Laboratory of Virology and Chemotherapy1 and Laboratory of Clinical and Epidemiological Virology2, Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Leiden Institute of Chemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands3
Unité des Virus Emergents, Faculté de Médecine de Marseille, 27 Boulevard Jean Moulin, 13005 Marseille cedex 5, France4
Author for correspondence: Johan Neyts. Fax +32 16 33 73 40. e-mail johan.neyts{at}rega.kuleuven.ac.be
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Abstract |
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Introduction |
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We present here: (i) the complete genome sequence (coding and non-coding regions) of MMLV; (ii) the particular characteristics (including the phylogeny) of this genome; and (iii) a detailed and comparative study of the organization and the secondary structure of the 3' UTRs of MMLV and three other NKV flaviviruses (RBV, MODV and APOIV). Furthermore, we report that the pentanucleotide sequence CACAG, which is conserved in the 3' UTR of all arboflaviviruses, is replaced by the sequence C(C/U)(C/U)AG in NKV flaviviruses.
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Methods |
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Generation of PCR fragments.
Viral RNA was extracted from 140 µl of supernatant medium of virus-infected cells, using the QIAamp Viral RNA kit (Qiagen). A reverse transcription (RT) reaction was designed employing the reverse primer 5' GGGTCTCCTCTAACCTCTAG 3'. Three sets of degenerated primers were designed based on the alignment of full-length genome sequences of different flaviviruses (Table 1). Other primers were designed based on: (i) the sequence of the amplicons generated by the first primer sets (a list of the primers is available upon request); and (ii) the sequence of a fragment of MMLV of approximately 1 kb (nt 89299939; Kuno et al., 1998
) (Fig. 1
). All PCR amplifications were achieved under standard conditions using Taq polymerase (HT Biotechnology) and 30 cycles including long polymerization steps (12 min depending on the expected size of the amplicons).
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Cloning and sequence analysis.
PCR products were cloned into the TOPO Cloning vector (Invitrogen) or the pGEM-T Vector System I (Promega) and One Shot competent E. coli cells (Invitrogen) were used for transformation. The cloned inserts were sequenced in a cycle sequencing reaction with fluorescent dye terminators and analysed using an ABI 373 automatic sequencer (PerkinElmer).
Prediction of RNA secondary structure.
The RNA secondary structure of the 3' UTRs of MMLV and of the three other NKV flaviviruses (MODV, RBV and APOIV) was analysed using the STAR program (Gultyaev et al., 1995 ). After folding each of the four sequences separately, the resulting structures were searched for common structural elements as shown by the occurrence of covariations in the stem regions in one or more of the other three sequences. These sequences, containing proven structures, were excised and replaced by five non-pairing nucleotides. The shortened sequences were then submitted to a new cycle of folding and this was repeated until no further common elements were detected.
Alignments and phylogenetic analysis.
The complete amino acid sequences of the flaviviruses listed above were aligned using the ClustalW (1.74) software (Monath & Lipman, 1988 ) and default alignment parameters, and manually edited in McClade (Maddison & Maddison, 1989
). Conserved motifs allowed an unambiguous control of validity for alignment as previously reported (Billoir et al., 2000
).
Genetic distances were estimated using maximum-likelihood calculation in TreePuzzle-5.0 (Strimmer & Von Haeseler, 1996 ) based on the Blosum62 (Henikoff & Henikoff, 1992
) model of substitution and taking into account a rate heterogeneity among sites with a discrete gamma distribution of eight categories. An unrooted phylogenetic tree based on the inferred distance matrix was constructed with NEIGHBOR in PHYLIP 3.5. Bootstrap analysis was performed according to the following algorithm: 1000 replicates were generated by SEQBOOT (PHYLIP) and redirected with Puzzleboot to TreePuzzle 5.0 where distance matrices were estimated. These distance matrices were subsequently used to infer phylogenetic trees in NEIGHBOR (PHYLIP) and a bootstrap consensus tree was generated with CONSENSE (PHYLIP).
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Results |
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Comparison of the amino acid sequence of MMLV with that of other flaviviruses revealed the presence of homologous protease cleavage sites (Table 2), internal signal sequences and transmembrane sequences (the C-terminal domains of the C, M, E and NS4A proteins are hydrophobic). As is the case for APOIV and RBV, the mature MMLV virion C protein, envelope and NS4A genes are markedly shorter than the corresponding genes of arthropod-borne flaviviruses (Table 3
).
