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Short Communication |
1 Molecular Biology Program, New Mexico State University (NMSU), Las Cruces, NM 88003, USA
2 School of Life Sciences, Queensland University of Technology, Brisbane, QLD 4001, Australia
3 Department of Biology, New Mexico State University (NMSU), Las Cruces, NM 88003, USA
Correspondence
Kathryn A. Hanley
khanley{at}nmsu.edu
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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A supplementary table showing post-transfection titres of wild-type DEN (rDEN4) and viruses carrying mutations in the 3' untranslated region is available in JGV Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Weaver & Barrett, 2004). Despite the urgent need to control this escalating pandemic, neither antiviral therapies nor a licensed vaccine for DEN are available (Halstead & Deen, 2002). Rational vaccine design, which employs targeted mutations in the DEN genome to generate vaccine candidates, is being actively pursued (Blaney et al., 2006; Pugachev et al., 2005). However, the success of this strategy depends upon detailed knowledge of both structure and function of the region to which mutations are added (Alvarez et al., 2005b; Martin, 1994; Proutski et al., 1997).
The current study investigates the secondary structure of the DEN4 3' untranslated region (UTR). DEN possesses a single-stranded, positive-sense RNA genome that encodes a single polypeptide and is organized as follows: 5' UTR–C–prM–E–NS1–NS2A–NS2B–NS3–NS4A–NS4B–NS5–3' UTR (C, capsid protein; prM, membrane precursor protein; E, envelope protein; NS, non-structural protein) (Rice, 1996). The 3' UTR is approximately 450 nt long and encompasses three regions: (i) a variable region (VR) immediately 3' of the stop codon, (ii) a core region downstream of the VR and (iii) a 3'-terminal region, which contains the cyclization sequence (CS) and a terminal long stable hairpin (LSH) (Markoff, 2003). The 3' UTR is involved in circularization of the genome through interactions with the 5' UTR (Alvarez et al., 2005a).
The function of the flavivirus 3' UTR depends on both primary sequence and secondary structure (Alvarez et al., 2005b; Elghonemy et al., 2005; Markoff, 2003; Thurner et al., 2004; Tilgner & Shi, 2004; Tilgner et al., 2005; Yu & Markoff, 2005), thus structural analysis of the DEN 3' UTR may contribute to understanding of several aspects of flavivirus biology. The nearly 80 flavivirus species group into three lineages: (i) mosquito-borne, (ii) tick-borne and (iii) those with no known vector (Cook & Holmes, 2005). Conserved regions of both primary sequence and secondary structure that vary among these three lineages have been identified in both the core and 3'-terminal regions of the 3' UTR (Leyssen et al., 2002; Markoff, 2003). Such conserved elements may potentially influence a variety of virus properties, including virus replication, host-cell tropism, vector specificity, pathogenicity and virulence (Alvarez et al., 2005b; Blaney et al., 2006; Markoff, 2003; Yu & Markoff, 2005; Zeng et al., 1998).
Although the secondary structure of the DEN 3' UTR has been predicted by using a variety of computer models (Olsthoorn & Bol, 2001; Proutski et al., 1997; Shurtleff et al., 2001), experimental verification of the formation of these structures in vitro (e.g. Chen & Frey, 1999; Tuplin et al., 2004) is lacking. Thus, the current study had three objectives: to (i) generate computer predictions of the DEN4 3' UTR core region; (ii) compare these predictions with patterns generated by nuclease maps of this region; and (iii) assess the secondary structure and viability of DEN4 viruses in which conserved elements in the 3' UTR have been either deleted or replaced with homologous sequence from a tick-borne virus.
Mfold (Zuker, 2003) was used to predict the secondary structure of the core region of the wild-type DEN4 3' UTR (isolate Dominica 1981; GenBank accession no. AF326825
[GenBank]
). To maintain consistency with previous predictions for this region, Mfold predictions were constrained to maintain the structures of elements upstream and including repeat CS2 (RCS2) and the 3' LSH (stem–loops 6 and 7) (Markoff, 2003; Proutski et al., 1997; Shurtleff et al., 2001; Thurner et al., 2004). The lowest free-energy structure was compared with a nuclease map of this region generated by single- and double-strand-specific RNases (Fig. 1b). This RNase map was generated following methods described by Tuplin et al. (2004); briefly, DEN4 cDNA p4 (Durbin et al., 2001) was transcribed as described previously (Hanley et al., 2002) and the transcript was treated with RNases T1 (0.05 U µl–1), A (0.01 U µl–1) and V1 (0.005 U µl–1) (Ambion) to cleave the 3' end of single-stranded guanines (Gs), the 3' end of single-stranded uracils and cytosines (Us and Cs) and double-stranded nucleotides, respectively. Reverse IR-700 primer D4.3'UTR.TR.001 (Li-Cor Biosciences) (Fig. 2) was used for reverse transcription of the digested RNA constructs and sequencing of the undigested cDNA constructs. Primer-extension controls using undigested template were included to detect non-specific cleavage and premature transcriptional termination (intrinsic stops) during reverse transcription. All products were analysed with a Li-Cor Biosciences 4200 DNA sequencer.
