VIROLOGY
225, 163 –171 (1996)
0584
ARTICLE NO.
Efficiency
of Sendai
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Primary Sequence Independent of Terminal Complementarity
CAROLINE TAPPAREL and LAURENT ROUX1
Department of Genetics and Microbiology, University of Geneva Medical School, CMU, 9 avenue de Champel, 1211 Geneva 4, Switzerland
Received May 16, 1996; accepted August 27, 1996
From the cDNAs of two defective RNAs naturally exhibiting a large difference in replication efficiency, a series of Sendai
virus RNA chimeras were constructed by reciprocal exchanges of their 3* end primary sequences. Using a reverse genetics
system, the ability of these RNAs to replicate when expressed from cDNAs in the context of the viral proteins N, P, and L,
also expressed from plasmids, was analyzed. First the extent of potential RNA 3*/ 5* end complementarity was tested by
disrupting and restoring the terminal 110-nucleotide complementarity of a copy-back RNA. Alternatively, this base pairing
potential was gradually increased from 12 to 57 or to 98 nucleotides by continuous substitutions. In all cases, the restoration
or the creation of more extended base pairing potential had no effect on RNA replication. Reciprocal exchanges were then
made in order to identify cis-acting sequences that could induce high replication efficiency. It was found that nucleotides
1 –31 of the antigenome 3* end were sufficient to confer a high replication property (more than a 10-fold increase), regardless
of the sequence adjacent to these terminal nucleotides. It is concluded that one of the most important features that
modulate replication efficiency is contained in the promoter end primary sequence and that this feature is likely to operate
independently of the ability to form a potential terminal base pairing. q 1996 Academic Press, Inc.
INTRODUCTION
polymerase obeys to start, stop, and restart templated
cis signals at the borders of each transcription unit. In
replication, the RNA polymerase is made insensitive to
these cis signals and, consequently, synthesizes a full
complement of the template. At the antigenome 3* end,
the RNA polymerase will eventually produce only a full
copy of its template, i.e., the RNA genome. At the genome
and antigenome 3* ends lie two regions of about 50 to
60 nucleotides called, respectively, the leader plus (le/)
and leader minus (le0) template regions (Leppert et al.,
1979; Vidal and Kolakofsky, 1989; Banerjee and Barik,
1992). These two regions encode respectively the le/
and le0 RNAs, which are thought to contain the initiation
sites for encapsidation (Blumberg et al., 1983). These
leader RNAs have further been proposed to be the sites
where the decision to replicate is made. Replication
would be tied to the leader RNA encapsidation rate, this
rate depending itself on the unassembled N protein concentration (Kolakofsky and Blumberg, 1982; Vidal and
Kolakofsky, 1989). This model emphasises the importance of the genome and antigenome RNA ends, acting,
on one hand, as binding sites for the RNA polymerase
(template 3* ends) and, on the other hand, as check
points to confirm replication (nascent strand 5* ends).
For convenience, we refer here to these RNA 3* ends
(still not precisely defined) as promoters. These, for the
time being, include indistinctly the template 3* end nucleotides as well as their templated 5* end nascent strands.
In the case of the Paramyxoviruses, these promoters are
likely to extend past the limit of the leader template regions (see text of Results and Discussion). In contrast, for
Negative-stranded RNA viruses initiate the viral multiplication cycle with viral messenger RNA synthesis templated by the infecting genome. This primary transcription leads to de novo synthesis of viral proteins, required
in turn for viral RNA replication. Genome replication begins with the copying of the incoming viral RNA of negative polarity, i.e., the genome, into its full-length complement of positive polarity, the antigenome, which will
serve in turn as template for viral genome amplification.
The viral RNA-dependent RNA polymerase is first
brought in by the infecting virus, before it is expressed
de novo following primary transcription and translation.
The functional template for transcription and replication
is not the naked RNA, but the N:RNA nucleocapsid of
helical symmetry.