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The flavivirus NS5 protein (the largest of the flavivirus-encoded proteins) encodes an RNA-dependent RNA polymerase and also contains a putative methyltransferase domain (Koonin, 1993 ). A heptapeptide sequence, containing the characteristic GDD sequence motif (Kamer & Argos, 1984
; Poch et al., 1989
), is also conserved in the MMLV NS5 gene (3136-SGDDCVV-3142). The same heptapeptide motif is also present in APOIV and RBV.
Predicted cleavage sites in the MMLV polyprotein
The N-termini of the proteins were defined, based on the position of the cleavage sites of other flaviviruses. Cleavage by the viral protease generally occurs following two dibasic residues and before an amino acid with a short side chain, whereas processing with a host protease occurs at sites obeying the (-3,-1) rule (Von Heijne, 1984 ). Table 2
summarizes the cleavage sites for the processing of the MMLV polyprotein. At the N-termini of prM, E and NS1 of MMLV, predicted signalase cleavage sites are detected, which are also contributed by the C-terminal hydrophobic regions of anchored C, prM and E, respectively. A signal sequence also precedes the N-terminus of NS4B, suggesting that this hydrophobic protein is processed in association with endoplasmic membranes. The N-terminus of NS2A follows a cleavage site defined by the sequence ValXAla (where X is Ser, Thr, Gln, Asn or Asp) (Cammisa-Parks et al., 1992
; Von Heijne, 1984
). In the case of MMLV, the sequence consists of ValSerAla.
Five of the flavivirus polyprotein cleavages take place after two basic amino acids (either LysArg or ArgArg or ArgLys) (Chambers et al., 1990 ): i.e. anchored Cvirion C, NS2ANS2B, NS2BNS3, NS3NS4A and NS4BNS5. In the case of MMLV, an ArgArg sequence is present at the C-termini of NS2B, NS3 and NS4B. For the other two cleavages (anchored Cvirion C and NS2ANS2B), suitable dibasic sequences could not be identified. For anchored Cvirion C, we suggest a cleavage site following a GlnArg pair. For NS2ANS2B cleavage may take place immediately after GlnPro (Gln is also present in the DEN2 and DEN4 NS2ANS2B cleavage site) (Mandl et al., 1998
). These sites were chosen based on the sequence alignment with the polyproteins of other flaviviruses (Table 1
) and on the notion that the dibasic sequences are usually flanked by amino acids with short side-chains, most commonly Gly, Ser or Ala. The prM protein is a glycoprotein precursor, which undergoes delayed cleavage to form M and the N-terminal pr segment. Akin to all flavivirus sequences, the N-terminus of the M protein of MMLV immediately follows a pair of basic amino acids believed to represent a cleavage site for either a viral or a host protease. The two amino acids are flanked by an amino acid with a short side-chain (Chambers et al., 1990
).
Characteristics of the 5'- and 3'-terminal nucleotide sequences
The ORF of the flavivirus genome is flanked by short non-coding regions, which may contain elements involved in the regulation of essential functions such as translation, replication or encapsidation of the genome (Cammisa-Parks et al., 1992 ). The 5' UTR of MMLV is 108 nucleotides long. The MMLV 3' UTR contains 460 nucleotides. As in other flaviviruses, the 3' UTR is not extended by a poly(A) tract. At each end of the genome, two terminal nucleotides, which are conserved among members of the whole Flavivirus genus, were detected, i.e. 5' AG and CU 3'. Besides these conserved terminal nucleotides, there is only one nucleotide sequence motif conserved among the mosquito- and tick-borne flaviviruses described. This conserved motif is a pentanucleotide sequence (5' CACAG 3') located approximately 4561 nucleotides from the 3' terminus (Wengler & Castle, 1986
). It is predicted that it is located on a side-loop of a conserved 3'-terminal secondary structure, suggesting that this motif would induce the formation of a circular RNA molecule, which could be important during replication or encapsidation (Chambers et al., 1990
; Khromykh et al., 2001
). From all 21 vector-borne flaviviruses (Table 4
) that were analysed, only Murray Valley encephalitis virus (MVEV) had a different pentanucleotide sequence, i.e. an A
C change at position 4 (CACCG). We confirmed the presence of this deviating pentanucleotide sequence in the MVEV genome by sequencing this particular area of the genome of this virus. An A
C change was also noted at position 4 of this pentanucleotide sequence in the genome of cell fusing agent virus. The second position of this pentanucleotide (at an analogous position, i.e. within the loop of a 3'-terminal stem and loop structure) was either a U or a C instead of an A for all four NKV flaviviruses. APOIV had, in addition, a C
U change at position 3. This pentanucleotide motif thus allows us to discriminate between NKV and vector-borne flaviviruses.