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); and (ii) RNases T1 and A cut at 3' ends of single-stranded nucleotides and could be recognizing these ends as single-stranded.
Three areas of inconsistency between Mfold predictions and the RNase map were identified. First, nuclease cleavages at nt U10420
[GenBank]
and G10429
[GenBank]
indicated that these were single-stranded when they were predicted to be double-stranded by Mfold. RNase V1 cleavage between nt A10505 and A10506 indicated double-stranded nature in this region and RNase T1 cleavage of nt G10516
[GenBank]
showed it to be single-stranded. The initial Mfold prediction was revised accordingly. Moreover, nt C10473
[GenBank]
and C10474
[GenBank]
were constrained to be single-stranded in this revised structure to achieve a more favourable free-energy value. The revised Mfold prediction is depicted in Fig. 1(a)
. Second, predictions at nt C10462
[GenBank]
and U10511
[GenBank]
were ambiguous, as cleavages occurred in both RNase A and V1 lanes. Bands were absent in the nuclease-free controls, indicating that these were not intrinsic stops. These regions may participate in dynamic folding, as discussed in detail below. Third, regions 1 and 2 (Fig. 1a, b) were predicted to be single-stranded by Mfold, but were shown to be double-stranded by RNase mapping. Both regions have previously been predicted to contribute to RNA pseudoknots, designated pk2 and pk1, that pair with regions designated pk2' and pk1' (Olsthoorn & Bol, 2001). Mfold is unable to predict pseudoknots (Zuker, 2003). Thus, seven nuclease cleavages out of 30 total cleavages were not predicted by Mfold, resulting in 23 % inconsistent cleavages.
To expand the relevance of this study for vaccine design, an Mfold prediction and nuclease map were also generated for p4delta30 (GenBank accession no. AY376438
[GenBank]
), the construct used to generate vaccine candidate rDEN4delta30 (GenBank accession no. AF326827
[GenBank]
), which carries a deletion of nt 10477–10507 in the 3' UTR (Fig. 2) (Durbin et al., 2001; Men et al., 1996). For this construct and all remaining constructs, Mfold predictions utilized constraints to maintain previously predicted structure in RCS2 and elements upstream of RCS2 because cleavages in these two areas were identical to p4. The 3' LSH was also constrained in all structures. The remainder of the predicted region was unconstrained (Fig. 3a). Seventy-one per cent of enzymic cleavages in the nuclease map of p4delta30 corresponded to the Mfold prediction. Ambiguous cleavages were detected at nt C10462
[GenBank]
and U10480.
[GenBank]
Regions 1 and 2 (Fig. 3a) identify the same putative pseudoknots as seen in Fig. 1(b). Nucleotide U10487
[GenBank]
, detected as single-stranded by RNase mapping, was located in an area predicted to be double-stranded by Mfold.
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): (i) deletion of conserved element CS2 (Hahn et al., 1987; Mandl et al., 1993), which includes nt 10508–10530 of DEN4, as well as a second conserved element termed conserved difference A (CD-A), which includes nt 10536–10545 of DEN4 and which was identified by alignment of flavivirus 3' UTR sequences [K. A. Hanley and others, unpublished data; this region was not identified as being homologous by Olsthoorn & Bol (2001)]; or (ii) replacement of these regions with homologous sequence from tick-borne Langat virus (LGT) (GenBank accession no. AF253419
[GenBank]
; Pletnev et al., 2001). DEN4 CS2 was replaced with LGT nt 10508–10528 and DEN4 CD-A was replaced with LGT nt 10536–10551. Mutations were generated via Kunkel mutagenesis and inserted individually into p4 by using previously described methods (Hanley et al., 2002) to create the following constructs: p4deltaCS2, p4deltaCD-A, p4LangatswapCS2 and p4LangatswapCD-A. Mfold predictions and nuclease maps were generated for each construct (Fig. 3b–e).