According to a current model (reviewed in Kingsbury,
1974; Kolakofsky and Blumberg, 1982; Vidal and Kolakofsky, 1989), which applies mainly to viruses of the Rhabdoand Paramyxoviridae families, the viral RNA synthesis
starts with the RNA polymerase entering at the genome
or the antigenome 3* ends. At the genome 3* end, the
RNA polymerase has the apparent choice to synthesize
the antigenome or to initiate the sequential synthesis of
the messenger RNAs. The RNA polymerase is thought
to be in different functional modes, defined, to this day,
by the reaction end products. For transcription, the RNA
1
To whom correspondence and reprint requests should be addressed. Fax: 41.22. 702.5702. E-mail: Roux@cmu.unige.ch.
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FIG. 1. Schematic representation of the defective RNA templates. The DI-H4 and E307 RNAs are schematically depicted as minus strands oriented
3* to 5* from left to right. H4 is a copy-back RNA which contains only a 5* end portion of the L gene (from nt 14,084), the last SeV RNA transcription
unit, followed, at its 5* end, by the minus-strand leader template region (le0), present at the 5* end of the SeV RNA. At its 3* end, an inverted
complementary repeat over 110 5* end nucleotides (15,384* –15274*) gives the RNA its copy-back feature. This includes the possibility for the
extremities of the naked RNA to form an intramolecular hybrid over 110 nucleotides (shown on the second line far right) and implies that both the
H4 minus- and plus-strand 3* ends are identical over 110 nucleotides. This latter feature is pictured on the second line (center), where the two
boxes refer to the 3* end sequences of, respectively, the H4 minus (left)- and plus (right)-strand promoters (of yet undefined length, but likely to be
shorter than 110 nucleotides) involved in the control of RNA replication (see Introduction). E307 is an internal deletion RNA, which has conserved
the full-length SeV RNA ends. At the 3* end, the leader plus (le/) template region is followed by a 3* end portion of the N gene fused to a 5* end
portion of the L gene. As a naked RNA, E307 can potentially form an intramolecular hybrid over only 12 nucleotides, since the first 12 nucleotides
of the plus- and minus-strand 3* ends are identical (as in the full-length SeV RNA). These are schematized on the line above, along with the two
boxes referring to the 3* end sequences of, respectively, the minus- and the plus-strand promoters, of yet undefined length , involved in the control
of RNA replication. Note that E307 and H4 RNAs have the same plus-strand 3* end, but differ in their minus-strand 3* ends. The black bar in the
middle of the promoter regions refer to the le/ or le0 template borders at positions respectively, 55 and 57 nucleotides.
the Rhabodviruses, they appear to span only the leader
regions (Schubert et al., 1979; Pattnaik et al., 1992, 1995;
Conzelmann and Schnell, 1994; Wertz et al., 1994). Early
in infection, replication must first produce antigenomes
from the incoming genome to build up replication intermediates. Later, an excess of genomes over antigenomes is generally observed (Schincariol and Howatson, 1972; Soria et al., 1974; Kolakofsky and Bruschi,
1975; Simonsen et al., 1979). Therefore, a preferential
amplification of the genome over the antigenome has to
be orchestrated late in infection. To account for this, the
antigenome, template for genome synthesis, has been
proposed to contain a stronger replication promoter than
the genome, on the rationale that this latter template
promoter is weakened by its implication in both replication and transcription.
In a previous work, we have addressed the question
of promoter characterization (Calain and Roux, 1995). Using a reverse genetics system, Sendai virus (SeV) defective interfering (DI) RNAs, expressed from cDNA, were
replicated in the context of the viral proteins expressed
from N, P, and L gene carrying plasmids. Two types of
template RNAs, identical to naturally occurring DI RNAs,
were used. An internal DI E307-RNA, with both ends of
the viral genome conserved (Fig. 1), replicated poorly in
the system. This DI genome also produced a functional
mRNA. In contrast, a copy-back DI H4-RNA exhibited a
20-fold higher replication efficiency, consistent with the
presence of the antigenome promoter on both the minus
and plus strands (Fig. 1). Reciprocal sequence exchanges between E307 and H4 promoters generated
RNA derivatives whose study led to two main conclusions. First, the ability to transcribe an mRNA did not
prevent efficient replication. Thus, in this reconstituted
system at least, the replication efficiency of the promoter
was found not to be hindered by its participation in both
transcription and replication. Second, the efficiency of
replication could be then strictly correlated with the promoter primary sequence, since a gradual replacement
of H4 promoter (high replication) with the corresponding
sequence from the E307 promoter (low replication) resulted in a gradual decrease in RNA replication and vice
versa (Calain and Roux, 1995). However, the gradual promoter replacement also diminished the base pairing potential of the H4 RNA 3*/5* ends which extents for 110
nucleotides, due to the copy-back nature of this RNA
(see Fig. 1). As the extent of the RNA 3*/5* end complementarity has been recently found to affect the replication
efficiency of a VSV defective RNA studied in a similar
system (Wertz et al., 1994), the question was raised as to
whether it was the variations of the extent of the terminal
complementarity that was also responsible for the differences of the SeV RNA replication efficiency.
