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1 Radboud University Medical Centre Nijmegen, Nijmegen Centre for Molecular Life Science, Department of Medical Microbiology, PO Box 9101, 6500 HB Nijmegen, The Netherlands
2 University of California, San Francisco, Mission Bay Genentech Hall, UCSF Department of Microbiology, 600 16th Street, PO Box 2280, San Francisco, CA 94143, USA
3 Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA
4 Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA
Correspondence
Willem J. G. Melchers
w.melchers{at}ncmls.ru.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Methods detailing plasmid construction are available as supplementary material in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Essential for viral RNA replication are cis-acting replication elements (CREs) located at both the 5' and 3' NCRs of the viral genome. The cloverleaf structure present at the 5' terminus of the virus genome is a multifunctional element that interacts with viral and cellular proteins to form a ribonucleoprotein (RNP) complex that is required for the initiation of both negative- and positive-strand RNA synthesis (Andino et al., 1990; Herold & Andino, 2000). Additionally, this RNA structure has been suggested to be involved in mediating the switch from viral translation to replication (Gamarnik & Andino, 1998) and also plays a role in the uridylylation of the viral protein VPg (Lyons et al., 2001). Studies have shown that the 3' NCR forms a complex ‘kissing’ RNA interaction that is required for virus replication (Melchers et al., 1997, 2000; Mirmomeni et al., 1997; Pilipenko et al., 1996; Wang et al., 1999). Interaction between the RNP complexes formed at the 5' and 3' termini of the viral genome has been proposed to induce a circular conformation that is required for the initiation of negative-strand RNA synthesis (Barton et al., 2001; Herold & Andino, 2001).
The identification of a CRE located within the coding region of the picornaviral genome (Gerber et al., 2001; Goodfellow et al., 2000; Lobert et al., 1999; Mason et al., 2002; McKnight & Lemon, 1998) presented an additional RNA structure that is required for virus replication. The conformation of these CREs, based on computational and biochemical analysis, is a hairpin structure containing a terminal loop, most commonly of 14 nt (Goodfellow et al., 2003a), confirmed recently by nuclear magnetic resonance spectroscopy (Thiviyanathan et al., 2004), although in different picornaviruses the size of the loop may vary (Gerber et al., 2001; Yang et al., 2002; Yin et al., 2003). Sequence similarity within the loop present in all CREs described thus far is consistent with the consensus sequence 5'-AAACA-3' (Rieder et al., 2000). In poliovirus and human rhinovirus 2, the first two adenosine residues within this consensus are the residues essential for the covalent linkage of UMP nucleotides to VPg, resulting in the extended viral peptide primer VPgpU(pU) (Gerber et al., 2001; Paul et al., 2000, 2003; Rieder et al., 2000). By studying the human rhinovirus 14 CRE(VP1), Yang et al. (2002) extended this research and postulated a common R1NNNAAR2NNNNNNR3 motif (R, A/G; N, any nucleotide) for the loop of rhinovirus and enterovirus CREs, a conclusion confirmed for poliovirus (Yin et al., 2003). Because positive as well as negative RNA strands are VPg-linked (Nomoto et al., 1977a; Pettersson et al., 1978), it was proposed that the CRE is required for the initiation of both steps of RNA replication (Paul et al., 2000; Rieder et al., 2000). Recent reports, however, showed that a structurally disrupted CRE mutant retained the capacity to induce negative-strand RNA synthesis in a cell-free translation/replication system, suggesting that the CRE is only required for positive-strand RNA synthesis (Goodfellow et al., 2003b; Morasco et al., 2003; Murray & Barton, 2003).
