Institut für Virologie, Klinikum der Friedrich-Schiller-Universität, Winzerlaer Str. 10, D-07745 Jena, Germany1
Institute for Molecular Biotechnology, Beutenbergstr. 11, D-07745 Jena, Germany2
School of Biology and Biochemistry, The Queen's University of Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK3
Author for correspondence: Roland Zell.Fax +49 3641 65 7202. e-mail i6zero{at}rz.uni-jena.de
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Abstract |
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Introduction |
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Phylogenetic studies also suggest that the 5'-non-translated region (NTR) of all enteroviruses and human rhinoviruses contains highly conserved RNA secondary structures. So far, the existence of such secondary structures was proven only for a few representatives of both genera, e.g. the polioviruses (PV) and human rhinoviruses 2 and 14. The secondary structures of PV constitute two genetic elements; one of which serves in the initiation of positive-strand RNA synthesis while the other facilitates the cap-independent translation initiation. Of special interest is a conserved cloverleaf-like domain at the very 5'-end of the genome. This secondary structure was previously demonstrated to be part of a ribonucleoprotein complex necessary for the initiation of positive-strand RNA synthesis. It interacts with virus-encoded proteins like the 3CD proteinase (3CDpro) and 3AB, the precursor of the VPg peptide (Andino et al., 1990 , 1993
; Leong et al., 1993
; Harris et al., 1994
; Walker et al., 1995
; Xiang et al., 1995
). Although the binding site of the 3CDpro was not defined precisely, some evidence indicates that it may map to stemloop D of the cloverleaf. Interaction was also demonstrated with the cellular protein p36, which was identified as a processed form of the eukaryotic elongation factor 1
(Andino et al., 1993
; Harris et al., 1994
; Roehl & Semler, 1995
; Roehl et al., 1997
). Binding of the poly(rC) binding proteins 1 and 2 (PCBP1, PCBP2) to the PV cloverleaf seems to up-regulate translation initiation (Gamarnik & Andino, 1997
, 1998
). Cap-independent translation initiation, the other function of the 5'-NTR, is facilitated by a type I internal ribosome entry site (IRES). The IRES region was demonstrated to be a cis-acting element that directs in vivo the binding of ribosomal subunits and several cellular protein factors to the viral RNA in order to accomplish internal translation initiation (for recent reviews, see Jackson & Kaminski, 1995
; Belsham & Sonenberg, 1996
). Although some experiments support the idea of two genetic elements which are physically separated and function independently (e.g. Rohll et al., 1994
), more recent data indicate that RNA synthesis of PV depends on sequences of both elements (Borman et al., 1994
), suggesting a dual role of the IRES sequences. Due to the high degree of sequence homology of the 5'-NTR, it is believed that the similar mechanisms of replication initiation and translation initiation apply to the other enteroviruses and rhinoviruses.
Although the general RNA folding pattern appears to be very similar, the 5'-NTRs of the bovine enteroviruses (BEVs; see Fig. 1), the human enteroviruses (see Fig. 2
) and the human rhinoviruses differ significantly from each other. Unique features of the BEVs are (i) the presence of sequences encoding a second putative cloverleaf-like secondary structure (domain I*) separated from the 5'-cloverleaf (domain I) by a small stemloop structure (domain I**), (ii) the size and shape of the putative domains III and VI of the IRES region, and (iii) the characteristic nucleotide sequence of the 3'-NTR (Zell & Stelzner, 1997
). With the exception of stemloop D, domain I* of the BEV 5'-NTR exhibits only little homology to the cloverleaf domains of the other enteroviruses and rhinoviruses (compare Figs 1
and 2
). The function of domains I* and I** is still unclear.
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Methods |
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Amplified DNA fragments were digested with the appropriate enzymes to generate compatible ends and ligated to the corresponding vector fragments of pCVB3-M2 derivatives. The following full-length cDNA plasmids were constructed (Fig. 3): pCVB3(BEV-A) (BEV domains II**I* in CVB3 background), pCVB3(BEV-B) (BEV domains II* in CVB3 background), pCVB3(BEV-C) (BEV domain I in CVB3 background), pCVB3(BEV-D) (BEV domains II** in CVB3 background), pCVB3(BEV-E) (BEV domain I* in CVB3 background), pCVB3(BEV-F) (BEV domains II**CVB3 domain I in CVB3 background), pCVB3(BEV 5'-NTR) (BEV 5'-NTR in CVB3 background), and pCVB3(BEV-G) (BEV IRES in CVB3 background). The structural intactness of all plasmid clones was verified by DNA sequencing.
