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Short Communication |
Centre for Ecology and Hydrology, Mansfield Road, Oxford OX1 3SR, UK
Correspondence
T. S. Gritsun
tsg{at}ceh.ac.uk
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ABSTRACT |
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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According to the Seventh Report of the ICTV (Heinz et al., 2000
), the flaviviruses are subdivided into three ecological groups, the mosquito-borne, tick-borne and no-known-vector flaviviruses (designated MBFV, TBFV and NKV, respectively). However, this classification scheme does not take into account three additional viruses, Kamiti River virus (KRV), Cell fusing agent virus (CFAV) and Tamana bat virus (TBV), that are antigenically unrelated to the other flaviviruses but share 
47 % polymerase protein sequence identity and a similar genome strategy. Moreover, these three viruses are relatively closely related to each other, with 81 % polymerase protein sequence identity (Cammisa-Parks et al., 1992; Crabtree et al., 2003; de Lamballerie et al., 2002; Sang et al., 2003). They are currently classified as tentative species in the genus Flavivirus and will be referred to as the non-classified flaviviruses (NCFV). Two of these viruses, CFAV and KRV, are not arboviruses, they are insect viruses, being found only in mosquitoes. Under laboratory conditions they replicate only in mosquito cell lines (Crabtree et al., 2003; Sang et al., 2003). Phylogenetically, they occupy the most divergent lineages in the genus Flavivirus, having diverged from the other flaviviruses before the separation of the MBFV and TBFV groups (Cammisa-Parks et al., 1992; Chastel et al., 1995; Cook & Holmes, 2006; Crochu et al., 2004). An unusual feature of KRV and CFAV is that they appear to be able to integrate their RNA into mosquito cell genomes as cDNA (Crochu et al., 2004). Flaviviruses are small, 50 nm diameter, enveloped viruses with positive single-stranded RNA genomes approximately 11 000 bases long. The genomic RNA contains a single open reading frame (ORF) that encodes a polyprotein of approximately 3400 amino acids which is co-translationally processed into the individual structural (C, M and E) and non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) proteins. The non-structural proteins have proteolytic and replicative functions (Lindenbach & Rice, 2001).
The ORF is flanked by the 5' and 3' untranslated regions (UTRs). These UTRs have attracted significant interest because genetic modifications within their sequence can attenuate flaviviruses without altering their antigenic specificity, making them potential candidates for live attenuated virus vaccines (reviewed by Markoff, 2003). They are also efficient targets for antisense oligonucleotides that may act as antivirals by preventing virus replication (Deas et al., 2005; Kinney et al., 2005). Computer-simulated predictions show that the RNA of the UTRs forms stable secondary structures that are conserved between different virus groups and are thought to be the sites for initiation of virus translation, replication and assembly (Charlier et al., 2002; Gritsun et al., 1997; Leyssen et al., 2002; Proutski et al., 1997a, b, 1999; Rauscher et al., 1997; Thurner et al., 2004).
The 3'UTRs of flaviviruses exhibit interesting features that reflect their evolutionary characteristics, such as repeat sequence elements. These direct repeats were originally described in the MBFV group as CSs (conserved sequences) and RCSs (repeated conserved sequences), 20–40 nucleotides long. Some of the MBFV CS/RCSs form tandem repeat sequences, whereas others are separated by hundreds of nucleotides (Hahn et al., 1987; Mutebi et al., 2004). Similar direct repeat elements have also been described for the NKV, TBFV and NCFV (Charlier et al., 2002; Crabtree et al., 2003; Gritsun et al., 1997; Mandl et al., 1991; Wallner et al., 1995), although no homology has been described between direct repeats from different flavivirus groups. The functions of these repeat sequences are not clear. Experimental deletion of CSs or RCSs in infectious clones did not abolish virus infectivity, although it reduced virus replication rates (Bredenbeek et al., 2003; Khromykh & Westaway, 1997; Lo et al., 2003; Mandl et al., 1998; Men et al., 1996; Pletnev, 2001; Tilgner et al., 2005). These repeat sequences probably function as signals for a variety of molecular interactions that facilitate virus transmission and dissemination, a function that has not been evaluated in the laboratory (Gritsun et al., 2006).
Two direct repeats were described as 67-nucleotide long elements in the CFAV and KRV 3'UTRs (KRV-R). They were almost identical, but their linear arrangement was different. In the CFAV 3'UTR, they occurred virtually in tandem, being separated by only 24 nucleotides, whereas in the KRV 3'UTR, these repeats were separated by a sequence of 534 nucleotides (Crabtree et al., 2003; Sang et al., 2003). The origin of the 534-nucleotide sequence was not clear.
