JOURNAL OF VIROLOGY, Sept. 2003, p. 9147–9155
0022-538X/03/$08.00⫹0 DOI: 10.1128/JVI.77.17.9147–9155.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 17
Competition between the Sendai Virus N mRNA Start Site and the
Genome 3⬘-End Promoter for Viral RNA Polymerase
Philippe Le Mercier, Dominique Garcin, Eduardo Garcia, and Daniel Kolakofsky*
Department of Genetics and Microbiology, University of Geneva School of Medicine, CH1211 Geneva, Switzerland
Received 3 March 2003/Accepted 30 May 2003
The genomic and antigenomic 3ⴕ-end replication promoters of Sendai virus are bipartite in nature and
symmetrical, composed of le or tr sequences; a gene start or gene end site, respectively; and a simple hexameric
repeat. The relative strengths of these 3ⴕ-end promoters determines the ratios of genomes and antigenomes
formed during infection and whether model mini-genomes can be rescued from DNA by nondefective helper
viruses. Using these tests of promoter strength, we have confirmed that tr is stronger than le in this respect.
We have also found that the presence of a gene start site within either 3ⴕ-end promoter strongly reduces 3ⴕ-end
promoter strength. The negative effects of the gene start site on the 3ⴕ-end promoter suggest that these closely
spaced RNA start sites compete with each other for a common pool of viral RNA polymerase. The manner in
which this competition could occur for polymerase off the template (in trans) and polymerase on the template
(in cis) adds insight into how the viral RNA polymerase switches between its dual functions as transcriptase
and replicase.
Sendai virus (SeV), a Respirovirus of the Paramyxovirinae
subfamily, is a model nonsegmented negative-strand ([⫺])
RNA virus. The first step in the SeV replication cycle is the
production of mRNA from a helical subviral structure, the
nucleocapsid (NC), in which the genome RNA is tightly and
stoichiometrically associated with the viral NC protein (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 (16).
The synthesis of [⫺] RNA genomes (or [⫹] antigenomes) and
their assembly with N protein is coupled, and these viral RNAs
are only found as nucleocapsids (10). Electron micrographs of
SeV nucleocapsids show a flexible helical assembly with 13 N
subunits per turn and variable pitch, in which each N subunit
binds 6 nucleotides (nt) (5) (Fig. 1). For viruses of the
Paramyxovirinae, efficient replication of model mini-genomes
in transfected cells requires that their total length be a multiple
of six, and viruses of this group whose genome has been entirely sequenced mostly have genome lengths that are multiples
of six (2, 15). Inefficient replication of non-hexamer-length
mini-genomes was not due to the lack of encapsidation of the
mini-genome but apparently was due to the inability of viral
RNA-dependent RNA polymerase (vRdRP) to initiate at the
mini-nucleocapsid 3⬘ end (2). It has been suggested that nucleocapsid assembly begins with the first 6 nt at the 5⬘ end of
the nascent chain and continues by assembling 6 nt at a time
until the 3⬘ end is reached. The efficiency of the 3⬘-end promoter then presumably depends on the position of these cisacting sequences relative to the N subunits, and this hexamer
or N-subunit “phase” is determined by the precise length of the
genome chain (15, 26). The requirement for hexameric ge-
nome length of the Paramyxovirinae has recently been underscored for human parainfluenza virus type 2 (hPIV2), a rubulavirus, and measles virus, a morbillivirus. hPIV2 recovered
from non-hexameric-length cDNAs were found to contain a
biased distribution of mutations that all restored hexameric
genome length (15, 22). Similarly, an obligatorily diploid measles virus was recovered from DNA that encodes a defective H
protein, again via mutations that restore hexameric genome
length (21).
The genomic and antigenomic promoters (G/Pr and AG/Pr)
of the Paramyxovirinae are found within the terminal 96 nt or
16 N subunits of each RNA and are composed of leader (le) or
trailer (tr) sequences; a gene start or gene end site, respectively; and a simple hexameric repeat (Fig. 1; see also Fig. 5).
Although G/Pr and AG/Pr are similar in overall structure, they
carry out separate functions. G/Pr is associated with le RNA,
antigenome RNA, and mRNA synthesis, while AG/Pr is associated with tr RNA and genome RNA synthesis. The relative
abundance of the various viral RNAs throughout the infection
is regulated in large part via G/Pr and AG/Pr, and this ensures
as well that a preponderance of [⫺] genomes are produced
during infection. These 3⬘-end promoters are (at least) bipartite in nature (11, 18, 20, 24). There is both an end element
comprising ca. the first 30 nt at the 3⬘ ends (in which the first
12 nt are conserved between G/Pr and AG/Pr and across each
genus), and a downstream element within the 5⬘ untranslated
region of the N gene ([⫺] nucleocapsids) or the 3⬘ untranslated
region of the L gene ([⫹] nucleocapsids). For SeV and hPIV3
and apparently all respiro- and morbilliviruses, the downstream element is a simple but phased hexameric repeat (3⬘
[C1N2N3N4N5N6]3, bound to the 14th, 15th, and 16th N protein
subunits [11, 24]). For rubulaviruses like SV5, [N1N2N3N4G5C6]3
is repeated in subunits 13, 14, and 15, such that all these
conserved nucleotides are adjacent to the first 12 nt in the
helical nucleocapsid with 13 subunits per turn (19) (Fig. 1).