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Viruses were usually propagated in GMK cells. Virus titres were determined by TCID50 assays according to Reed & Muench (1938) . Plaque morphology was determined by plaque assays. CVB3 (Nancy strain), CVB3(SnaBI) and the hybrid viruses CVB3(BEV-A), CVB3(BEV-B) and CVB3(BEV-F) were generated upon transfection of GMK cells with pCVB3-M2 and mutated full-length cDNA constructs, respectively. For transfection, GMK monolayers were incubated with 15 µg plasmid DNA per 6 cm Petri dish together with 15 µl Lipofectin (Gibco BRL Life Technologies) as liposome reagent in 2 ml DMEM for 3 h according to the manufacturer's instructions. Using wild-type cDNA, virus-induced cytopathic effect (CPE) was visible within 48 h post-transfection. If no CPE was visible, cells were passaged at day 3 and, if necessary, again at days 6 and 9. Cells were observed for at least 14 days. All viable chimeric viruses of this study appeared after passage at day 3. Hybrid cDNA constructs were considered to be non-infectious when at least three transfection experiments did not yield viable virus within 14 days at 37 °C and 33 °C, respectively. Mutations and the hybrid 5'-NTRs of virus chimeras were verified by reverse transcription followed by PCR and DNA sequencing.
Construction of plasmids supporting IRES-driven translation and in vivo translation experiments.
Starting from HindIII-linearized plasmid pD6 (Niepmann et al., 1997 ), amplified DNA fragments of the 5'-NTRs of PV1 (template DNA pT7-XL), CVB3 (template DNA pCVB3-M2), BEV1 (template DNA pGEM-3Z-BEV1) and the various hybrid constructs, respectively, were cloned 3' to the chloramphenicol acetyltransferase (CAT) gene and 5' to the firefly luciferase reporter gene. The following synthetic oligodeoxyribonucleotides were used for the amplification: pD6-BEV1 (5' GCAGGAAAGCTTGTAACTTAGAAGTACTAGCAA 3'); pD6-BEV2 (5' GCAGGAAAGCTTTTCAAATGTCTTGTGTRATGCTG 3'); pD6-PV1 (5' GCAGGAAAGCTTGTAACTTAGACGCACAAAACC 3'); pD6-PV2 (5' GCAGGAAAGCTTTATGATACAATTGTCTGATTG 3'); pD6-CVB1 (5' GCAGGAAAGCTTGTAACTTAGAAGTAACACAC 3'); pD6-CVB2 (5' GCAGGAAAGCTTTTGCTGTATTCAACTTAACAA 3'); pD6-BEV-CL (5' GCAGGAAAGCTTTTAAAACAGCCTGGGGGTTG 3'); pD6-BEV-CL2 (5' GCAGGAAAGCTTCTCTACCAATGTGGGGAGTAG 3'); and pD6-CVB-CL (5' GCAGGAAAGCTTTTAAAACAGCCTGTGGGTTG 3').
The following pD6 derivatives were constructed (see also Figs 5 and 6
): pD6(CVB IRES), pD6(PV IRES), pD6(BEV IRES), pD6(CVB 5'-NTR), pD6(BEV 5'-NTR), pD6(BEV-A) (BEV domains II**I* followed by a CVB IRES), pD6(BEV-B) (BEV domains II* followed by a CVB IRES), pD6(BEV-C) (BEV domain I followed by a CVB IRES), pD6(BEV-D) (BEV domains II** followed by a CVB IRES), pD6(BEV-E) (BEV domain I* followed by a CVB IRES), pD6(BEV-F) (BEV domains II** followed by CVB domains IVII) and pD6(BEV-G) (CVB cloverleaf followed by a BEV IRES). The correct DNA sequence of the inserts was verified prior to translation experiments.
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Results |
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The relevant genome region of the hybrid 5'-NTR of both cDNA-generated virus chimeras was sequenced after reverse transcription and amplification of a 450 nt DNA fragment. As a result, no sequence deviation from the expected sequence was observed (data not shown). For further characterization, the plaque phenotypes and the one-step growth curves at 37 °C were determined (Fig. 4). The chimeric virus CVB3(BEV-A) replicated normally, as estimated from the large-plaque phenotype and the one-step growth curve. In contrast, the other chimera CVB3(BEV-B) had a pinpoint plaque phenotype. The one-step growth curve revealed slow growth and a replication efficiency of about 10% when compared to the wild-type virus. Since both virus chimeras replicated in different cell lines of human and simian origin (data not shown), the cell type specificity seems to be unchanged in comparison to the CVB3 wild-type virus.