To address this question, new H4 RNA derivatives ex-
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hibiting different potential for 3*/5* end base pairing were
constructed and tested for replication. The results obtained do not support the effect of terminal base pairing
on replication efficiency. In consequence, a closer examination of the promoter was made, in order to further
identify primary sequences controlling SeV RNA replication efficiency. In the end, nucleotides 1 to 31 at the
antigenome 3* end were shown to be sufficient to confer
high replication efficiency, regardless of the adjacent
downstream sequence.
MATERIAL AND METHODS
Virus and cells
Hela and CV1 cells were grown in regular MEM supplemented with 5% fetal calf serum in a 5% C02 atmosphere. Vaccinia recombinant virus expressing the T7
RNA polymerase, vTF7-3, a gift from Bernard Moss (National Institutes of Health, Bethesda, MD) has been described by Fuerst and colleagues (1986) and used accordingly. vTF7-3 stocks were grown in HeLa cells with
titers ranging from 5 1 108 to 109 PFU/ml.
Sequence and plasmids
165
(GP) and the 3* end of the antigenome, the antigenomic
promoter (AGP). In the case of H4 RNA, which contains
the same antigenomic sequence (AGP) on both the minus- and the plus-strand RNA, the distinction between
the two promoters had to be made [(AGP(0) or (/)]. For
example, H4-AGP(0)120 refers to a H4 RNA derivative
where the minus-strand 3* end has been substituted over
the first 120 nucleotides with the corresponding nucleotides coming from the E307 RNA minus-strand 3* end
(see also the diagrams presented in the figures).
Replication system
The replication system has been described previously
(Calain and Roux, 1995). In brief, CV1 cells (about 107)
were infected (m.o.i. Å 3) with vTF7-3. One hour later,
the cells were transfected with 5 mg of pGem4-NP, 5 mg
of pGem4-P/c, 1 mg of pGem4-L, and 5 mg of the plasmid
carrying the DI RNA sequence. Cytoplasmic extracts
were prepared 40 hr later, and CsCl gradient-purified
nucleocapsids were analyzed by Northern blotting (Mottet and Roux, 1989). 32P-labeled riboprobes of positive or
negative polarity, spanning 1453 nt in the L gene from
nucleotides 13,452 to 14,905 (two XbaI sites) and stopping at 477 nt from the genome 5* end, have been described before (5*ex probe, Mottet and Roux, 1989). This
probe was shortened by 474 nt by a further internal HpaI
cut at position 14,433, to eliminate a cross-reactivity with
a nonspecific RNA band appearing unexpectedly in some
experiments (as shown in Fig. 5). To prepare the probes,
routinely, 1 mg of linearized plasmid was transcribed under standard conditions in the presence of 50 mCi of
[a-32P]uridine and 106 Cherenkov counts per minute of
Sephadex G-50 purified radioactive RNA was added per
milliliter of hybridization solution (10 ml total). Calibration
of the Northern blot analysis was made using increasing
amounts of unlabeled in vitro-made transcripts to ensure
a linear RNA/signal (not shown), and the 20- to 50-fold
signal differences observed reflect this proportionality.
The signals were routinely quantitated by PhosphoImager scanning (Molecular Dynamics).