This common surface, comprising the conserved sequences of
* Corresponding author. Mailing address: Department of Genetics
and Microbiology, University of Geneva School of Medicine, CMU, 9
Ave. de Champel, CH1211 Geneva, Switzerland. Phone: 41 22 379
5657. Fax: 41 22 379 5702. E-mail: Daniel.Kolakofsky@medecine
.unige.ch.
9147
9148
LE MERCIER ET AL.
J. VIROL.
FIG. 1. Genome 3⬘-end promoter (G/Pr) in resting nucleocapsids. A model of the helical N-subunit assembly of the SeV resting genome
nucleocapsid is shown, based on the electron micrographic reconstruction of Egelman et al. (5), with a pitch of 5.3 nm and 13 subunits per turn.
The position of the genome RNA within this structure is unknown; the RNA line is placed on the outside surface of the N assembly for clarity
and because it is just long enough when extended to occupy this position. The N subunits (and the 6 nt positions within each subunit) are numbered
from the genome 3⬘ end (lower right). An enlargement of the bipartite 3⬘-end promoter (dark subunits on the left) is shown on the right, where
the 3⬘-terminal 96 nt are placed within each subunit in groups of six. The 3⬘-terminal 12 nt and the [C1N2N3N4N5N6]3 repeat conserved in all
genomes and antigenomes of respiro-and morbilliviruses are highlighted. The position of the N gene start site (gs-1) is also indicated.
both elements of the bipartite promoter on two turns of the
helix, is thought to mediate the initial interaction of vRdRP
with resting nucleocapsids to initiate RNA synthesis at the
genome 3⬘ end (see below).
The remarkable sequence simplicity of the downstream promoter element is presumably possible because its hexamer
phase also contributes to its activity (19, 24). This phase effect
is thought to stem from the different chemical environments of
the 6 nt bases associated with each N subunit, as revealed by
chemical attack studies of resting SeV nucleocapsids (12). Adenosines in any hexamer phase are largely protected from
dimethyl sulfate, whereas cytosine reactivity is highly variable
and strongly depends on hexamer phase. Cytosines are highly
reactive only in hexamer positions 1 and 6, precisely the positions of the conserved cytosines in the downstream element of
G/Pr and AG/Pr (Fig. 1). If this correlation is not coincidental,
this suggests that the conservation of hexamer phase of this
cis-acting sequence serves to make these cytosines accessible to
vRdRP in resting NCs. Another feature strictly conserved
among respiro-, morbilli-, and rubulaviruses is that the first (N)
mRNA starts precisely opposite U56 (underlined) within the
decameric gene start signal 3⬘ 55A/UCCCA NUUUC/N66 (for
SeV, hexamer phase indicated). We refer to this mRNA initiation signal as gene start 1 (gs-1). Curiously, none of the
conserved cytosines of gs-1 are in hexamer positions 1 and 6.
However, once vRdRP has initiated RNA synthesis at the
genome 3⬘ end and begun to elongate, we assume that conformational changes occur within N:RNA that alter vRdRP’s
interaction with the nucleotide bases, including gs-1.
Since SeV RdRP is minimally a coiled-coil tetramer of P (4,
25) and a single L protein (with an aggregate molecular mass
equivalent to 8 N-subunits) and this polymerase initiates RNA
chains at two such closely spaced sites at the genome 3⬘ end
(only 56 nt apart), one would expect these two events to interfere with each other, at least under some conditions. However,
detailed studies involving modified DI mini-genomes did not
uncover any indication that gs-1 affected G/Pr strength; only
the first 30 nt of le/tr regions were found to influence this
property. We now report studies that show that in some cases
exactly the same situation applies, namely, that only the le/tr
sequences appear to influence promoter strength. More importantly, we find other cases in which gs-1 clearly decreases
3⬘-end promoter strength. The manner in which gs-1 negatively
influences G/Pr strength is discussed.
MATERIALS AND METHODS
Generation of recombinant SeV (rSeV). Briefly, one 9-cm-diameter dish of
BSR-T7 cells that endogenously express T7 RNA polymerase (1) were trans-
VOL. 77, 2003
PROMOTER COMPETITION FOR SENDAI VIRUS RNA POLYMERASE
fected with 1.5 g of pGEM-L, 5 g of pGEM-N, 5 g of pGEM-PHA (which
does not express C proteins), and 15 g of the various pSeV infectious constructs. After 24 h the cells were scraped into their medium and injected directly
into the allantoic cavity of 9-day-old embryonated chicken eggs. Three days later,
the allantoic fluids were harvested and reinjected undiluted into eggs. For further
passages, the viruses were diluted 1/500 before injection. The presence of viruses
was determined by pelleting allantoic fluids through a TNE (10 mM Tris-HCl
[pH 8.0], 100 mM NaCl, 1 mM EDTA)–25% glycerol cushion for 20 min at
14,000 rpm in an Eppendorf 5417C centrifuge. Virus pellets were resuspended in
sample buffer, and the proteins were separated by sodium dodecyl sulfate–10%
polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue.
