Scanning mutagenesis identifies critical residues in the rinderpest virus genome promoter

Valerie Miouletb,1, Thomas Barrett1 and Michael D. Baron1

Division of Molecular Biology, Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, GU24 0NF, UK1

Author for correspondence: Michael Baron. Fax +44 1483 232 448. e-mail michael.baron{at}bbsrc.ac.uk


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Short regions at the 3' and 5' ends of the genome of Rinderpest virus (RPV) contain signals that regulate transcription of the viral genome, known as the genome promoter and the (complement to the) antigenome promoter, respectively. An RPV minigenome construct carrying the CAT coding sequence was used as a reporter to investigate residues in the 3'-terminal region of the genome important for these functions. Single-base scanning mutagenesis showed that modifications to nucleotides 1, 3, 4, 10 and 19 of the RPV leader had an extremely inhibitory effect on transcription and/or encapsidation of the minigenome, with CAT expression reduced to 0–10% of control values. Changes in any of the other first 22 nucleotides reduced the efficiency of the minigenome to 20–80% of the wild-type control, with the exception of nucleotides 16, 17 and 20, where mutations did not affect CAT expression significantly. Mutagenesis in blocks identified critical residues in positions 23–26, but changes to leader residues 27–48 had no major effect on CAT expression. A region of about 16 nucleotides (49–65) located around the start of the nucleocapsid gene, including the intergenic triplet CTT, was identified as essential for minigenome function. Mutations further into the nucleocapsid gene (nt 66–89) had a moderate effect (CAT activity 20–60% of control), while at least one critical residue was found in positions 93–96. The importance of four highly conserved G residues at positions 79, 85, 91 and 97 was also investigated. G79 was found to be optimal, though not critical, while a purine was required at 85 and 91. Although G97 is conserved in morbilliviruses, all bases were equally effective at this position.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Rinderpest is one of the oldest known diseases of domestic livestock and it can affect all species of the Order Artiodactyla (even-toed ungulates). The causative agent, Rinderpest virus (RPV), is a member of the genus Morbillivirus of the family Paramyxoviridae. It is closely related to Measles virus, Canine distemper virus and Peste des petits ruminants virus. Like all the paramyxoviruses, RPV is a non-segmented, negative-sense, single-stranded RNA virus. Viral RNA synthesis requires the viral nucleocapsid (N), phospho- (P) and large (L) proteins as well as an encapsidated viral RNA template (reviewed in Moyer & Horikami, 1991 ). The RPV genome is 15882 nt long (Baron & Barrett, 1995b ); short regions (approximately 100 nt) at each end of the genome contain all the signals necessary for transcription and replication (Baron & Barrett, 1997 ) and therefore the promoters for the virus polymerase must be located in these regions. Transcription of viral mRNA, and of the antigenome template from which new genomes will be transcribed, is initiated at the 3' end of the genome-sense RNA. The first 55 nucleotides transcribed form the ‘leader’ sequence, followed by the start of the N gene transcript (N mRNA) (Baron & Barrett, 1995b ). After the last of the viral genes, there are 37 nucleotides of ‘trailer’, believed to be the promoter for genome RNA synthesis (antigenome promoter). In the morbilliviruses, only the last 15–18 bases of the trailer show strong sequence similarity to the 3' end of the genome RNA, although another region of similarity has been identified, 80–90 bases from each 3' end (Crowley et al., 1988 ; Blumberg et al., 1991 ), which may also have a role in regulating or promoting transcription. We have used scanning mutagenesis to determine the critical residues in the genome promoter of RPV, by using a minigenome construct carrying a CAT reporter gene in place of the normal virus protein-coding regions (Baron & Barrett, 1997 ).


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Cells and virus.
Cells of a human embryonic kidney cell line, HTK293, were used for the transfection experiments. Cells were grown in DMEM containing 10% foetal calf serum (FCS), penicillin (200 U/ml) and streptomycin (200 µg/ml).

RPV Saudi/81 (Taylor, 1986 ), a virulent strain of RPV used as a helper virus, was grown and titrated in B95a cells, a marmoset-derived lymphocyte cell line immortalized by Epstein–Barr virus transformation (Kobune et al., 1991 ). B95a cells were grown in RPMI–HEPES, containing 5% FCS, penicillin (200 U/ml) and streptomycin (200 µg/ml).