This study was undertaken to examine the structural and functional requirements of the coxsackie B3 virus (CVB3) CRE(2C) in RNA replication. CRE(2C) loop mutants were analysed for their capacity to serve as template for VPg uridylylation and to induce negative- and positive-strand synthesis using HeLa S10 cell extracts. Here, we provide evidence that the CVB3 CRE(2C) is involved in the initiation of both negative- and positive-strand RNA synthesis.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). HeLa S3 cells (ATCC CCL 2.2) were grown either (i) in tissue-culture flasks in Dulbecco's modified Eagle's medium–nutrient mixture F-12 (Ham) (1 : 1), supplemented with 2 mM L-glutamine, 100 U penicillin and streptomycin ml–1 and 10 % fetal calf serum, or (ii) in suspension using suspension MEM (Joklik-modified) supplemented with 2 mM L-glutamine, 100 U penicillin and streptomycin ml–1 and 10 % newborn calf serum.
Construction of CRE mutants.
All CRE mutations were introduced into the CVB3 infectious clone (Klump et al., 1990) by using the Altered Sites in vitro mutagenesis system (Promega). Selected clones were verified for the correct mutation by using sequence analysis and a BssHII/XbaI digestion was used to ligate the mutated CREs into pRibCB3/T7. For details of the construction, see Supplementary Material (available in JGV Online).
Oligonucleotide site-directed mutagenesis.
Oligonucleotide-directed mutagenesis was performed by using phagemid pALTER-1 as described previously (Melchers et al., 1997). Synthetic oligonucleotides (Biolegio) were used to introduce site-specific mutations (Table 1). The mutated fragments were cloned into the infectious cDNA clone (pRibCB3/T7) and sequence analysis was used to verify the mutated nucleotide sequence.
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). The transcript RNA was transfected into BGM cells as described previously (Melchers et al., 1997). Virus yields were determined in BGM cells by end-point titrations using eight replicates of serial 10-fold dilutions in 96-well plates containing BGM cell monolayers (van Kuppeveld et al., 1997). TCID50 was calculated according to Reed & Muench (1938).
Luciferase assay.
BGM cells, grown in six-well plates to a confluence of 80 %, were transfected as described previously (Melchers et al., 1997) with 4 µg T7 RNA polymerase-generated cRNA derived from MluI-linearized replicon plasmid, containing the firefly luciferase gene. Ten hours post-transfection, cells were washed twice with PBS, prior to lysis using 200 µl lysis buffer (Promega). Luciferase activity was measured on a BioOrbit 1251 luminometer using the Luciferase Assay system according to the recommendations of the manufacturer (Promega).
Expression and purification of coxsackie B3 viral 3Dpol and 3CDpro–6His.
Proteins were expressed by using the pET26-Ub-based prokaryotic expression system as reported previously (Gohara et al., 1999), which generates N-terminally linked yeast ubiquitin-fusion proteins. Overexpression of protein is performed in the BL21(DE3)pCG1 strain of Escherichia coli. CVB3 3Dpol and 3CDpro–6His were expressed essentially in the same manner as the poliovirus proteins described previously (Gohara et al., 1999) from the polyethyleneimine precipitation. The protein concentration of each collected fraction (0·5 ml) was determined by measuring the A280 in the presence of 6 M guanidine hydrochloride and using the following absorbance coefficients: 3D, 69 270 M–1 cm–1; 3CD, 82 640 M–1 cm–1. Samples were aliquotted and stored at –80 °C.
In vitro uridylylation assay.