Virus infection and passage. A549 or BSR T7 cells were infected at a multiplicity of infection (MOI) of 10 in Dulbecco’s modified Eagle medium. After 1
to 2 h of absorption, the inoculum was removed and replaced with fresh medium
containing 10% fetal bovine serum. Virus supernatant from 48-h-infected cells
was cleared by filtration, treated for 1 h with trypsin (1.2 g/ml), and used to
reinfect fresh A549 or BSR T7 cells.
Real-time PCR. A 9-cm-diameter dish of A549 cells was infected with SeV at
an MOI of 10. After 24 h, medium was removed, the cells were scraped, and total
RNA was extracted using Trizol. One-tenth of this RNA was mixed with 0.5 g
of specific primer and converted to cDNA with Moloney murine leukemia virus
reverse transcriptase for 1 h at 37°C. One-tenth of the cDNA was used for
real-time PCR, using TaqMan Universal Master mix and the ABI Prism 7700
sequence detector. Several dilutions of cDNA were tested to construct a standard curve that was used to obtain relative numbers for the samples.
DI particle rescue with helper virus. Dishes of BSR-T7 cells (diameter, 9 cm)
were infected with either wild-type SeV (SeV-wt) or SeV-AGP55 at an MOI of
10. One hour later, the medium was removed and the cells were transfected with
5 g of the DI-encoding plasmid. Twelve hours later the medium was replaced
with fresh Dulbecco’s modified Eagle medium containing 10% fetal bovine
serum. The ND and DI particles present in the culture medium were then
passaged on fresh cells as described above.
DI particle rescue with plasmids. Dishes of BSR-T7 cells (diameter, 9 cm)
were transfected with 1.5 g of pGEM-L, 5 g of pGEM-N, 5 g of pGEM-PHA
(which does not express C proteins), and 5 g of the various DI-encoding
plasmids.
RESULTS
The ratio of genomes to antigenomes was previously determined by (32)P-primer extension analysis. This ratio can also
be determined by real-time reverse transcriptase PCR (RTPCR) analysis, as follows (Fig. 2A) (and as described in Materials and Methods). Four pairs of primers that span gene
junctions are used; one primer of each pair specifically extends
on genomes and lies upstream of the junction, and the other
specifically extends on antigenomes and lies downstream of the
junction. The four genome-specific and four antigenome-specific primers are used in individual reactions to synthesize
cDNA with RT. Samples of the RT reaction mixture are then
used in real-time PCR amplification along with the reverse
primer located across the junction. This approach eliminates
the contribution of mRNAs to the amount of antigenomes
determined (only discistronic mRNAs that are very rare in SeV
infections will be scored). At least three sets of primers (and
fluorescent probes) were used for each determination, and the
average genome/antigenome ratio from at least two infections
of each SeV is shown. The ratios determined by RT-PCR agree
well with those determined by primer extension analysis (in
parentheses in Fig. 2), and require considerably less viral RNA
as starting material.
Relative to SeV-wt infections, where ca. 10 times as many
genomes as antigenomes are found intracellularly, SeVAGP55 infections (in which the entire 55 nt of tr is replaced
with the le sequence) contain equal levels of the two (17) (Fig.
2). (AGP refers to a virus or DI particle. When “AGP” is
9149
followed by a single number, as in AGP55, this indicates that
positions 1 to 55 of AG/Pr have been exchanged with le sequences.) SeV-AGP65 infections, which contain gs-1 in addition to the le sequence at AG/Pr, yielded similar results (Fig.
2). The same genome-to-antigenome ratios were found in virions (data not shown), since, similar to rabies virus infections
(7), SeV genomes are not being selectively exported during
virion assembly (14). Genome/antigenome ratios intracellularly should then simply reflect the frequencies with which
vRdRP productively initiates at the template 3⬘ ends and begins to encapsidate nascent le/tr RNAs, to produce genome or
antigenome nucleocapsids. The efficiency of this process is one
measure of promoter strength. By this criterion, the replacement of the tr region of AG/Pr with the le sequence alone
(SeV-AGP55) strongly decreases AG/Pr strength.
