Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, Church Street, Glasgow G11 5JR, UK
Correspondence
Richard M. Elliott
r.elliott{at}vir.gla.ac.uk
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ABSTRACT |
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These authors contributed equally to the work described in this publication.
Present address: Department of Biochemistry, Dartmouth Medical School, 7200 Vail Building, Hanover, NH 03755, USA.
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INTRODUCTION |
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Bunyamwera virus (BUN; genus Orthobunyavirus) has been used in our laboratory as the prototype virus for studying replication of this important family of viruses. The BUN L segment encodes an RNA-dependent RNA polymerase called the L protein, whilst the M segment encodes a precursor to the virion glycoproteins Gn and Gc, as well as a non-structural protein of unknown function (NSm). The S segment encodes two proteins in different, overlapping reading frames: the nucleocapsid protein, N, and a second non-structural protein, NSs, which are translated from the same mRNA. The N protein encapsidates viral genomes and antigenomes (complementary or replicative-intermediate RNA) to give RNAN complexes termed ribonucleoproteins or RNPs (Elliott, 1996). Recent studies have shown that BUN NSs is an interferon antagonist and, in mammalian but not mosquito cells, NSs mediates host-protein shut-off (Kohl et al., 2004
; Weber et al., 2001
, 2002
).
A feature of bunyavirus genome RNAs is the conservation of the 3'- and 5'-terminal sequences, from which genus-specific consensus sequences can be derived (Elliott et al., 2000). For the genus Orthobunyavirus, the consensus extends to 11 nt that are complementary apart from a conserved U-G pairing at position 9 (Fig. 1a
). These terminal 11 nt are followed by 3 (M segment) or 4 (S and L segments) nt that are complementary and conserved on a segment-specific basis (Fig. 1a
). Beyond these terminal 14 or 15 nt, variable lengths of complementarity are found, which are generally unique to an individual virus and segment (Elliott et al., 1991
). The non-coding regions (NCRs) vary among different segments and different viruses; for the BUN S segment, the 3' NCR (genome sense) is 85 nt and the 5' NCR is 174 nt (Elliott, 1989
). Signals within these NCRs are poorly understood.
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METHODS |
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Plasmids.
Plasmids pTM1-BUNN, pTM1-BUNL and pTM1-BUNNSs, expressing bunyavirus proteins, and the reporter RNAs pBUNSCAT, pT7riboBUNLRen(), pT7riboBUNMRen(), pT7riboBUNSRen(), pT7riboBUNSRen()mut16 and pTM1-FF-Luc have been described previously (Dunn et al., 1995; Kohl et al., 2003
, 2004
; Weber et al., 2001
). Transcripts generated from pT7ribo plasmids mimic bunyavirus RNAs and can be encapsidated, transcribed and replicated after transfection into cells expressing the BUN N and L proteins (Dunn et al., 1995
).
Plasmids pT7riboBUNS(+), pT7riboBUNM(+) and pT7riboBUNL(+) used in virus rescue have been described by Bridgen & Elliott (1996). Plasmids pT7riboBUNSRen()mut946 and pT7riboBUNS(+)mut946, carrying the C946A point mutation used in mini-replicon assays and virus rescue, were generated by using a QuikChange mutagenesis kit according to the manufacturer's instructions (Stratagene). All constructs were verified by DNA sequencing. Details of cloning strategies and oligonucleotide sequences can be obtained from the authors on request.
Transfection, mini-replicon reconstitution and reporter-gene assays in BSR-T7/5 cells.
Approximately 5x105 cells in 35 mm Petri dishes were transfected with 1 µg pTM1-BUNL, 0·5 µg <1?show=[to]>pTM1-BUNN and 0·1 µg pTM1-FF-Luc, together with 0·5 µg pT7riboBUNLRen(), pT7riboBUNMRen() or pT7riboBUNSRen() DNA, by using 5 µg DAC-30 (Eurogentec). Transfection efficiencies were normalized by measuring luciferase expression from co-transfected pTM1-FF-Luc. Luciferase activities were determined by using a Dual-Luciferase Assay kit (Promega); cells were lysed in a total volume of 200 µl lysis buffer at 24 h post-transfection and luciferase activity was measured in 1 µl cell extract.