{blacksquare} DNA manipulations.
All DNA manipulations were carried out using standard methods. The basic minireplicon plasmid (pMDB8A) has been described previously (Baron & Barrett, 1997 ). Mutations were introduced into pMDB8A using the method described by Deng & Nickoloff (1992) or (in the case of mutants XVIII, G97->C and G97->T) using the QuickChange site-directed mutagenesis system (Stratagene), according to the supplier’s instructions. Except in the case of nucleotides 5, 79, 85, 91 and 97, mutations were always transitions, and their locations and the nomenclature used for each mutation are shown in Fig. 1. RNA was transcribed from plasmid DNA using the RiboMAX large-scale RNA production kit (Promega) following the manufacturer’s instructions. RNA was purified using a Qiagen RNeasy mini kit, again following the manufacturer’s instructions.



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Fig. 1. Mutations introduced into the RPV minigenome sequence. All sequences are shown in antigenome sense. Conserved residues are in white on black. A map of the functional region of plasmid pMDB8A is shown above the sequence alignment. The T7 promoter (T7) allowed transcription, by the T7 RNA polymerase, of an antigenome-sense RNA consisting of the RPV leader plus the start of the N gene (RPV 3' end), the coding sequence of the CAT protein (CAT ORF), followed by the end of the RPV L gene and the trailer sequence (RPV 5' end). Transcription was stopped by the two T7 terminators (T{Phi}), while the self-cleaving ribozyme sequence of the hepatitis delta virus ({delta}) ensured that the newly synthesized RNA was cleaved at the exact 3' end of the minigenomes. In the alignment of known leader sequences of morbilliviruses, the positions of the point mutations (arabic numerals) and block mutations (roman numerals) introduced in the leader and N gene start are indicated, as are the start of the N mRNA (N gene start) and the positions of the four conserved G residues. Sequences from the RBOK vaccine strain of RPV (RPV-R), the virulent Kabete ‘O’ RPV strain (RPV-K), from which vaccine was derived, RPV-Kuwait/82 strain (RPV-Kw; Taylor, 1986 ), measles virus Edmonston strain (MV) and canine distemper virus Onderstepoort strain (CDV) are compared.

 
{blacksquare} Transfections.
Transfections were performed on sub-confluent HTK293 cells in 25 mm wells (12-well plates). The cells were infected with RPV Saudi/81 at an m.o.i. of 1 for 2 h at 37 °C in a volume of 200 µl. Next, 0·5 ml DMEM (Life Technologies) containing 10% FCS, penicillin (200 U/ml) and streptomycin (200 µg/ml) was added to each well and incubation at 37 °C was continued for a further 1 h. The inoculum was removed and the cells were rinsed with 0·5 ml Optimem I (Life Technologies) and transfected with 2·5 µg in vitro-synthesized RNA using 5 µl Lipofectin (Life Technologies) in 0·5 ml Optimem I. After incubation overnight at 37 °C, cell monolayers were lysed in 150 µl NP40 lysis buffer [120 mM NaCl, 50 mM Tris–HCl, pH 8·0, 0·5% (v/v) NP40 (ICN Biomedicals Inc)]. The lysates were centrifuged at 13000 r.p.m. for 1 min in a microcentrifuge and the supernatants were transferred to new tubes. The cleared lysates were assayed for the presence of CAT protein.

{blacksquare} CAT assays.
An ELISA kit obtained from Boehringer Mannheim was used, following the manufacturer’s instructions, to determine how much CAT protein was synthesized in transfected cells. ELISAs were performed on 20 µl aliquots of cell lysate. Absorbances were read at 405 nm using a Labsystems Multiskan Plus. The data from all the assays were initially obtained as A405. They were subsequently expressed as percentages relative to the absorbance obtained for the minigenome of the unmutated plasmid pMDB8A (wild-type; referred to below as 8A). The calculation was carried out using the formula: percentage of wild-type=[A405(mutant+RPV)-A405(8A)]/[A405(8A+RPV)-A405(8A)]x100.

