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Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands
Correspondence
Eric J. Snijder
e.j.snijder{at}lumc.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The predicted RNA secondary structures of the deletion mutants produced in this study are available as supplementary material in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Positive-strand RNA viruses replicate in the cytoplasm of infected cells, a process that is mediated by an enzyme complex containing the viral RNA-dependent RNA polymerase (RdRp). Genomic RNA serves as template for the production of negative-strand RNA, which is subsequently used as template for the synthesis of new plus strands. The amplification of viral RNA requires the recruitment of the RdRp complex to specific cis-acting sequence motifs or structures (‘replication signals’) within the termini of the viral RNA, where the RdRp complex initiates the synthesis of plus or minus strands (Buck, 1996; van Dijk et al., 2004).
In addition to the production of progeny genome copies, replication of arteriviruses and coronaviruses entails the synthesis of a nested set of 3' and 5' co-terminal subgenomic (sg) mRNAs that are generated by a unique mechanism involving a discontinuous step. Accumulating evidence supports a model in which the discontinuous step in sgRNA production occurs during negative-strand RNA synthesis (Sawicki & Sawicki, 1995, 2005; van Marle et al., 1999; Baric & Yount, 2000; Sawicki et al., 2001; Pasternak et al., 2001, 2006; Zúñiga et al., 2004; van den Born et al., 2004). Thus, for nidoviruses both genome replication and sgRNA synthesis would initiate at the 3' end of the viral genome. Therefore, it is likely that at least part of the regulatory signals for these processes is implicit in the RNA sequence and structure of the genomic 3' untranslated region (UTR).
Among positive-stranded RNA viruses, there is considerable variety of 3' terminal structures: tRNA-like elements, poly(A) tails and terminal structures that fit neither of these two categories are found (Dreher, 1999). Nidoviruses have 3'-polyadenylated genomes and the structure of the 3' UTR upstream of the poly(A) tail has been the focus of several studies. For the coronavirus Murine hepatitis virus (MHV), the structure of the 3' UTR has been probed and several stem–loop structures involved in virus replication were identified (Liu et al., 2001). The minimal signal needed for initiation of MHV negative-strand RNA synthesis has been mapped to the last 55 bases of the 3' UTR (Lin et al., 1994). The 3'-proximal domain of the genome folds into a stem–loop structure that has been identified as the binding site for host proteins (Yu & Leibowitz, 1995). For Bovine coronavirus, a pseudoknot interaction between two stem–loop structures upstream in the 3' UTR has been demonstrated to be required for virus replication (Williams et al., 1999). A similar pseudoknot interaction was found for MHV (Hsue & Masters, 1997; Hsue et al., 2000; Goebel et al., 2004) and predicted for Severe acute respiratory syndrome coronavirus (Goebel et al., 2004).
For the arterivirus PRRSV, a kissing-loop interaction was proposed between a stem–loop structure in the 3' UTR and an upstream hairpin located in open reading frame (ORF) 7 (Verheije et al., 2002). Recently, the structure of the 3' UTR of SHFV was investigated by ribonuclease probing experiments (Maines et al., 2005) and a stem–loop structure was identified that contains binding sites for two cellular proteins. To gain insight into the molecular details of EAV RNA synthesis, we developed an RNA secondary structure model for the 3'-proximal region of the genome using chemical and enzymic probing in combination with computer-assisted structure prediction. Based on this structure model, and using an EAV reverse genetics system, several deletions were engineered in the 3'-proximal domain of the genome. These mutants were tested in vivo for their effect on virus replication in general and RNA synthesis in particular. Two RNA domains were identified that are essential for virus replication and most probably play a key role in viral RNA synthesis.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) on the MFOLD web server (Zuker, 2003).
Site-directed mutagenesis, RNA transfections and analysis of virus replication.
