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Department of Microbiology, University of Alabama School of Medicine, BBRB Room 360/Box 17, 845 19th Street South, Birmingham, AL 35294-2170, USA
Correspondence
John N. Barr
jbarr{at}uab.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A supplementary table showing the wild-type and altered nucleotide sequences used in this study is available in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The BUNV genome comprises three segments of negative-sense RNA, designated small (S), medium (M) and large (L). The S segment utilizes overlapping open reading frames (ORFs) to encode the BUNV nucleocapsid (N) and non-structural (NSS) proteins (Fuller & Bishop, 1982
; Fuller et al., 1983; Elliott, 1989b). The M segment encodes a polyprotein that is cleaved into the glycoproteins Gn and Gc and the non-structural NSM protein (Gentsch & Bishop, 1979; Fuller & Bishop, 1982; Elliott, 1985), and the L segment encodes the RNA-dependent RNA polymerase (RdRp) (Elliott, 1989a). Each negative-sense segment serves as template for two distinct RNA-synthesis activities: (i) transcription to generate a single mRNA; and (ii) replication to generate a complementary, positive-sense antigenome that can in turn be replicated to generate more genomic strands (Fig. 1a).
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). The 5' ends of bunyavirus mRNAs possess capped extensions of 12–17 nt that are derived from host-cell mRNAs (Jin & Elliott, 1993) and resemble the well-characterized 5' extensions present on influenza virus mRNAs (Bouloy et al., 1978; Krug et al., 1979; Plotch et al., 1981). The 3' ends of bunyavirus mRNAs are truncated relative to their corresponding templates. This truncation is by approximately 100 nt for the S segment and by approximately 40 nt for the M and L segments (Patterson & Kolakofsky, 1984; Eshita et al., 1985; Cunningham & Szilágyi, 1987; Bouloy et al., 1990; Jin & Elliott, 1993).
Transcription termination has been studied in detail for many negative-sense RNA viruses, in particular for viruses having non-segmented negative-sense (NNS) genomes. The best-studied examples within this group are Vesicular stomatitis virus (VSV), Human respiratory syncytial virus and Simian virus 5. Despite belonging to different taxonomic families, these viruses possess termination signals that have two common elements, namely a U tract and a short upstream A/U-rich sequence (Whelan et al., 2004). Much evidence suggests that this U-tract element is transcribed reiteratively to generate the 3' poly(A) tails present on all NNS virus mRNAs. Studies with VSV, the prototypic NNS RNA virus, show that a U7 tract is specifically required to support reiterative transcription and suggest that poly(A)-tail formation is a critical step in the termination process itself (Barr et al., 1997). The upstream A/U-rich sequence is also known to play a critical role in termination (Barr et al., 1997; Hwang et al., 1998) and more recent work has indicated that this element regulates the extent of reiterative transcription (Barr & Wertz, 2001).
Within the SNS RNA viruses, transcription termination has been best studied for Influenza virus. Despite belonging to a different taxonomic family, Influenza virus also possesses a U-tract element within its termination signal (Li & Palese, 1994) and this U tract has been shown to be functionally analogous to the U tract of NNS RNA viruses (Poon et al., 1999).
In contrast, transcription termination for other SNS RNA viruses, such as members of the families Arenaviridae and Bunyaviridae, is poorly understood. Although the cis-acting signals responsible for signalling transcription termination of arena- and bunyaviruses have not been defined, the U-tract and A/U-rich sequences that comprise the NNS termination sequences are not found consistently within their non-translated regions (NTRs). Coinciding with this observation, the mRNAs generated by many arena- and bunyaviruses do not possess 3' poly(A) tails. Therefore, arenavirus and bunyavirus transcription termination may utilize novel mechanisms unrelated to those of other negative-sense RNA viruses.