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Region II of the 3' UTR is predicted to form a Y-shaped structure. The 3' arm of the Y structure contains the CS2 sequence [5' G(A/U)CUAGAGGUUAGAGGAGACCC 3'], which was present in all four of the NKV viruses studied. The 5' hairpin on the main stem of region II varies considerably in length for the four NKV viruses, in contrast with other flaviviruses. Upstream from region II, a repeated structure (IIbis) is formed in the 3' UTR of APOIV, which contains the repeated conserved sequence 2 (RCS2) [5' GACUAG(A/C)GGUUAGAGGAGACCC 3']. Except for the CS2 sequence, the primary structure of region IIbis is not identical to the sequence of region II; however, the secondary structure is well conserved.
Three very similar hairpins (a, b and c) are predicted between regions I and II for MMLV and RBV, the two NKV flaviviruses that are also, according to the phylogenetic analysis (see below), most related. These consist of a stemloop (b), flanked by two shorter stemloops (a and c); loop b may form a pseudoknot at the 5' side. The existence of this pseudoknot is not only supported by its prediction with the STAR program but also by the presence of one covariation in each of the two stems of the pseudoknot.
Region III folds into a Y shape for all four NKV viruses studied. The two loops of the Y structure of region III of the 3' UTRs are formed by a conserved stretch of nucleotides. However, the sequences of the stems carrying these loops are not conserved, but rather show a large number of compensatory base changes, strongly supporting the proposed secondary structure.
In region IV, the 3'-terminal nucleotides of the 3' UTR of the NKV flaviviruses form a long stable hairpin structure (3' LSH), which preserves its shape despite significant differences in sequence. This 3' LSH was calculated to fold in the genome of the four NKV flaviviruses with a similar position of the conserved C(C/U)(C/U)AG motif (4561 nucleotides from the 3' terminus). At the 5' side of the 3' LSH, a small stemloop (belonging to region IV and probably coaxially stacking with the long 3'-terminal hairpin) is calculated for the four NKV flaviviruses.
Inspection of the 3' UTRs revealed the existence of a 6979 nucleotide long sequence motif (e.g. 5' GCUUUUGCUCCCGC G U U U U U C AA A U U G C C U C A U C U U G A A U G G - G G GGCGGCGUGGAUAUAUACUCCAGCC 3' for MMLV) located approximately 50 nucleotides away from the 3' terminus and representing an inverted repeat of another conserved sequence element located approximately 54 nucleotides from the 5' terminus (including the last 40 nucleotides of the 5' UTR and the first 29 nucleotides of the coding region). This may suggest a role in genome circularization. A similar but much smaller cyclization sequence has been observed for the tick-borne flaviviruses (Khromykh et al., 2001 ). The predicted folding of the four regions in the 3' UTR as reported here is supported by the fact that a large number of covariant and semi-covariant sites occur in base-paired regions.
Phylogenetic analysis
A phylogenetic analysis was performed using complete coding sequences (Fig. 3). Compared with the other flaviviruses, CFAV showed a similarity below 30% and was therefore unreliable as an outgroup for phylogenetic analysis of the complete genome of the flaviviruses. An unrooted phylogenetic tree including the complete ORF sequences of 19 flaviviruses, and constructed with the neighbour-joining method, was supported by high bootstrap values (ranging from 99·2 to 100%). Three major branches were observed: (i) the mosquito-borne virus branch; (ii) the tick-borne virus branch; and (iii) the NKV virus branch. This confirms the presence of MMLV in the group of the NKV viruses, as predicted by Kuno et al. (1998)
using a 1 kb fragment in NS5. The bootstrap value of 100% allows us to conclude that MMLV belongs to the RBV branch, which is consistent with the fact that both viruses have the bat as their vertebrate host.
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Discussion |
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Moreover, the maximum bootstrap value allowed us to conclude that MMLV belongs to the RBV branch, which is consistent with the fact that both viruses have the bat as their vertebrate host. APOIV and MODV (both isolated from rodents) are located in two distinct evolutionary branches, MODV being more closely related to MMLV and RBV than to APOIV.
The deduced amino acid sequence of MMLV revealed conservation of the main features of flaviviruses, i.e. cleavage and glycosylation sites of virus-specific proteins, and the presence of highly conserved motifs important for protease, helicase, methyltransferase and RNA-dependent RNA polymerase activity (Monath & Heinz, 1996 ). The fact that the genome of MMLV has the same organization as flaviviruses that are infectious to humans, as well as the same conserved regions in genes that can be considered as antiviral targets, further points to the relevance of this model in antiviral studies. Indeed, using MMLV we have established a convenient infection model for flavivirus encephalitis in SCID mice. This model may be particularly attractive for the in vivo evaluation of agents with anti-flavivirus activity (accompanying paper: Charlier et al., 2002
).