Infection, metabolic labelling and Western blotting.
BHK-21 cells (1x106) grown in 35 mm Petri dishes were infected at an m.o.i. of 1 with wild-type (wt) BUN or BUN S(C946A) virus. Cells were labelled with 50 µCi [35S]methionine as indicated. Extracts were prepared by using RIPA buffer containing Complete protease inhibitor mix (Roche) and analysed by SDS-PAGE. Quantification was performed by using a Molecular Dynamics phosphorimager and ImageQuant software. For Western blotting, cell extracts were separated by SDS-PAGE and blotted on to Hybond-C pure membrane, followed by incubation with appropriate antibodies, as described previously (Kohl et al., 2004).
Recovery of recombinant BUN from cDNA.
Recombinant BUN was recovered from cDNAs by using recent modifications (A. C. Lowen, C. Noonan, A. McLees & R. M. Elliott, unpublished results) to our original protocol (Bridgen & Elliott, 1996). In brief, BSR-T7/5 cells were transfected with 1 µg each of pT7riboBUNM(+), pT7riboBUNL(+) and either pT7riboBUNS(+) for recovery of wt virus or pT7riboBUNS(+)mut946 for recovery of BUN S(C946A) virus by using 5 µg DAC-30. Support plasmids pTM1-BUNN, pTM1-BUNL and pTM1-BUNM were co-transfected (1 µg each). After 1 week or when cytopathic effects were visible, supernatants were plated onto BHK cells and several plaques were isolated and grown up. The presence of the C946A mutation was confirmed by RACE (rapid amplification of cDNA ends) analysis of the viral S RNA segment terminal sequences. One plaque isolate of each virus was chosen for further characterization.
Purification of RNA transcripts by using spin columns.
RNAs were transcribed in vitro from BbsI-linearized plasmids (Dunn et al., 1995) and purified on RNeasy mini spin columns (Qiagen) as described by the manufacturer. The eluted RNA transcripts were either used immediately or aliquotted and stored at 70 °C. The concentration of the RNA transcripts was determined by measuring A260.
Transfection of CV-1 cells and CAT assay.
Procedures for transfection of plasmid DNA and in vitro-transcribed RNA were as described previously (Dunn et al., 1995). In brief, subconfluent monolayers of CV-1 cells in 35 mm Petri dishes were infected with vTF7-3 at an m.o.i. of 10 for 1 h at 37 °C. After washing, cells were transfected with plasmid DNAs that expressed BUN proteins, followed 2·5 h later by in vitro-transcribed RNA. Cells were harvested 16 h later and chloramphenicol acetyltransferase (CAT) enzyme activity was determined as described previously (Gorman et al., 1982
). For quantitative purposes, extracts were diluted as described previously (Dunn et al., 1995
) and chromatography plates were analysed by using a Molecular Dynamics phosphorimager and ImageQuant software.
Northern blots.
BHK-21 cells in 35 mm Petri dishes were infected with BUN at an m.o.i. of 1. At 18 h post-infection, total cellular RNA was extracted by using TRIzol reagent (Invitrogen). RNA was quantified by spectrophotometry and 10 µg was combined with 5·5 µl formaldehyde, 15 µl deionized formamide and 1·5 µl 10x MOPS buffer in a total volume of 30 µl. Samples were heated at 55 °C for 15 min and then cooled on ice. Aliquots of 10x loading buffer (3 µl; 40 mg bromophenol blue ml1, 40 mg xylene cyanol ml1, 2·5 µg Ficoll 400 ml1) were added and samples were loaded on a 1·5 % agarose/2·2 % formaldehyde gel. Electrophoresis was performed at 75 V for 5 h in MOPS buffer. RNAs were transferred by capillary blotting overnight in 20x SSC on to a positively charged nylon membrane (Nytran). After UV cross-linking, 150 ng digoxigenin (DIG)-labelled RNA probe complementary to either the S genome or antigenome segment was hybridized to the membrane in 4 ml 50 % formamide buffer overnight at 68 °C. Synthesis and quantification of probes using a DIG Northern starter kit (Roche), washing of the membrane and subsequent detection were as directed by the manufacturer. Blots were exposed to X-OMAT UV film (Kodak) for up to 2 min.