For short RNA transcripts of wild- type and CRE(2C) loop mutants, a PCR product was generated, using wild-type or mutant infectious clones as template, with reverse primer 5'-CAGGCGCAAACATACAGGTTCAA-3' and forward primer 5'-GGGGGGTAATACGACTCACTATAGGGCGAATGAGCAATTACATACAGTTCAA-3', containing a T7 polymerase sequence (italic type). For the generation of genomic wild-type and mutant RNA transcripts, infectious clone p53CB3/T7 was linearized by using MluI. Synthesis of VPgpU(pU) was measured by using a reaction mixture (20 µl) containing 50 mM HEPES (pH 7·5), 8 % (v/v) glycerol, 3·5 mM magnesium acetate, 0·7 µM 3CDpro, 2 µM 3Dpol, 40 µM synthetic CVB3 VPg, 0·75 µCi (0·277 MBq) [
32P]UTP (3000 Ci mmol–1) and 25 µM unlabelled UTP. Amount of template for the reaction was 0·5 µg for CRE(2C) transcript RNA and 2 µg for genomic transcript RNA. Reaction mixtures were incubated for 1 h at 34 °C and the reaction was quenched by adding 5 µl gel-loading buffer. The samples were analysed by Tris/Tricine SDS-PAGE (Bio-Rad) with 13·5 % polyacrylamide. The gels were dried and autoradiographed for visualization of product. Reaction products were quantified by measuring the amount of [32P]UMP incorporated into the product by using a phosphorimager (Storm 860; Molecular Dynamics) and converting it into c.p.m. by using a radioactive marker.
In vitro translation and replication assay.
Preparation of HeLa S10 cell extract and initiation factors has been described previously in detail by Barton et al. (1995). Negative- and positive-strand RNA synthesis was analysed as described by Herold & Andino (2000) with some minor modifications: 2 µg coxsackievirus transcript RNA was mixed with 30 µl HeLa S10 cell extract, 2 µl initiation factors, 5 µl 10x NTP/energy mix (Herold & Andino, 2000) and 1 µl 100 mM guanidine hydrochloride in a total volume of 50 µl. After incubation at 30 °C for 4 h and centrifugation, the pre-initiation complexes were resuspended in 50 µl labelling mix, containing 30 µl HeLa S10 cell extract, 5 µl 10x NTP/energy mix and 4 µl [-32P]UTP (10 µCi µl–1). After incubation at 30 °C for 2 h, the samples were mixed with 150 µl TENSK buffer [50 mM Tris/HCl (pH 7·5), 5 mM EDTA, 100 mM NaCl, 1 % (v/v) SDS, 200 µg proteinase K ml–1] to stop the reaction. RNA was extracted by using phenol/chloroform and RNA was precipitated by using ethanol. The pellet was resuspended in RNA-storage solution (Ambion) and gel-loading buffer was added prior to loading on a 0·8 % agarose gel. After drying the gel, products were visualized by using autoradiography. In order to quantify the accumulated replicative form (RF) bands, irrespective of loading and RNA-extraction efficiency, the amount of [32P]UMP incorporated in both the RF and the 28S rRNA was determined by using a phosphorimager (Storm 860; Molecular Dynamics). The amount of 28S rRNA in each lane represents the amount of RNA loaded on the gel and was normalized to wild-type. Subsequently, the accumulated RF was corrected for the amount loaded on the gel.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) (Goodfellow et al., 2000; Paul et al., 2000; Rieder et al., 2000; Witwer et al., 2001). Experimental evidence, however, has not yet been provided. The corresponding region was predicted by MFOLD (Zuker et al., 1999) to form a hairpin structure with a terminal loop region of 14 nt (Fig. 1b). In order to investigate whether this RNA structure is indeed required for CVB3 replication, silent mutations designed to disrupt the stem were introduced [distortion mutant, CRE(2C)-DM, Fig. 1c]. Transfection of this RNA into susceptible BGM cells abolished virus production, an observation indicating the significance of this RNA structure for virus replication (data not shown). Testing CRE(2C)-DM in a construct in which the capsid-coding region was replaced by a luciferase gene showed no increase in luciferase activity above translational levels of the input RNA as determined in the presence of guanidine hydrochloride, thereby indicating a defect in RNA replication (Fig. 2a).
