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1 Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, NL-2300 RC Leiden, The Netherlands
2 Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, B-3000 Leuven, Belgium
Correspondence
Peter J. Bredenbeek
p.j.bredenbeek{at}lumc.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Department of Medical Microbiology, Academic Medical Center, Amsterdam, The Netherlands. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Thiel et al., 2005). Phylogenetic analysis of the genus Flavivirus has grouped these viruses into three major clusters: (i) the mosquito-borne viruses; (ii) the tick-borne viruses; and (iii) the no known vector (NKV) viruses (Cook & Holmes, 2006; Kuno et al., 1998). It is unknown whether the inability of NKV flaviviruses to infect arthropod vectors is due to a block at the level of entry, replication or assembly (Lawrie et al., 2004).
Flaviviruses are small, enveloped viruses containing a positive, single-stranded RNA genome of approximately 11 kb in length with a 5' cap structure and a 3' non-polyadenylated terminus. The genomic RNA encodes a single large open reading frame flanked by 5' and 3' untranslated regions (UTRs) of approximately 100 and 400–600 nt, respectively. Translation of the genome results in the synthesis of a large polyprotein, which is co- and post-translationally processed by viral and cellular proteases into three structural proteins (C, prM and E) and seven non-structural proteins that are primarily involved in the replication of the viral RNA (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) (Lindenbach & Rice, 2001).
The 3' UTR of the mosquito-borne flaviviruses contains several conserved sequences and is predicted to fold into a complex structure including well-conserved secondary and tertiary RNA elements that are involved in the initiation and regulation of genome amplification and translation (reviewed by Markoff, 2003). This conservation of RNA structure is especially obvious in the stem–loop (SL) that is predicted to be formed at the 3' end of every flavivirus genome. This structure involves 80–90 nt that are not well conserved in primary sequence, except for the pentanucleotide (PN) sequence CACAG (Brinton et al., 1986; Hahn et al., 1987; Wengler & Castle, 1986) in the bulge at the top of the SL and the dinucleotide CU at the end of the genome (Fig. 1a). Deletion of the SL is lethal for flavivirus RNA synthesis (Bredenbeek et al., 2003; Elghonemy et al., 2005; Nomaguchi et al., 2004; Tilgner et al., 2005). The SL structure is also required for efficient translation of the flavivirus genome (Chiu et al., 2005; Holden & Harris, 2004).
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). In addition, several host proteins such as translation elongation factor-1
(Blackwell & Brinton, 1997; Nova-Ocampo et al., 2002), Mov34 (Ta & Vrati, 2000), La and PTB (Kim & Jeong, 2006; Nova-Ocampo et al., 2002) have been shown to interact with the SL of several flaviviruses.
Sequence comparison within the genus Flavivirus reveals that the CACAG sequence is only well conserved when the vector-borne viruses are aligned (Fig. 1b
, numbering according to Fig. 1a). When the NKV flaviviruses are included in this comparison, the PN sequence was shown to be more variable. Rio Bravo virus (RBV) contains a C residue at the second position, whereas Montana myotis leukoencephalitis virus (MMLV) and Modoc virus (MODV) have a U (Charlier et al., 2002). In addition to the sequence variation at the second position, Apoi virus (APOIV) and Yokose virus (YOKV) also contain different nucleotides at the third and fourth positions (CCUAG and CGCCG, respectively) (Charlier et al., 2002; Tajima et al., 2005).
The conservation of the PN motif suggests that it is an important element for the replication of arthropod-borne flaviviruses. Mutagenesis of this CACAG sequence in replicons of West Nile virus (WNV) revealed that only the A at the fourth position could be replaced by another nucleotide without affecting virus replication (Tilgner et al., 2005). These data were partially confirmed in a study using a full-length WNV cDNA instead of a replicon (Elghonemy et al., 2005). However, in contrast to the results obtained using the WNV replicon, mutagenesis of the A residue at the second position of the PN motif did not impair RNA synthesis in the background of WNV full-length genomic RNA.
