 ends of the Sendai virus replication promoters are bi-partite
in nature. They are symmetrically composed of leader or trailer sequences, a gene start (gs) or
gene end (ge) site, respectively, and a simple hexameric repeat. Studies of how mRNA synthesis
initiates from the first gene start site (gs1) have been hampered by the fact that gs1 is located
between two essential elements of the replication promoter. Transcription initiation, then, is
separated from the replication initiation site by only 56 nt on the genome, so that transcription and
replication may sterically interfere with each other. In order to study the initiation of Sendai virus
mRNAs without this possible interference, Sendai virus mini-genomes were prepared having
tandem promoters in which replication takes place from the external one, whereas mRNA synthesis
occurs from the internal one. Transcription now initiates at position 146 rather than position 56
relative to the genome 39 end. Under these conditions, it was found that the frequency with
which mRNA synthesis initiates depends, in an inverse fashion, on the strength of the external
replication promoter. It was also found that the sequences essential for replication are not required
for basic mRNA synthesis as long as there is an external replication promoter at which viral
RNA polymerase can enter the nucleocapsid template. The manner in which transcription and
replication initiations influence each other is discussed.
INTRODUCTION
The first step in the Sendai virus (SeV) multiplication cycle
is the production of mRNA from a helical subviral nucleocapsid, in which the viral genome RNA is tightly and
stoichiometrically associated with the viral nucleocapsid
(N) protein. This N-subunit assembly, together with multiple attached viral polymerases (a complex of the P and L
proteins) is the minimum subviral unit that is thought to
retain infectivity (Lamb & Kolakofsky, 2001). The synthesis
of negative-stranded RNA genomes (or antigenomes) and
their assembly with N protein is coupled, and these viral
RNAs are only found as nucleocapsids (Gubbay et al.,
2001). SeV nucleocapsid appears as a flexible helical assembly complex of variable pitch, with 13 N-subunits per turn,
in which each N-subunit binds 6 nt (Bhella et al., 2002;
Egelman et al., 1989). For viruses of the subfamily Paramyxovirinae, efficient replication of model mini-genomes
in transfected cells requires that their total length be a
multiple of six (Calain & Roux, 1993; Vulliémoz & Roux,
2001). The requirement for hexameric genome length for
viruses of the subfamily Paramyxovirinae has recently
been underscored using reverse genetic systems for Human
parainfluenza virus 2, a rubulavirus, and measles virus,
Morbillivirus (Rager et al., 2002; Skiadopoulos et al., 2002).
0008-0435 G 2005 SGM
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 39 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 59-UTR of the N gene or the 39-UTR of
the L gene (Fig. 1). For SeV and Human parainfluenza virus
3, PrE-II is a simple but phased hexameric sequence repeat
(39 [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 PrEII 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
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D. Vulliémoz and others
Fig. 1. Primary structure of the SeV replication promoters. The 96 nt of the genomic (G/Pr) and antigenomic (AG/Pr) are
presented as RNA sequence in ‘hexamers’, numbered 1–16 from the 39 end (-OH 39). PrE-I and PrE-II refer to the two
elements described as essential for viral RNA replication (see text), with the three times repeated motif in PrE-II indicated as
C*. In G/Pr, the leader coding sequence (leaderC, 55 nt) is outlined, as well as the N gene transcription start signal (nt
56–65), which constitutes the first transcription start signal (N gs1) seen by the RdRp. In AG/Pr, the trailer coding sequence
(trailerc, 57 nt) is shown, as well as the complement of the L gene polyadenylation site (L gec). For more details see text of
Introduction.
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.
Besides the bi-partite nature of G/Pr and AG/Pr, another
feature conserved among the viruses of the subfamily
Paramyxovirinae (respiro-, morbilli- and rubulaviruses) is
that the first (N) mRNA always starts precisely opposite
U56, within the conserved decamer mRNA start signal 39
55-AU2CCCA NUUUCN-66 (for SeV, phase indicated).