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; Goodfellow et al., 2003a; Paul et al., 2000; Yang et al., 2002). This prompted us to establish whether the CVB3 CRE(2C) is also the primary site for VPgpU(pU) synthesis. Full-length genomic RNA transcripts, containing either wild-type CRE(2C) or the DM mutant, were used as templates together with purified CVB3 3Dpol, 3CDpro, VPg and Mg2+ in an in vitro uridylylation assay. Using full-length genomic RNA transcripts, wild-type RNA was able to support VPg uridylylation in the presence of both 3Dpol and 3CDpro, whereas no detectable amount of uridylylated VPg was observed upon distortion of the CRE(2C) structure (Fig. 2b
). These results suggest that the CVB3 CRE(2C) is indeed the primary template for VPg uridylylation.
Disrupting the CVB3 CRE(2C) stem–loop structure does not affect negative-strand RNA synthesis
A cell-free translation/replication system, originally described for poliovirus (Molla et al., 1991), has been used to analyse the effect of gross distortions of the CRE structure on negative-strand RNA synthesis (Goodfellow et al., 2003b; Morasco et al., 2003; Murray & Barton, 2003). To ascertain whether CVB3 transcript RNA is able to function as a template for RNA replication using the cell-free system, CVB3 transcript RNA containing different 5' ends was used (Fig. 2c). RNA containing the 5' hammerhead ribozyme (HR) sequence (Herold & Andino, 2000), which, after transcription, produces RNA with authentic 5' termini, efficiently produced both negative-stranded (RF) and positive-stranded [replicative intermediate (RI) and ssRNA] RNA. Absence of this HR sequence produced CVB3 transcripts that contained two non-viral guanosine residues at their 5' terminus, which only produced RF RNA (Herold & Andino, 2000) and therefore efficiently blocked the accumulation of RI and ssRNA during the 2 h incubation period (Fig. 2c). No bands were observed when CVB3 HR transcripts were incubated in the presence of guanidine hydrochloride, indicating that the cell-free system only displays viral RNA production (Fig. 2c). It has previously been described that rearrangements of the CRE structure did not interfere with the accumulation of negative strands (Goodfellow et al., 2003b). In order to quantify the amount of complementary strands produced by coxsackievirus RNA using HeLa S10 cells extracts, we used RNA transcripts containing two non-viral guanosine residues at their 5' terminus. Wild-type RNA produced a band corresponding to the RF of RNA replication. Accumulation of this band was abolished upon addition of guanidine hydrochloride, an observation demonstrating that this band is indeed of viral origin (compare WT with WT+GuHCl, Fig. 2d). In HeLa S10 cell extracts, CRE(2C)-DM RNA also accumulated RF, indicating that negative-strand RNA synthesis in this mutant is not inhibited (Lane DM, Fig. 2d). Quantification indicated CRE(2C)-DM RNA to accumulate amounts of RF at the measured time point comparable to those of wild-type (Fig. 2d). In contrast to wild-type transcript containing a cis-acting HR sequence, pre-initiation replication complexes programmed with CRE(2C)-DM RNA containing a HR sequence produced only the RF band, but no progeny positive-strand RNA accumulation was observed (Fig. 2e).
The results described above suggest that, under the conditions of the experiments, CRE(2C)-derived VPgpU(pU) is required for the initiation of positive-strand, but not negative-strand, RNA synthesis.