In view of the observed sequence variation in the PN motif of the NKV flaviviruses and the contradicting results concerning the PN sequence requirements in WNV replicon RNA versus genomic RNA, we performed an extensive mutagenesis of the PN motif of yellow fever virus (YFV) using an infectious full-length YFV and replicon RNA to determine the PN sequence requirements for replication in animal cells.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and Vero E6 cells were grown at 37 °C in 5 % CO2 in Dulbecco's modified Eagle's medium (DMEM; Cambrex) supplemented with 8 % fetal calf serum (FCS; Bodinco).
Recombinant DNA techniques and plasmid constructions.
Standard nucleic acid methodologies were used (Ausubel et al., 2000; Sambrook et al., 1989). Chemically competent Escherichia coli DH5 cells (Inoue et al., 1990) were used for cloning. Plasmid pACNR-MODV/YFV-pnMODV, a derivative of pACNR-MODV/YFV (Charlier et al., 2004) in which the YFV PN motif CACAG was mutated to CUCAG, mimicking the sequence found in MODV, was digested with SfiI and XhoI. The 644 bp fragment corresponding to the MODV/YFV cDNA 3' end was cloned into pBluescript-YFV9845–10861 to yield pBlscrpt-3'YFV-pnCUCAG. This plasmid was used as template for mutagenesis of the PN sequence using the QuickChange Site-directed Mutagenesis strategy (Stratagene). The inserts were sequenced to verify the mutations and to exclude unintended mutations. The mutant pBlscrpt-3'YFV derivatives were digested with XbaI and XhoI and the DNA fragment containing the mutated PN motif was cloned into pACNR-FLYF17Dx (Bredenbeek et al., 2003).
Renilla luciferase-expressing YFV replicons (YFRPs) containing a mutated PN motif were created by cloning the YFV 3' UTR from the full-length YFV cDNA harbouring the mutated PN motif using SfiI and XhoI into pYF-R.luc2A-RP (Jones et al., 2005).
RNA secondary structure prediction.
RNA secondary structure was predicted using MFOLD version 3.1 (Mathews et al., 1999; Zuker, 2003).
In vitro RNA transcription.
Plasmid DNA for in vitro run-off RNA transcription was purified with a Qiagen Plasmid Midi kit. pYF-R.luc2A-RP or pACNR-FLYF17Da (Molenkamp et al., 2003) and their derivatives containing the mutated PN sequence were linearized with AflII and purified by proteinase K digestion and phenol/chloroform extraction. Approximately 1–2 µg DNA was used as a template for in vitro transcription using the mMESSAGE mMACHINE SP6 kit (Ambion). Trace amounts of [3H]UTP were added to the reaction mixture to determine the yield (Bredenbeek et al., 2003). Genomic full-length transcripts were used for transfection without any additional purification. In vitro-transcribed replicon RNA was purified according to the protocol supplied with the mMESSAGE mMACHINE kit and the yield was quantified using a Nanodrop photospectrometer.
RNA transfection.
BHK-21J cells were prepared for electroporation as described by Lindenbach & Rice (1997). Immediately after preparation, 5 µg in vitro-transcribed RNA was mixed with 600 µl cell suspension and electroporated using an Easyject electroporator (Eurogentec) (van Dinten et al., 1997).
Labelling and analysis of viral RNAs.
Viral RNA synthesis was analysed by in vivo labelling of the transfected cells with [3H]uridine at 18–24 h post-electroporation (p.e.) (Bredenbeek et al., 2003). At 24 h, total RNA was isolated, denatured with glyoxal and analysed on a 0.8 % MOPS/agarose gel (Sambrook et al., 1989).
Virus stocks, infections and plaque assays.
Medium was harvested from transfected cells to obtain virus stocks when complete cytopathogenic effect (CPE) was observed. For infections, the cells were washed once with PBS and infected with virus using the m.o.i. indicated in the relevant figure legends. After 1 h, the inoculum was replaced by DMEM containing 2 % FCS. For analysis of virus growth kinetics, the medium was collected and replaced by the same volume of fresh medium at the indicated times. Virus titres were determined as described previously (Bredenbeek et al., 2003) except that Vero cells were used instead of SW13 cells in the plaque assays.
RT-PCR.
Total RNA was isolated using Trizol at 30 h p.i. from a 10 cm2 dish containing Vero or BHK-21J cells infected with either YFV-17D or the mutant viruses. RNA was dissolved in 30 µl H2O and 5 µl was used for RT-PCR to amplify the 3' UTR of the YFV genome using the ThermoScript RT-PCR system (Invitrogen). Primer sequences are available on request.