Deletions in the 3'-proximal region of the EAV genome were generated by standard site-directed PCR mutagenesis and introduced into full-length cDNA clone pEAV211 (van den Born et al., 2004). The mutations were verified by sequence analysis. Following in vitro transcription from the full-length cDNA clones, the RNA concentration was measured by UV spectroscopy and the integrity of the RNA was verified by agarose gel electrophoresis. Equal amounts (30 µg) of full-length EAV RNA were transfected into BHK-21 cells by electroporation as described previously (van Dinten et al., 1997). Immunofluorescence dual labelling assays with EAV-specific antisera for non-structural protein 3 (nsp3) (rabbit) and GP5 (mouse monoclonal antibody) were performed as described previously (van der Meer et al., 1998) at different time points after transfection. For RNA isolation, cells were lysed at 14 h post-transfection and intracellular RNA was isolated using the acidic phenol method, as described previously (Pasternak et al., 2000). Viral RNA was analysed on denaturing formaldehyde/agarose gels and hybridized with the radioactively labelled oligonucleotide probe E868 (antisense, nt 12270–12289), which recognizes both genomic and subgenomic positive-strand RNA.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Nucleotides sensitive to the chemicals DMS and CMCT or the RNases T1 and T2 are assumed not to be involved in base-pairing or base-stacking interactions, whereas RNase V1 is specific for structured or double-stranded regions (Ehresmann et al., 1987). The sites of modification or cleavage were determined by primer-extension analysis. Control experiments with untreated RNA were performed in parallel to detect natural pauses in reverse transcription on the EAV template.
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. Two representative chemical probing experiments are shown in Fig. 2. Computer-based thermodynamic analysis of the EAV 3' UTR (nt 12646–12704) predicted a stem–loop structure with a large loop region (SL5 in Fig. 1). The structure-probing experiments were in agreement with the predicted structure, as the loop region was found to be highly accessible to single-strand-specific probes. Probing of the region upstream of SL5 (nt 12600–12658) demonstrated that this region was highly exposed to the chemicals DMS and CMCT. Although there were several base-pairing possibilities predicted by MFOLD analysis in this region, the probing experiments were consistent with the presence of a large single-stranded region and the small stem–loop structure SL4. Thermodynamic analysis of nt 12401–12600 predicted a large extended stem–loop structure in this region with several protruding bulges, internal loops and small stem–loops. The probing results were in good agreement with the MFOLD prediction. The predicted top of the extended stem–loop structure was formed by stem–loop structure SL3. The loop region was highly sensitive to single-strand-specific probes and, in addition, several predicted bulges in the stem were exposed. The upper part of the stem was cleaved by RNase V1, confirming the double-stranded nature of this region. Upstream of SL3, two protruding stem–loops could be recognized, SL2 and SL1. The loop region of both structures was found to be sensitive to single-strand-specific probes, whereas the stem region was cleaved by RNase V1.
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. However, sequence variation appeared limited in this region of the EAV genome and co-variations supporting the predicted structure were not identified. Most nucleotide variations were found in single-stranded or loop regions. Three nucleotide changes (nt 12440, 12476 and 12534; shaded grey in Fig. 3) that did not disrupt base pairing were identified within base-paired regions and only two nucleotide changes in SL3 (nt 12543 and 12546; indicated in black in Fig. 3) were identified that were predicted to disrupt base pairing. Although these changes affected base pairing in the lower part of the stem, they did not interfere with formation of the SL3 structure. Therefore, similar RNA secondary structures could be predicted for all EAV strains.
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) were engineered in an EAV full-length cDNA clone (van Dinten et al., 1997), which has been used as the basis for reverse genetics experiments. The deletions were designed carefully so as not to affect the folding of other structural elements in this region. The predicted RNA secondary structure of the deletion mutants is shown in Supplementary Fig. S1 (available in JGV Online). The small protruding SL1 and SL2 structures were deleted in mutants 
SL1 and SL2, respectively, whereas SL3, representing the top of the large extended stem–loop structure, was deleted in mutant SL3. Interestingly, this region is followed by a lengthy single-stranded region containing only the small SL4 structure. In mutant SL4, SL4 and the single-stranded flanking sequences on either side of this structure were deleted. The 3' UTR folds into SL5, a stem–loop structure with a large loop region. To disrupt this RNA secondary structure, the left side of the stem region was deleted in mutant S5. In addition, mutant L5 was generated, in which the large loop region was deleted and replaced by four A residues to maintain the SL5 stem.
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), which served as indicators for genome replication and sg mRNA synthesis, respectively. As SL1–SL4 are located within ORF7 (Fig. 4a), encoding the nucleocapsid protein, we anticipated that the production of infectious progeny would be affected for these deletion mutants, if they proved to be replication competent. Indeed, positive IFA results were obtained for mutants SL1, SL2 and SL3, but, in contrast to the wild-type control transfection, no spread of virus to (initially untransfected) neighbouring cells was observed (Fig. 4b). In addition, supernatants harvested from the transfected-cell cultures were tested for the presence of progeny virus using plaque assays. Even at low dilutions, plaques could not be detected, confirming that these mutants were unable to produce infectious progeny (results not shown). No IFA signal was observed for mutants SL4, S5 and L5 up to 56 h post-transfection (Fig. 4b) and no infectious progeny virus could be detected using plaque assays (results not shown), suggesting that important replication signals had been deleted or affected in these mutants.