To investigate how these linear repeat elements (KRV-R) and the 534 nucleotide sequence arose, we aligned KRV and CFAV, initially using CLUSTAL X (Thompson et al., 1997). Since evolution of the flavivirus 3'UTR involves both deletions and substitutions (Gritsun et al., 1997; Mandl et al., 1998; Mutebi et al., 2004; Wallner et al., 1995), we manually introduced long and short gaps to reveal homology between distantly located regions (Fig. 1).
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). This could have occurred by the dissociation of the RNA-dependent RNA polymerase (RdRp) from the template, upstream of the first repeat (partition site P), subsequently reassociating immediately downstream of the second repeat (anchoring site A) (Fig. 2a), resulting in the replication of the second repeat and shortening the nucleotide sequence between the two repeats. This is consistent with the mechanisms described previously (Pilipenko et al., 1995).
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. In this scenario, the RdRp would have had to skip twice; in the first instance dissociating from the original template and reassociating with the foreign template, followed by the amplification of approximately 500 nucleotides. Subsequently, the RdRp would have to reassociate with the original template, anchoring the growing strand to the region downstream of the second repeat. In either case, all the molecular events would be random, not requiring any special mechanisms except for the presence of short repeated nucleotide sequences in partition/anchoring sites as described previously (Pilipenko et al., 1995). The origin of the insertion, whether or not it is foreign, might indicate which of the above possibilities is the most likely. Considering the first possibility, i.e. a KRV-like virus being the precursor of CFAV, the question arises, how were these repeats originally separated by a very long sequence? One possibility is that they could represent the remains of longer repeats that subsequently evolved, very slowly in the 64-nucleotide region and more rapidly in the loci outside the repeats.
We attempted to trace the remnants of the longer repeated sequences that flank the KRV repeats. This was done by aligning the regions around the repeats, using CLUSTAL X (Thompson et al., 1997), followed by manual introduction of deletions and insertions between regions of high sequence identity (Fig. 3a). The BioEdit Sequence Alignment Editor (Hall, 1999) was used to determine the identity between different regions along the alignment. The identity between the approximately 600-nucleotide duplicates (without gaps) was 62 % and the KRV repeat sequence, comprising 67 nucleotides, showed 96 % nucleotide identity. The alignment in Fig. 3(a) shows a gradual decrease in nucleotide identity either upstream or downstream of the 67-nucleotide KRV repeats. Thus, the region of 200 nucleotides immediately downstream of the repeats shows 71 % identity, which decreased to 60 % in the terminal 100 nucleotides. Even after removal of the KRV repeat, the level of nucleotide identity along the alignment was 60 %. For comparison, the overall level of nucleotide identity between the genomes of KRV and CFAV was 60 %. This level of nucleotide identity is significantly higher than otherwise might be expected in random nucleotide sequence alignments, which should be about 25 %.
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), there was approximately 80 % identity between the 3'UTRs of KRV and CFAV, but the identity was 60 % in the corresponding region of the KRV self-alignment. This implies that the KRV 3'UTR duplication event preceded evolution of the CFAV 3'UTR and the subsequent deletion of 510 nucleotides between the repeats.
Thus, the 3'UTR of KRV appears to have arisen by duplication of a precursor sequence (Fig. 3b) that subsequently evolved, leaving the KRV/CFAV repeats conserved, probably because they have an essential function in virus replication. The 510 nucleotide deletion between the repeats that occurred in CFAV (Fig. 2a) was a secondary event, again suggesting that the duplicated conserved sequences are vital for virus survival in the environment. Duplication of 216 nucleotides has been previously described in the 3'UTR for one natural strain of YFV isolated in South America (Bryant et al., 2005).
In conclusion, we have demonstrated that the KRV 3'UTR was formed as the result of duplication of a 3'UTR sequence that probably represented an early lineage in the evolution of KRV and CFAV. Following this duplication event, the subsequent evolution of the KRV 3'UTR probably involved a combination of mutations, substitutions, deletions and insertions. However, the 67-nucleotide repeat sequence was virtually completely conserved, implying that it provides an important biological function. This is confirmed by the preservation of these repeats in CFAV, which lost a long region of sequence in the 3'UTR, leaving the 60-nucleotide repeats intact. Further research designed to predict and compare the secondary RNA structures of MBFVs and NKVs is currently being carried out to extend our understanding of the structure and function of the flavivirus 3'UTRs.
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Received 14 February 2006;
accepted 12 May 2006.
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