Renilla luciferase activity.
Electroporated BHK cells (800 µl) were seeded per well of a 12-well plate. At 2 and 18 h post-transfection, the cells were lysed in 200 µl passive lysis buffer (Promega). Luciferase activity was determined using the Renilla luciferase assay system (Promega) and a LB9507 luminometer (Berthold). Protein concentrations of the lysates were determined using the Bradford method (Bio-Rad Laboratories).
Virus competition experiments.
Vero E6 cells were infected simultaneously with the mutant virus and YFV-17D at an m.o.i. of 5 and 0.5, respectively (ratio 10 : 1). After 72 h, 200 µl medium was used to infect fresh Vero cells. Intracellular RNA was isolated from the infected Vero cells at the tenth passage and used for RT-PCR. The RT-PCR products were cloned using the TA Cloning kit (Invitrogen). Plasmid DNA was isolated from bacteria cultures and sequenced to determine the nucleotide sequence of the PN motif.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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pnCACAG, the complete PN sequence was deleted. Computer-aided RNA folding indicated that this deletion could significantly change the RNA structure at the top of the 3' SL. Therefore, an additional mutant was constructed in which the CACAG sequence was changed to UGUGA. RNA modelling predicted that the 3' SL structure of this mutant would adopt a similar structure to the wt YFV-17D (Fig. 2a). Viral RNA synthesis was detected only in BHK-21J cells transfected with YFV-17D RNA (Fig. 2b). No viral RNA was detected in cells electroporated with either YFV-pnCACAG or YFV-pnUGUGA RNA. Even after prolonged incubation (96–120 h), no virus could be detected in the medium of cells transfected with YFV-pnCACAG or YFV-pnUGUGA RNA by plaque assay (data not shown). These results demonstrated that the PN motif is absolutely required for YFV replication.
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, the PN sequence is not absolutely conserved in flaviviruses. Variation is observed in the second, third and fourth positions. To verify whether other nucleotides were tolerated at these positions in the YFV PN motif, a set of mutants was created in which the A at the second position, the C at the third position or the A at the fourth position was replaced by alternative nucleotides. Some of these mutations resulted in PN motifs mimicking the sequence of NKV flaviviruses such as MODV and MMLV (CUCAG) or RBV (CCCAG). In addition to these YFV mutants containing a single mutation, a mutant was created in which the nucleotides at the second and third position were mutated, thereby mimicking the PN motif of the NKV APOIV (CCUAG).
As shown in Fig. 3(a), the mutants in which the A residue at the second position was replaced by either a C, G or U synthesized RNA at a similar rate to YFV-17D. In addition, the viral growth curves of these viruses showed similar growth kinetics when compared with YFV-17D (Fig. 3d).
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). The titre in the medium of cells transfected with YFV-pnCAGAG RNA was approximately 105 p.f.u. ml–1 when CPE was complete. In addition, heterogeneity in plaque size was observed. Most of the plaques were small and turbid and therefore hardly visible, but larger plaques were also observed. RT-PCR on RNA isolated from Vero cells infected with this virus revealed that the introduced G at the third position was replaced by a U. The original PN sequence contains a C residue at this position. However, YFV-pnCAUAG was also shown to replicate efficiently (Fig. 3b, e). Given the limited genetic stability of YFV-pnCAGAG, this mutant was excluded from the growth curves. The growth kinetics of YFV-pnCAUAG did not differ significantly from the parental virus, whereas the growth of YFV-pnCAAAG was slightly delayed (Fig. 3e). In agreement with the above results, the mutant YFV-pnCCUAG mimicking the PN motif of APOIV was able to synthesize viral RNA efficiently and showed similar growth kinetics to YFV-17D, despite the fact that it contained two mutations within the PN motif.
Mutagenesis of the A residue at the fourth position had no significant effect on virus replication. The mutants YFV-pnCACCG, YFV-pnCACGG and YFV-pnCACUG all synthesized RNA at comparable levels (Fig. 3c) and showed similar growth kinetics (Fig. 3f) when compared with YFV-17D.