Since SeV-AGP55 replicates to levels similar to those of
SeV-wt, it appears that G/Pr is inherently weak only in the
presence of the stronger AG/Pr. Thus, as described for rabies
virus infections (7), competition between G/Pr and AG/Pr for
vRdRP appears to be the primary determinant of the relative
amounts of SeV genomes and antigenomes formed (Fig. 1B)
The genome/antigenome ratio of 1 in AGP55/65 infections
suggests that promoter strength is primarily determined by tr
versus le sequences themselves, in agreement with previous
studies of DI genome replication (3). However, it is also possible to construct SeV in which the first 42 or 48 nt of the le
sequence of G/Pr is replaced with the equivalent tr sequences
(SeV-GP42/48) without seriously affecting viral mRNA synthesis (8, 9). SeV-GP42 and SeV-GP48 are notable in that their
infections fail to induce apoptosis. Nevertheless, SeV-GP42/48
infections still accumulate 10 times as many genomes as antigenomes intracellularly, like SeV-wt (only SeV-GP42 is shown
in Fig. 2). This result was unexpected because, if promoter
strength is determined primarily by the nature of first 30 nt of
le/tr sequence, the modified G/Pr of SeV-GP42/48 should have
been equivalent in strength to AG/Pr.
Possible explanations for why G/Pr of SeV-GP42/48 remains
weaker than AG/Pr include (i) the remainder of the le region
(positions 49 to 55) is specifically important for G/Pr strength
in the context of nondefective infections and (ii) in some situations, the presence of gs-1 within G/Pr does indeed reduce
G/Pr strength, despite the presence of 48 nt of tr sequence. To
directly examine this question, a matched series of rSeV isolates were prepared that bridged the transition from SeV-wt to
SeV-AGP65 (Fig. 2). Except for the ambisense SeV (i.e., SeV
that expresses ambisense mRNA) that grow less well than
SeV-wt, all the other SeV grow similarly to SeV-wt; i.e., they all
accumulate similar levels of viral macromolecules intracellularly. SeV-wt⫹MCS contains a cassette at the L gene end/tr
junction with three elements, a poly(A)-stop site that terminates mRNAs transcribed from the antigenome 3⬘ end, a multicloning site (MCS), and a duplicated L gene end site. SeVwt⫹MCS infections produce genome/antigenome ratios of 10
like SeV-wt; thus, the inclusion of this cassette appears not to
have affected relative promoter strengths. SeV-AGP56-65 is
identical to SeV-wt⫹MCS except that the 10 nt adjacent to tr
(i.e., the L gene end site) are converted to the complement of
gs-1. Remarkably. the simple inclusion of these 10 nt (gs-1) in
AG/Pr of AGP56-65 leads to transcription of a short (80 nt)
ambisense mRNA from this antigenome that terminates just
9150
LE MERCIER ET AL.
J. VIROL.
FIG. 2. 3⬘-end promoter mutations of nondefective SeV and the ratio of genomes to antigenomes formed during infection. (A) The SeV
genomes are shown as a series of boxes (not drawn to scale). (B) The 96-nt genome 3⬘-end promoter (on the left) and antigenome 3⬘-end promoter
(right) are shown as narrow boxes. The remainder of the genome (from the N gene AUG codon to the UAA that terminates the L open reading
frame) is shown as wider boxes. The genome and antigenome 3⬘-end promoters, G/Pr and AG/Pr, are composed, respectively, of 55 nt of le (white
box) or tr sequence (black box), gs-1 (white circle with forward arrow) or the complement of the L gene end signal (black circle with stopped arrow),
a short spacer sequence, and the [C1N2N3N4N5N6]3 repeat or its complement (BB box). The cassettes added at the L gene end/tr junction to control
ambisense mRNA expression are marked with a dotted line. White and grey indicate the various G/Pr and AG/Pr sequences present at each 3⬘-end
promoter. In naming the viruses, GP and its associated numbers refer to those nucleotides of G/Pr that have been replaced with the equivalent
sequences of AG/Pr, or vice versa for AGP viruses. Total RNA from cytoplasmic extracts of A549 cells infected for 24 h with 20 PFU/cell of the
various ND-SeV was used to estimate the relative amounts of genomes and antigenomes by real-time RT-PCR. Four sets of primers and probes
that spanned different gene junctions (A) were used (to minimize the contribution of mRNA to the antigenome determination [see Materials and
Methods]). At least three sets of primers and probes were used for each determination, and the average G/AG ratio from at least two infections
of each SeV is shown. The numbers in parentheses show the ratios determined by 32P-labeled primer extension.
before the opposing L gene. SeV-AGP56-65 infections, however, now accumulate almost as many antigenomes as genomes. Thus, gs-1 placed within AG/Pr not only functions well
but also apparently acts to equalize the two promoters, presumably by weakening AG/Pr. SeV-AGP52-65 and AGP48-65
differ from AGP56-65 by the further conversion of the 4 and 8
nt adjacent to the ambisense gene start site. The exchange of
these nucleotides further increases the relative amounts of
antigenomes, but only slightly. Thus, AG/Pr can be weakened
relative to G/Pr in one of two ways: by exchanging the resident
tr sequences for le sequences or by introducing an ectopic gs-1
within this 3⬘-end promoter.