Curiously, none of the conserved cytidines of this cis-acting
signal are in hexamer positions one and six. The N gene
mRNA start site (abbreviated as gs1) is also on another
face of the helix than the bi-partite replication promoter
in resting nucleocapsids. Since virus RNA-dependent RNA
polymerase (vRdRp) initiates chains at two closely spaced
start sites on the N : RNA template, one would expect that
these two events would interfere with each other, at least
under some conditions, and evidence of the negative
influence of gs1 on SeV replication promoter strength has
recently been presented (Le Mercier et al., 2003). The study
of the influence of the replication promoters on mRNA
synthesis from gs1, however, is complicated by its location
between the two essential elements of the replication
promoter, and the fact that the two RNA initiation events
are only 56 nt apart on the N : RNA. In order to study the
initiation of SeV mRNA in the absence of the above
complications, we have prepared SeV mini-genomes with
tandem 96 nt long G/Pr in which replication takes place
172
from the external G/Pr, whereas mRNA synthesis occurs
from the internal G/Pr, having initiated at position 146
rather than position 56.
METHODS
Virus and cells. BSR-T7 cells, a BHK cell line constitutively
expressing a T7 RNA polymerase, a gift from K.-K. Conzelmann
(Max-von-Pettenkofer Institute & Gene Center, LudwigMaximilians-University Munich, Munich, Germany) (Buchholz et al.,
1999), were propagated in Dulbecco minimal Eagle medium
(DMEM) supplemented with 5 % fetal calf serum (FCS) in a 5 %
CO2 atmosphere. The AGP-55 recombinant Sendai virus (rSeVAGP55) was constructed and rescued as described previously
(Le Mercier et al., 2002). AG/Pr of this virus has the first 55 nt of
the trailer sequence replaced with the 55 nt of the leader sequence
(see Fig. 1). Virus stocks, prepared in 9-day-old embryonated
chicken eggs from three times plaque purified virus, reached titres
ranging between 56108 and 109 p.f.u. ml21.
Sequence and plasmids. All the plasmids harbouring the mini-
replicons expressing the green fluorescent protein (GFP) are derived
from pSV-DI-H4D96 described in Vulliémoz & Roux (2001, 2002).
In all derivatives used in this study, the 39 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
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Journal of General Virology 86
Paramyxovirus replication and transcription
Fig. 2. Generation of the ectopic transcription start signal. (a) Schematic representation of the mini-replicons expressing
GFP. First line: derivative of pSP65 plasmid containing the mini-genome encoding sequences, flanked by the Hepatitis delta
virus ribozyme (Rbz) and the T7 RNA polymerase promoter (T7p). Encapsidated T7 RNA transcripts which constitute proper
mini-genome templates for synthesis of GFP mRNAs are portrayed below (lines 1–5) in a 39 to 59 orientation. In the double
promoter constructs, a 6 nt intervening sequence (XhoI site) is inserted between the two promoters (short black line). The 59
end extremity of the mini-replicons is invariably the AG/Pr complementary sequence (AG/Prc), meaning that the anti minigenome RNAs invariably harbours an AG/Pr. GFP, green fluorescent protein ORF. Right-angle arrow, viral transcription start
signal (in text, gs1). White cross and flush end right angle bar in G/Pr, C58A mutation portraying transcription arrest. Open
triangle, deletion at the 39 end of G/Pr. The extent of the deletion is indicated by the denomination of the construct to the left:
d12 means a 12 nt deletion. (b) Representative Northern blot analysis of the encapsidated RNAs produced in the rescue of
the mini-replicons presented in (a). About 107 BSR T7 cells were infected with rSeV-AGP55 (helper virus) and transfected
with the plasmids harbouring the corresponding mini-genome sequences as described in Methods. Encapsidated RNAs were
purified and analysed by Northern blotting using a specific 32P-labelled riboprobe of positive polarity (see Methods). ND,
rSeV-AGP55 genome. DI-RNA, mini-replicons RNAs, templates for GFP messages. The numbers below refer to levels of
replication (arbitrary units, measured as described in Methods). Note the relative position of the mini-replicons DI-RNAs
consistent with replication initiation at the external promoter only. (c) Relative GFP fluorescence normalized to the corrected
replication (see Methods) averaged from three independent experiments with [AGP-GP] taken as the series standard (100 %).
Deviation of the mean is indicated by bars.
