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

Abstract

The paramyxovirus genome, a nonsegmented, negative-polarity, single-stranded RNA of approximately 15 kb, contains six transcription units flanked at the 3' and 5' ends by a short (approximately 50- to 60-nucleotide) extracistronic sequence, dubbed the positive and negative leader regions. These leader template regions, present at the 3' end of the genome and the antigenome, have been shown to contain essential signals governing RNA replication activity. Whether they are sufficient to promote replication is still open to question. By using a series of Sendai virus defective interfering RNAs carrying a nested set of deletions in the promoter regions, it is shown here that for both the genomic and antigenomic promoters, a 3'-end RNA sequence of 96 nucleotides is required to allow replication. Sequence comparison of active and inactive promoters led to the identification of a set of three nucleotide hexamers (nucleotides 79 to 84, 85 to 90, and 91 to 96) containing a repeated motif RXXYXX [shown as 5'-3' positive-strand]. Sequential mutation of each hexamer into its complementary sequence confirmed their essential role. The three hexamers are required, and their relative positioning is important, since displacing them by 6 nucleotides destroyed promoter function. RNAs carrying degenerate nucleotides in the three hexamers were used as replication templates. They led to the selection of actively replicating RNA species exclusively carrying the basic motif (GNNNNN)3 from nucleotides 79 to 96. These results clearly show that, apart from the region from nucleotides 1 to 31, previously identified as governing Sendai virus replication activity, a second element, spanning at the most nucleotides 79 to 96, appears essential. Thus, the paramyxovirus replication promoters are not confined to the leader template regions, as seems to be the case for the rhabdoviruses.

FIG. 1

SeV RNAs and template plasmid structure. (A) The SeV genome (negative-strand RNA of 15,384 nt) is presented 3′ to 5′ as a grey rod, with the main transcription units indicated (not to scale). At each end, le⁺ and le⁻ refer to the extracistronic regions, with the promoter for antigenome synthesis indicated (GP). The DraIII site, central to this study, is shown. Above, the transcription products include the le⁺ RNA and the six mRNAs, whose synthesis sequentially proceeds from the same GP region. Below, the antigenome is schematized exactly complementary to the full-length genome, with the AGP at its 3′ end. (B) The structure of the internal deletion DI RNA E307 is shown as negative and positive strands to emphasize the remnant GP and AGP promoter sequences, as in the SeV genome. A deletion from nt 607 to 14197 results in the fusion of the N and L genes, so that transcription produces on top of the le⁺ RNA a fused N/L mRNA. The E307 sequence is used in this study only as the donor of GP sequences. (C) The copy-back DI-H4 RNA structure emphasizes the presence, at the negative- strand 3′ end, of the same promoter sequence region (110 nt, nt 15275 to 15384) as that of the positive-strand 3′ end. Both strands contain in this way the same promoter, named AGP^L and AGP^R, respectively. No mRNA is transcribed from DI RNA. The box in the middle of the sequence scheme is there to specify the backbone sequence of the viral RNA, i.e., L sequence only for H4, N and L sequences for E307 (see panel B). Numbering for panels B and C corresponds to that of the full-length genome in panel A. (D) The H4 sequence is shown in the pSP65 plasmid, where it has been cloned along with the T7 promoter sequence. T7 transcription, which starts at the exact viral RNA 5′ end, produces an exact positive-strand DI RNA through endolytic cleavage at the 3′ end by the hepatitis delta virus ribozyme. Relevant restriction sites and positions are indicated. The numbering has been adjusted here to the H4 sequence inserted in plasmid pSP65.

FIG. 2

Primers. The primers (RT and PCR), used for the RT-PCR amplification of a fragment covering the AGP^R region of DI RNA derivatives in verification of the selection experiment results (see Fig. 7 and 8), are shown below the schematized H4 sequence, along with the IRD primer used for the sequencing. Below the H4-(+)ΔDra/AGP96 schematized sequence, the series of oligonucleotides used to construct the plasmids containing the degenerate nucleotides are outlined, with Rib3 commonly used. Numbering refers to positions in the pSV-DI-H4 plasmid (Fig. 1D).

FIG. 3

Minimal genomic promoter length. The H4-ΔDra/GP series (obtained as described in Materials and Methods) in which various GP lengths have been fused to the DraIII site, replacing the original AGP^R sequence, are shown with (from top to bottom) decreasing lengths of the GP sequence. The promoter on the 3′ end of the H4 negative strand (AGP^L) is the same for all the derivatives. H4-AGP(+)57 is particular in that the GP 57 nt exactly replaces the AGP^R 57 nt, introducing an le⁺ template region in place of le⁻ one. E307 RNA is shown for general DI RNA structure information and because it is the original source of the GP sequences. To the right-hand side, a Northern blot analysis of the encapsidated viral RNAs recovered from in vivo replication assays (see Materials and Methods) primed with the RNAs described to the left is shown. The P + or − lanes refer, respectively, to fully competent replicating assays and to assays done in the absence of the P protein as negative controls. The RNA probe (5′ ex) (44) is of positive polarity to score the RNA species complementary to the T7 RNA transcript.

