1 Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, Church Street, Glasgow G11 5JR, UK
2 Department of Microbiology, University of Alabama School of Medicine, 845 19th Street South, Birmingham, AL 35294, USA
Correspondence
Richard Elliott
r.elliott{at}vir.gla.ac.uk
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ABSTRACT |
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Present address: Department of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine BT52 1SA, UK.
Present address: Department of Biochemistry, Dartmouth Medical School, 7200 Vail Building, Hanover, New Hampshire 03755, USA.
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MAIN TEXT |
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Following infection viral mRNAs are transcribed from genomic RNAs that contain a non-templated, host-derived capped primer at the 5' end and are truncated but not polyadenylated at the 3' end (Bouloy et al., 1990; Jin & Elliott, 1993
, Patterson & Kolakofsky, 1984
). For replication, the negative-stranded genome RNAs serve as template for the synthesis of full-length, positive-sense RNAs called antigenomes, which in turn serve as templates for synthesis of progeny negative-stranded genomes. Genomes and antigenomes are encapsidated by the N protein to form biologically active structures called the viral ribonucleoproteins or RNPs. The 3' and 5' genome termini are complementary and interact to form a panhandle structure (Hacker & Kolakofsky, 1991
). Reverse genetics systems developed for BUN and other members of the Bunyaviridae (Dunn et al., 1995
; Lopez et al., 1995
; Flick & Pettersson, 2001
) have shown that transcription and replication of artificial minigenomes require only two viral proteins, the polymerase (L) and the N protein.
For bunyaviruses the N and NSs proteins are translated from a single mRNA but use different start codons and are encoded in different open reading frames (ORF), the NSs ORF being located within the N ORF. Previously we described recovery of infectious bunyavirus entirely from cDNA (Bridgen & Elliott, 1996) and subsequently we reported the creation of BUNdelNSs, a BUN virus that no longer expresses NSs (Bridgen et al., 2001
). Point mutations were introduced into the S segment to ablate the NSs ORF but maintain the N ORF intact (Fig. 1
A). BUNdelNSs showed increased levels of N expression, an impaired ability to shut-off host protein synthesis, a small plaque phenotype and slower growth in tissue culture. BUNdelNSs, as opposed to wtBUN, was also found to be an inducer of the interferon-
promoter (Bridgen et al., 2001
).
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Following passage of BUNdelNSs 9a virus in BALB/c mice (Bridgen et al., 2001) it was observed that the plaque sizes were not homogeneous (data not shown). For further analysis, one virus displaying a larger plaque phenotype (subsequently called BUNdelNSs vL) was plaque purified, and the 3' end of the S segment RNA was sequenced using RACE. As shown in Fig. 1(B)
, this virus had a sequence identical to wtBUN in its 3' S segment terminus (i.e. U at position 16), but still maintained the mutations that abrogated the NSs ORF.
To analyse protein expression profiles of these viruses, CV1 cells were infected at an m.o.i. of 1 and labelled for 3 h at 16 h post-infection with 50 µCi [35S]methionine per dish. Equal amounts of cell extracts were analysed by 10 % SDS-PAGE (Fig. 2A), and quantification of labelled proteins was carried out by phosphorimager analysis (Bio-Rad; Quantity One software). The G1 glycoprotein band was used to normalize radioactive protein levels. The levels of N expressed by BUNdelNSs vL were similar to wtBUN, whereas N levels of BUNdelNSs 9a were 2-fold higher than wtBUN. N levels expressed in BUN132CT-infected cells were 1·6-fold higher than those of wtBUN. Virus yields from Vero cells infected by these BUN viruses were measured by plaque assay on BHK-21 cells at 72 h post-infection as previously described (Bridgen et al., 1996
). BUNdelNSs vL gave titres only slightly lower than those of wtBUN (8x106 p.f.u. ml-1 vs 11x106 p.f.u. ml-1), and significantly higher than the titre achieved by BUNdelNSs 9a (1x106 p.f.u. ml-1). Virus yields measured at earlier time-points showed a similar pattern.