We have investigated the process of transcription termination for BUNV, the prototype member of the family Bunyaviridae. Through mutagenesis of both 3' and 5' NTRs, we have defined the transcription termination signal of the BUNV S segment. Related sequences are found within the S segments of many other orthobunyaviruses, suggesting that the process of mRNA transcription termination is conserved functionally across the genus.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Barr et al., 2003) contained the BUNV S-segment sequence flanked by the T7 RNA polymerase promoter and the hepatitis delta virus self-cleaving ribozyme. Following ribozyme cleavage, the T7 transcript represented the S-segment antigenome, except for two non-BUNV G residues at the 5' end. The previously described plasmid pBUN-S(ren) (Barr et al., 2003) was derived from pT7riboBUN-S by exchanging precisely the BUNV S segment-coding region for the 936 nt coding region of the Renilla luciferase protein, leaving the 3' and 5' NTRs of the BUNV S segment unaltered (Fig. 1b). Plasmids designed to generate the genome analogues 
3' and 5' were derived from pBUN-S(ren) by using PCR-based mutagenesis. For template 3', oligonucleotides (Operon Inc.) were designed such that nt 26–85 of the 3' NTR were replaced with a single XhoI site (Fig. 1b). To generate template 5', oligonucleotides were designed such that nt 788–936 of the 5' NTR were replaced with a single BglII site (Fig. 1b). Alterations within the 5' NTR of BUN-S(ren) to generate BUN-S(ren)-term1 to BUN-S(ren)-term8 were performed by using PCR-based mutagenesis on plasmid pBUN-S(ren). For these templates, oligonucleotides were designed to replace designated nucleotides with a sequence having an equivalent nucleotide composition.
Plasmid pS-BUN-T1-UP, designed to generate template S-BUN-T1-UP, was generated by inserting a fragment containing nt 829–880 of the S segment into the naturally occurring NsiI site (nt 794) of the BUNV S-segment cDNA (see Fig. 5a). The T1-UP cDNA fragment was generated by NsiI digestion of annealed oligonucleotides containing the appropriate sequences. Derivatives of pS-BUN-T1-UP with altered T1-UP termination sequences were generated by ligating NsiI-digested oligonucleotides incorporating the altered T1-UP sequences. All nucleotide changes were confirmed by DNA sequence analysis.
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; Barr & Wertz, 2004). Briefly, 4 µg cDNAs expressing BUNV S- and L-segment ORFs and 6 µg genome analogue-expressing cDNA were transfected into BHK-21 cells previously infected with vaccinia virus recombinant vTF7-3, which expresses T7 RNA polymerase (Fuerst et al., 1986). BUNV RNAs were labelled metabolically by using [3H]uridine (33 µCi ml–1; Perkin Elmer) in the presence of actinomycin D (10 mg ml–1; Sigma). After labelling for 6 h, cellular RNAs were harvested by using the RNeasy procedure (Qiagen).
RNA analysis.
Metabolically radiolabelled BUNV-specific RNAs were separated using agarose/urea gel electrophoresis and visualized by using fluorography and autoradiography, as described previously (Barr et al., 2003).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) (Barr et al., 2003). This template directed transcription of characteristically 3'-truncated BUNV S-segment mRNAs, showing that signals responsible for S-segment transcription termination are located entirely within the 3' and 5' NTRs (Fig. 1c, lanes 1 and 2). We have previously shown that signals for the separate activities of mRNA transcription and RNA replication comprise nucleotides from both 3' and 5' ends of BUNV templates (Barr & Wertz, 2004, 2005; Barr et al., 2005). Therefore, we wanted to investigate whether sequences in both the 3' and 5' NTRs were also required for transcription termination. We therefore generated two altered templates based on the genome analogue BUN-S(ren). Template 3' (Fig. 1b) was generated by removing nt 26–85 of the genomic 3' NTR and template 5' (Fig. 1b) was generated by deleting nt 788–936 of the genomic 5' NTR. In each case, the deletions were made such that the extreme 25 nt of the 3' and 5' NTRs were left intact. We have previously shown that these nucleotides allow abundant replication and transcription of BUNV segments (Barr & Wertz, 2004).
We then tested the ability of these templates to generate the characteristically 3'-truncated S-segment mRNAs by resolving metabolically labelled BUNV-specific RNAs using agarose/urea gel electrophoresis. RNA analysis revealed that template 3' generated characteristically truncated BUNV mRNAs (Fig. 1c, lane 3), showing that the nt 26–85 region within the BUNV 3' NTR did not contain the termination signal. In contrast, mRNAs generated by template 5' exhibited mobility equal to that of the antigenomic RNAs, indicating that the termination signal that truncates transcription was eliminated (Fig. 1c, lane 4). Taken together, these results showed that signals essential for transcription termination are located within nt 788–936 of the S-segment genomic 5' NTR.