The background for these assays was the A405 from a lysate of uninfected cells that had been transfected with unmutated minigenome RNA transcribed from pMDB8A. This background value was not, in fact, different from the A405 of the zero point of the CAT standard curve, showing that the helper virus was absolutely required for CAT expression. In each case, the amount of CAT produced was estimated from three independent CAT assays performed on cell lysates from independent sets of transfections.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
The RPV minigenome plasmid pMDB8A (Baron & Barrett, 1997 ) was used in these experiments (see Fig. 1). In this minigenome, all the virus coding regions and intergenic regions have been replaced by the ORF of a single reporter protein, CAT. The plasmid contains a T7 RNA polymerase promoter, followed by the RPV (RBOK strain) leader and N gene start (residues 1–107 of the antigenome), the CAT ORF, the L gene end and trailer sequence (antigenome residues 15744–15882), the hepatitis delta virus ribozyme and a tandem repeat of T7 polymerase terminators. This allows transcription, by the T7 RNA polymerase, of an antigenome (positive-sense) RNA consisting of the RPV leader and N gene start, the CAT ORF and the RPV L gene end and trailer. Transcription by T7 polymerase starts at the exact 5' end of the RPV leader, while the self-cleaving ribozyme sequence of hepatitis delta virus ensures that the newly synthesized RNA was cleaved at the exact 3' end of the viral sequences. We have shown previously that RNAs transcribed in vitro from this minigenome, when transfected into 293 cells previously infected with helper RPV, resulted in the synthesis of CAT protein (Baron & Barrett, 1997 ). This requires the RNA to be encapsidated by the viral N protein and copied to genome-sense (replication) by the N, P and L proteins supplied by the helper virus and the CAT mRNA is then transcribed, also by P and L. The transfected RNA was not translated efficiently in the absence of helper virus, even though it is in the positive sense, as it is not capped.

For the present studies, mutations were introduced throughout the RPV leader and beyond it, into the start of the N gene (Fig. 1). Note that, for ease of comparison with other studies, the original sequence and all mutations are described in terms of the antigenome sequence, despite the fact that the functional promoter is, of course, the complement to that sequence. RNA was transcribed from each mutant in vitro and these RNAs were transfected into RPV-infected cells and the amount of CAT synthesized was measured after 24 h. Since no mutations were introduced into the antigenome promoter region, it is reasonable to assume that all the mutants were copied with the same efficiency into genome-sense RNA, once they were encapsidated. The levels of CAT produced in each case therefore depend on the combination of the efficiency of encapsidation of the minigenome by the N protein and of transcription from the modified genome promoter. Clearly, some of the transcription from the genome promoter will be of CAT mRNA and some will be of fresh antigenome-sense minireplicon, which can give rise to further genome-sense template, indirectly increasing the amount of CAT mRNA. Alterations to the production of antigenome could therefore also contribute to the overall level of CAT protein.

The relative efficiency of these processes in the mutants was determined by measuring the amount of CAT produced by each mutant minigenome RNA relative to that produced by the standard minigenome RNA. RNAs were transcribed in vitro as this allowed us to transfect the same amount of all mutant and control antigenomes in all studies. Although we have shown that minigenomes can be replicated when transcribed from a plasmid in transfected cells, using viral proteins expressed from co-transfected plasmids, the changes made to bases at the beginning of the leader, and therefore adjacent to the transcriptional start of the T7 RNA polymerase, had a significant effect on transcription efficiency (not shown), meaning that this procedure could not be used for these studies.

Effects of point mutations in nt 1–22
Conservation of sequence between the genome and antigenome promoters is limited to the first 15–18 bases in paramyxoviruses (Blumberg et al., 1991 ), although a longer region is conserved in the known leader sequences of morbilliviruses (Fig. 1). We elected to examine in detail the first 22 nucleotides of the leader region, with mutation of larger blocks of sequence for more distal regions (see below). HTK293 cells were transfected with mutant minigenome RNAs where each of these first 22 nucleotides had been mutated individually to create transitions. The results are shown in Fig. 2, and can be divided into three groups. Changes at nucleotide positions 16, 17 and 20 reduced the relative amounts of CAT only slightly compared to the unmutated minigenome RNA, and the differences were not statistically significant. Mutations at nucleotide positions 2, 5–9, 11–15, 18, 21 and 22 had a moderate effect, decreasing CAT production to between 23 (C2->U) and 61% (G21->A) of the control. Nucleotide positions 1, 3, 4, 10 and 19 were found to be critical for encapsidation or transcription, as transition mutations at these positions reduced CAT production dramatically to between 0 (A4->G) and 10% (A10->G).