The complete SeV RNA primary sequence (15,384 nucleotides) was taken from Shioda et al. (1983, 1986) with
the correction by Neubert et al. (1991). The plasmids
expressing the SeV NP, P, and L proteins (respectively,
pGem-NP, pGem-P/c, and pGem-L) under the control of
the T7 RNA polymerase have been described previously
(Curran et al., 1991; Kast et al., 1991). The cloning of the
natural SeV DI RNAs H4 and E307 in pSP65 under the
control of the T7 RNA polymerase promoter has been
described previously (Calain et al., 1992; Engelhorn et al.,
1993; Calain and Roux, 1995). The nucleotide exchanges
performed to generate the DI H4 and E307 RNA derivatives were done by fusion PCR (Ho et al., 1989) with a
judicious use of previously existing derivatives and their
particular restriction sites. A first round of PCR products
were obtained using amplimers composed of about eighteen 3* end nucleotides complementary to the donor
strand and about eighteen 5* end dangling nucleotides
complementary to the recipient strand, along with amplimers common to both DI-H4 and E307 RNA, including a
Sal 1 site to the left of the T7 RNA polymerase promoter.
These first PCR products were then combined adequately and amplified with the amplimers common to
both RNAs. The recombined final products flanked by
the adequate sites were finally subcloned into pSV-H4
or pSV-E307 in replacement of the original fragments. All
the sequence modifications were verified by sequencing.
The derivatives were identified as having an H4 or E307
backbone. The promoter accepting the substitution was
than indicated, as well as the range of nucleotides coming from the donor promoter. For convenience, the 3*
end of the viral genome was called genomic promoter
The replication system based on the transfection of
the plasmids expressing the viral DI RNAs along with
the functions needed for replication (see Materials and
Methods) has been found adequate to compare the replication efficiency of various SeV RNAs. This conclusion
was reached before, when we examined the effect on
RNA replication of reciprocal exchanges of primary sequences present in the viral genome and antigenome
promoters (Calain and Roux, 1995): to gradual substitution of sequences corresponded consistent gradual
changes in replication efficiency. In this study, the same
approach is used to test whether the presence of RNA
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RESULTS
The effect of possible 3*/ 5* RNA complementarity
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FIG. 2. Disruption and reestablishment of 3*/5* end complementarity, by intercalation of discontinuous GP sequences into AGP. The H4 RNA
plus- or/and minus-strand 3* ends (dark boxes) were modified by the substitution of nucleotides 24 to 55 with the corresponding nucleotides of
the E307 RNA minus-strand 3* end (GP, light gray box). This substitution was introduced at the 3* end of the minus or the plus strand as in,
respectively, H4-AGP(0)24/55 and H4-AGP(/)24/55, or at both 3* ends simultaneously, as in H4-AGP(0/ /)24/55. The consequence of these
substitutions for the 3*/5* end complementarity is schematically indicated at the right end side of the figure. These RNAs were expressed from
plasmids as transcripts of positive polarity by the T7 RNA polymerase in the replication system, as described under Materials and Methods. The
replication was monitored by Northern blot analysis of the nucleocapsid RNAs using the probe 5* ex (see Methods) of positive polarity which reacts
with the RNA-strand complementary to the T7 RNA transcript. The 0/ / L lanes correspond to replications performed in the absence or in the
presence of the viral RNA polymerase L. Duplicate samples are shown for the RNA derivatives. The Northern blot was quantified by PhosphoImager
and the numbers refer to the efficiency of replication relative to that obtained for the H4 RNA, arbitrarily set at 100. The arrow indicates the direction
of the agarose gel migration.
