In conclusion, in cases where the N phase context of the promoter was conserved, the 3′-OH congruence of the template was not critical for replication. On the other hand, when the N phase context was changed so as to obliterate replication, the 3′-OH congruence could not restore replication. Therefore, it appears that the SeV RNAp is likely to be sensitive to the nucleotide N phase context. The observance of the rule of six, then, must depend on the recognition of certain nucleotides properly positioned, in the promoter region, with regard to the N interaction points (pockets).
The first 12 nucleotides of the promoter, conserved in GP and AGP, are obvious candidates for recognition in the N phase context, although it is not yet clear whether every nucleotide has a significant role. A nucleotide addition at position 13, shifting the phase of nucleotides 1 to 12 by 1, was shown to abolish expression of a reporter gene, possibly by preventing replication of the minireplicon (22
). Other phase shifts were accompanied by a nucleotide deletion, making the interpretation of the results difficult. For VSV and RSV (48
; reference 62
and references therein), detailed studies have come to the conclusion that not all the nucleotides in the promoter sequence are relevant for replication. Whether these results apply to SeV (and the Paramyxoviridae
family) is open to question, since RSV and VSV do not conform to the rule of six (or to the rule of any number [52
]), a fact that may indicate that the nucleotides are not seen in the context of N (see below). This assertion, however, is counterbalanced by the finding that the chemical probe reactivities of the bases in the VSV nucleocapsid are not uniform. Some are protected and others are hyperreactive, implying a discriminate association of certain nucleotides with N (29
The C residues in the repeated motif C1
NNNNN (or the NNNNC5
residues of the corresponding Rubulavirus
SV5 motif [42
]), on the other hand, are likely to constitute such signals. Indeed, the appearance of these motifs three times in the same context speaks for the importance of the phase in their recognition by the viral RNAp (vRNAp). The start-stop transcription sequence motifs, as well as the editing sites (D. Kolakofsky, personal communication), have been proposed to represent other examples of such signals, on the basis of their nonrandom positioning with regard to the phase context (33
). Recently, a recombinant measles virus (a Paramyxovirus of the Morbillivirus
genus) series with modifications resulting in shifting of these regions into all possible phases exhibited an abnormal pattern of readthrough at the M/F and F/H gene boundaries (1
). Similarly, all mutants showed slightly altered P transcript editing behavior, suggesting that the nucleotides participating in these signals might be recognized differently depending on the N phase context. The difference between the editing patterns of constructs #5 and #3 (no +2 or +3 addition in #3 [Fig. C]) may also add to the argument, since the Swap editing sequence is not in a similar N phase context in the two constructs (N6
for constructs #5 and #3, respectively).
In the absence of precise structural information on the N-RNA complex, one is reduced to mere speculations as to how the signal recognition operates. The nucleotide signal could be properly accessible to the vRNAp only when in the proper N pocket. Alternatively, the nucleotide could induce a conformational change in the N protein only when in a defined pocket. This second possibility is particularly attractive for the (C1
motif, since the C's in phase context N1
appear to signal independently of the primary sequence environment (N2
). If it is not repeated, the signal must obviously be formed by more than one nucleotide, since the probability is increased that any nucleotide is going to be presented many times in each N phase context. If the signal is made by a local conformational change in the nucleocapsid, this change should propagate through more than one N subunit. The picture could be more complicated, considering that the signal recognition mechanism might have to be flexible, if, for instance, it is confirmed that the N phase context is relevant for transcription. Indeed, the transcriptional start-stop signals remain ignored during replication. The vRNAp could be made insensitive to the signals, subsequent to a structural modification (phosphorylation or conformational change induced by encapsidation of the nascent RNA chain or by interaction with a viral cofactor, etc.). Alternatively, the signal could be masked to the vRNAp by a modification of the N-RNA complex itself, although a biochemical or conformational difference between the transcription and replication templates has not been considered so far. At early times of infection, the nucleocapsids could conform better to transcription. Later, following interaction with a viral partner that accumulates with time, or following a slow chemical modification, they could conform better to replication. The C proteins, shown to modulate the selectivity of the vRNAp for the replication signals, may in fact act by modifying the template. Indeed, the different C protein-to-template ratios required to inhibit replication at AGP or at GP—inhibition seen here as the price of selectivity—argue for a stoichiometric rather than an enzymatic role of the C's (57
). It is noteworthy in this context that the polR
mutants of VSV, producing more leader-N junction readthrough transcripts, harbor a mutation in N (6
). This is compatible with the idea that the modification of the template could be responsible for the lower stringency with which the signal is observed. Finally, nucleocapsid subpopulations with different sensitivities to RNase or harboring a cleaved N have been reported (5
The ability to add a sequence extension at the 3′-OH end of the T7 RNA template without losing the ability to replicate is surprising. Similar extensions are not tolerated for VSV and RSV, in contrast to 5′-end extensions (guanosine residues generally added to improve T7 RNA polymerase activity) (9
; D. Garcin, unpublished data). Whether the discrepancy between SeV and VSV or RSV reflects a significant difference in the template structure or in the template recognition mechanism is open to question. The vRNAp could recognize the nucleocapsid 3′-OH end as such, regardless of the RNA sequence, or regardless of 3′-OH congruence. After binding, the vRNAp would gain access to the nucleotides and scan the template in search of the promoter sequence to start RNA synthesis. Evidence for forward, and even backward, scanning by negative-stranded RNA virus polymerases has been provided (18
). Alternatively, the vRNAp could bind internally to the promoter region, a mechanism that implies a vRNAp capable of nucleotide sequence discrimination before binding. We propose that, when the rule of six applies, the phase context provides the vRNAp with all the information needed to bind the template at the promoter and to initiate replication, without the need for 3′-OH-end recognition. The 3′-OH-end recognition would then be the prerogative of the templates (VSV and RSV) lacking the phase context conditions and thus requiring the information given by the 3′-OH end. The question of access to internal replication promoters, although it is academic considering that the replication promoters are naturally present at the nucleocapsid 3′-OH end, may still be of interest for understanding of the general manner in which the vRNAp operates. The achievement of the present work, namely, the use of double promoter constructs, may lead to the design of more-appropriate experiments to contribute to the understanding of transcription initiation or of the mechanism of defective interfering RNA generation.
The conditions used to perform these experiments, i.e., the absence of the C proteins, may raise concerns about the validity of the results, since the C proteins are known to influence SeV RNAp activity by increasing selectivity (57
). However, even if the absence of C tones down the stringency of the rule of six, the rule still applies (see Fig. B). In the absence of C, observations not possible in the presence of C, where constructs of 6n
≠ 0) nucleotides do not replicate to detectable levels, can now be made. Extending sequences past the promoter (Fig. ) and adding two promoters in tandem (Fig. ) represent other artificial conditions. These clearly impose more penalties on the replicating system, so that a distinction can now be perceived between 3′-OH congruence and a change in the N phase context. This distinction was, for instance, not possible in Fig. , where the constructs still replicated efficiently. Thus, the application of both sets of conditions appears to widen the window of observation so that more recognition steps can be identified.