Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
Correspondence
Andrew J. Easton
a.j.easton{at}warwick.ac.uk
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ABSTRACT |
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MAIN TEXT |
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RNA synthesis in pneumoviruses requires a ribonucleoprotein complex comprising the nucleocapsid (N) protein bound to the genomic RNA together with the phosphoprotein (P) and large (L) polymerase protein. Efficient, progressive, transcription is enhanced by the M2-1 protein, a feature that is unique to the members of the subfamily Pneumovirinae (Collins et al., 1995; Naylor et al., 2004
). Transcription of virus mRNA is carried out by the virion-associated polymerase complex in a progressive stopstart manner from the 3' to the 5' end of the genome (Dickens et al., 1984
; Pringle & Easton, 1997
), and is directed by gene start and gene end sequences that flank the transcription units. The gene order in non-segmented negative-strand RNA viruses is significant, as their transcription strategy results in more mRNA transcripts from genes at the 3' end of the genome, with levels of mRNA progressively decreasing in a step-wise manner to the 5' end. In Human respiratory syncytial virus (HRSV) and APV, the gene start sequence is conserved in all of the genes except for the L gene (Li et al., 1996
; Ling et al., 1992
, 1995
; Randhawa et al., 1996
; Yu et al., 1991
, 1992a
), although the HRSV gene start sequence is 10 nt in length (Collins et al., 1986
; Kuo et al., 1996
). The APV consensus is GGGACAAGU in mRNA sense (Li et al., 1996
; Ling et al., 1992
, 1995
; Randhawa et al., 1996
; Yu et al., 1991
, 1992a
) and the L gene start sequence has three differences (underlined), giving a sequence of AGGACCAAU (Randhawa et al., 1996
). A consensus gene end sequence at the 3' end of the transcription unit is thought to be involved in termination and polyadenylation (Bukreyev et al., 1996
; Jacobs et al., 2003
; Ling et al., 1992
; Randhawa et al., 1996
). Termination and polyadenylation of the mRNA occur at the gene junction, followed either by progression of the polymerase across the non-transcribed intergenic region to the next gene or relocation of the polymerase complex to the genome 3' terminus to begin the process again. The reinitation of transcription of the next gene then occurs (Collins et al., 1986
).
Here, we report the effect of mutation of the APV gene start sequence on gene expression, and identify the key residues in the gene start sequence that control transcription. Each of the nine bases of the conserved APV gene start sequence was mutated to the three other possible nucleotides. A dicistronic minigenome for APV, similar to the dicistronic HRSV minigenome (Kuo et al., 1996), was kindly provided by Dr J. Smith, University of Warwick, UK. The minigenome was constructed using the APV leader and trailer sequence flanking genes for chloramphenicol acetyltransferase (CAT) and firefly luciferase (Luc). The conserved gene start sequence was located at the beginning of both reporter genes. The CAT gene end transcription termination sequence was taken from the APV P gene and the Luc gene was terminated with the L gene end sequence. The intergenic region between the two reporter genes was UCGAU. Placing this next to the last A residue of the polyadenylation site of the P gene end sequence generated a ClaI site that was used for cloning. The newly created intergenic region contains a pyrimidine as the first base, which is common with APV intergenic regions, but does not otherwise resemble any of the wild-type regions. The consensus gene start sequence at the beginning of the Luc gene was followed with a 4 nt AACC sequence (in mRNA sense) to preserve the Kozak sequence (Kozak, 1986
) around the Luc AUG start codon. Mutagenic primers of 2229 nt in length were designed to create each of the 27 desired mutants of the Luc gene start sequence in the APV dicistronic minigenome. PCRs were carried out using a mutagenic primer and a standard T7 promoter primer that annealed with the T7 promoter sequence in the vector. In addition, a non-consensus APV L gene start sequence and an intermediate mutant with two of the three bases unique to the non-consensus L gene start sequence were also created using mutagenic primers. All mutations were confirmed by sequencing. Details of the primer sequences are available upon request from the authors.