complementary termini is important for replication efficiency. In a first series of experiments the complementary termini of the H4 RNA are interrupted by the replacement of nucleotides 24 to 55 of its minus strand 3* end
with the corresponding nucleotides coming from the
E307 minus strand 3* end (Fig. 2). This replacement,
creating derivative H4-AGP(0)24/55 (for an explanation
of the derivative nomenclature see Materials and Methods), decreases the complementary termini from 110 nucleotides down to 71 nucleotides. Figure 2 shows that
H4-AGP(0)24/55 replication efficiency decreases correspondingly by about 10-fold relative to H4. Whether the
substitution is made in the minus strand 3* end, as in
H4-AGP(0)24/55, or in the plus strand 3* end, as in H4AGP(/)24/55, the results are identical. Restoring now the
complete H4 RNA end complementarity by introducing
the substitution in both 3* ends [H4-AGP(0/ /)24/55]
does not restore high replication efficiency. On the contrary, the double substitution has a more pronounced
negative effect on replication. Figure 2 presents the results obtained by detecting the minus-strand RNAs. This
could, at first, appear inadequate in the cases where
modifications are made at the minus-strand 3* ends and
therefore are expected to reflect on the synthesis of the
plus-strand RNA. In practice, this possible bias is minimized by the multiple rounds of replication which occur
in the system. As a consequence, the probing of the
experiment in Fig. 2 with a negative polarity probe gives
similar results (not shown).The negative polarity probe,
however, can detect, in cases of poor replication, a signal
due to the T7 RNA transcript. This is the reason for the
preferential use of the positive polarity probe.
At first glance, the results of Fig. 2 do not support the
postulate that RNA end complementarity represents a
determinant factor of replication efficiency. However, the
substitutions may have disrupted the integrity of the H4
RNA promoters, so that the effect of complementarity is
now counterbalanced by the creation of weaker promoters. New derivatives are therefore constructed, with substitution of the H4 RNA promoter with noninterrupted
sequences coming from the E307 promoter. Since the
extent of the template 3* end RNA sequence necessary
to promote replication is yet not defined, nucleotides 1
to 57 or 1 to 98 are substituted at once (Fig. 3). The
former region just exceeds the plus leader template region, while the latter extends past the putative BB3 box
proposed, on theoretical ground, to be part of the Paramyxovirus promoter (Crowley et al., 1988; Blumberg et
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FIG. 3. Generation of further 3*/5* end complementarity by transfer of continuous GP sequences into AGP in the context of H4 RNA. The experiment
was carried out exactly as in Fig. 2, with the 3* end region substitutions pictured in the middle and the extent of possible 3*/5* end complementarity
drawn on the right end side. The derivatives H4-AGP(0)57 and 120 are taken from Calain and Roux (1995), where they were referred to as,
respectively, AGP57 and 120. Arrow as in Fig. 2.
al., 1991). Ninety-eight nucleotides also correspond
roughly to the shortest inverted repeat ever described
for a Paramyxovirus copy-back RNA (Enami et al., 1989;
Calain et al., 1992; Sidhu et al., 1994) and may thus correspond to the minimal Paramyxovirus promoter length. As
expected from results obtained previously with similar
RNA constructs (in particular H4 AGP65, Calain and
Roux, 1995), RNA derivative H4-AGP(0)57, with the minus-strand 3* end first 57 nucleotides replaced with the
corresponding E307 nucleotides, exhibits a decreased
replication efficiency (7- to 10-fold compared to the H4
RNA parent, top Fig. 3), which coincides with a decrease
in possible base pairing potential (continuous 110 down
to discontinuous 12 plus 53). Again, restoring the 110
parental base pairing by introducing the substitution in
both the minus and the plus strands [derivatives H4AGP(0/ /)57] does not restore parental replication efficiency. Identical results are obtained with the longer substitutions. When disruption of H4 end complementarity
is achieved by a first 120-nucleotide substitution [H4AGP(0)120], resulting in a more than 10-fold decrease in
replication, a second substitution [H4-AGP(0)120/(/)98],
restoring continuous possible end complementarity over
98 nucleotides, does not restore high replication. Similar
results are obtained with the corresponding derivatives
of E307 RNA. In this case it is enough to replace the
plus-strand 3* end with 57 or 98 nucleotides from the
minus-strand 3* end to create a potential base paring
over 57 or 98 nucleotides (E307-AGP57, E307-AGP98).