Effect of CRE(2C) point mutations on replication efficiency
A functional analysis was undertaken to determine whether the consensus sequence for the apical loop postulated by Yang et al. (2002) is also applicable to the CVB3 CRE(2C). Subsequently, every nucleotide involved in this consensus sequence was substituted for almost all other nucleotide possibilities and, additionally, the coxsackie B-specific A5AAUG9 loop sequence was substituted for the consensus AAACA sequence (consensus mutant, CM) to ascertain the relevance of the CVB3-specific U8G9 residues (Fig. 3b) (Rieder et al., 2000). As point-mutational analysis of the CRE(2C) at its original 2C location faces complications, due to amino acid changes in the 2C protein, and the CRE function is described to be position-independent (Goodfellow et al., 2000; Yin et al., 2003), a HR-CRE(Art) construct was devised containing the CRE(2C) distortion mutant at the original 2C position and a second, artificial CRE at the junction of the P2 region and the luciferase gene, which replaces the capsid-coding region (Fig. 3a). Also, a cis-acting hammerhead ribozyme sequence was introduced in order to generate RNA transcripts containing authentic 5' ends (Herold & Andino, 2000). This construct enabled a mutational analysis of the CVB3 CRE(2C) for its function in RNA replication, without altering the amino acid sequence of the CVB3 2C protein. In vitro-transcribed RNA was transfected into susceptible BGM cells and, at 10 h post-transfection, luciferase activity was measured as described previously (van Kuppeveld et al., 1995). Substituting the coxsackievirus-specific AAAUG sequence for the consensus AAACA motif showed wild-type-like luciferase activity (Lane CM, Fig. 3b). Alterations affecting the adenosine triplet showed that A7 can only be replaced by another purine, whilst for A5 and A6, no substitutions were permitted (Fig. 3b). Substituting A5 for a uridine residue, which results in the introduction of a stop codon (UAA), is included as a negative control for further studies described below. In this assay, however, no conclusions can be drawn for this mutation with respect to virus replication. Purine residues at the extreme ends of the CRE(2C) loop sequence were proposed by Yang et al. (2002) to be required for rhinovirus and enterovirus replication. Substitutions regarding G1 were in accordance with this consensus, as only a transition to an adenosine conferred the ability to replicate. Interestingly, residue A14 can be substituted by both a guanosine and a uridine, although the uridine substitution displayed reduced replication efficiency compared with the guanosine substitution. This difference in replication efficiency, when comparing the A14U with the A14G transition, might be explained by alternative folding of the loop as a result of non-canonical base-pair formation (MFOLD prediction), as opposed to changes in the primary CRE sequence (data not shown).
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, with the exception of nucleotide A14, which can be substituted by a guanosine or a uridine residue.
Effect of CRE(2C) point mutations on VPg uridylylation efficiency
The capacity of CVB3 CRE(2C) mutants to support VPg uridylylation was tested by using short mutant CRE(2C) transcript RNAs, representing only the CRE(2C). Uridylylation efficiencies were quantified as described in Methods. Consistent with the results obtained by using genomic RNA transcripts, a short transcript containing only the CRE(2C) distortion mutant (Fig. 1c) was unable to support uridylylation above detectable levels (Lane DM, Fig. 3c). Substituting the AAAUG9 sequence for the AAACA9 consensus motif showed only a slight decrease in uridylylation efficiency compared with wild-type (Lane CM, Fig. 3c). In general, mutants that were able to support RNA replication (Fig. 3b) also showed efficient VPg uridylylation (G1A, A7G, U8C/A, A14G/U and CM, Fig. 3c). Interestingly, lower levels of uridylylation did not always correlate with reduced levels of RNA replication, as shown for mutants U8C and U8A (compare Fig. 3b with Fig. 3c). The reason for this might be that the uridylylated products are made in large excess over what is used for RNA synthesis (Murray & Barton, 2003). No VPgpU(pU) synthesis above background levels, however, could be detected for G1C, A7C/U or A14C substitutions or for any of the A5 or A6 mutants (Fig. 3c), which were also defective for RNA replication (Fig. 3b). Therefore, it can be concluded that uridylylation of VPg is essential for viral RNA replication.
Effect of CRE(2C) point mutations on negative-strand RNA synthesis
To ascertain the effect of CRE(2C) point mutations on RF synthesis, we used a construct from which RNA transcripts containing two non-viral guanosine residues at their 5' termini, termed GG-CRE(Art), are derived (Fig. 4a), allowing us to specifically evaluate and quantify the effect of CRE(2C) mutations on RF synthesis (Herold & Andino, 2000). A very good correlation was observed between the [32P]UMP labelling of the 28S rRNA band and the ethidium bromide staining of the gel, which allowed us to quantify RF accumulation irrespective of loading by determining the ratio between the 28S rRNA band (Herold & Andino, 2000) and synthesized viral RF.