Nuclease maps were highly consistent with Mfold predictions for all four constructs (64, 83, 75 and 75 %, respectively). p4deltaCS2 had the lowest consistency, due to a relatively low number of cleavages; a low number of cleavages may be attributable to steric radii in the nucleases that sometimes block access to solvated nucleotides (Christiansen & Garrett, 1988). Moreover, the two putative pseudoknots identified in p4 (regions 1 and 2) were also identified in each of the four constructs (p4LangatswapCD-A had only region 2). In p4deltaCS2, secondary structure at nt C10462
[GenBank]
was ambiguous. In p4deltaCD-A, secondary structure at nt U10511
[GenBank]
was ambiguous. In p4LangatswapCD-A, nt 10543 and 10546 (region A) were predicted to be single-stranded by Mfold, but were shown to be double-stranded in the nuclease map.
These data are consistent with the existence of RNA pseudoknots predicted by Olsthoorn & Bol (2001) in regions 1 and 2. The observation that pk1', the pk1 complement, has single-stranded cleavages at nt G10489
[GenBank]
and U10491
[GenBank]
must therefore be explained. These band intensities were exceptionally low, suggesting that some transcripts were folded incompletely. The secondary-structure predictions of Proutski et al. (1999) for the DEN4 3' UTR did not identify pseudoknots pk1 and pk2, but did identify pseudoknots at the 3' terminus of the region. An additional pseudoknot may occur in region A (Fig. 3e) of p4LangatswapCD-A. Interestingly, nt 10542–10547 (CUCCUC) in this construct complement nt 10520–10525 (GAGGAG), the latter located in a loop of a branched internal stem–loop. Positive-sense RNA viruses tend to fold into extensive quaternary structures (Simmonds et al., 2004), thus discrepancies between Mfold and nuclease maps could represent genome-scale ordered RNA structure (GORS) rather than pseudoknots and reflect long-range interactions in the genome that could not be characterized fully with the techniques used in this study. Based on the work of Alvarez et al. (2005a), interactions between the 3' and 5' UTRs seem a likely possibility for such long-range interactions.
Ambiguous cleavages were observed at either C10462
[GenBank]
or U10511
[GenBank]
(U10480
[GenBank]
in p4delta30) or both in most transcripts. Ambiguous cleavages often occur where a sequence may be sensitive to both V1 and a single-strand-specific nuclease (Christiansen & Garrett, 1988; Lowman & Draper, 1986; Ziehler & Engelke, 2000). C10462
[GenBank]
is the 3' nucleotide of the pk2 pseudoknot (Olsthoorn & Bol, 2001), thus a double-stranded cleavage is expected. Single-stranded cleavage at this nucleotide may be due to a lack of complete folding or to recognition of the 3' end of C10462
[GenBank]
as single-stranded. Alternatively, this may be a dynamic region where pseudoknots form transiently. In p4, U10511
[GenBank]
is located in a 2 bp stem within a large stem–loop structure and is predicted by Mfold to be double-stranded. It is cleaved by both V1 and RNase A; as mentioned previously, the latter sometimes cuts at double-stranded regions. In p4delta30, U10480
[GenBank]
is located at the top of a stem–loop structure at the 5' end of the loop. It is predicted to be single-stranded by Mfold and is cleaved by both RNases A and V1 in the RNase map. RNase V1 can cleave adjacent to, rather than within, helices (Lowman & Draper, 1986), which may account for this ambiguous cleavage. In p4deltaCD-A, U10511
[GenBank]
is predicted to be double-stranded by Mfold, but the structure is kinked between the major stem–loop structure and an internal stem–loop branching off it. Such a kink may also produce an ambiguous cleavage.
Each of the six transcripts described above was transfected in duplicate into both simian (Vero) and mosquito (C6/36) cells and the presence of virus in the cell supernatant was detected as described previously (Hanley et al., 2002). All six constructs produced viable virus in at least one of the two cell types. Titres following transfection ranged from 2.2 to 5.4 log10(p.f.u. ml–1) in Vero cells and from <0.7 to 7.9 log10(p.f.u. ml–1) in C6/36 cells (see Supplementary Table S1, available in JGV Online). Thus, despite their substantial impacts on secondary structure, neither the deletions nor the substitutions described in this study abolished viability completely. Future research will assess the impacts of these mutations on the phenotypes, particularly host-cell tropism and vector specificity, of the viruses carrying them.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 5 May 2006;
accepted 26 June 2006.
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) indicate cleavages used to revise the Mfold prediction in (a).
) indicate a nucleotide found to be single-stranded in the RNase map and double-stranded in the Mfold prediction. Single-stranded regions 1 and 2 (circled) in the Mfold prediction were found to be double-stranded in the nuclease map. In RNase maps, CS2 and CD-A are indicated with a dashed and a solid box, respectively. RNase maps and Mfold structures were cropped to focus on regions affected by mutations.