E307-AGP98, for instance, with a 98-nucleotide base
pairing potential does not replicate better than E307 (not
shown). In summary, even when care is taken to create
The observation made in the above experiments rather
favors a direct effect of the primary sequence on replication efficiency. Therefore, further substitutions of the H4
RNA minus-strand 3* end with the corresponding E307
RNA sequence were made in attempts to identify the
minimal region carrying the high replication property exhibited by the H4 RNA. In Fig. 2, H4-AGP(0)24/55, with
its nucleotides 24 to 55 replaced by the corresponding
E307 RNA nucleotides, exhibited a 10-fold decreased
replication. This suggested that nucleotides 24 to 55 of
the H4 minus-strand 3* end contains a sequence that
confers high replication. In an attempt to more precisely
delineate this sequence, the extent of the substitution
was first gradually trimmed from nucleotide 55 toward
nucleotide 24 to generate derivatives H4-AGP(0)51, H4AGP(0)48, H4-AGP(0)42 and H4-AGP(0)31 (Fig. 4).
Down to the 24 to 31 substitution, all the derivatives
exhibit a reduced replication efficiency, showing that it
is sufficient to replace the H4 minus-strand 24 –31 nucleotides to lose high replication efficiency. Next, the border
of the substitution was analyzed from nucleotide 24 toward nucleotide 55 (Fig. 5). The first derivative produced
with substitution extending from nucleotide 31 to 55 [H4AGP(0)31/55] exhibits an almost full parental replication
efficiency (Fig. 5). Therefore, if nucleotides 1 to 24 of
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a potential base pairing with an uninterrupted promoter
sequence, we are unable, in this system, to find support
for the notion that the extent of terminal complementarity
affects replication efficiency.
Assessing the high replication property of H4
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FIG. 4. Analysis of the right end side limit of the AGP sequence required to confer high replication. The experiment was carried out as in Fig. 2,
with the H4 derivatives pictured on the right end side. Only the H4 minus-strand 3* end region has been substituted by the E307 corresponding
sequence, and, in consequence, only the H4 minus-strand 3* end is schematically presented. Arrow as in Fig. 2.
the H4 minus-strand 3* end region appear insufficient
to confer high replication, the extension of these end
nucleotides to nucleotide 31 is sufficient. These results
agree well with those obtained before, when nucleotides
1 to 33 of the H4 minus-strand 3* end region, substituting
for the corresponding nucleotides in E307 RNA, were
found sufficient to increase the E307 RNA replication up
to the level of H4-RNA (E307-GP33, Calain and Roux,
1995). In that series of experiments a substitution of 1 to
30 (E307-GP30) had an intermediate effect, close to the
FIG. 5. Left end side limit of the AGP sequence required to confer high replication. As in Fig. 4, but this time H4 derivative AGP(0)31/55 is
analyzed in parallel with two derivatives for which the left end border of the substitution of the E307 corresponding sequence is at position 24. The
band in lane H4-AGP(0)24/51 L-, marked by a black dot, is explained in the text under Results. Arrow as in Fig. 2.
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one exhibited here by H4-AGP(0)31/55. In conclusion,
high replication is attached to the H4 minus-strand 3*
end sequence extending from nucleotide 1 to nucleotide
31, regardless of the sequence downstream.
In Fig. 5, an RNA band is visible in lane 0L of derivative
H4-AGP(0)24/51 (marked by a dot). This band (also visible in the /L lane above the major signal), which migrates at an intermediate position between the H4 and
the E307 RNAs, is likely to result from a nonspecific
cross-reactivity with the 5* ex probe. This parasite signal,
which appeared unexpectedly, is eliminated by shortening the 5* ex probe (not shown, see Materials and
Methods).
169
In the infected/transfected cell system that we use, the
copy-back H4 RNA replication property is compared to
that of the internal deletion E307 RNA, and this latter
replicates 20-fold less efficiently (Calain and Roux, 1995).
As the two RNAs share common plus-strand 3* ends, but
differ in their minus-strand 3* end, it was reasonable to
assume that different replication efficiencies would result
from the different minus-strand 3* end sequences. It is
on that basis that the reciprocal replacement approach
has been used, assuming, moreover, that there are interchangeable blocks of sequence with different properties.