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To confirm that the U-to-G mutation at position 16 in the S segment 3' terminus influences N levels in infected cells, we used a minireplicon system (Weber et al., 2001). This consists of expression plasmids for N (pTM1-BUNN) and L (pTM1-BUNL) proteins as well as a plasmid, pT7riboBUNSREN(-), containing a reporter gene (Renilla luciferase) cloned in antisense direction between the 3' and 5' noncoding regions of the BUN S segment. Using site-directed mutagenesis, the mutation at position 16 was introduced into pT7riboBUNSREN(-) to create pT7riboBUNSREN(-)mut16. The plasmids were co-tranfected into BHK-SinT7 cells (Agapov et al., 1998
), together with an internal reporter encoding firefly luciferase (pTM1-FF-Luc) to measure transfection efficiency, and dual luciferase activities were measured as previously described (Weber et al., 2001
). As shown in Fig. 3(A)
, Renilla luciferase activity obtained with pT7riboBUNSREN(-)mut16 was about 3-fold higher than with pT7riboBUNSREN(-). Firefly luciferase activities were similar in all assays, showing that transfection efficiencies were similar.
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The effects of this mutation on the panhandle structure formed by interaction of the non-encapsidated 3' and 5' ends of the genome on the predicted RNA secondary structures were determined using Mfold (Zucker et al., 1999) (Fig. 3C
). The presented structures were formed using 25 bases of the S segment terminus in either the genomic (vRNA) or antigenomic (cRNA). Introduction of the mutation leads to a loop structure and a decrease in free energy for genome (mutant -29·2 kcal mol-1; wt -25·5 kcal mol-1) and antigenome (mutant -22·1 kcal mol-1; wt -21·8 kcal mol-1) RNAs. The predicted panhandle structures were identical if more nucleotides from each terminus were used (data not shown).
Our data further characterize BUNdelNSs, a genetically engineered BUN virus that does not express the NSs protein, described previously (Bridgen et al., 2001). Using RACE, we have found that this virus differed from wtBUN in that it carries a U-to-G mutation at position 16 (3' end) of the viral genome. When introduced into a minireplicon system, this mutation lead to a 3-fold increase in reporter gene activity, and radioactive labelling of RNAs indicated an increase in antigenome and mRNAs levels.
These results suggest that the single U-to-G mutation at position 16 in the 3' noncoding region is responsible for higher N levels in BUNdelNSs-infected cells as the result of increased transcription to make more S segment mRNA, and not to an increase in translatability of the viral mRNA. Indeed, the 3' terminus of the BUNdelNSs vL S genome segment is identical to that of wtBUN, yet this virus also contains the mutations introduced around the NSs initiation codon(s). Moreover, we recovered a variant of wtBUN carrying the mutation at position 16 that overexpresses N. Increased N expression appears to be responsible for the lower titres obtained with those viruses. It has been shown for both BUN and Rift Valley fever virus that the relative molar rations of L and N proteins are critical for optimal viral RNA replication (Elliott, 1996; Lopez et al., 1995
), and in the case of the viruses containing the mutation at position 16 described here the optimal ratio would be distorted.
The origin of the mutation in the S segment 3' end remains unknown. It might have been introduced through selection or mutation during rescue of the virus from cDNA, and possibly the U-to-G mutation confers a short-term selective advantage. However, in the long term viruses that revert to the wild-type sequence appeared that make normal levels of N. The same mutation was also identified in stocks of wtBUN, so some biological significance cannot be excluded, though because bunyaviruses that are diploid with regard to the S segment have been reported (see Pringle, 1996) the effect of the mutation might be masked in a population. The U-to-G mutation is predicted to give a more stable panhandle structure. In the case of La Crosse bunyavirus, it has been proposed that increased complementarity would lead to less mRNA synthesis because separating the paired ends would be more difficult (Hacker & Kolakofsky, 1991
). However, for BUN virus we have found that increasing the complementarity of chimeric minireplicons containing L- and M-segment genome ends leads to an increase in minireplicon activity (E. Dunn & R. M. Elliott, in preparation).
For further experiments we will use BUNdelNSs vL as a virus lacking NSs since this expresses wild-type levels of N protein and has a wild-type 3' genome end sequence.
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ACKNOWLEDGEMENTS |
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Received 7 November 2002;
accepted 17 December 2002.