We have studied the particular characteristics of the 3' UTR of NKV flaviviruses and therefore also included the 3' UTR secondary structures of MODV, RBV and APOIV in our analysis. The 3' UTR structures of flaviviruses have previously been suggested to be organized into two distinct regions: (i) the 3'-terminal core element (approximately 330400 nucleotides in length for mosquito- and tick-borne flaviviruses), which is, within the different serogroups, highly conserved in its primary sequence and RNA folding pattern (Wallner et al., 1995 ; Proutski et al., 1999
); and (ii) the variable region, which is inserted between the core element and the coding region of the genome. This distinction corresponds with functional differences between these two regions (Proutski et al., 1999
). It was suggested that the variable region could possibly act as a spacer separating the folded 3' UTR structure from the rest of the genome (Blackwell & Brinton, 1995
). Mandl et al. (1998)
showed that deletion mutants of TBEV, which lack the entire variable region, replicate as efficiently as wild-type virus in cell culture and mice. Akin to the situation in mosquito- and tick-borne flaviviruses, the NKV flaviviruses also contain a variable region in their 3' UTR, and this region also varies substantially in length. Most of this variability is probably due to deletions or duplications in the region immediately following the NS5 stop codon, as suggested for the mosquito-borne flaviviruses (Shurtleff et al., 2001
). The core element, with its stems and loops, would constitute specific binding sites recognized by the virus-encoded replicase, cellular proteins or viral capsid proteins, and would play an important role in virus-specific transcription, translation and encapsidation (Mandl et al., 1998
; Gritsun et al., 1997
; Proutski et al., 1997b
; Blackwell & Brinton, 1995
, 1997
; Chen et al., 1997
). Using deletion mutants of DEN4, Proutski et al. (1999)
proposed two parts within the core element: (i) the most 3'-terminal structures/sequences that would act as a viral promoter critical for the initiation of minus-strand RNA synthesis; and (ii) more 5' proximal structures/sequences that may function as enhancers of viral RNA replication. MODV has the shortest 3' UTR (366 nucleotides) of the NKV flaviviruses discussed here and of all flaviviruses sequenced so far. The folding pattern of this virus contains possibly the (almost) basic 3' UTR pattern that is necessary for replication of an NKV flavivirus.
The phylogenetic tree based on the UTRs of NKV flaviviruses showed similar topology to those constructed from the coding regions (data not shown), indicating that the genetic information in these regions reflects the evolutionary history of MMLV and the other NKV flaviviruses. Analysis of the folding of the 3' UTR points to the relatedness of MMLV, MODV and RBV through a common folding pattern. Folding of the 3' UTR of these three viruses revealed four conformationally conserved structural elements (regions IIV) that are supported by compensatory mutations, which is suggestive for their functional importance. Six of the eight loops expose conserved sequence motifs. Furthermore, at the 3' end of region I, a conserved motif of 22 nucleotides [5' UUGUAAAUA(C/A)UU(U/G)(G/A)GCCAGUCA 3'] was observed. This motif has not been described for the mosquito- or the tick-borne flaviviruses and may be a particular characteristic of New World NKV flaviviruses (MMLV, MODV, RBV). APOIV, which can be considered an NKV flavivirus of the Old World, lacks region I and thus this motif. Phylogenetically, APOIV is the most distantly positioned flavivirus within the NKV flavivirus cluster. The particular characteristics of the secondary structure of the 3' UTR of APOIV (absence of region I and presence of a duplicated region II) corroborates this observation. Interestingly, MMLV and RBV, which both have the bat as their natural host, share a common pseudoknot structure (located between regions I and II). Moreover, the sequence of region I in the 3' UTR of MMLV and RBV is very similar, whereas the stems contain compensatory mutations.