The introduction of gs-1 within AG/Pr automatically destroys the L gene end site. It thus remains possible that the
destruction of this sequence is responsible (at least in part) for
weakening AG/Pr. We therefore attempted to prepare derivatives of wt⫹MCS in which the L gene end site is mutated to
something other than the complement of gs-1. The complement of the L gene end site (5⬘ UAAGAAAAA) was mutated
to 5⬘ UGCCGCAUG and UUUCUUUUU. However, we were
VOL. 77, 2003
PROMOTER COMPETITION FOR SENDAI VIRUS RNA POLYMERASE
unable to prepare these rSeV in three separate attempts, and
we sought another manner to examine this question. The mutated L gene end sites were built into mini-genomes expressing
green fluorescent protein (GFP) (Fig. 3), and these were recovered from DNA by coinfection with SeV-AGP55 (which
contains two weak replication promoters [see below]). We
were again unable to recover the 5⬘ UGCCGCAUG minigenome (DI-Ge-1; replicable genomes may not tolerate certain
sequences here). However, DI-Ge-3 (5⬘ UUUCUUUUU) could
be recovered almost as well as the wt. When RNA from these
mixed infections were examined for their ratio of DI genomes
to antigenomes, DI-Ge-3 and DI-wt were found to contain the
same excess of genomes over antigenomes (approximately
threefold in this case; the ratio of DI genomes and antigenomes are not necessarily the same as that of ND genomes and
antigenomes). Thus, the loss of the complement of the L gene
end site per se does not alter AG/Pr strength. We conclude
that it is the presence gs-1 within AG/Pr that is responsible for
weakening this 3⬘-end promoter.
Competition between DI genomes and ND helper genomes
for vRdRP. The relative amounts of genomes and antigenomes
formed during infection is one measure of the relative
strengths of G/Pr and AG/Pr of nondefective SeV. This manner of characterizing promoters stems from early studies of
copy back DI genomes, which have deleted most of the genome and simultaneously exchanged ⬎96 nt of G/Pr with the
equivalent sequences of AG/Pr (13). Copy back DI genomes
do not transcribe mRNAs and have a clear replicative advantage over ND virus. Moreover, in contrast to their ND helper
virus, these coinfections accumulate equal amounts of copy
back DI genomes and antigenomes. Further studies suggested
that their replicative advantage did not stem from their shorter
length, and that relative promoter strength is determined by
the terminal 96 nt of G/Pr and AG/Pr.
Internal-deletion (int-⌬) DIs that maintain the wt promoter
constellation can also be generated on high-MOI passage of
SeV stocks. However, these DIs are considerably more difficult
to generate, and the reasons for their replicative advantage
over their ND helper virus are unknown. In further contrast to
copy back DIs, int-⌬ DI genomes cannot be recovered from
DNA using SeV-wt as helper, even though they are efficiently
amplified when the replication substrates (N, P, and L) are
provided from plasmids. More importantly, the same int-⌬ DIs
are efficiently recovered from DNA using SeV-AGP55 or
AGP65 as helper virus (Fig. 3 and 4). Thus, although int-⌬ DIs
presumably have no advantage in competing with wt helper
virus for limiting RdRP during recovery from DNA, int-⌬ DIs
can clearly compete with ND helpers whose AG/Pr is weakened (as defined by ND genome/antigenome ratios). We have
adapted this system to study promoter strength by including a
GFP transcription unit within int-⌬ DIs. BSR T7 cells infected
with various helper viruses are transfected with plasmids expressing various mini-antigenomes (see time line in Fig. 3).
The particles released 36 to 48 h postinfection are then used to
infect fresh cultures that are examined by fluorescent microscopy for GFP expression. This simple test reveals whether the
DI particles have been generated from plasmid DNA during
the initial infection/transfection, and amplified by the helper
virus during subsequent coinfection (P1) (Fig. 3).
Using this DI reporter system, we have reexamined whether
9151
gs-1 within G/Pr affects the ability of G/Pr to compete for
vRdRP during coinfection. A series of mini-genome constructs
that varied only in their G/Pr was used to determine what
changes were required so that the int-⌬ DI could be recovered
and amplified by SeV-wt helper (5 top constructs, Fig. 4). We
first exchanged the first 42 or 55 nt of le of G/Pr with the
equivalent tr sequences (GP42/wt and GP55/wt), but only background fluorescence was found at passage 1. We then added a
further 12 nt of the trailer sequence but maintained the remainder of DI GP55/wt, so that the GFP expression was retained and now started at nt 68 rather than nt 56 (DI
GP68⫹start/wt). However, this DI as well could not be recovered (in six attempts) with SeV-wt. Only when the mRNA start
site was eliminated from G/Pr of DI GP55/wt, by exchanging it
for the equivalent AG/Pr sequence (DI GP96/wt), was this DI
recovered with SeV-wt. (The elimination of gs-1 of course also
eliminates GFP expression, and DI RNA levels were determined by real-time RT-PCR [Fig. 4, bottom].) Thus, int-⌬ DI
genomes successfully compete for vRdRP with wt helper genomes only when the mRNA start site within G/Pr has been
eliminated; simply moving gs-1 downstream by 12 nt (DI
68⫹start/wt) has no effect.