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Fig. 2. Quantification of the effects on CAT production of point mutations in the first 22 nucleotides of the leader. Mutated minigenome RNAs were transfected into RPV-infected HTK293 cells. After incubation overnight, the cells were lysed and the cleared lysates assayed for the presence of CAT protein as described in Methods. CAT production relative to that of the control pMDB8A RNA is shown. Numbers on the horizontal axis refer to the nucleotide positions in the RPV-RBOK leader. Error bars indicate the standard errors for three independent experiments.

 
In addition to a transition (G->A), the fifth nucleotide was also changed to either a C or a T. These additional mutations were introduced because, in the known morbillivirus leader sequences, this nucleotide is always a purine and it is the only one of the first 10 nucleotides to differ between morbilliviruses, or even between strains of the same morbillivirus (see Fig. 1). All changes at this position were moderately inhibitory to CAT production in the RPV (RBOK strain) minigenome used; G5->A or G5->U were roughly equivalent, reducing the level of CAT to 49·5 and 44·8% of standard minigenome, respectively, while G5->C was slightly more inhibitory, reducing CAT to only 17·7% of the standard level (data not shown).

Effects of block mutations in nt 23–96
The remainder of the leader shows only limited sequence conservation (Fig. 1), apart from the CUU that marks the start of the N gene. This region was mutated in overlapping blocks of six nucleotides, all changes being transitions. This block mutagenesis was continued through the conserved N gene start up to position 96 in order to investigate the possibility that important replication signals exist outside the leader proper. In addition, it was of interest to see how much of the gene start sequence was important since, although this region is well conserved between the N genes of different morbilliviruses, it is not well conserved between different genes, the general pattern AGGR being all that is conserved (Baron & Barrett, 1995a ). Mutated RNAs were therefore prepared and transfected into RPV-infected cells as described above and CAT protein synthesis was measured. In the leader region, only block mutations I and VII had any effect on CAT synthesis, mutations II–VI having no effect or only slightly negative or positive effects (Fig. 3). Block mutation I is situated at the start of the less conserved area of the leader, but modified the last nucleotide of the highly conserved region at the start of the leader. It also modified two nucleotides (G24 and U26) that are invariant in known morbillivirus leader sequences. These modifications led to a reduction in CAT synthesis to 6·5% of wild-type. As expected, block mutations VIII, IX and X all effectively abolished CAT synthesis; these mutations cover the leader end and N gene start and would be expected to affect mRNA transcription. Interestingly, this critical region clearly extends beyond the first four residues of the gene, as block mutation XI also showed almost no CAT synthesis. Changes in this overall region reduced CAT levels to between 0·1 (block mutation IX) and 5·4% (block mutation VIII) of wild-type.



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Fig. 3. Quantification of the effects on CAT production of block mutations in leader positions 23–96. Mutated minigenome RNAs were transfected into RPV-infected HTK293 cells. After incubation overnight, the cells were lysed and the cleared lysates assayed for the presence of CAT protein. CAT production relative to that of the control pMDB8A RNA is shown. Roman numerals on the horizontal axis refer to the blocks of six nucleotides (see Fig. 1) that were mutated in the RPV-RBOK leader and in the start of the N gene sequence. Error bars indicate the standard errors for three independent experiments.

 
Block mutations XII to XVII (nt 67–92) each led to a moderate decrease in CAT production, with relative CAT levels varying from between 25·8% for block mutation XVI to 57·2% for block mutation XV. Notably, block mutation XVIII essentially abolished CAT synthesis. These mutations were all situated after the start of the N gene and before the AUG initiation codon for the N protein, in a poorly conserved region. However, some of them overlapped a series of conserved G residues at positions 79, 85, 91 and 97. The same positions are Gs in the genome sequence, and a similar group of G residues was shown to be important for replication of Sendai virus (SeV) (Tapparel et al., 1998 ). We therefore mutated these residues individually to determine their importance in RPV replication.

Effects of mutations at positions 79, 85, 91 and 97
Mutant minigenomes were created where the four conserved Gs at positions 79, 85, 91 and 97 were each mutated in turn to A, C or U. The amount of CAT produced by each mutant is shown in Fig. 4. All changes to G79 were found to have moderate effects, since when this position was changed to A, C or U, CAT production was between 57·7 (G79->A) and 41·7% (G79->C or G79->U) of standard levels. Mutating the G residues at positions 85 and 91 to A or the fourth conserved G at position 97 to A or C did not affect the amount of CAT produced significantly (from 88·6% for G91->A to 103·8% for G85->A), while mutation G97->U led to an increase in CAT production, to 128·5% of the control. In contrast, transversions at positions 85 and 91 essentially abolished CAT production (between 0·7% for G91->U and 3·7% for G85->U).



