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
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Abstract |
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Introduction |
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Methods |
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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 EpsteinBarr virus transformation (Kobune et al., 1991
). B95a cells were grown in RPMIHEPES, containing 5% FCS, penicillin (200 U/ml) and streptomycin (200 µg/ml).
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 suppliers 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 manufacturers instructions. RNA was purified using a Qiagen RNeasy mini kit, again following the manufacturers instructions.
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CAT assays.
An ELISA kit obtained from Boehringer Mannheim was used, following the manufacturers 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.
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Results |
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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 122
Conservation of sequence between the genome and antigenome promoters is limited to the first 1518 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, 59, 1115, 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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Effects of block mutations in nt 2396
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 IIVI 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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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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Discussion |
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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 G5A 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 14, 69, 11 and 12 were all found to be critical, with moderate effects on reporter gene expression of changing positions 5, 10 and 1416, 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 2328), 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 (IIVI) 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 5166 (block mutations VIIXI) 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 6974), and block mutations XIIIXVII, in the less well conserved region (nt 7592), all reduced the minigenome efficiency to approximately 3060% 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 80100 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 4060% 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 XVIXVIII, 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
).
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Acknowledgments |
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Footnotes |
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References |
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Received 23 May 2001;
accepted 10 August 2001.