Mapping of the transcription termination signal within the BUNV S-segment 5' NTR
Having showed that the BUNV transcription termination signal resides within nt 788–936 of the S segment, we next wanted to determine which nucleotides within this 149 nt region comprised this signal. To achieve this aim, we divided the 149 nt sequence into eight regions designated term1–term8 (Fig. 2a). The eight regions were individually altered to generate eight templates named BUN-S(ren)-term1 to BUN-S(ren)-term8. The regions were altered such that each nucleotide was changed, but overall nucleotide composition was maintained (altered sequences are shown in Supplementary Table, available in JGV Online). However, due to their particular sequences and the cloning strategy used, the nucleotide composition of term3, -4 and -8 differed from their original sequences by a single nucleotide. Maximal preservation of nucleotide composition was important, as our agarose/urea gel system separated RNAs based on both size and sequence, and thus by maintaining nucleotide composition, we could relate changes in RNA mobility accurately to changes in RNA size. The eight templates were analysed for their ability to generate characteristically 3'-truncated mRNAs (Fig. 2b).
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, compare lanes 4, 5 and 6 with wt lanes 1 and 10). This finding showed that BUN-S(ren)-term3, BUN-S(ren)-term4 and BUN-S(ren)-term5 possessed altered transcription termination signals. As this phenotype was exhibited by all three templates, we concluded that the corresponding term3, term4 and term5 regions contained sequences that contributed to the termination signal. These regions comprised nt 829–880. All other templates within this series generated mRNAs with a mobility that was indistinguishable from that of mRNAs generated by BUN-S(ren).
Identification of a second transcription termination signal within the BUNV S segment
Above, we showed that templates BUN-S(ren)-term3, BUN-S(ren)-term4 and BUN-S(ren)-term5 did not perform authentic transcription termination, showing that nt 829–880 were involved in forming the BUNV termination signal. However, we noticed that mRNAs made by these templates did not co-migrate with the antigenomic RNA, indicating that transcription was terminated before the RdRp reached the 5' end of the genome. This phenotype was different to that exhibited by template 5', which generated mRNAs that co-migrated with the antigenome (Fig. 1c, lane 4). We propose two possible explanations for this. Firstly, the termination signal may comprise select nucleotides within regions term3, term4 and term5 and, unless these nucleotides are altered simultaneously, the signal may still be active, but may cause termination before the RdRp reaches the 5' end of the genome. Secondly, a second termination signal may exist downstream of the region encompassed by nt 829–880 and would only be evident when the upstream termination signal is inactivated.
To test the first possibility, we generated template BUN-S(ren)-829–880, in which the nt 829–880 region comprising term3, term4 and term5 was replaced with a scrambled sequence of the same length and nucleotide composition (Fig. 3a; see also Supplementary Table, available in JGV Online). Agarose/urea gel electrophoresis showed that mRNAs generated by BUN-S(ren)-829–880 were indistinguishable in mobility from mRNAs generated by templates BUN-S(ren)-term3, BUN-S(ren)-term4 and BUN-S(ren)-term5 (data not shown) described above (Fig. 3b, lane 2). This showed that simultaneous removal of nucleotides in term3, term4 and term5 (nt 829–880, Fig. 3a) did not alter the position at which transcription termination occurred.
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829–880 as a parental template to generate BUN-S(ren)-829–936, in which the nucleotides within regions term6, term7 and term8 (nt 881–936, Fig. 3a) were exchanged for a scrambled sequence of equal nucleotide composition (see Supplementary Table, available in JGV Online). RNA analysis showed that this template generated mRNAs with mobility equal to that of antigenomic RNAs (Fig. 3b, lane 3), showing that a second termination signal resided within nt 881–936 of the S segment, which was evident only when the upstream termination signal was removed. We also determined that the two termination signals acted independently, as disruption of the downstream termination signal alone (within nt 881–936) did not affect termination signalling at the upstream site (Fig. 3b, lane 4).