To analyse whether reversion of the introduced mutations to the original YFV-17D PN sequence could have influenced the outcome of these experiments, mutant viruses from the 60 h time point of the growth curves were used to infect Vero cells. At 30 h p.i., total RNA was isolated and used for RT-PCR. All viruses had maintained the original mutation. However, second-site reversions in other regions of the genome could not be excluded.
Mutational analysis of the first position of the PN motif reveals the importance of base pairing
The C residue at the first position of the PN motif appears to be truly conserved in all flaviviruses. This C residue is predicted to base pair with an equally well conserved G four positions downstream of the PN motif (Fig. 1b). This position will be referred to as the ninth position. To determine the importance of this C residue and the potential role of the C-G base pair in YFV replication, the C was replaced by each of the other three nucleotides. Mutagenesis to either an A (YFV-pnAACAG9G) or G (YFV-pnGACAG9G) was predicted to disrupt the base pairing, whereas this base pair was predicted to be maintained when the C was replaced by a U (YFV-pnUACAG9G). As shown in Fig. 4, no RNA synthesis was detected in cells transfected with YFV-pnAACAG9G, whereas viral RNA synthesis was significantly impaired in cells transfected with YFV-pnGAGAC9G. YFV-pnUACAG9G synthesized RNA with an efficiency that was similar to the parental virus. These data suggested that the formation of the base pair between the first and ninth positions is more important than the actual nucleotides. To verify this hypothesis, additional mutants were created by introducing mutations at the ninth position in combination with the first position mutants described above. This resulted in YFV-pnAACAG9A, YFV-pnCACAG9C, YFV-pnUACAG9U, YFV-pnGACAG9C, YFV-pnAACAG9U and YFV-pnUACAG9A. In the first three mutants, the G residue at the ninth position was changed to the same nucleotide as in the first position of the mutated PN motif, thus impairing base-pair formation. In the last three mutants, the potential for base pairing was restored, albeit it with different nucleotides compared with the parental virus. No YFV RNA was detected in cells transfected with either YFV-pnAACAG9A or YFV-pnCACAG9C, and viral RNA synthesis was significantly impaired in cells transfected with YFV-pnUACAG9U (Fig. 4a). In contrast, the mutant viruses YFV-pnGACAG9C, YFV-pnAACAG9U and YFV-pnUACAG9A in which base pairing was restored showed efficient RNA synthesis and viral growth kinetics similar to that of the parental virus (Fig. 4). Taken together, these data clearly demonstrated that base-pair formation between the first nucleotide of the PN motif and the nucleotide at the ninth position is more important for efficient virus replication than the nature of the nucleotides at these positions.
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). Second-site reversions in other regions of the genome could not be excluded.
The G residue at the fifth position is essential for virus replication
The well-conserved G residue at the fifth position of the PN motif was replaced by one of the other nucleotides. As shown in Fig. 5, viral RNA synthesis was only detected in cells transfected with the parental YFV-17D transcript; no RNA was detected in cells electroporated with YFV-pnCACAA, YFV-pnCACAC or YFV-pnCACAU RNA, and no virus could be detected by plaque assay in the medium of the transfected cells (data not shown). These data demonstrated that the G at the fifth position of the PN motif is absolutely required for YFV replication.
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Viruses with a mutation at the second position of the PN motif, such as YFV-pnCCCAG and YFV-pnCUCAG, were clearly outcompeted by YFV-17D within ten passages (Table 1). A similar result was also obtained for the third-position mutant YFV-pnCAUAG. These results were supported by the fact that the virus mimicking the APOIV PN motif (YFV-pnCCUAG) was also not detected after ten passages. Compared with viruses with mutations at the second and third positions, YFV-pnCACCG replicated relatively well and was still the dominant virus after ten passages. However, the ratio of 12 : 7 for YFV-pnCACCG versus YFV-17D at the tenth passage indicated that the parental virus was slowly outcompeting the mutant virus. These data demonstrated that viruses with a mutation at the second, third or fourth position of the PN motif were less fit than the parental YFV-17D in Vero cells, despite the fact that these mutant viruses showed similar replication efficiency and growth kinetics in individual infection experiments.