The hybrid BEVCVB double cloverleaf is also functional in CVB3
Construction of hybrid plasmids with a deletion of either the first or the second putative cloverleaf of BEV1 resulted in non-infectious DNAs, indicating the necessity of a genome region encoding the putative double cloverleaf. To test the hypothesis of whether the function of the second BEV cloverleaf can also be performed by the CVB3 cloverleaf, a plasmid with a hybrid double cloverleaf was constructed. Starting from the non-infectious plasmid pCVB3(BEV-D), which has a deletion of the second BEV cloverleaf, the genome region representing the CVB3 cloverleaf was inserted 3' to the BEV domain I**. This construct contained BEV domains I and I** followed by the CVB domain I and the remnant of the CVB3 genome (Fig. 3F). Transfection experiments with this plasmid yielded viable virus [designated CVB3(BEV-F)], which was further characterized (Fig. 4
). The chimera had a normal plaque phenotype and replicated like wild-type CVB3. Sequencing of viral RNA revealed that the nucleotide sequence was as expected (data not shown).
Exchange of the IRES region
As depicted in Fig. 1, the putative BEV IRES (nt 215700) exhibits significant differences in domains III and VI, which may result in altered properties of the IRES region. In order to investigate the capability of the BEV1 IRES region to substitute for the CVB3 IRES, domains IIVII of CVB3 were exchanged with the corresponding BEV1 domains IIVI. This construct [pCVB3(BEV-G)] contained the complete putative BEV IRES and the remnant of the BEV 5'-NTR up to the start codon (nt 215821; Fig. 1
). Although the exchanged region of this plasmid construct showed no deviation from the published sequence, it was not possible to rescue viable virus. Since a functional BEV IRES may require specific interactions with other parts of the 5'-NTR, the exchanged region was expanded to the complete 5'-NTR of BEV1 [pCVB3(BEV 5'-NTR), nt 1821]. This plasmid construct was also non-infectious.
IRES-driven luciferase expression in GMK cells
Translation experiments were designed to define the BEV1 and CVB3 IRES, respectively, and to assay the translation efficiency of hybrid NTRs leading to a null-phenotype of full-length cDNA constructs. For this purpose, several pD6 plasmids allowing the in vitro transcription of bicistronic RNAs were constructed and used to transfect GMK cells. The pD6 plasmids contain two reporter genes separated from each other by a DNA fragment derived from the 5'-NTR of either BEV1, PV1, CVB3 or the hybrid constructs. These DNA fragments were cloned 3' to the CAT reporter gene and 5' to the luciferase reporter gene (Figs 5 and 6
).
The first experiment was intended to demonstrate the ability of the putative IRES regions of BEV1 and CVB3 to direct cap-independent translation of the firefly luciferase message in vivo (Fig. 5). GMK cells were transfected with 1 µg in vitro-transcribed uncapped RNA per Petri dish. After incubation at 37 °C for 8 h, the cells were lysed and the cell extracts were assayed for CAT and luciferase activity. Since uncapped RNA was used for transfection, no CAT expression was traceable, neither with a CAT activity assay nor with a CAT-specific ELISA. Therefore, read-through activity was considered to be negligible. However, specific luciferase activities were detectable when the luciferase messenger was under control of IRES-encoding BEV1 sequences (nt 215821) and CVB3 sequences (nt 110743). Mutated or inverted IRES sequences cloned into the pD6 vector did not promote translation initiation (data not shown). Comparison of the CVB3 IRES with the PV IRES (as positive control) and the BEV IRES revealed slightly superior efficiency of the former element.
Recent experiments ofGamarnik & Andino (1997 , 1998
) have indicated that binding of PCBP to the cloverleaf region of PV seems to up-regulate translation initiation. Since sequences of the putative BEV double cloverleaf may exhibit a similar effect on IRES-driven translation, a pD6 construct with the BEV 5'-NTR was tested and compared to the translation efficiency of the CVB3 5'-NTR. The translation initiated by the complete 5'-NTRs of both viruses was significantly enhanced, as demonstrated in Fig. 5(B)
. Since a full-length cDNA construct containing a replacement of the CVB IRES with the BEV IRES was non-infectious, the possibility could not be excluded that translation initiation of this construct was severely affected. Therefore, the translation efficiency of the corresponding pD6 derivatives was also assayed. Fig. 5(B)
indicates that translation driven by the BEV IRES was enhanced by the presence of the CVB cloverleaf.