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Fig. 4. Quantification of the effects on CAT production of point mutations in the four conserved G residues situated downstream of the start of the N gene. Each G was mutated to A, C or U as indicated and RNAs were transcribed in vitro. Mutated minigenome RNAs were transfected into RPV-infected HTK293 cells. After incubation overnight, the cells were lysed and the cleared lysates assayed for the presence of CAT protein. CAT production relative to that of the wild-type pMDB8A RNA is shown. Error bars indicate the standard errors for three independent experiments.

 

   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
In minigenomes, also known as minireplicons, the viral coding and intergenic regions are replaced by the ORF of a reporter protein, most commonly the bacterial CAT protein. The minimal replicon requires (3' to 5') a genomic promoter, a viral gene start, a reporter ORF, a viral gene stop and the complement of the antigenome promoter. In structure, such minigenomes thus resemble internal deletion defective-interfering (DI) particles. Artificial or naturally occurring DIs have been used successfully to study replication signals in several negative-stranded RNA viruses such as SeV (Park et al., 1991 ), respiratory syncytial virus (Collins et al., 1991 ; Fearns et al., 2000 ; Peeples & Collins, 2000 ), vesicular stomatitis virus (Li & Pattnaik, 1997 , 1999 ; Whelan & Wertz, 1999a , b ), Human parainfluenza virus 3 (HPIV-3) (Hoffman & Banerjee, 2000a ), Simian virus 5 (Murphy et al., 1998 ; Murphy & Parks, 1999 ) and Rabies virus (Conzelmann & Schnell, 1994 ). Replication of these plasmid-derived constructs can only be achieved in the presence of the viral N protein, required to encapsidate the RNA, and the transcriptase/replicase proteins (P and L). These can be provided by a helper virus or in the form of plasmids that are transcribed by a suitable enzyme, usually T7 polymerase supplied by infecting with a recombinant poxvirus, to produce the required mRNAs. The same polymerase can also be used to transcribe the minigenome RNA from a co-transfected plasmid.

In the experiments reported here, a helper virus was used to provide the viral proteins and the minigenome RNA was synthesized in vitro, allowing us to ensure that identical amounts of each mutant were available for replication. The in vitro-synthesized minigenome RNAs were transfected as antigenome-sense RNA, but could not act as a message to produce CAT as they were not capped. This is shown by the fact that no CAT protein was produced by the transfected RNA in the absence of helper virus (Baron & Barrett, 1997 ). To obtain CAT protein in these assays, the antigenome-sense minigenome RNA had to be encapsidated by the N protein and replicated to genome-sense RNA, which could then be transcribed to produce a mRNA capable of being translated to give CAT protein or transcribed in replicative mode to produce more antigenome-sense minireplicon. Where the level of CAT synthesis was altered, any of these minigenome functions (i.e. encapsidation, genome-sense RNA synthesis or transcription from the genome promoter) could have been affected by the mutation; since genome-sense template synthesis will be dependent on the antigenome promoter, which was not modified in any of these experiments, the effects we have observed must be due to alterations in the efficiency of encapsidation or of RNA synthesis from the genome promoter. We attempted to extend the observations made on overall minigenome function by looking for (encapsidated) genome and antigenome-sense RNA using Northern blots or direct incorporation of 32P, in order to distinguish between effects on encapsidation and those on polymerase function. However, these studies were unsuccessful (not shown), suggesting that, in RPV, or with the replicon used here, the levels of minigenome replication are too low for direct visualization of the RNAs made. These findings also indicate that the amount of transfected antigenome RNA was always in large excess over any synthesized in the transfected cell.