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). The result obtained for YFV-pnUACAG9A was essentially similar to that of the mutant virus YFV-pnCACCG. At the tenth passage, the YFV-pnUACAG9A was still the dominant virus, but the ratio indicated that the mutant would eventually be outcompeted. A more interesting picture was observed for both YFV-pnGACAG9C and YFV-pnAACAG9U. At the tenth passage, they were clearly the dominant viruses, suggesting that equilibrium was possible between the mutant and the wt virus. As expected, a poorly replicating mutant such as YFV-pnUACAG9U, in which the base pairing was disrupted, was easily outcompeted by YFV-17D. Although the results obtained with YFV-pnGACAG9C and YFV-pnAACAG9U indicated that some of the mutants were as fit as the wt virus, it was obvious that the wt PN sequence still had an as yet undefined advantage over most of the mutant PN sequences when analysed in animal cells.
Mutations in the PN motif do not affect translation
To analyse whether the effect of the PN mutations was due to a direct effect on RNA synthesis or an indirect effect by influencing virus RNA translation, a selected set of mutations was cloned into pYF-R.luc2A-RP. RNA transcribed from these plasmids was transfected into BHK cells, which were subsequently analysed for Renilla luciferase expression at 2 h (the peak time for translation of input RNA) and 18 h p.e. (when only virus-synthesized RNA is translated). At 2 h p.e., all of the replicons expressed luciferase at a comparable level (Fig. 6) including replicons such as YFRP-pnUGUGA, YFRP-pnCAGAG, YFRP-pnCACAU and YFRP-pnGACAG9G for which no or hardly any RNA synthesis could be detected in the background of the full-length YFV RNA (Figs 2, 3, 4 and 5). From these data, it was concluded that the mutations in the PN motif had at best a relatively minor effect on translation. No luciferase was detected in cells transfected with YFRP-pnUGUGA, YFRP-pnCAGAG or YFRP-pnCACAU at 18 h p.e., whereas YFRP-pnGACAG9G showed low luciferase activity. The replicons YFRP-pnCCCAG, YFRP-pnCACCG, YFRP-pnUACAG9G and YFRP-pnAACAG9U showed a high level of luciferase activity. These mutations also allowed efficient replication in the background of the full-length clone, demonstrating that mutagenesis of the PN motif in either the replicon or the full-length YFV RNA yielded identical results.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). However, when the NKV flaviviruses are included in this comparison, the PN sequence is far less conserved. None of the NKV viruses sequenced to date contains the sequence CACAG as the PN motif. Other nucleotides are observed at either the second or third position and some viruses even contain substitutions at the second and third or the second and fourth positions (Charlier et al., 2002; Tajima et al., 2005). These nucleotide substitutions in the NKV viruses are rather surprising and suggest that mutations of some PN positions may be tolerated in arthropod-borne flaviviruses. These observations prompted us to analyse the requirement for the YFV PN sequence.
YFV mutants in which the PN sequence CACAG is either deleted or completely changed (YFV-pnUGUGA) were unable to replicate, demonstrating that at least part of this sequence is absolutely required for YFV replication. These data are in agreement with the observation for WNV in which deletion of the PN motif is also lethal (Tilgner et al., 2005).
To determine whether other nucleotides are tolerated in the YFV PN motif, we performed an extensive mutagenesis study using in vitro-transcribed YFV genomic and replicon RNA. Our data showed that mutations at either the second, third or fourth position of the PN motif had no significant effect on YFV replication, except when the C at the third position was replaced by a G, which severely impaired viral RNA synthesis and growth. Except for the fourth position, our data are clearly different from the published results on the WNV PN motif (Tilgner et al., 2005). Using WNV replicon RNA, it was shown that the A and C residues at the second and third positions were absolutely required for WNV RNA synthesis. Another study using full-length WNV genome RNA transcripts instead of replicon RNA confirmed the data of Tilgner et al. (2005) concerning the third position of the PN motif. However, in contrast to the WNV replicon, replacement of the A at the second position by a U residue was tolerated in the complete WNV genome (Elghonemy et al., 2005).
