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Short Communication |
1 Radboud University Medical Centre Nijmegen, Nijmegen Centre for Molecular Life Sciences, Department of Medical Microbiology, PO Box 9101, 6500 HB Nijmegen, The Netherlands
2 Radboud University Nijmegen, Institute for Molecules and Materials, Laboratory of Biophysical Chemistry, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands
Correspondence
Willem J. G. Melchers
w.melchers{at}ncmls.ru.nl
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ABSTRACT |
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A rotating version of Fig. 1(c)
is available as supplementary material in JGV Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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).
The enterovirus oriL is involved in virus replication and translation (Andino et al., 1990; Barton et al., 2001; Gamarnik & Andino, 1998). This conserved element folds into a cloverleaf structure and interacts with diverse viral and host proteins (Andino et al., 1990; Gamarnik & Andino, 2000; Harris et al., 1994; Parsley et al., 1997). The existence and functional importance of a ribonucleoprotein (RNP) complex, involving oriL interacting with the virus protein 3CD and the cellular factor PCBP, are required for genome circularization and RNA synthesis (Barton et al., 2001; Herold & Andino, 2001).
The enterovirus oriR is involved in the initiation of (–)-strand RNA synthesis (Melchers et al., 1997; Pilipenko et al., 1996). We have shown that sequences within the loop of domain X base pair with complementary sequences in the loop of domain Y to form an intramolecular tertiary structural element, designated a ‘kissing’ interaction (K domain) (Fig. 1a). The functional significance of the K domain is evident, as mutations introduced to destabilize this structure resulted in inactivated viruses, severely impaired mutants or the accumulation of revertants with a restored kissing interaction (Mirmomeni et al., 1997; Pilipenko et al., 1996; Wang et al., 1999). The K domain can be stacked onto the helix of domain X to form one coaxial helical domain that, connected to domain Y, determines the overall structure of the oriR (Melchers et al., 1997; Pilipenko et al., 1996). Thus, the enteroviral oriR can be viewed as two elongated helices (Fig. 1a–c). We previously found that the kissing structure is relatively stable and that it may serve as an anchor to orient the attached helical elements (Wang et al., 1999). We suggested that the mutual orientation of the X and Y helices is critical to ensure a proper oriR function and that certain areas of these two helical domains should crosstalk (Melchers et al., 2000). This crosstalk might be important for the simultaneous interaction with ligands, probably required to form an RNP composed of viral (Harris et al., 1994) and host (Mellits et al., 1998; Todd et al., 1995; Waggoner & Sarnow, 1998) proteins. Here, we hypothesize that the single-stranded nucleotides bridging the coaxial helices (Y–X and K–Y linkers; Fig. 1a–c) are important to determine this spatial orientation.
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–d). Covariance analysis showed that the length and sequence of the spacers (K–Y and Y–K) are conserved in the human B enteroviruses (Fig. 1d). Although also conserved, the K–Y spacer is 12 nt in poliovirus-like enteroviruses, but lacks the single bridging nucleotide (Fig. 1d). To investigate the significance of the single-stranded linkers, a series of mutants was designed in which the spacers were either shortened or enlarged. Mutations were introduced into the pRibCB3/T7-cDNA clone (an infectious cDNA clone derived from the coxsackievirus B3 Nancy strain; van Ooij et al., 2006), by PCR using forward primer 5'-ACTCTACGCTTTAAATGGTTGGACTCCTTTTAG-3', containing a blunt DraI restriction site (underlined), in combination with mutagenic oligonucleotides containing SalI restriction sites (Table 1). PCR products were digested with DraI and SalI and subcloned in pRibCB3/T7-Stu digested with StuI and SalI. In the first two mutants, the spacer bridging the top of the K domain and the Y helix was shortened by 3 (Y6, deleting GAU65–67) or 6 (Y3, deleting GAUAAC65–70) nt. Another mutant was engineered in which the length of the spacer was increased by 3 nt (Y12) by duplicating the CCA stretch, resulting in a mutant with a poliovirus-like spacer of 12 nt (5'-CCACCAGAUAAC-3') (Fig. 1d). Also, a mutant was constructed in which the single adenosine (A55) bridging the bottom Y helix to the K domain was deleted.
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a, Y6), whereas the GAUAAC65–70 deletion (Y3) only showed plaques after extended incuation periods, indicative of a very severe defect in virus replication. In contrast, the poliovirus-like coxsackievirus mutants with the enlarged linker (Y12) or the A55 deletion exhibited growth characteristics similar to those of wild-type coxsackievirus B3 at both 36 and 39 °C (Fig. 2a).