(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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Rescue of mini-replicons in the presence of the helper
rSeV-AGP55. BSR-T7 cells were seeded at 16106 cells in 9 cm
diameter Petri dishes. The next day, 10 p.f.u. per cell of rSeV-AGP55
in 1 ml basic salt solution (BSS) was added (33 uC, 1 h). The infectious medium was replaced drop by drop with a pre-prepared mix
containing 12 mg plasmid harbouring GFP-mini-genome and 24 ml
Fugene (Roche) in a total of 400 ml BSS. After 6 h, 10 ml of
DMEM-10 % FCS replaced the transfection mix. The next day, fresh
FCS-free DMEM was added. At day 4, medium and cells were
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D. Vulliémoz and others
collected after trypsinization (3 mg ml21 acetylated trypsine), and
overlaid onto subconfluent fresh BSR-T7 cells, infected with
10 p.f.u. per cell of rSeV-AGP55. After 4 h, floating cells and
medium were replaced by 10 ml FCS-free DMEM. At day 6, the
cells were trypsinized, resuspended in DMEM-5 % FCS to inactivate
the trypsin, pelleted and finally resuspended in 1 ml PBS. A fraction
of the cell samples was used for analysing GFP expression by flow
cytometry. The remainder was pelleted and used to characterize the
extent of replication or transcription by Northern blotting and
primer extension, respectively.
distribution among the different templates as follows: Replication/
%gated6100=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).
Replication of mini-replicons in the presence of support
plasmids. Confluent BSR-T7, seeded as described above the day
RESULTS
before, were transfected with a mixture of plasmids including the
plasmid harbouring the mini-genome (5 mg), the pTM1-N (1?5 mg),
pTM1-P/Cstop (1?5 mg), pTM1-L (0?5 mg), 20 ml 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 20–40 % w/w CsCl gradients (Beckman SW60).
After centrifugation (40 000 r.p.m., 12 uC, 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
32
P-labelled riboprobe contained promoter and GFP-specific sequences,
which allow detection of the helper virus genome. To score nonencapsidated RNAs present in the CsCl gradient pellets, a GFPspecific 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 pre-
viously (Vulliémoz & Roux, 2001). An infrared dye-labelled oligonucleotide (IRD 800, 59-CAGCTTGCCGTAGGTGGCATCGCCC-39)
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 BectonDickinson 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
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A transcription start site positioned downstream
of a replication-only promoter
In this study, SeV transcription was followed using a series of
mini-replicons expressing GFP, and GFP fluorescence was
measured by flow cytometry. Since GFP expression depends
on the efficiency of mini-genome transcription, but also
on template availability, it was normalized to mini-genome
abundance as estimated by Northern blotting. Moreover,
the fraction of the cells harbouring these templates in the
culture will vary according to the replication ability of the
mini-genomes. Since the mean fluorescence of the FACS
analysis is that of the cells that are gated, these results
should also be normalized for the different distribution.
For example, when a certain amount of template (Northern
blot signal) is distributed in 20 or 90 % of the cells, the
amount of template per cell is 4?5 times higher in the
former case. Consider these two situations: 100 templates
distributed in 100 cells (one template per cell), or 100
templates distributed in 20 cells (five templates per cell);
if, in both situations, the mean fluorescence of the gated
cells is the same, then the five templates in the 20 cells
transcribe fivefold less efficiently than the template in the
100 cells. In the end, crude GFP measure by flow cytometry
was standardized to the amount of template corrected for
their distribution in the culture (see also Methods). This
approach was, finally, validated by direct measurement of
GFP mRNAs (see below).
Transcription downstream of AG/Pr
To create mini-replicons in which GFP mRNAs do not
initiate from within the elements used for replication, minireplicons 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 minireplicons 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 [AGPGPd1258A], 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
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Journal of General Virology 86
Paramyxovirus replication and transcription
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).
As shown in Fig. 2(c), construct [AGP] only expressed
background levels of GFP, indicating that GFP expression
depends on the presence of gs1. Construct [AGP-GP], on
the other hand, did so significantly. The 12 nt deletion
of G/Print, which results in replication suppression (see
[GPd12]) had no deleterious effect on GFP expression (see
[AGP-GPd12]). Inhibition of this expression by the C58A
mutation (construct [AGP-GPd1258A]) indicates that mRNA
initiation takes place at gs1. In conclusion, an ectopic gs1
within an internal, replication-incompetent G/Pr, is functional downstream of AG/Pr. This raises the question of
how vRdRp reaches gs1. Does it enter the template at AG/
Prext and proceed to gs1, or is direct entry at G/Pr possible?
Internal gs1 still functions without the two
replication promoter elements
The 12 nt deletion at the 39 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 ([AGPGPd48], Fig. 3), transcription was decreased ca. fivefold,
indicating that nucleotides 12–48 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 39 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.