The leader and trailer-complement sequences of all members of the Paramyxoviridae begin ACCA, and this sequence is believed to contain the landing site for the viral RNA-dependent RNA polymerase. Changes to this sequence would be expected to have a major effect on transcription and we indeed found that mutations in nucleotides 1, 3 and 4 almost abolished CAT production; surprisingly, mutation of nucleotide 2 had a less severe effect, resulting in more than 20% of the control activity. Position 5 appears to be much less critical in forming the viral promoter, since all four bases are tolerated here to some extent, although A, G and U were better than C. It is interesting to note that, although G5->A reduced CAT expression by about 50%, this change is found naturally in another morbillivirus (measles virus) and in two virulent strains of RPV (Baron & Barrett, 1995b ). It may be that some changes at this position can be offset or balanced by changes at another, as has been found for HPIV-3 (Hoffman & Banerjee, 2000b ). Mutation of nucleotides 10 or 19 also led to a dramatic loss of CAT production. Nucleotide 10 is conserved in the genome and antigenome promoters but nucleotide 19 is not. Along with nucleotides 1, 3 and 4, these two nucleotides appear to be the only ones in which the exact residue is essential for minigenome function. In the remainder of the nucleotides mutated individually, transition mutations appear to be tolerated at least partially, although the base observed in the normal sequence is required for fully efficient minigenome function, as any changes reduced the amount of CAT produced to 60%, or less, of wild-type. These findings contrast with a similar recent study of HPIV-3 (Hoffman & Banerjee, 2000b ), where positions 1–4, 6–9, 11 and 12 were all found to be critical, with moderate effects on reporter gene expression of changing positions 5, 10 and 14–16, and no effect of mutation at position 13. However, the mutations used in that study were transversions, rather than the transitions used here.

Block mutation I, which altered nucleotides just beyond this region (positions 23–28), severely reduced minigenome function. This might be due to the change of the normally conserved U23, though changes of the preceding two positions had only moderate effects on CAT expression, or the change of G25, which is conserved, or to the combination of changes to more than one base in this block. Mutations in the rest of the leader region (II–VI) had little or no effect until blocks (VII and VIII) that affected the conserved sequence 52ACUUAGG, which marks the end of the leader and the start of the N gene. Although only the ACUUAGG motif is absolutely conserved, mutations introduced in nucleotide positions 51–66 (block mutations VII–XI) all reduced severely or abolished CAT production from the RPV minigenome. Clearly, some of the individually conserved positions in this region are critical for controlling mRNA transcription or encapsidation, and further analysis will be required to determine which. Block mutation XII, located at the end of the highly conserved N gene start region (nt 69–74), and block mutations XIII–XVII, in the less well conserved region (nt 75–92), all reduced the minigenome efficiency to approximately 30–60% of normal levels, indicating that there are residues in this region that, even if not absolutely critical, do play a role in encapsidation or transcription.

Early observations on the sequences of the 5' and 3' ends of paramyxovirus genomes showed that there was, in addition to the similarities between the immediate 3' ends of genome and antigenome (the leader complement and the trailer), a region roughly 80–100 bases from each end (and therefore inside the N and L genes) that also showed similarity between the genome and antigenome (Crowley et al., 1988 ; Blumberg et al., 1991 ). This led to the suggestion that signals important in transcription or encapsidation are found outside the leader and trailer regions, a suggestion supported by the finding that naturally occurring DIs have a minimum run of 94 nucleotides derived from the original genome (Calain et al., 1992 ). It has been shown recently that the genomic and antigenomic promoters of paramyxoviruses require a conserved motif in this region, although the motif appears to differ between the members of Rubulavirus and Respirovirus (Tapparel et al., 1998 ; Murphy & Parks, 1999 ; Hoffman & Banerjee, 2000a ). In both groups of viruses, this motif is position-sensitive (Murphy et al., 1998 ; Tapparel et al., 1998 ) and appears to be on the same side of the coiled nucleocapsid as the 3' terminus of the template strand. The motif identified in the respiroviruses includes three G residues (in the transcribed strand, C in the template strand) spaced six nucleotides apart at positions 79, 85 and 91 (Tapparel et al., 1998 ). In the morbilliviruses, there are four conserved G residues situated in similar positions in the non-coding region of the N gene, at positions 79, 85, 91 and 97 (see Fig. 1). As in the case of SeV and HPIV-3, the conserved G nucleotides in the N gene of RPV possess counterparts situated at the same positions in the genome RNA, where they are in the complement to the terminal non-coding region of the L gene. The four G residues in the N gene untranslated region were each mutated in turn to A, C or U. Changing the first conserved G residue, at position 79, to any other nucleotide led to a decrease in CAT production to 40–60% of the control (Fig. 4). This level of reduction was also seen in block mutations XIV and XV, which also altered this G nucleotide to an A. This G residue appears to be important for efficient minigenome function. When the G residues at positions 85 and 91 were mutated individually to A (transition), CAT production was not affected significantly. However, when these two nucleotides were mutated to either C or U (transversions), CAT production was abolished, indicating that a purine is essential at these positions. This is consistent with the presence of an A residue in these positions in some paramyxoviruses and also with a consensus hexamer repeat RNNNNN (Tapparel et al., 1998 ). However, A residues have only been found in these positions in sequences of rubulaviruses, in which group of viruses C is also sometimes found at the same positions relative to the ends of the genome. A different motif has been proposed for this group of viruses (Murphy & Parks, 1999 ). It may be that, although an A can function at these positions in our minigenome assay, other factors favour a G in a full genome. In comparison to the effect of the single G->A mutations at 85 and 91, block mutations XVI–XVIII, which also altered these G residues to A, had a greater effect on minigenome efficiency. Less than 40% of control activity was obtained with these mutations, indicating that other residues in these regions are important. It is unclear why block mutation XVIII was so detrimental to minigenome activity since, apart from G91->A, which is not itself deleterious, there were no other conserved residues in this region of the genome. Modifications to the fourth G residue (G97) conserved in the morbillivirus sequences did not have any effect on the levels of CAT produced; if anything, there was a slight increase in CAT production when the G was mutated to a U. Therefore, this nucleotide does not appear to be involved directly in minigenome replicative functions; a G in this position is not conserved in other paramyxoviruses.