RNA folding.
Predicted RNA structures at 28 and 37 °C (temperatures at which the virus replicates in mosquito and mammalian cells, respectively) were determined by using Mfold (Zuker et al., 1991); a minimum of 25 nt from the 5' and 3' termini (unless otherwise indicated) was analysed.
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RESULTS |
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Mutagenesis of the terminal regions of the S-segment 3' and 5' NCRs
It has previously been shown that a CG mutation at position 12 of the BUN S genome segment strongly reduces CAT activity in the reporter assay (Dunn et al., 1995
). That a single point mutation could exert such an effect on the functionality of the reporter RNA suggested that conservation of the sequence and/or the structure of the BUN segment NCRs might be of great importance.
A series of single point mutations was made in pBUNSCAT (using a PCR-based mutagenesis strategy) in the 3' and 5' NCRs at each of the 15 nt at either end. In vitro-transcribed RNA was transfected into vTF7-3-infected CV-1 cells expressing BUN L and S proteins and CAT activities were compared with that of wt BUNSCAT. The results are summarized in Fig. 2(a). It can be seen that most single point mutations in either the 3' or 5' terminus resulted in a marked reduction in CAT activity (all <11 %, with most <4 % of wt activity), except for mutations at positions 1, 4, 8, 9 and 15 at the 5' end and positions 1 and 15 at the 3' end, where the mutant RNAs maintained significant CAT activity (>40 % that of wt BUNSCAT RNA).
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Effect of deletions within S-segment NCRs
The NCRs of the L, M and S segments contain cis-acting signals for the initiation of transcription and replication, as well as for nucleocapsid assembly and perhaps packaging into virions. To determine the minimum length of the 5' and 3' NCRs in the BUN S segment that is needed for expression of CAT activity, a series of pBUNSCAT constructs containing varying lengths of the terminal sequences was produced (Fig. 3a).
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Generation of chimeric BUNCAT constructs
A recent report (Barr & Wertz, 2004) suggested that cooperation between the 3' and 5' NCRs of the BUN M, L and S segments is necessary to allow RNA synthesis and that complementarity of the NCRs extending beyond the conserved 11 terminal residues is required. However, the sequences and structures presented in that paper (see Fig. 1A
of Barr & Wertz, 2004
) do not represent authentic BUN sequences (these are compared directly in Fig. 4
); thus, we made constructs containing the correct sequences to verify these findings. A series of chimeric mini-replicons containing the antisense CAT ORF flanked by heterologous combinations of NCRs were constructed; the sequences of the terminal 40 nt of these constructs are shown in Fig. 5(a)
. When transfected into vTF7-3-infected cells expressing BUN L- and S-segment proteins, no CAT activity was detected with any chimeric construct (Fig. 5b
), whereas constructs with homologous NCRs gave different activities of CAT, depending on the origin of the NCR, as reported previously (Barr et al., 2003
; Kohl et al., 2004
). Comparison of the predicted base-pairing between homologous segment termini (Fig. 1a
) and the heterologous termini (Fig. 5a
) shows disruption of predicted complementarity beyond the conserved terminal 11 nt. A consistent feature was disruption of the segment-specific base-pairing over nt 1214 or 15; these residues are highlighted in Fig. 5(a)
. To investigate whether increasing complementarity across this region resulted in a functional template, mutagenesis was performed on the pBUNL/M-CAT construct, so that the 5' NCR (L segment-derived) was mutated to resemble the sequence of the M-segment NCR (the pBUNL/M-CAT construct was chosen as the L- and M-segment NCRs are similar in length). A series of mutants was constructed by PCR-directed mutagenesis and is shown in Fig. 6(a)