FIG. 4

Minimal AGP length. (A) As in Fig. 3, except that the proper AGP^R region of DI-H4 RNA has been increasingly truncated from the DraIII site position toward the plus-strand 3′ end, leaving the number of nucleotides indicated in the denomination of the derivatives (102, 96, and 90 nt). The numbers to the right refer to the total number of nucleotides in the RNA. (B) Northern blot as in Fig. 3, with the RNA derivatives with AGP truncations shown in panel A.

FIG. 5

Importance of the postulated motif in hexamers 14, 15, and 16. (A) SeV AGP and GP are aligned (as positive-strand DNA from 5′ to 3′) in groups of 6 nt (hexamers), to emphasise a sequence similarity repeated in the three hexamers 14, 15, and 16. The four sequences presented below the AGP and GP sequences originate from modified versions of GP, which led to successful (competent Yes) or unsuccessful (competent No) replication (reference and unpublished data). The postulated motif is the nucleotide sequence predicted to be required for competent replication in the three hexamers. (B) H4(+)ΔDra/AGP96 derivatives carrying, in AGP^R, substitutions introduced at positions marked with an asterisk. Every mutation consists of the replacement of a nucleotide(s) by its (their) complement(s). Yes and No refer to the ability of these RNA derivatives to be replicated, as shown in panel C. (C) Northern blot analysis of the encapsidated RNAs described in panel B recovered from replication assays as described in the legend to Fig. 3.

FIG. 6

The three hexamers are required for competent replication. (A) H4(+)ΔDra/AGP96 derivatives carrying, in AGP^R, deletions of one hexamer as indicated. (B) Outline of the relevant sequence in the derivatives schematically presented in panel A. Note that in derivatives 2 and 3, the sequence in hexamer 14 is different. In derivative 4, the three hexamers are present but displaced toward the end (left) by 6 nt. (C) Replication ability of the RNAs presented in panels A and B measured by Northern blot analysis, as in Fig. 3C. For derivative 1, the result is shown in triplicate.

FIG. 7

Selection applied to the first nucleotide of each of the three hexamers. Three oligonucleotides were created to contain two degenerate positions; one common to all three at position 73, and the other at position 79, 85, or 91, respectively, corresponding to the first position of each of hexamers 14, 15, and 16 (see the outlined sequence in panel B). PCR products, obtained with each of these three degenerate oligonucleotides with, as the other amplimer, the Rib3 primer (Fig. 2), were cloned into the DraIII and BamHI sites of H4(+)ΔDra/AGP96 to prepare the Selec 73/79, Selec 73/85, and Selec 73/91 plasmids with modified AGP^R sequences. Pooled plasmid preparations were obtained from bacterial colonies scraped from a large LA petri dish (diameter, 13 cm) plated with 9/10 of a transformation reaction mixture; the remaining 1/10 was plated to pick individual colonies, allowing the sequencing of individual species (10 to 15 clones analyzed) to characterize the starting pool of plasmids (Pre distribution in panel C). After replication, using as the template plasmid each of the three pools, an RT-PCR fragment covering the AGP^R region was obtained and cloned (see Materials and Methods) and 10 to 15 individual clones were sequenced to characterize the nucleotide (Post) present at the original degenerate position. (A) Schematized representation of the RNAs, with the degenerate positions indicated by N. (B) Northern blot analysis of the replicated encapsidated RNAs (as in Fig. 3), shown in duplicate for H4(+)ΔDra/AGP96, Selec 73/79, and Selec 73/85. (C) Nucleotide distribution at the positions of interest. The number accompanying the nucleotide designation refers to the number of clones containing this base at the indicated position. Pre, nucleotide distribution in the original plasmid preparation; Post, nucleotide distribution in the RT-PCR products after replication.

FIG. 8

Selection applied to the 6 nt of each of the three hexamers. Three oligonucleotides were designed to contain six degenerate positions corresponding to hexamers 14, 15, and 16. Pooled plasmid preparations were obtained, as in Fig. 7. This time, Pre sequence characterization was done by sequencing the pooled plasmid preparation. After replication, RT-PCR products were obtained as in Fig. 7, and Post sequence characterization was done by directly sequencing the RT-PCR products of the replicated RNA. At the sides of each sequence panel, the Pre and Post sequences are outlined. At the bottom is shown the AGP^R sequence from 5′ to 3′ (a) and the sequence in the sequencing gel (complement of that in a) (b).

FIG. 9

Prevalence of (GNNNNN)₃ among paramyxoviruses. The GP and AGP sequences (as positive-strand DNA from 5′ to 3′) of nine paramyxoviruses are aligned to emphasize the sequence similarity in the three hexamers under focus here. SeV, BPIV3 (bovine parainfluenza type 3), and HPIV3 (human parainfluenza type 3) are closely related in the SeV subgroup. MEAS (measles virus), RIND (rinderpest virus), and DOLPHIN belong to the subgroup of the morbilliviruses. MUMPS, SV41, and SV5 (simian virus 41 and 5) belong to the subgroup of the rubulaviruses. The AGP sequence of the dolphin morbillivirus is not available.

FIG. 10

Frequency of the presence of each nucleotide in the first hexamer position. The frequency with which a particular nucleotide is found in the first hexamer position is plotted as a function of the hexamer number in the GP and AGP of the SeV and morbillivirus subgroups (Fig. 9), which are known to contain a viral RNA with the total number of nucleotides being a 6n + 0 number.