Characterization of the termination signal within nt 881–936
The above results showed that the S segment contains two independent transcription termination signals: an upstream signal located within nt 829–880 and a second signal within nt 881–936. To aid description, we named these two termination signals T1 (upstream) and T2 (downstream). To characterize T2 more precisely, we used BUN-S(ren)-829–880 as a parental template lacking the upstream termination signal, and divided the region containing the downstream termination signal (nt 881–936) into five regions of between 10 and 12 nt each. By altering these regions individually, we generated templates SEC1, SEC2, SEC3, SEC4 and SEC5 (Fig. 4a). For each template, the nucleotide blocks were removed and replaced with a different sequence of equal length and minimal change to nucleotide composition (see Supplementary Table, available in JGV Online). Agarose/urea gel electrophoresis revealed that mRNAs transcribed from SEC1, SEC3, SEC4 and SEC5 (Fig. 4b, lanes 4, 6, 7 and 8) co-migrated with mRNAs from BUN-S(ren)-829–880 (Fig. 4b, lane 2), indicating that these templates still contained sequences that signalled termination before the RdRp reached the 5' end of the genome. However, template SEC2 (Fig. 4b, lane 5) generated mRNAs that co-migrated with the antigenomic RNAs, showing that template SEC2 had lost all ability to terminate transcription. Confirmation that SEC2 generated both mRNAs and antigenomic RNAs was made by primer-extension analysis using a negative-sense oligonucleotide (results not shown).
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SEC2. To do this, we divided the 12 nt SEC2 sequence into four regions of 3 nt and each region was altered independently to generate four new templates named SEC2A, SEC2B, SEC2C and SEC2D (Fig. 4c). RNA analysis showed that the T2 termination signal of template SEC2A was active and generated mRNAs that were correspondingly truncated (Fig. 4d, lane 2). Template SEC2B showed a reduced ability to terminate at the T2 location, such that the quantity of transcripts terminated at the T2 position was reduced (Fig. 4d, lane 3). In contrast, templates SEC2C and SEC2D generated no transcripts terminated at T2 and, consequently, all mRNAs co-migrated with the antigenomic RNA (Fig. 4d, lanes 4 and 5). This analysis showed that the T2 termination signal is contained within the genomic sequence 3'-UCUGUCGCC-5'.
Defining the boundaries of the BUNV S-segment upstream T1 termination signal
Having localized the upstream T1 signal to within 52 nt spanning regions term3, term4 and term5 (nt 829–880), we wanted to define the T1 signal more accurately. However, first we wanted to confirm that we had defined the boundaries of the T1 region correctly.
To achieve this, we inserted the 52 nt of T1 at an internal site within the authentic BUNV S-segment sequence to generate template S-BUN-T1-UP (Fig. 5a). These 52 nt were positioned at a naturally occurring NsiI site located 30 nt upstream of the existing T1 sequence. This template now possessed two T1 termination signals in tandem: the original T1 sequence and an identical upstream termination sequence, T1-UP (Fig. 5a). We reasoned that, if T1-UP contained the intact T1 termination signal, transcription termination would now occur in response to this upstream signal, leading to synthesis of a correspondingly shorter mRNA.
RNA analysis revealed that mRNAs generated by S-BUN-T1-UP were correspondingly truncated, indicating that transcription was terminated in response to the T1-UP signal (Fig. 5b, lane 2). We detected no mRNAs terminating at the downstream T1 signal, showing that T1-UP was an effective termination signal. Further confirmation that regions term3, term4 and term5 were all required to form T1 was provided by the finding that T1-UP sequences containing only term4 and term5 (nt 844–880, Fig. 5c, lane 3), only term3 and term4 (nt 829–860, Fig. 5c, lane 4) or term5 alone (nt 861–880, Fig. 5c, lane 5) were unable to terminate with wt ability.
Analysis of the role of conserved sequences within the BUNV 5' NTR in transcription termination
Above, we showed that 52 nt between positions 829 and 880 of the S-segment genomic 5' NTR contained the T1 termination signal. Consistent with this finding, the 3' end of the BUNV mRNA end has been shown previously to map within this region, specifically between nt 851 and 861 (Jin & Elliott, 1993). Next, we wanted to perform a more detailed mutagenesis of these 52 nt to define more accurately the minimal sequence required for signalling BUNV transcription termination.
To direct our mutagenesis, we first compared these 52 nt with S-segment sequences of several other orthobunyaviruses. This comparison revealed several regions that were conserved, suggesting that these sequences may perform conserved functions in the termination process (Fig. 6a, shaded boxes). In addition to these conserved sequences, the 52 nt sequence contains a potential stem–loop structure that is also predicted to be conserved structurally in many orthobunyaviruses (Fig. 6a, underlined with arrows). Aided by the identification of these conserved motifs, we divided the 52 nt into seven regions that spanned the entire fragment (Fig. 6a, regions A–G).