Deletions in the 3' terminus of the EAV genome affect RNA synthesis
To investigate the synthesis of viral RNA (genomic and subgenomic), intracellular RNA was isolated from transfected cells at 14 h post-transfection. At this time point, the wild-type control virus had not yet spread to untransfected cells (first-cycle analysis) and could therefore be compared with the non-spreading mutants carrying deletions in the nucleocapsid protein gene. We did not anticipate an effect of these deletions on viral RNA synthesis, as our previous studies had demonstrated that all EAV structural proteins, including the nucleocapsid protein, are dispensable for both genome replication and sg mRNA synthesis (Molenkamp et al., 2000).
The isolated intracellular RNA was separated in denaturing formaldehyde/agarose gels and hybridized to an oligonucleotide probe detecting all virus-specific plus-strand RNA molecules. As shown in Fig. 5, for the wild-type virus the full-length genome (RNA1) as well as sg mRNAs 2–7 were detected. All of these RNAs were also produced by mutants SL1, SL2 and SL3, albeit at reduced levels compared with wild-type. Both genome replication and sg mRNA synthesis were affected to the same extent and a reduction to approximately 40 % of the level of the wild-type control was measured for all RNA species by phosphorimager analysis.
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SL4, S5 and L5. These results indicated that key signals for viral RNA synthesis are located in the 3' terminal 100 nt of the genome and that a moderate role in viral RNA synthesis can be attributed to the more upstream sequences deleted in mutants SL1, SL2 and SL3. Mutant SL4 contained a large deletion of 44 nt that included both SL4 and its flanking sequences. Two smaller and more specific deletions were introduced in the SL5 structure. Replacing the loop sequence by four A residues in mutant L5 completely blocked RNA synthesis, suggesting that the sequence and/or conformation of the loop are essential for RNA synthesis. Deletion of the left side of the stem in mutant S5 also inhibited RNA synthesis. This deletion was predicted to disrupt the SL5 structure, suggesting that the stem region is important for the formation and correct presentation of the loop. Alternatively, the structure of the stem region itself or a sequence motif in this region may be important for viral RNA synthesis. The contribution of distinct sequences or structural elements within SL5 to the process of EAV RNA synthesis is the subject of ongoing studies.
Role of the identified RNA signals in virus replication and RNA synthesis
Thermodynamic analysis also predicts stem–loop structures at the 3' terminus of other arterivirus genomes, although these structures are not identical to the SL5 structure in the EAV genome. For SHFV, structure-probing experiments provided evidence for a stem–loop structure at the genomic 3' terminus, which also contains a top region highly sensitive to single-strand-specific RNases (Maines et al., 2005). Two cellular proteins were found to bind to this domain and were identified as polypyrimidine-tract-binding protein and aldolase A (Maines & Brinton, 2001; Maines et al., 2005). The same proteins have been reported to interact with the 3' UTR of EAV and PRRSV (Maines et al., 2005). The biological role and importance of the binding of these proteins to the 3' UTR remains to be investigated. As there is no obvious sequence conservation between the 3' UTRs of different arteriviruses, the binding sites of these proteins may be determined by RNA secondary structure. For other nidovirus 3' UTRs, various interactions involving structure elements have been reported that are important for RNA synthesis and virus replication. For PRRSV, a kissing-loop interaction has been proposed between a predicted stem–loop structure in the 3' UTR and an upstream hairpin (Verheije et al., 2002). For several coronaviruses, a pseudoknot interaction between two stem–loop structures in the 3' UTR has been reported (Hsue & Masters, 1997; Williams et al., 1999; Hsue et al., 2000; Goebel et al., 2004). Whether the SL5 structure in the EAV genome is involved in such tertiary interactions is not currently known. Since SL5 is located in the 3' UTR of the EAV genome, it is tempting to speculate that it acts as a direct recognition signal for the initiation of minus-strand RNA synthesis. Alternatively, RNA synthesis may initiate on the poly(A) tail, in which case the RNA structure near the 3' terminus of the viral genome could provide the specificity to the process. This would allow the RdRp complex to discriminate between the polyadenylated viral RNA genome and polyadenylated cellular mRNAs, which are abundantly present in the host cell's cytoplasm.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 8 December 2005;
accepted 14 March 2006.
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