In addition to these point mutations, a YFV mutant mimicking the NKV APOIV PN motif (CCUAG) was constructed. This mutant replicated efficiently and showed only slightly slower growth kinetics compared with the YFV-17D. As a control for these results for the second, third and fourth positions, the mutant YFV-pnCGUGG was created. As expected from our previous observations, this mutant was able to replicate, although less efficiently than YFV-17D (data not shown). These results confirmed our initial finding that point mutations at the second, third or fourth position of the YFV PN motif are tolerated, although the almost undetectable effects of individual mutations became more obvious when mutations were combined.
Only the first and fifth positions of the PN motif appear to be truly conserved among all flaviviruses. Replacing the G at the fifth position of the YFV PN motif for another nucleotide was lethal. A similar result was obtained for the WNV PN motif (Elghonemy et al., 2005; Tilgner et al., 2005), suggesting that this G residue has a critical role in the replication of all flaviviruses.
Analysis of the sequence surrounding the PN motif revealed that the G residue at the ninth position is also strictly conserved. RNA folding of the flavivirus 3' SL structure predicted that this G will base pair with the well-conserved C residue at the first position of the PN motif, suggesting that the formation of this base pair is essential. Mutants at the first and/or the ninth position that disrupted this predicted base pair were either unable to replicate or were significantly impaired in virus replication, whereas mutants that allowed the formation of this base pair showed RNA synthesis and viral growth with similar kinetics to the parental virus. From these data, we concluded that the formation of a base pair between the first nucleotide of the PN motif and the nucleotide at the ninth position is a critical determinant for efficient virus replication. The importance of this base pair was also recognized for WNV (Tilgner et al., 2005). However, WNV replicons with an alternative base pair showed only 10–20 % of the luciferase activity of the wt replicon, whereas the comparable YFV mutants in this study were virtually indistinguishable from the wt virus or replicon. Using these luciferase-expressing YF replicons, we also demonstrated that the PN mutations only had a direct effect on viral RNA synthesis and did not affect virus RNA translation.
Flavivirus RNA replicons have been used extensively to study virus replication (Alvarez et al., 2005; Holden et al., 2006; Khromykh & Westaway, 1997; Lo et al., 2003; Molenkamp et al., 2003; Shi et al., 2002) and so far no significant differences have been observed when analysing the effect of 3' UTR mutations on virus replication using flavivirus RNA replicons versus full-length genomic RNA. This is also true for the data presented in this study and we have no explanation as to why the sequence requirements of the YFV PN motif are so different from those determined using either a WNV replicon or a full-length RNA.
It has been stated that the C at the eighth position in WNV (Elghonemy et al., 2005) is critical for replication. Substitution of the U at this position in YFV for C yielded a virus with similar characteristics to wt YFV-17D, indicating that in YFV the nucleotide at this position is not critical for replication (data not shown).
The variability that can be introduced in the YFV PN sequence is somewhat surprising when we take into account the fact that the PN CACAG is well conserved within the arthropod-borne flaviviruses. To evaluate the importance of the wt sequence for replication in animal cells, competition experiments between YFV-17D and a set of mutants were performed. Although the results showed that some of the base-pair mutants appeared to be as fit as the parental virus, the wt PN sequence had an advantage over most of the mutant PN sequences in animal cells. These results are to some extent similar to what has been observed for tick-borne encephalitis virus mutants where point mutations that seem to have little or no effect in animal cell culture were shown to have a clearly different phenotype in a relevant small-animal model (Gritsun et al., 2001).
Taken together, our data support the fact that the PN CACAG is quite variable in sequence when analysed in animal cell culture systems. Individual point mutations at the second, third and fourth positions are generally well tolerated in the YFV PN motif, whereas the G residue at the fifth position is truly conserved. In addition to this G, base pairing between the nucleotides at the first and ninth positions is also essential for efficient replication. Despite this sequence variability that can be introduced, there appears to be a preference for the parental CACAG sequence in animal cell culture. The reason for this is currently unclear. The PN motif may be part of either a host or viral protein RNA-binding site. The G at the fifth position would then be crucial for protein binding, whilst the formation of the base pair might be required to form the proper RNA structure. Given the mutations that can be introduced into the PN sequence, it is unlikely that the PN motif is involved in an RNA–RNA interaction.
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ACKNOWLEDGEMENTS |
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Received 20 December 2006;
accepted 19 February 2007.
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