Image reconstitution from electron micrographs of negatively stained preparations indicates that the SeV nucleocapsid is a left-handed helix containing 13 N protein subunits per turn, with each predicted to contact six nucleotides (Egelman et al., 1989 ), a prediction that was corroborated by the ‘rule of six’ (Calain & Roux, 1993 ). The three GNNNNN motifs would thus form hexamers 14, 15 and 16, counting from the start of the genome, with the Gs at positions 79, 85 and 91. Assuming similar physical relationships in the morbilliviruses (which are also bound by the ‘rule of six’), hexamers 14, 15 and 16 will be positioned on the same face of the helix and stacked exactly above the first three hexamers (Tapparel et al., 1998 ). It is unclear at present whether the spatial arrangement of these hexamers indicates that the two elements are involved in promoter function in the paramyxoviruses, possibly binding to the viral P protein (Murphy & Parks, 1999 ), or whether the internal motif forms part of the encapsidation signal for the virus (Tapparel et al., 1998 ).


   Acknowledgments
 
V.M. was the recipient of an Institute for Animal Health studentship.


   Footnotes
 
b Present address: Centre for Applied Microbiology Research, Porton Down, Salisbury, Wiltshire SP4 0JG, UK.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Baron, M. D. & Barrett, T. (1995a). The sequence of the N and L genes of rinderpest virus, and the 5' and 3' extra-genic sequences: the completion of the genome sequence of the virus. Veterinary Microbiology 44, 175-185.[Medline]

Baron, M. D. & Barrett, T. (1995b). Sequencing and analysis of the nucleocapsid (N) and polymerase (L) genes and the terminal extragenic domains of the vaccine strain of rinderpest virus. Journal of General Virology 76, 593-602.[Abstract]

Baron, M. D. & Barrett, T. (1997). Rescue of rinderpest virus from cloned cDNA. Journal of Virology 71, 1265-1271.[Abstract]

Blumberg, B. M., Chan, J. & Udem, S. (1991). Function of paramyxovirus 3' and 5' end sequences in theory and practice In The Paramyxoviruses , pp. 235-247. Edited by D. W. Kingsbury. New York & London:Plenum.