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The extent to which termination was affected by changes within these seven regions varied considerably. Alterations to regions A, C and G (Fig. 6b, lanes 3, 5 and 9) neither reduced the abundance of mRNAs terminated at T1-UP, nor resulted in the corresponding generation of mRNAs terminated at the downstream T1 site, showing that regions A, C and G play no detectable role in forming the T1-UP signal. This finding was surprising, as regions A and G display extensive conservation with other members of the genus Orthobunyavirus (Fig. 6a). In contrast, template S-BUN-T1-UP-F (Fig. 6b, lane 8) showed complete loss of termination signalling at the T1-UP site, showing that corresponding F-region nucleotides are crucial for forming the termination signal. Alterations within the remaining B, D and E regions affected the termination signalling ability of the upstream T1-UP signal only slightly, as evidenced by a low abundance of mRNAs terminated at the downstream T1 site (Fig. 6b, lanes 4, 6 and 7). Although these mRNAs were present at low abundance, they were detected reproducibly in repeated experiments. This analysis showed that the sequences involved in forming the T1 termination sequence were contained in regions B, D, E and F, a region spanning a 33 nt linear sequence between nt 841 and 873.
Definition of the minimal sequence of the BUNV T1 transcription termination signal
To determine whether all nucleotides within the region nt 841–873 contributed to the termination signal, we performed systematic mutagenesis of successive nucleotide triplets throughout the 33 nt region of the upstream T1-UP termination signal of template S-BUN-T1-UP. We chose to substitute each nucleotide triplet with the corresponding complementary sequence, thus maintaining any possible spacing requirements that might exist between nucleotides within this sequence. This approach generated 11 templates, named S-BUN-T1-UP1 to S-BUN-T1-UP11 (Fig. 7a). We then analysed whether these alterations affected the termination ability of the T1-UP signal.
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, lanes 9 and 10) and consequently resulted in exclusive termination at downstream T1. Alterations at triplets 1 and 2 resulted in slight reductions in T1-UP termination signalling ability such that a band corresponding to the downstream T1 termination product was visible (Fig. 7b, lanes 1 and 2). Alterations to all other triplets resulted in a low abundance of downstream T1-terminated mRNAs, indicating that these alterations caused minor, if any, reductions in T1-UP termination signalling ability.
Comparison of the BUNV S-segment T1 and T2 termination signals with 5' NTR sequences of BUNV M and L segments
Above, we determined the nucleotide sequence of the BUNV S-segment T1 and T2 termination signals. Interestingly, a comparison of the T1 and T2 sequences revealed a common pentanucleotide, 3'-UGUCG-3' (Fig. 8a), suggesting that the T1 and T2 signals may utilize similar mechanisms to affect RdRp function.
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, vertical lines), one of which included the pentanucleotide sequence shared between the T1 and T2 signals (Fig. 8b, shaded area). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The majority of negative-sense RNA virus transcription termination signals that have been characterized directly or identified indirectly through sequence analysis have revealed a common sequence composition that comprises a U tract preceded by a short A/U-rich region directly upstream (Whelan et al., 2004). For VSV, these two elements allow reiterative transcription on the U tract to generate the characteristic VSV mRNA 3' poly(A) tails (Barr et al., 1997; Barr & Wertz, 2001). There is also evidence to suggest that generation of poly(A) sequences is a prerequisite for allowing termination to occur, suggesting that reiterative transcription and transcription termination are mechanistically dependent processes (Barr et al., 1997). Due to the conservation of NNS RNA virus termination signals (Whelan et al., 2004), this may be a universal scenario.
The BUNV termination signal does not contain a U tract and this finding is consistent with the observation that BUNV mRNAs do not possess 3' poly(A) tails. Taken together, we suggest that the BUNV termination signal not only represents a new class of termination signal, but probably acts to terminate transcription by using a mechanism that is unrelated to that of other negative-sense RNA viruses that polyadenylate their mRNAs.