This obviously may have oversimplified the problem. For
instance, the displacement of a putative block with a
certain property may create a new environment (borders)
that can modify the box property. Despite this possible
drawback, the reciprocal exchange approach was validated by the results obtained with a series of derivatives
for which the replication properties correlated directly to
the extent of each other promoter sequence (Calain and
Roux, 1995). Moreover, this approach allowed us to test
a possible countereffect of transcription over replication
by the production of E307 RNA derivatives that exhibited
an equally high replication property, independent of their
transcription ability. These results failed to support a major effect of transcription on replication in this system
(Calain and Roux, 1995).
Encouraged by these previous results, we used this
same approach to further define the feature which confers high replication on the H4 RNA. As this RNA contains, on both the minus and the plus strands, the 3*
end sequence of the viral antigenome, the experiments
presented here characterize, in essence, the replication
promoter present on the antigenome. Moreover, for the
H4 RNA, the partial replacement approach has the inherent consequence of partially disrupting the H4 RNA copyback feature. The study of the RNA derivatives produced
to investigate the consequence of this change clearly
shows that the extent of the terminal base pairing potential has no consistent effect on replication efficiency. The
derivatives presented in Fig. 3 are particularly relevant.
The restoration of complete (110 nt) or almost complete
(98 nt) H4 base pairing potential in H4-AGP(0/ /)57 and
H4-AGP(0)120/(/)98 RNAs does not increase their replication above the level of their respective parents, H4AGP(0)57 and H4-AGP(0)120. These had lost replication
efficiency after a putative disruption of their base pairing
potential. Also, a disruption of only 7 nucleotides of the
base pairing potential has a 20-fold down effect on replication [see H4-AGP(0)24/31, Fig. 4], while a disruption
of 24 nucleotides has virtually no effect [see H4AGP(0)31/55, Fig. 5]. In the case of SeV, therefore, the
extent of 3*/5* end complementarity per se does not represent a factor that can influence replication efficiency.
The data obtained with the various derivatives can be
better interpreted as a direct role of the promoter primary
sequence. The new derivatives addressing this question
(Figs. 4 and 5) identify the first 31 nucleotides present at
the antigenome 3* end as an important determinant for
the high replication property. Indeed, the derivatives containing various lengths of genome promoter sequence
but containing only the first 24 nucleotides of the antigenome promoter (Figs. 4 and 5) replicate poorly. Only
H4-AGP(0)31/55, which has acquired the first 31 nucleotides of the antigenome, recovers high replication (Fig.
5). The question can be raised as to whether the 7 nucleotides (24 –31) of the antigenome promoter are sufficient.
In fact, when these 7 nucleotides are introduced at the
same position in the genome promoter of E307, no increase in replication is observed (E307 GP24/53, Cadd
et al., 1996), arguing that the signal for high replication
is not confined to these 7 nucleotides, but extends in the
1 –24 nucleotides toward the 3* end. Since high replication depends on the presence of the 31 nucleotides at
both the minus- and the plus-strand 3* ends, this creates
the potential for a terminal complementarity over 31 nucleotides. In this respect, it is impossible to formally exclude a participation of this potential complementarity in
the high replication property. Were this the case, however, then the effective complementarity could only be
created by base pairing of nucleotides 1 –31 of the antigenome. Therefore, at the least, terminal complementarity per se would not be enough to confer high replication,
since this would have to be generated by a specific sequence.
The identification of the 1 –31 nucleotide determinant
does not exclude the presence of other features governing replication efficiency. In fact, in the absence of these
3* end 31 nucleotides, i.e, when they are replaced by
nucleotides coming from the viral genome 3* end, a second region of the antigenome 3* end, extending from
nucleotides 48 to 65, or better 48 to 98, also exerts a
positive effect on replication (not shown). This effect,
however, is minor (2- to 3-fold) compared to that achieved
by the first 31 nucleotides (10- to 50-fold) and is only
apparent when the major effect is not present (not
shown). All these observations now agree well with those
made before with substitutions extending linearly form
the 3* ends for different lengths (Calain and Roux, 1995).