Transcription downstream of G/Pr
As transcription from gs1 can initiate downstream of AG/
Pr, we next asked whether the nature of the external replication promoter influenced this transcription. We therefore
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Fig. 3. GFP expression in the absence of the PrE-I and PrE-II
elements. (a) Schematic representation of the mini-replicons
used as in Fig. 2(a). dBB, deletion of PrE-II element in the
internal G/Pr. (b) A representative Northern blot analysis of the
negative polarity encapsidated RNAs of the mini-replicons
presented in (a) (rescue and purification as in Fig. 2b). (c)
Relative GFP fluorescence averaged from three independent
experiments as in Fig. 2(c). [AGP-GP] was taken as the series
standard.
replaced AG/Prext with G/Pr-58A, as this minimal mutation
has little or no effect on promoter strength but strongly
suppresses mRNA synthesis from gs1 so that transcription from this minimally mutated gs1 was suppressed
(Le Mercier et al., 2003). The second series of constructs
carrying G/Pr-58Aext is presented in Fig. 4(a). Fig. 4(b)
illustrates their replication ability as measured by Northern
blotting. Two points are noteworthy. First, the difference in
migration of the single or double G/Pr mini-replicons is
visible, and shows that only G/Prext is used for replication
(compare lanes 1 and 3, with 2, 4 and 5), as shown before
(Vulliémoz & Roux, 2002). Fig. 4(c) presents the level of
GFP fluorescence normalized to replication relative to the
single GP mini-genome. Comparison of [GP] and [GP58A]
shows that the C58A mutation strongly inhibits GFP
expression and has no deleterious effect on replication
[Fig. 4(b), compare 1 and 3]. Second, the comparison of
[GP-GP] and [GP58A-GPd12] shows, as in the case of the
AG/Prext series, that G/Print deleted of its first 12 nt
remains competent for transcription. This observation was
confirmed by [GP58A-GPd1258A], in which GFP expression
was suppressed upon mutation of the internal gs1 as well.
By analogy with the AG/Prext constructs, G/Pr-58Aext of
[GP58A-GPd12] can apparently recruit vRdRp that initiates
at the genome 39 end, and which in this case leads to leader
RNA synthesis. Upon release of leader RNA, vRdRp would
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Fig. 4. GFP expression downstream of G/Pr. (a) Mini-genomes
used, portrayed as in Fig. 2(a). In this series, the external promoter is G/Pr or its mutated version. (b) A representative
Northern blot analysis of the replicated RNAs of negative polarity
of the mini-replicons portrayed in (a). The mini-replicons were
rescued in BSR-T7 cells with the help of rSeV-AGP55 and the
encapsidated RNAs purified as described in Fig. 2(b). Note the
relative position of the mini-replicons DI-RNAs consistent with
replication initiation at the external promoter only. (c) Relative
GFP fluorescence averaged from three independent experiments
as in Fig. 2(c). [GP] was taken as the series standard.
again be free to scan the template for a downstream, ectopic
gs1 (see also Discussion).
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 minireplicons which contain either AG/Prext or G/Pr -C58Aext
(Fig. 5a). The three AGP constructs were amplified (20–50fold) 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
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Fig. 5. Comparison of GFP expression downstream of AG/Pr
or G/Pr in the presence of helper virus rSeV-AGP55. (a)
Schematic representation of the mini-replicons used as in
Fig. 2(a). (b) A representative Northern blot analysis of the
negative polarity encapsidated RNAs of the mini-replicons presented in (a) (rescue and purification as in Fig. 2b). (c) Relative
GFP fluorescence averaged from four independent experiments.
[GP58A-GP] was taken as the series standard. (d) Northern
blot analysis of the total non-encapsidated RNAs recovered
from the CsCl gradient pellets as described in Methods, using
a probe of negative polarity positioned in the GFP ORF. Lanes
1–6 as in (a). Lanes 7 and 8, rSeV-AGP55 and mock infected
BSR-T7 cells, respectively. (e) As in (d), but after selection for
the poly adenylated RNAs (see Methods).
normalization to template levels, the G/Prext constructs
expressed 20–50-fold more GFP than the AG/Prext counterparts (Fig. 5c).
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Journal of General Virology 86
Paramyxovirus replication and transcription
Direct estimation of the various levels of GFP mRNA
present intracellularly was also carried out, by Northern
blotting and primer extension. The pellets of the CsCl
gradients used to purify the encapsidated mini-replicons
(Fig. 5b) contain the bulk of cellular and non-encapsidated
viral RNAs. These CsCl pellet RNAs were first analysed by
Northern blotting (using a GFP probe of negative polarity)
either directly (Fig. 5d) or after oligo-dT selection (Fig. 5e).