Sequence comparison of T1 and T2 signals showed that, whilst these sequences are different in length, they are related in sequence, with both containing the conserved pentanucleotide 3'-UGUCG-5'. This pentanucleotide is conserved in all sequences shown in Fig. 6(a) with the exception of Inkoo virus, which exhibits a single nucleotide deviation to give the sequence 3'-CGUCG-5'. This 3'-CGUCG-5' sequence is also present in the corresponding regions of La Crosse, Germiston and snowshoe hare viruses. These viruses are not represented in Fig. 6 due to their slightly lower overall nucleotide identity across this region; however, the presence of the related pentanucleotide 3'-CGUCG-5' suggests that these orthobunyaviruses probably utilize termination signals that are closely related to that of BUNV. It is interesting to note that, for BUNV, La Crosse, Germiston and snowshoe hare viruses, this conserved pentanucleotide lies downstream of their experimentally determined mRNA 3' ends (Patterson & Kolakofsky, 1984; Eshita et al., 1985; Cunningham & Szilágyi, 1987; Bouloy et al., 1990; Jin & Elliott, 1993).
Mutagenesis of the T1 signal (Figs 6 and 7) showed that the conserved pentanucleotide plays a major role in forming the termination signal, whilst other nucleotides within T1 play minor roles not readily detected when changed individually. We suggest that individual nucleotides outside the conserved pentanucleotide exert a minor effect on termination signalling ability; however, their role is important due to their cumulative effect. Direct evidence that these minor-role nucleotides are components of the termination signal was shown by our findings that changes to regions S-BUN-T1-UP-B, -D and -E, which included nucleotides outside the pentanucleotide, all affected T1 signalling activity (Fig. 6b, lanes 4, 6 and 7), as did templates having T1-UP sequences truncated from the full term3, term4 and term5 sequences (nt 829–880) to term3 and term4 alone (nt 829–860, Fig. 5b, lane 4).
It is well established that poly(A) tails of eukaryotic mRNAs regulate both mRNA turnover and translation. BUNV may have developed mechanisms to generate alternative structures or modifications to the 3' mRNA end that act as substitutes for poly(A) tails. To search for a potential structure, we analysed the mRNA 3' end by using Mfold version 3.1 (Zuker, 2003). This analysis predicted a stem–loop structure positioned just upstream of the BUNV S mRNA 3' end (Jin & Elliott, 1993). Interestingly, whilst the sequence of this stem–loop is not well conserved in other orthobunyaviruses, the predicted stem–loop is conserved in both structure and position (Fig. 6a, horizontal arrows), supporting the possibility that this sequence may be functionally important. The finding that nucleotide changes to this stem–loop only affected termination slightly allows the possibility that it may instead provide mRNA stability and translation enhancement. We are currently investigating this possibility.
The finding that the BUNV S segment possesses two termination signals in tandem was surprising. We are curious as to why BUNV would maintain two termination sequences, especially given the high termination ability of the upstream T1 signal. Perhaps T2 is an evolutionary relic that has been functionally replaced by the T1 sequence. This explanation may be particularly likely if the T1 sequence contains a stability or translation enhancer that is absent from the T2 signal.
Comparison of the S-segment T1 and T2 signals with the M- and L-segment 5' NTRs identified a related sequence within the L segment. This sequence contained the conserved pentanucleotide sequence and was located 40 nt from the genomic 5' terminus. In contrast, no related sequences were found within the same region of the M segment. These findings are consistent with our previous results showing that the truncation of mRNAs generated from S, M or L segment-specific genome analogues was greatest for the S segment and lowest for the M segment, with the L segment exhibiting an intermediate level of truncation. The finding that the M segment does not possess a related termination signal also allows the possibility that BUNV M mRNAs are not truncated and are in fact co-terminal with the M segment antigenomic strand.
Comparison of the T1 and T2 signals with those of other orthobunyaviruses revealed that four regions were especially well conserved: an upstream region (Fig. 6a, region A), the potential hairpin (regions B and D), the pentanucleotide sequence (region F) and the sequence 3'-CCCACCC-5' (region G). Our analysis showed that, whilst the hairpin and pentanucleotide sequences were components of the termination signal, the conserved 3'-CCCACCC-5' sequence was not. Remarkably, this motif is also present in a corresponding location within the S segments of several members of the genus Hantavirus (Hutchinson et al., 1996). This conservation suggests functional importance and we are currently investigating what this function might be.
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ACKNOWLEDGEMENTS |
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Received 18 July 2005;
accepted 29 September 2005.
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