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) (Morasco et al., 2003; Murray & Barton, 2003), it was rather unexpected that the majority of the CRE(2C) substitutions regarding the enterovirus consensus sequence did not accumulate wild-type-like RF levels. Mutants positive for both luciferase activity (Fig. 3b) and VPg uridylylation (Fig. 3c) accumulated, albeit with different efficiencies, the highest levels of RF synthesis (G1A, A7G, U8C/A and A14G/U, Fig. 4b). Only the U8 mutants, a residue not belonging to the consensus sequence, showed wild-type RF production (U8C/A, Fig. 4b). In general, a good correlation was observed between the luciferase activity and the efficiency of RF synthesis using these mutants (compare Fig. 3b with Fig. 4b). VPgpU(pU)-defective point mutations accumulated RF synthesis only to approximately 20 % of that of wild-type CVB3 RNA (G1C, A5C, A6G/C/U, A7C and A14C, Fig. 4b), whereas the VPgpU(pU)-defective A5G mutant was even unable to support RF synthesis above detectable levels (Fig. 4b). The fact that point mutants reduced in VPgpU(pU) accumulation were affected in RF accumulation already suggests that CRE(2C)-derived VPgpU(pU) is also utilized to initiate negative-strand RNA synthesis. Because inhibiting RF synthesis by introducing a stop codon (A5U, Fig. 4b) displayed results similar to those for the A5G transition, it was concluded that this latter mutant was defective for negative-strand RNA synthesis. This, again in contrast to previous reports (Goodfellow et al., 2003b; Morasco et al., 2003; Murray & Barton, 2003), implies the necessity for the CRE(2C) in positive-strand, but also in negative-strand, RNA synthesis. Additionally, GG-CRE(Art) transcript RNA, containing a completely distorted CRE(2C) with an additional A5G mutation, still produced wild-type RF levels, concluding that the dominant-negative effect of the A5G transition is only functional in the context of a wild-type CRE(2C) structure (Fig. 4c). In summary, all CRE(2C) point mutants affecting the enteroviral CRE consensus sequence, so excluding U8A/C, showed a marked decrease in their capacity to support negative-strand RNA synthesis. Therefore, it can be concluded that these results ascribe an important role to the coxsackievirus CRE(2C) in the initiation of both positive- and negative-strand RNA synthesis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Yang et al., 2002), did not affect VPg uridylylation efficiency or RNA replication significantly. Characterization of the CVB3 CRE(2C) loop showed that the proposed entero- and rhinoviral consensus sequence (R1NNNAAR2NNNNNNR3), extrapolated from sequence-alignment studies and extensive mutational analysis of the rhinovirus 14 CRE(VP1) loop region (Yang et al., 2002; Yin et al., 2003), is also applicable to the CVB3 CRE(2C) loop sequence, because mutations that interfered with this consensus sequence affected CVB3 VPg uridylylation and RNA replication to a similar extent.
Consistent with previous reports, a mutant in which the CRE(2C) structure was disrupted completely still supported efficient negative-strand RNA synthesis in vitro (Goodfellow et al., 2003b; Morasco et al., 2003; Murray & Barton, 2003). This suggested that, under the conditions of the experiment, CRE-mediated VPg uridylylation is required for positive-strand, but dispensable for negative-strand, RNA synthesis. However, we also found a direct correlation between CRE(2C) point mutants producing lower, but detectable, amounts of VPgpU(pU) and reduced RF levels, affected to a similar extent. These latter results suggest that CRE(2C)-derived VPgpU(pU) is also involved in the initiation of negative-strand RNA synthesis. In correlation, CRE(2C) point mutants unable to support VPg uridylylation to a detectable level also showed a severe defect in RF accumulation. In addition, the A5G transition that affects the first templating adenosine residue was found to abolish RF synthesis completely (Fig. 4b).