Translation experiments employing artificial bicistronic plasmids with BEV sequences fused to CVB3 IRES sequences were constructed to determine whether the putative BEV cloverleaf domains enhance CVB IRES-driven translation initiation. The results of these experiments are presented in Fig. 6. The translation efficiency of the CVB3 IRES is significantly enhanced in the presence of the BEV double cloverleaf. Since the non-viability of certain full-length constructs (Fig. 3CE
) may be due to a lack of translation initiation, their efficacy of translation initiation was also assayed. As shown in Fig. 6
, all constructs stimulated IRES-driven translation, albeit in various amounts. A possible correlation was observed between the non-viability of the full-length plasmids pCVB3(BEV-C), pCVB3(BEV-D) and pCVB3(BEV-E) (Fig. 3
) and little luciferase expression induced by the respective pD6 constructs.
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Discussion |
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Enhancement of reporter gene translation
Phylogenetic studies as well as translation experiments described in this study suggest the presence of functional IRES elements within the BEV1 and CVB3 5'-NTR, respectively. While the latter is very similar to the well-studied PV 5'-NTR, significant differences in the former 5'-NTR were described which may affect functional properties of the IRES element. However, translation efficiencies of both elements are comparable (Fig. 5B). In our in vivo system, the BEV sequence from nt 215 to 821 and the CVB3 sequence from nt 105 to 743 are sufficient to drive translation of the luciferase message (Fig. 5B
). Since the IRES-encoding sequences were dissected from the virus background and fused to the heterologous firefly luciferase gene, efficient translation initiation seems not to require essential sequences far distant from the 5'-NTR. Significant enhancement of the luciferase activity was observed when the IRES element was fused to the CVB3 cloverleaf or the putative BEV double cloverleaf. The intact 5'-NTRs of both viruses as well as hybrid 5'-NTRs with reciprocal exchanges of cloverleaf-encoding sequences yield translation efficiencies which are four to six times higher than the respective IRES regions (Figs 5
and 6
). This translation stimulation was significantly reduced in the case of hybrid NTRs with deletions of either the first or second BEV cloverleaf. The low values of luciferase activity correlated with the non-viability of the respective full-length cDNA plasmids (Fig. 3CE
). Therefore, one may assume that reduced translation efficiency is the cause of non-viability. However, disturbed replication could also explain the null-phenotype. To address this question, experiments with suitable bicistronic full-length cDNA clones are presently being performed. It has to be investigated whether the enhancement of translation is based on PCBP-binding to the cloverleaf. In PV,Gamarnik & Andino (1997
, 1998
) have observed an up-regulation of translation after binding of these proteins to the cloverleaf.
Non-complementation of the IRES function
Phylogenetic analyses as well as the translation assays described in this study indicate that in analogy to the PV IRES, the BEV IRES spans a genome region ranging approximately from nt 215 to 696 followed by the downstream AUG start codon at nt 819 (Fig. 1). The putative domains III and VI of the BEV IRES differ significantly in size and shape from the corresponding domains of the enteroviruses and rhinoviruses. Also, the BEV IRES has no putative domain VII, which is conserved in all the other enteroviruses (Zell & Stelzner, 1997
). Therefore, it may be considered as a third specimen of the so-called type I IRES. Whereas previous experiments proved that type I and type II IRES regions of certain picornaviruses can substitute for the IRES function of other related viruses (e.g. Johnson & Semler, 1988
; Rohll et al., 1994
; Xiang et al., 1995
; Todd et al., 1997
), there is still a lack of information as to whether the BEV IRES also has this ability. In this study, rescue of viable chimeric virus was not successful after transfer of the IRES region (nt 215821) and the complete 5'-NTR (nt 1821). The translation efficiency of both constructs was not affected. The null-phenotype may indicate that the BEV IRES fails to functionally substitute for the coxsackieviral IRES. However, non-viability of both full-length constructs could also be a result of disturbed replication. Borman et al. (1994)
have demonstrated that sequences of domains IV and V are essential for RNA synthesis of PV. This may also be the case for BEV1 and CVB3. The significance of this observation for other enteroviruses is unknown. A third explanation could be a failure of other steps of the virus life-cycle (e.g. encapsidation). At present, one cannot exclude the removal of unknown functional sequences of CVB3 located 3' to the IRES element. Experiments addressing the replication efficiency of the BEVCVB chimeric constructs are currently being performed to resolve these questions.
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Acknowledgments |
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References |
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Received 19 February 1999;
accepted 17 May 1999.