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They also support the conclusion that in the case of SeV,
and maybe in the case of all the Paramyxoviruses, the
promoter regions involved in replication may be longer
than the leader template regions (BB3 box, Crowley et
al., 1988; Blumberg et al., 1991). From a series of experiments where the genomic promoter, this time, has been
under scrutiny through various insertions, a similar conclusion has been drawn: the replication promoter appears to extend past the le/ template region and may
be discontinuous in nature (Pelet et al., submitted for
publication).
The identification of the 1 –31 nucleotide determinant
does not explain the mechanism controlling replication
efficiency. This could apply on the template, if the 31
first nucleotides of the promoter contain a higher-affinity
binding site for the RNA polymerase. Alternatively, the
encapsidation of the 5* end nascent strand could be
more efficient, allowing the RNA polymerase, which
would bind equally to the genome and the antigenome,
to proceed more efficiently toward productive replication.
Attempts to clarify this mechanism by measuring the ratio
of leader RNAs to their final replication products using
representative RNA derivatives are under way. The present results can be interpreted by attributing a positive
effect on replication efficiency to the identified nucleotides. There is, however, no way to tell whether one deals
with a direct positive effect or whether these nucleotides
are the sites where a negative control cannot apply. In
fact, a recent study by Cadd and co-workers (1996), as
well as unpublished experiments, would tend to favor
this latter interpretation, since conditions can be found
where the replication of most of the poorly replicating
RNAs is improved in assays where the C protein is not
expressed. This suggests that the replication of these
RNAs is under a negative control that can be relieved by
the absence of C. Further work is under way to more
clearly understand the role of C. In regard to the derivatives presented in this paper, however, we stress that
the absence of C does not change the conclusions. For
example, if the absence of C results in a significant increase in H4-AGPP(0/ /)57 replication, this, however, is
paralleled by a similar increase in H4-AGP(0)57 replication under the same conditions, so that the restoration of
the end complementarity has still no effect on replication
efficiency (not shown).
In the end, our results diverge from those obtained
previously with a VSV derivative le47 (analogous to H42AGP57) for which the terminal complementarity had a
positive effect on replication (Wertz et al., 1994). This
result was interpreted as evidence that the nucleocapsid
ends communicate to promote efficient replication. Such
a control mechanism is believed to apply in the case of
the influenza viruses for which partial 3*/5* end complementarity of the segmented RNAs is described (Robertson, 1979; Desselberger et al., 1980) and proposed to
operate in transcription control (Fodor et al., 1994). Partial
3*/5* RNA end complementarity has been identified as
well for the Arenaviruses and the Bunyaviruses over, respectively, about 80 and 20 –30 nucleotides. In all these
cases, evidence for actual base pairing (Raju and Kolakofsky, 1989) or for the formation of circles has been
readily observed in a large proportion of nucleocapsids
(Pettersson and von Bonsdorff, 1975; Samso et al., 1975;
Obijeski et al., 1976; Palmer et al., 1977; Young and Howard, 1983; Bishop and Auperin, 1987). Although the extent
of the H4 RNA base pairing potential is larger than that
in any of the cases mentioned above, H4 circular nucleocapsids have been detected with an exceedingly low
probability estimated to be 1 in 10,000 (Kolakofsky and
Boy de la Tour, unpublished). Whether the results obtained here represent another indication that the nucleocapsid ends do not interact in the cases of the nonsegmented negative-stranded RNA viruses or whether a putative RNA termini interaction takes place via a
mechanism that does not involve termini base pairing
but rather protein interactions, as suggested more recently by the results of Baudin and colleagues (1994) for
influenza virus, is open to question. The answer to this
question is likely to come from further studies involving
other nonsegmented negative-stranded RNA viruses,
such as rabies, measles, and respiratory syncytial viruses, for which reverse genetics now allows this type
of analysis (Conzelmann and Schnell, 1994; Garcin et al.,
1995; Lawson et al., 1995; Radecke et al., 1995; Whelan
et al., 1995).
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ACKNOWLEDGMENTS
The authors are indebted to Daniel Kolakofsky for extensive advice,
discussions, and criticism and to Philippe Calain for his advice and
encouragement to C.T. This work was supported by a grant from the
Swiss National Foundation for Scientific Research and by supply money
from the de Reuter, Sandoz, and Ciba Geigy Foundations.
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