Similar experiments were carried out with DI wt/AGP55,
which contains le in place of tr sequences at AG/Pr (bottom
three constructs in Fig. 4). Just as DI-wt/wt cannot be rescued
by coinfection with SeV-wt, DI wt/AGP55 cannot be rescued
by coinfection with SeV-AGP55 (presumably because both
G/Pr and AG/Pr of the DI and ND-helper virus are again
equivalent). When the first 42 nt of G/Pr are exchanged for the
equivalent tr sequences, this DI GP42/AGP55 cannot be rescued by SeV-AGP55. However, DI GP96/AGP55, in which the
first 96 nt are trailer sequence and gs-1 is eliminated, can be
rescued with SeV-AGP55. Taken together, these results indicate that gs-1 within G/Pr clearly decreases the ability of this
3⬘-end promoter to compete for vRdRP during recovery from
DNA with helper SeV and subsequent coinfection.
DISCUSSION
The 96-nt G/Pr contains 55 nt of le sequence (light color,
weak), gs-1 (oval with arrow) and a simple hexamer repeat (BB
box), whereas the symmetrical AG/Pr contains tr sequence
(dark color, strong), the L gene end site (empty oval), and a BB
box (Fig. 5). AG/Pr is a stronger replication promoter than
G/Pr, as defined by the preponderance of [⫺] genomes formed
during virus replication. In these and other experiments, the
two BB boxes appear to be interchangeable without effect
(data not shown). The major finding of this work is that gs-1
within 3⬘-end promoters negatively influences promoter strength.
Our results are summarized schematically in Fig. 5.
G/Pr contains two elements that weaken this 3⬘-end promoter relative to AG/Pr; the presence of the le as opposed to
the tr sequences, and gs-1. Simply replacing the tr sequence of
AG/Pr with le sequence reduces AG/Pr strength such that
SeV-AGP55 infections now accumulate equal amounts of genomes and antigenomes (Fig. 2). Similarly, introducing gs-1
into AG/Pr of SeV-wt⫹MCS (SeV-AGP56-65) reduces the
genome/antigenome ratio from 12 to 2.5 (Fig. 2B). Thus, the
stronger AG/Pr apparently can be weakened in two different
ways. G/Pr has been examined via DI mini-genomes as well as
9152
LE MERCIER ET AL.
J. VIROL.
FIG. 3. Defective-interfering mini-genomes expressing GFP. (A) DI-wt, in which the entire viral coding sequences (from the N gene AUG to
the UAA of the L open reading frame) were replaced with the GFP open reading frame, is shown schematically as for Fig. 2. The sequences present
at L gene end site of DI-wt, DI-Ge-1 and DI-Ge-3 (see text) are indicated. (B) A time line of DI particle recovery from DNA is shown (see text
for details). Green fluorescent photos of the first passage cultures (P1) that contained equal densities of cells are shown. To measure viral RNA
levels, two additional virus passages were carried out to minimize spill-over from biased genome/antigenome ratios during DI recovery from DNA.
Genome/antigenome levels were then determined by real-time RT-PCR with the two primers pairs and probe as indicated in panel A. Transfection
of DI-wt without helper virus (first photo in panel A), and DI-Ge-1 transfection with helper virus (third photo in panel A) serve as negative controls
that show the amplification specificity for the DI RNAs. Error bars, standard deviations.
VOL. 77, 2003
PROMOTER COMPETITION FOR SENDAI VIRUS RNA POLYMERASE
9153
FIG. 4. DI particle recovery from DNA with various nondefective helper SeV. The various DI constructs are shown schematically and are
named as for Fig. 2 and 3. For those constructs with a functional gs-1 driving GFP expression, recovery was scored (⫹ or ⫺) by fluorescent
microscopy at P1 (Fig. 3). For those constructs without functional gs-1 (marked with asterisks), recovery was scored by determining DI genome
levels by real-time RT-PCR and is plotted in the bar graph below. Error bars, standard deviations.
ND-SeV, because gs-1 is not essential for DI-genome replication. Exchanging the first 42 or 48 nt of the le region of G/Pr of
ND viruses with the equivalent tr sequences (SeV-GP42/48)
does not diminish the genome/antigenome ratio of 10, nor
does the analogous exchange of the first 55 nt of G/Pr improve
the ability of int-⌬ DI genomes to be rescued by SeV-wt (Fig.
4). Thus, simply exchanging le for tr sequences apparently
cannot overcome the negative effects of gs-1 within G/Pr. The
simultaneous elimination of the mRNA start site within G/Pr
of int-⌬ DI genomes (DI GP98/wt and DI GP98/AGP55) was
9154
LE MERCIER ET AL.
J. VIROL.
FIG. 5. A model for promoter competition in cis for vRdRP. The various 96-nt G/Pr and AG/Pr are shown schematically as for the other figures,
with the weak le sequences shown by light shading and the strong tr sequences shown by dark shading. The first 12 nt of each promoter are
conserved and are shown as a separate shading. The larger oval near the end of the le/tr regions represents vRdRP that has initiated at the 3⬘ end,
has cleared this promoter, and has just released the le/tr RNAs. If vRdRP remains attached to the N:RNA without a nascent chain, it is free to
scan the template (in either direction) for another RNA start site, which can be either the 3⬘-end promoter or gs-1 (horizontal arrows below oval).