Several characteristics of the 3' UTRs of the NKV flaviviruses are comparable with those of either mosquito-borne or tick-borne flaviviruses or both. A characteristic feature similar for mosquito-borne (with the exception of YFV), but not tick-borne, flaviviruses is the presence of duplicated conserved RNA sequences (called CS2 and RCS2) (Chambers et al., 1990 ; Proutski et al., 1997b
). It was assumed that they play an important role in initiating viral transcription as cis-acting signals, either by virtue of their exact nucleotide sequence (Hahn et al., 1987
; Mangada & Igarashi, 1997
) or through the interaction of secondary RNA structures with cellular proteins (Blackwell & Brinton, 1995
). The CS2 sequence is present in region II of the 3' UTR folding pattern of NKV flaviviruses and is located in a loop, as in the mosquito-borne flaviviruses,. According to Proutski et al. (1999)
, a single copy of either CS2 may be sufficient for normal virus replication of DENV4. However, deletion of both stemloop structures containing CS2 and RCS2 sequences led to an inability of the mutants to replicate in mammalian cells. As is the case for the mosquito-borne flaviviruses, APOIV contains both CS2 and RCS2, whereas MMLV, MODV and RBV carry only one such sequence. This would be in line with the observation of Proutski et al. (1999)
that only one CS2 sequence is required for the efficient replication of mosquito-borne flaviviruses. The mosquito-borne and NKV flaviviruses thus share a common factor that may possibly be important for replication in mammalian cells.
The 3' UTR of NKV flaviviruses share also particular characteristics with tick-borne flaviviruses. Like the tick-borne viruses, NKV flaviviruses lack the small stemloop located in region I of the 3' UTR of mosquito-borne flaviviruses (Proutski et al., 1997b , 1999
). Deletion of this structure led to a reduced efficiency of replication of DENV4 in mosquito cells. It was suggested that this structure may function as an enhancer of virus replication in mosquito cells. The fact that NKV flaviviruses probably do not (or inefficiently) replicate in mosquito cells may reinforce this hypothesis.
Region III of the NKV flaviviruses folds into a structure similar to the one predicted in the 3' UTR of tick-borne flaviviruses [where it is part of a larger structure with three different branches of hairpins (Mandl et al., 1998 ; Proutski et al., 1997b
)], but that is not present in the 3' UTR of mosquito-borne flaviviruses (Hahn et al., 1987
; Shurtleff et al., 2001
). As for the tick-borne flaviviruses, the two loops of the Y structure of region III of the NKV 3' UTRs are formed by a conserved stretch of nucleotides. These can be detected at analogous positions (5' AUUGGC 3' and 5' (G/U)(G/U)UU 3') (Gritsun et al., 1997
; Mandl et al., 1993
; Proutski et al., 1997a
, b
).
The very 3' terminus of the 3' UTR (region IV) folds in a manner typical for all flaviviruses, forming the 3' LSH structure and a small stemloop (belonging to region IV and probably coaxially stacking with the long 3'-terminal hairpin). The pseudoknot, which is predicted between the small stemloop and the 3' LSH of mosquito-borne but not tick-borne flaviviruses, is probably not formed in the 3' UTR of the NKV flaviviruses. The fact that the 3' LSH is detected in mosquito-, tick-borne (Grange et al., 1985 ; Brinton et al., 1986
; Hahn et al., 1987
; Mohan & Padmanabhan, 1991
; Mandl et al., 1993
; Wallner et al., 1995
; Shi et al., 1996
; Proutski et al., 1997a
, b
) and NKV flaviviruses strongly suggests that it plays a crucial role in the replication of all flaviviruses. In particular, the presence of the highly conserved pentanucleotide 5' CACAG 3' in the top loop has been suggested to play an important role in virus replication (Khromykh et al., 2001
). Analysis of the 3' UTRs of the NKV flaviviruses revealed a pentanucleotide sequence motif [5' C(C/U)(C/U)AG 3'] at an analogous position. However, this pentanucleotide sequence is, at positions 2 and 3, different from the 5' CACAG 3' motif of mosquito- and tick-borne flaviviruses and appears to be unique to NKV flaviviruses. Indeed, whereas the conserved CACAG motif was detected in all flaviviruses analysed so far [with the exception of MVEV (and CFAV), which carries an A/C change at position 4], all four NKV flaviviruses analysed carry a C(C/U)(C/U)AG pentanucleotide motif. From a taxonomic point of view, knowledge of the sequence of this pentanucleotide may thus be sufficient to allocate a flavivirus either to the vector-borne or to the NKV flaviviruses. It would be of interest to unravel whether this pentanucleotide sequence plays a role at the molecular level in the fact that NKV flaviviruses are not vector-borne. Construction of an infectious full-length clone, and the introduction of mutations within this pentanucleotide motif, may provide more insight into this fascinating observation. It would also be worth determining whether vector-borne viruses, in which the pentanucleotide sequence CACAG has been replaced by the pentanucleotide sequence that is typical for NKV flaviviruses, would have an altered efficiency of replication in either mosquito or tick cells.
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Acknowledgments |
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Footnotes |
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References |
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Received 14 December 2001;
accepted 10 March 2002.