. Run-off transcripts from each of the BbsI-linearized templates were transfected into vTF7-3-infected CV-1 cells expressing BUN L- and S-segment proteins. CAT activities for each mutant are shown in Fig. 6(b)
. Weak CAT activity was detected when nt 1216 of the L NCR were mutated to those of the M NCR (6 % of wt level). When the mutated region was extended to nt 1218 and 1221 of the M 5' NCR, as in mutants pBUNL/M-CAT(1218M) and pBUNL/M-CAT(1221M), CAT activity was found to increase dramatically (71 and 65 %, respectively, of wt pBUNMCAT activity). This indicated that base-pairing is required not only between the terminal 11 nt, but also, at least for the M segment, between nt 12 and 18 of the 5' and 3' ends. This was in agreement with the results shown in Fig. 2(b)
for double mutations that restored complementarity at nt 1315 in the S-segment-derived BUNSCAT RNA.
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DISCUSSION |
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We found that deletion of 13 nt from the 3' end of the S segment still allowed transcription (although this was very weak if more than 1 nt was removed), similar to what has previously been described for Rift Valley fever virus (Prehaud et al., 1997). This suggested that signals for polymerase recognition and interaction were still functional, although impaired. As deletion of nucleotides from the 5' end was tolerated much less well, the data presented here would lend support to a model of bunyavirus transcription similar to that described for influenza virus (Li et al., 1998
), in which the 5' terminus is involved in recognition by the L polymerase and the 3' end is involved in transcription initiation.
We also carried out mutagenic analysis of the first 15 nt of the BUN S 5' and 3' ends. As most of the changes made to these sequences of either end dramatically reduced CAT activity, we investigated whether restoring complementarity would restore CAT activity; our data showed that base-pairing was important, as well sequence specificity within the termini. The importance of complementarity was also shown by the use of S/L/MCAT chimeric RNAs that were inactive in our assay. CAT activity was only detected when at least 1416 nt was complementary between the 3' and 5' termini; further increasing the complementarity further increased the CAT signal. However, as the different segments have different complementary sequences after the conserved terminal 11 nt, complementarity per se is the important feature of this region. This is in agreement with a previously published report (Barr & Wertz, 2004) although, as mentioned above, the templates reported in that paper apparently contained base substitutions compared with the authentic BUN genome segments (Fig. 4
). These results indicated that complementarity that extends past the extreme conserved terminal 11 nt is critical, possibly for forming the correct secondary structure at the termini, to allow transcription and replication.
Mini-genomes containing flanking sequences of either 13 or 20 nt of the S-segment 5' and 3' termini resulted in insignificant CAT activity, but a signal close to wt activity was obtained by flanking the reporter with 32 nt of the S-segment 5' NCR and 33 of the S-segment 3' NCR. This suggested that the minimal requirement of nucleotides for the S-segment NCR is more than 20 and less than 32. Any encapsidation signal has to be localized within these 32 nt. Finally, comparison of known strong promoters allowed us to identify a structural motif within the terminal 20 nt of the M and S(U16G) RNAs. Whilst structural prediction has limitations, we successfully created a mutant S segment carrying this structure (C946A) that showed increased reporter-gene expression. When introduced into a viable virus, the mutant overexpressed the N and NSs proteins and produced more antigenomes and mRNAs in infected cells. This suggested that structure, rather than sequence, defined this element, as in the case of the U16G mutation described previously (Kohl et al., 2003).
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ACKNOWLEDGEMENTS |
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Received 30 June 2004;
accepted 3 August 2004.