The relative strength of the 3⬘-end promoter, i.e., its ability to compete for limiting vRdRP during infection relative to other 3⬘-end promoters,
is indicated by the size of bent arrow at the 3⬘ ends. Relative 3⬘-end promoter strength is conditioned both by the presence of the le/tr sequences
and that of gs-1 (circle with bent arrow).
found to be essential for their rescue by SeV-wt and SeVAGP55, respectively (Fig. 4). Thus, the ability of DI genomes
to compete with helper virus genomes for vRdRP is dominated
by the presence of gs-1 within the DI genome. We note that
there are also examples at AG/Pr where the converse situations applies, namely, where the presence of le as opposed to
the tr sequences predominates, and effects due to the presence
of gs-1 are not detected (e.g., SeV-AGP55 and SeV-AGP65
rescue int-⌬ DI genomes with equal efficiency).
Almost all our DI constructs were efficiently amplified when
their replication substrates were provided via plasmids (in the
absence of other competing SeV templates) (Fig. 4). The inability of some constructs to be rescued with SeV-wt, coupled
with their efficient rescue with SeV-AGP55/65, suggests that
DI rescue depends on the ability of the DI 3⬘-end promoters to
compete with the helper genomes for vRdRP. It is from these
experiments that the conclusion that gs-1 negatively affects
3⬘-end promoter strength comes through most clearly. On what
basis, though, does gs-1 within G/Pr diminish the ability of DI
genomes to compete with ND genomes for replication substrates? Simple competition of resting ND and DI N:RNAs for
a common pool of vRdRP (off the template) may be part of the
explanation. In this case, the competition implies that the eventual transcriptases and replicases cannot be distinguished off
the template.
However, it is also possible that this competition occurs in
cis, once vRdRP has engaged G/Pr, as this might offer an
explanation for why the presence of gs-1 within G/Pr is so
deleterious for 3⬘-end promoter strength. A model for this
in-cis competition is shown in Fig. 5. vRdRP in this cartoon
(elongated oval) has already initiated at the genome 3⬘ end and
cleared the promoter. In those cases where RNA synthesis and
assembly with N become coupled, vRdRP is committed to
replication and this P4-L does not reenter the pool until it has
completed N:RNA synthesis. G/Pr strength, or its relative abil-
ity to initiate RNAs that are subsequently coassembled with N,
is presumably conditioned by the presence of the le versus tr
sequences within G/Pr. These sequences as [⫺] genome would
promote vRdRP initiation, and as nascent [⫹] le RNA would
promote N assembly. When RNA synthesis and its assembly
with N do not become coupled, vRdRP releases the nascent le
chain at or near gs-1. If vRdRP is simultaneously released from
the template along with the le RNA, this P4-L rejoins the
vRdRP pool off the template and is free to interact with all the
available 3⬘-end promoters in the coinfected cell (Fig. 1B).
However, if vRdRP is not released from the template along
with le RNA, it may then be free to scan the template (in both
directions) for the nearby gs-1 as well as the genome 3⬘ end,
similar to vRdRP that has released its mRNA at gene junctions
(6, 23). Scanning confers an advantage to this 3⬘-end promoter,
precisely because vRdRP is restricted to reinitiating on this
template. The presence of gs-1 within G/Pr weakens this replication promoter by diverting these scanning vRdRP from
reinitiating at the genome 3⬘ end, thus decreasing its relative
replication promoter strength.
A corollary of this competition-in-cis model is that relative
initiation from gs-1 will similarly depend on competition with
the 3⬘-end promoter; i.e., gs-1 will be initiated more frequently
when present within or near G/Pr (near weaker le sequence)
than within or near AG/Pr (near stronger tr sequence). This
aspect has recently been tested with DI mini-genomes containing tandem 96-nt promoters in which gs-1 of the external promoter was corrupted and the replicative ability of the internal
promoter was disabled, such that gs-1 of the internal promoter
was now found at position 146, i.e., 50 nt downstream of the
external 96-nt replication promoter. In this location, gs-1 was
used 15 times more frequently when downstream of a weak
G/Pr than downstream of a strong AG/Pr (D. Vulliemoz, P. Le
Mercier, and L. Roux, personal communication). Again, the
obvious conclusion is that gs-1 and nearby replication promot-
VOL. 77, 2003
PROMOTER COMPETITION FOR SENDAI VIRUS RNA POLYMERASE
ers compete with each other, presumably in cis, for a common
pool of vRdRP in coinfected cells.
13.
ACKNOWLEDGMENTS
We thank Laurent Roux for numerous discussions.
This work was supported by a grant from the Swiss National Science
Fund.
REFERENCES
1. Buchholz, U. J., S. Finke, and K. K. Conzelmann. 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. 73:251–259.
2. Calain, P., and L. Roux. 1993. The rule of six, a basic feature for efficient
replication of Sendai virus defective interfering RNA. J. Virol. 67:4822–4830.