How can we explain these observations? Very recently, Crowder & Kirkegaard (2005) reported, by using poliovirus CRE(2C) mutants, that a dominant-negative effect is observed on wild-type poliovirus replication using CRE(2C) point mutants, but not when the CRE(2C) has been disrupted completely. This suggests that RNA containing a complete CRE(2C) distortion mutant, as opposed to point mutants, utilizes an alternative mode of RNA replication that is so distinct from wild-type poliovirus replication that it does not interfere. As RF synthesis, in the absence of detectable levels of VPgpU(pU), was observed in the cell-free system using RNA containing a completely distorted CRE(2C) structure, it was suggested previously that this alternative mode of replication might involve the 3' poly(A) tail. This homopolymeric stretch might serve as the alternative template for the covalent linkage of uracil residues to VPg (Goodfellow et al., 2003b; Morasco et al., 2003; Murray & Barton, 2003), which was based on the findings that negative-strand RNA synthesis is VPg-dependent (Nomoto et al., 1977a, b) and that poly(A) templates supported VPg uridylylation in vitro (Paul et al., 1998).
A wild-type CRE(2C) structure sequesters replication proteins (Yin et al., 2003) and the formation of such protein–RNA interactions is most probably affected upon distortion of the CRE(2C) structure. Absence of this RNP complex in a genome containing a completely distorted CRE(2C) structure might favour the 3' poly(A) tail as the alternative template for the covalent linkage of UMP to VPg in vitro. The inability to detect free VPgpU(pU) within purified replication complexes might imply that VPg is elongated immediately into complementary negative RNA strands (Murray & Barton, 2003). A number of CRE mutants unable to accumulate detectable levels of uridylylated VPg still retained the ability to induce RF synthesis (e.g. G1C, A6C/G/U, A7U/C and A14C). Therefore, it can be speculated that these mutants also utilize the proposed alternative, 3' poly(A) tail-dependent mechanism to induce negative-strand RNA synthesis. However, whereas the CRE(2C) distortion mutant showed wild-type levels of RF synthesis at the measured time point, RF accumulation was reduced severely in CRE(2C) mutations affecting the enterovirus consensus sequence. Also, in contrast to the CRE(2C) distortion mutant, the G1C, A6C/G/U and A7U/C substitutions all maintained a wild-type CRE(2C) conformation, which we propose still enabled them to sequester the proteins required for VPg uridylylation. As shown previously for poliovirus, A6 substitutions still produced VPgpU(pU), albeit with reduced efficiency, which might support this speculation (Paul et al., 2003). Although the A14C mutation is predicted by MFOLD to adopt an alternative conformation due to a GC base-pair formation between the G1 and C14 residue, this mutant was found in poliovirus not to be reduced in protein binding (Yin et al., 2003) and might therefore still be able to produce VPgpU(pU). Additionally, Nayak et al. (2005) showed that A6 and A7 mutations displayed higher uridylylation activities in the context of full-length RNA compared with short hairpin RNAs, concluding that the template used in the uridylylation reaction determines both the specificity and efficiency of the assay. In summary, we speculate that the CRE-dependent uridylylation mechanism prior to negative-strand synthesis is strongly favoured when the CRE structure is intact.