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). As we have described previously, analysis of mutants affecting the length of the helical domains X and Y allowed us to conclude that there are crosstalking events between the bottoms (probably the bottom U88 · A110 and C43 · G82 base pairs; Fig. 1) of the helices (Melchers et al., 2000). In other words, these bottom base pairs should be recognized simultaneously and in a coordinated way. We have also shown that changes in the length of the helical elements do not bring about changes in the spatial position of, among others, the 5'-GUAAA-3' spacer joining these two helical domains (Melchers et al., 2000). To assess whether the length of the single-stranded spacer itself might contribute to phenotypical changes possibly associated with the orientation of the helical elements, mutants were designed in which the length of the 5'-GUAAA-3' spacer, joining helical elements K–X and Y, was either deleted, shortened or enlarged (Fig. 2c). Following transfection, all designed mutations resulted in mutant viruses. Assessment of the replication kinetics of these mutant viruses at both 36 and 39 °C showed that a complete or partial deletion of the GUAAA spacer severely debilitated virus propagation at 36 °C (Fig. 2c). At 39 °C, minute plaques were only observed upon extended incubation periods for the complete GUAAA deletion and the GUA deletion mutant, whilst a small-plaque phenotype was observed for the GUAAA
GUAA mutant (Fig. 2c). Enlarging the Y–X spacer by an additional three residues (GUAAA to GUAAAAAA) resulted in a mutant virus that was severely temperature-sensitive, as evidenced by the pinprick plaques at 39 °C, but close to wild-type growth characteristics at 36 °C (Fig. 2c). Therefore, both increasing and decreasing the length of this spacer affected virus replication, possibly resulting from a disorientation of the bottom base pairs as described previously (Melchers et al., 2000), and thereby an altered geometry of the overall structure (Fig. 1c).
There are only limited data available on the functional aspects of interconnecting single-stranded regions, but the J/J5a linker that joins the two halves of the P4–P6 domain of the Tetrahymena self-splicing group I intron acts as a hinge, allowing the coaxially stacked elements on either side of it to interact via specific tertiary contacts (Szewczak & Cech, 1997). Indeed, shortening the coxsackievirus oriR Y–X and K–Y linkers resulted in highly disabled mutant viruses. Again, this may be the result of disturbed ligand binding. On the other hand, phenotypic effects of the local rotation of one or more base pairs in either domain X or domain Y suggested that the two distal base pairs of these domains serve as orientation-dependent recognizable signals (Melchers et al., 2000). Shortening the proximal spacer may increase the pressure on the distance and orientation of the distal base pairs, as in the ends of a clothes peg. Increasing the length, on the other hand, may increase the flexibility of the loop, which can be anticipated by the coaxial helical domains without affecting the overall geometry of the molecule. Indeed, this mutant did not manifest itself in phenotypic alterations at 36 °C (Fig. 2c).
To investigate the physiological importance of the primary sequence, a point-mutational analysis was undertaken in which each base of the K–Y and Y–X single-stranded spacers was mutated individually without affecting the overall secondary structure of the oriR (as determined by Mfold computer predictions; Zuker et al., 1999). The specific mutations are shown in Fig. 2(b, d). All mutated coxsackievirus B3 full-length transcripts rendered virus upon transfection. The mutant K–Y viruses could be grouped into three classes depending on their growth properties (Fig. 2b). In one group, the mutations did not result in a phenotypic alteration. This group consisted of the 5' mutations directly flanking the kissing interaction (i.e. Ypm1, Ypm2 and Ypm3) and mutant virus Ypm5, containing a mutation localized in the middle portion of the K–Y spacer. In the second group, mutant viruses Ypm4 and Ypm6 also had wild-type growth characteristics at 36 °C, but appeared to be thermosensitive (Fig. 2b). The last group, mutant virus Ypm7, Ypm8 and Ypm9, containing mutations flanking the Y-helical domain, were all severely affected in growth at both 36 and 39 °C (Fig. 2b). The negative effect on replication of a number of point mutations (especially those flanking the Y domain) may reflect the disturbance of a necessary local geometry. On the other hand, these mutations may disturb specific interactions with replication proteins that depend on such sequence-based recognition (Harris et al., 1994; Mellits et al., 1998; Todd et al., 1995; Waggoner & Sarnow, 1998). Of all the Y–X spacer point mutations introduced, only the AUAAA mutant was found to revert back to wild-type oriR sequence in two out of the three transfections performed, whereas all others had retained the engineered substitution after transfection. Interestingly, although each point mutation altering the sequence resulted in defective growth characteristics at 36 °C, the mutations did not manifest a phenotypic alteration at 39 °C, indicating a cold-sensitive mutant (Fig. 2d). The observation of wild-type growth characteristics at higher temperatures suggests that the 5'-GUAAA-3' spacer may adopt a specific structure. Although this observation is difficult to explain, this structure might be affected by the specific point mutations at the optimal temperature (36–37 °C), resulting in an adverse effect on replication due to the destabilization of recognition signals. At higher temperatures, these point mutations might be tolerated because of increased movement of the molecule, thereby compensating the effect on replication. A complete deletion of this GUAAA spacer in poliovirus has been shown to abolish RNA replication at the level of negative-strand RNA synthesis by using a cell-free replication system (Barton et al., 2001; Morasco et al., 2003). Although ultimately this mutation does give rise to (highly debilitated) viruses, from these results it can be concluded that the GUAAA sequence is of importance for an efficient initiation of RNA synthesis.
In conclusion, our results show the importance of the single-stranded regions linking the two coaxially stacked helices K–X–S and Y–Z in the coxsackievirus oriR and demonstrate that the intrinsic connection between oriR structure and function is fine-tuned by the spacing between these coaxial RNA helices. Obviously we are aware of the limitations to understand large RNA structures by studying the effects of altering structural domains using biochemical/virological approaches. However, as long as large, complex RNA molecules elude high-resolution structure determination by nuclear magnetic resonance or X-ray crystallography, virological/biochemical experiments are important to elucidate the structure–function relationship of RNA elements.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Received 26 September 2005;
accepted 19 November 2005.
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