3. Calain, P., and L. Roux. 1995. Functional characterisation of the genomic
and antigenomic promoter of Sendai virus. Virology 211:163–173.
4. Curran, J., R. Boeck, N. Lin-Marq, A. Lupas, and D. Kolakofsky. 1995.
Paramyxovirus phosphoproteins form homotrimers as determined by an
epitope dilution assay, via predicted coiled coils. Virology 214:139–149.
5. Egelman, E. H., S.-S. Wu, M. Amrein, A. Portner, and G. Murti. 1989. The
Sendai virus nucleocapsid exists in at least four different helical states.
J. Virol. 63:2233–2243.
6. Fearns, R., and P. L. Collins. 1999. Model for polymerase access to the
overlapped L gene of respiratory syncytial virus. J. Virol. 73:388–397.
7. Finke, S., and K.-K. Conzelmann. 1997. Ambisense gene expression from
recombinant rabies virus: random packaging of positive- and negative-strand
ribonucleoprotein complexes into rabies virions. J. Virol. 71:7281–7288.
8. Garcin, D., T. Pelet, P. Calain, L. Roux, J. Curran, and D. Kolakofsky. 1995.
A highly recombinogenic system for the recovery of infectious Sendai paramyxovirus from cDNA: generation of a novel copy-back nondefective interfering virus. EMBO J. 14:6087–6094.
9. Garcin, D., G. Taylor, K. Tanebayashi, R. Compans, and D. Kolakofsky.
1998. The short Sendai virus leader region controls induction of programmed cell death. Virology 243:340–353.
10. Gubbay, O., J. Curran, and D. Kolakofsky. 2001. Sendai virus genome
synthesis and assembly are coupled: a possible mechanism to promote viral
RNA polymerase processivity. J. Gen. Virol. 82:2895–2903.
11. Hoffman, M. A., and A. K. Banerjee. 2000. Precise mapping of the replication
and transcription promoters of human parainfluenza virus type 3. Virology
269:201–211.
12. Iseni, F., F. Baudin, D. Garcin, J. B. Marq, R. W. Ruigrok, and D. Kolakof-
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
9155
sky. 2002. Chemical modification of nucleotide bases and mRNA editing
depend on hexamer or nucleoprotein phase in Sendai virus nucleocapsids.
RNA 8:1056–1067.
Kolakofsky, D. 1976. Isolation and characterization of Sendai virus DIRNAs. Cell 8:547–555.
Kolakofsky, D., and A. Bruschi. 1975. Antigenome in Sendai virions and
Sendai virus-infected cells. Virology 66:185–191.
Kolakofsky, D., T. Pelet, D. Garcin, S. Hausmann, J. Curran, and L. Roux.
1998. Paramyxovirus RNA synthesis and the requirement for hexamer genome length: the rule of six revisited. J. Virol. 72:891–899.
Lamb, R. A., and D. Kolakofsky. 2001. Paramyxoviridae: the viruses and their
replication, p. 1305–1340. In D. M. Knipe and P. M. Howley (ed.), Fields
virology. Lippincott, Williams & Wilkins, Philadelphia, Pa.
Le Mercier, P., D. Garcin, S. Hausmann, and D. Kolakofsky. 2002. Ambisense Sendai viruses are inherently unstable but are useful to study viral
RNA synthesis. J. Virol. 76:5492–5502.
Murphy, S. K., Y. Ito, and G. D. Parks. 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. 72:10–
19.
Murphy, S. K., and G. D. Parks. 1999. RNA replication for the paramyxovirus simian virus 5 requires an internal repeated (CGNNNN) sequence
motif. J. Virol. 73:805–809.
Pelet, T., C. Delenda, O. Gubbay, D. Garcin, and D. Kolakofsky. 1996.
Partial characterization of a Sendai virus replication promoter and the rule
of six. Virology 224:405–414.
Rager, M., S. Vongpunsawad, W. P. Duprex, and R. Cattaneo. 2002. Polyploid measles virus with hexameric genome length. EMBO J. 21:2364–2372.
Skiadopoulos, M. H., L. Vogel, J. M. Riggs, S. R. Surman, P. L. Collins, and
B. R. Murphy. 2003. The genome length of human parainfluenza virus type
2 follows the rule of six, and recombinant viruses recovered from nonpolyhexameric-length antigenomic cDNAs contain a biased distribution of
correcting mutations. J. Virol. 77:270–279.
Stillman, E. A., and M. A. Whitt. 1999. Transcript initiation and 5⬘-end
modifications are separable events during vesicular stomatitis virus transcription. J. Virol. 73:7199–7209.
Tapparel, C., D. Maurice, and L. Roux. 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. 72:3117–3128.
Tarbouriech, N., J. Curran, R. W. Ruigrok, and W. P. Burmeister. 2000.
Tetrameric coiled coil domain of Sendai virus phosphoprotein. Nat. Struct.
Biol. 7:777–781.
Vulliemoz, D., and L. Roux. 2001. “Rule of six”: how does the Sendai virus
RNA polymerase keep count? J. Virol. 75:4506–4518.