The dicistronic minigenome, shown diagrammatically in Fig. 1, was transfected into BSR-T7 cells (Buchholz et al., 1999
) that had previously been infected with APV, as described by Marriott et al. (2001)
. To quantify the effect of the gene start mutations on gene expression, the levels of protein production from the reporter genes were determined as described previously (Marriott et al., 2001
) and the level of Luc gene expression was normalized to the CAT reporter gene expression. The values for the two reporter genes expressed from the dicistronic minigenome with an unaltered consensus gene start sequence for the CAT gene were determined. For each mutant, three to eight individual transfection experiments were averaged and a standard deviation determined.
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It should be noted that the A7U mutant introduces a translational start codon, AUG, which may alter the protein reading frame from the naturally occurring one. This will affect the observed activity of the Luc enzyme produced from these transcripts, and hence the level of expression for this mutant cannot be considered to be truly representative of transcriptional capacity. Results of CAT ELISA and Luc enzyme assays have previously been shown to closely match levels of mRNA transcription from minigenomes measured by Northern blot analysis (Marriott et al., 1999). To clarify the effect of the introduction of this start codon, and to confirm that the mutations were affecting mRNA transcription rather than having an indirect effect on another aspect of gene expression, Northern blot analysis was carried out using mRNA isolated from HEp2 cells previously infected with recombinant vaccinia virus expressing T7 RNA polymerase in a plasmid-based rescue system as described by Marriott et al. (1999)
, with plasmid amounts optimized for APV (0·4 µg of the N plasmid and dicistronic minigenome, 0·2 µg of the P and L plasmids and 0·02 µg of the M2-1 plasmid) transfected into approximately 2x106 cells using Lipofectin (Invitrogen). A range of mutants were chosen to represent the different levels of Luc activity seen in Fig. 2
and this included the A7U mutant (Fig. 3
). A riboprobe containing sequences from both CAT and Luc genes was generated by transcription from an APV dicistronic minigenome and the blots were processed as described by Marriott et al. (1999)
. It can be seen that the levels of Luc mRNA relative to those of CAT mRNA are consistent with the protein levels determined for the mutants. The mRNA of the mutant found to have the least effect, C5U, had both the CAT and Luc band at a similar relative intensity to that of the wild-type minigenome. The mutations with the most detrimental effect in the protein assay were G1C where no Luc protein was detected and mutant G1U which expressed Luc protein at levels of approximately 5 % of the wild-type control. From Fig. 3
it can be seen that no Luc mRNA can be detected for either of these mutants. In contrast, mutant G1A had an intermediate effect on Luc protein production and it can be seen that the Luc mRNA band is of moderate intensity compared with the wild-type Luc mRNA and their CAT mRNA levels are of similar intensity. It can be seen that mutant A7U did not direct the synthesis of detectable levels of Luc mRNA, indicating that this mutation had a significant effect on transcription.
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The APV gene start sequence mutation data presented here differ from those of HRSV in that the APV gene start sequence showed a trend to more readily accept changes from a G to an A. However, the APV gene start sequence was also more sensitive to mutation overall than that of HRSV and none of the mutations resulted in enhanced activity of the gene start sequence. The APV gene start sequence also tolerated best the changes at the positions found to differ from the consensus in the L gene start sequence over any other possible nucleotide. This trend was also found at positions 4 and 10 of the HRSV gene start sequence (mRNA sense) in the gene start sequence for the L gene if the A at position 9 of the HRSV gene start sequence is considered to be an insertion rather than a substitution (Kuo et al., 1997). The data presented here, together with those of Kuo et al. (1997)
for HRSV, indicate that there is a consistent pattern for the nucleotides important for initiating efficient transcription by pneumoviruses. In addition, the expression of the L gene is regulated not only by genome location but also by its unique gene start sequence, which is less efficient than the consensus present in the other genes.
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ACKNOWLEDGEMENTS |
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Received 15 July 2005;
accepted 15 September 2005.
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