By using purified components [CRE(2C), VPg, 3Dpol and 3CDpro], poliovirus VPg nucleotidylylation has been found not to be stringently dependent upon an AAACA motif in the CRE loop if one of the NTPs other than UTP was present. For example, VPgpG is formed by CAACA and GTP, as the first nucleotide, in this case a C residue, functions as a template (Gerber et al., 2001; Paul et al., 2003). Similarly, an A5C transversion or an A5G transition in the CVB3 A5AAUG motif yielded VPgpG and VPgpC, respectively, in the CVB3 CRE(2C) nucleotidylylation reaction (unpublished results). Nucleotidylylation is aborted at the stage of VPgpG or VPgpC because of the ‘slide-back’ mechanism to A6 (Gerber et al., 2001; Paul et al., 2003; unpublished results). If dual CRE(2C) CVB3 transcripts carry a 5'-terminal pppG group (Fig. 4a), only the A5C transversion mutant produced some RF, albeit in greatly reduced yield (Fig. 4b). The rationale for employing the pppGG transcript was to reduce positive-strand RNA synthesis in the cell-free assay, thereby shifting the complex to synthesize, if possible, predominantly negative-strand RNA (Herold & Andino, 2000). Virological characterization of both the CVB3 A5C and A5G mutants using a single CRE(2C) construct showed that the A5G mutation was quasi-infectious and reverted to a wild-type CRE sequence, whereas, in contrast to previous reports (Morasco et al., 2003; Yin et al., 2003), the A5C mutant proved to be a lethal mutation, probably caused by a dysfunctional 2C protein (unpublished results). On the basis of these data, we suggest that initiation of negative-strand RNA synthesis requires the A5 residue, but that, at least in assays performed using cell-free extract, this requirement is leaky (see below).
The stringency of sequence requirement does not appear to apply to the AA6AUG residue, as all mutations in A6 still produce RF, albeit also at very reduced yield (Fig. 4b). This corresponds to data from poliovirus, which have revealed that mutations in the corresponding adenosine residue (AAACA) yielded quasi-infectious phenotypes (Paul et al., 2003). These phenotypes were also found for the analogous point mutants in CVB3 CRE(2C) (unpublished data). Perhaps VPgpU formed on ANACA CRE(2C) mutants of the poliovirus or ANAUG CRE(2C) mutants of CVB3 can still function as primer for negative-strand RNA synthesis on the 3' poly(A) template.
RNA replication of human enterovirus genomes proceeds on membranes in RNP complexes of which the structures have not yet been entirely deciphered. Available evidence suggests that the 5'-terminal cloverleaf and the 3'-terminal poly(A) tail induce the formation of a circular RNP complex via protein–protein interaction (Barton et al., 2001; Herold & Andino, 2001). Moreover, it has been suggested that the cloverleaf structure is also involved in VPg uridylylation (Lyons et al., 2001). Thus, all three elements, the cloverleaf, the CRE and the 3' poly(A) tail, may interact for initiation of genome replication to occur. Elimination of the CRE by destroying its stem–loop structure may allow the system to bypass CRE requirement for negative-strand RNA synthesis and, by a default mechanism, make use of the genetically encoded 3' poly(A) tail as template for uridylylation in a cell-free in vitro system (Goodfellow et al., 2003b; Morasco et al., 2003; Murray & Barton, 2003; Paul et al., 1998). As viral negative-strand RNA was found to be absent in cells transfected with the complete CRE(2C) disruption mutant (Goodfellow et al., 2000), it is sensible to conclude that utilization of the 3' poly(A) tail for uridylylation can only be seen in the in vitro translation/replication system. In stark contrast, if the replication proteins are presented with a CRE element whose A5 has been mutated to a G residue, the aberrant nucleotidylylation product VPgpC might operate as a dominant-negative element. The nucleotidylylation complex, however, is not ‘frozen’ on the mutated CRE(2C) itself, as the addition of a second wild-type CRE restored the ability to initiate RNA replication (unpublished results; Yin et al., 2003). An alternative mechanism could be envisaged in which the replicase complex containing VPgpC is positioned on the 3' poly(A) tail and thereby might block the initiation of negative-strand RNA synthesis. We speculate that the A5C transition mutant, accumulating a VPgpG nucleotidylylation product, may form a less-stable RNP complex on the 3' poly(A) tail, allowing a severely reduced yield of complementary RNA strands via the alternative 3' poly(A) tail-dependent mechanism, although further investigation is required to elucidate the exact mechanism.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 29 June 2005;
accepted 18 September 2005.
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