Calain, P. & Roux, L. (1993). The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA. Journal of Virology 67, 4822-4830.[Abstract]

Calain, P., Curran, J., Kolakofsky, D. & Roux, L. (1992). Molecular cloning of natural paramyxovirus copy-back defective interfering RNAs and their expression from DNA. Virology 191, 62-71.[Medline]

Collins, P. L., Mink, M. A. & Stec, D. S. (1991). Rescue of synthetic analogs of respiratory syncytial virus genomic RNA and effect of truncations and mutations on the expression of a foreign reporter gene. Proceedings of the National Academy of Sciences, USA 88, 9663-9667.[Abstract]

Conzelmann, K.-K. & Schnell, M. (1994). Rescue of synthetic genomic RNA analogs of rabies virus by plasmid-encoded proteins. Journal of Virology 68, 713-719.[Abstract]

Crowley, J. C., Dowling, P. C., Menonna, J., Silverman, J. I., Schuback, D., Cook, S. D. & Blumberg, B. M. (1988). Sequence variability and function of measles virus 3' and 5' ends and intercistronic regions. Virology 164, 498-506.[Medline]

Deng, W. P. & Nickoloff, J. A. (1992). Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Analytical Biochemistry 200, 81-88.[Medline]

Egelman, E. H., Wu, S. S., Amrein, M., Portner, A. & Murti, G. (1989). The Sendai virus nucleocapsid exists in at least four different helical states. Journal of Virology 63, 2233-2243.[Medline]

Fearns, R., Collins, P. L. & Peeples, M. E. (2000). Functional analysis of the genomic and antigenomic promoters of human respiratory syncytial virus. Journal of Virology 74, 6006-6014.[Abstract/Free Full Text]

Hoffman, M. A. & Banerjee, A. K. (2000a). Precise mapping of the replication and transcription promoters of human parainfluenza virus type 3. Virology 269, 201-211.[Medline]

Hoffman, M. A. & Banerjee, A. K. (2000b). Analysis of RNA secondary structure in replication of human parainfluenza virus type 3. Virology 272, 151-158.[Medline]

Kobune, F., Sakata, H., Sugiyama, M. & Sugiura, A. (1991). B95a, a marmoset lymphoblastoid cell line, as a sensitive host for rinderpest virus. Journal of General Virology 72, 687-692.[Abstract]

Li, T. & Pattnaik, A. K. (1997). Replication signals in the genome of vesicular stomatitis virus and its defective interfering particles: identification of a sequence element that enhances DI RNA replication. Virology 232, 248-259.[Medline]

Li, T. & Pattnaik, A. K. (1999). Overlapping signals for transcription and replication at the 3' terminus of the vesicular stomatitis virus genome. Journal of Virology 73, 444-452.[Abstract/Free Full Text]

Moyer, S. A. & Horikami, S. M. (1991). The role of viral and host cell proteins in paramyxovirus transcription and replication In The Paramyxoviruses , pp. 249-274. Edited by D. W. Kingsbury. New York & London:Plenum.

Murphy, S. K. & Parks, G. D. (1999). RNA replication for the paramyxovirus simian virus 5 requires an internal repeated (CGNNNN) sequence motif. Journal of Virology 73, 805-809.[Abstract/Free Full Text]

Murphy, S. K., Ito, Y. & Parks, G. D. (1998). A functional antigenomic promoter for the paramyxovirus simian virus 5 requires proper spacing between an essential internal segment and the 3' terminus. Journal of Virology 72, 10-19.[Abstract/Free Full Text]

Park, K. H., Huang, T., Correia, F. F. & Krystal, M. (1991). Rescue of a foreign gene by Sendai virus. Proceedings of the National Academy of Sciences, USA 88, 5537-5541.[Abstract]

Peeples, M. E. & Collins, P. L. (2000). Mutations in the 5' trailer region of a respiratory syncytial virus minigenome which limit RNA replication to one step. Journal of Virology 74, 146-155.[Abstract/Free Full Text]

Tapparel, C., Maurice, D. & Roux, L. (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. Journal of Virology 72, 3117-3128.[Abstract/Free Full Text]

Taylor, W. P. (1986). Epidemiology and control of rinderpest. Revue Scientifique et Technique Office International des Epizooties 5, 407-410.

Whelan, S. P. J. & Wertz, G. W. (1999a). Regulation of RNA synthesis by the genomic termini of vesicular stomatitis virus: identification of distinct sequences essential for transcription but not replication. Journal of Virology 73, 297-306.[Abstract/Free Full Text]

Whelan, S. P. J. & Wertz, G. W. (1999b). The 5' terminal trailer region of vesicular stomatitis virus contains a position-dependent cis-acting signal for assembly of RNA into infectious particles. Journal of Virology 73, 307-315.[Abstract/Free Full Text]

Received 23 May 2001; accepted 10 August 2001.