The specificity of this probe is underlined in both cases by
the absence of signal for the RNAs isolated from uninfected
cultures (lane 8), or infected by the helper virus alone
(lane 7). The interesting point is that the level of GFP
mRNAs detected is similar regardless of the nature of the
external replication promoter (crude, Fig. 5d, and olig-dT
selected, Fig. 5e, compare lanes 1 and 2 with 4 and 5). Given
the 20–50-fold difference to which the templates for these
messengers had accumulated intracellularly (Fig. 5b), it
is clear that the G/Prext constructs produce significantly
more GFP mRNAs (20–50-fold) per mini-genome than
their AG/Prext counterparts.
Primer extensions were also performed to precisely determine the position of the RNA 59 ends. For instance, the
strong decrease in GFP expression due to the C58A
mutation introduced in the internal G/Pr (Fig. 2, [GP58AGPd1258A]; 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 59 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 59 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
Fig. 6. Identification of the transcription and
replication start positions. (a) Primer extension analysis using the non-encapsidated
RNAs presented in Fig. 5(d) as templates.
An infrared dye labelled (IRD) primer of
negative polarity positioned in the GFP ORF
was used (see Methods). Above and below,
schematic representation of relevant portions
of the two promoters present at the 39 end
of the GP58A-GP and AGP-GP minireplicons, respectively. The corresponding
portions of the nucleotide sequence, performed on plasmid DNA with the same IRD
primer as the one used for primer extension,
are presented and serve as size markers. In
the sequencing gel, the 39 end extremities
of the external promoters, and the gene
starts (mutated, gs1m or not, gs1) are highlighted. (b) The encapsidated RNAs purified
from CsCl gradients, as in Fig. 5(b), were
used as templates for primer extensions
using the IRD primer of negative polarity
cells as in Fig. 5(a). In the sequencing gel,
the 39 end extremities of the external promoters, and the gene starts (mutated, gs1m or
not, gs1) are highlighted.
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D. Vulliémoz and others
DISCUSSION
We have prepared mini-replicons in which mRNA synthesis initiates not from its normal position gs156 within G/Pr,
but at a gs1 downstream of the promoter used for replication, at position 146. In this manner, we have examined the
minimum sequence requirements for this ectopic gs1 to
function, and the effect of nearby cis-acting sequences on
the frequency with which this ectopic gs1 is used to express
GFP.
There are two views of how non-segmented negativestranded 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
39 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
39 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 39
end, gs1 of the highly deleted G/Print would still function.
Fig. 7. Comparison of GFP expression downstream from
AG/Pr or G/Pr in the presence of support plasmids. The minireplicons presented in Fig. 5(a) were replicated in the presence
of the helper functions L, P and N expressed from plasmids
(see Methods). (a) Mean GFP fluorescence expressed as the
mean of data from duplicate samples, shown with variation of
the mean. (b) Northern blot analysis of the purified encapsidated RNA as in Fig. 2(b). The cells of the duplicate samples
presented in (a) were pooled. (c) Relative GFP expression
corrected for template availability as measured in (b). [GP58AGP] was taken as the series standard.
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).
178
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 39 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 39 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
39 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
39 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
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Journal of General Virology 86
Paramyxovirus replication and transcription
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 39 end.
Nevertheless, the apparent competition between gs1 and the
39 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 39 end replication promoter
would divert vRdRp from scanning back to the genome 39
end, and the presence of a strong 39 end promoter would
disfavour vRdRp scanning to gs1. It will be of interest to
displace a minimal gs1 progressively away from the genome
39 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
56–65 of GPext were all substituted with the corresponding
nucleotides of AGP. These replicated two- to threefold
better than their [GP58A-GPd12] counterparts, but generated
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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.
ACKNOWLEDGEMENTS
We are indebted to Dominique Garcin, Philippe Le Mercier and Daniel
Kolakofsky for critical discussions in the course of the work. We are
moreover grateful to Daniel Kolakofsky for his substantial contribution
to the writing of the paper. This work was supported by a grant from
the Swiss National Foundation for Scientific Research and from the
Commission of the European Communities specific RTD programme
‘Quality of Life and Management of Living Resources’, QLK2-CT200101225, ‘Towards the design of new potent antiviral drugs: structure
and function analysis of Paramyxoviridae RNA polymerase’. It does
not necessary reflect the views of the Commission and in no way
anticipates future policy in this area.
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