1 Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, LUMC P4-26, PO Box 9600, 2300 RC Leiden, The Netherlands
2 AVI BioPharma Inc., 4575 SW Research Way, Corvallis, OR 97333, USA
Correspondence
Eric J. Snijder
e.j.snijder{at}lumc.nl
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this report, we describe the evaluation of the efficacy of P-PMOs designed to inhibit the amplification of Equine arteritis virus (EAV), the prototype of the family Arteriviridae, which is united with the families Coronaviridae and Roniviridae in the order Nidovirales (Snijder & Meulenberg, 2001; González et al., 2003
). In addition to the full-length RNA genome (Fig. 1
a), EAV and other nidoviruses produce a set of nested subgenomic (sg) mRNAs in infected cells. In the case of arteriviruses and coronaviruses, these sg mRNAs contain a common leader sequence, derived from the 5'-terminal region of the genome, that is fused to different but overlapping body sequences, which are derived from the 3'-proximal region of the genome. Thus, sg mRNAs are both 5'- and 3'-coterminal with the genomic RNA. Conserved transcription-regulating sequences (TRSs) precede every open reading frame in the 3'-proximal quarter of the genome (body TRSs) and an additional copy is found at the 3' end of the leader (leader TRS). Base pairing between the leader TRS (in the plus strand) and body TRS complements in the minus-strand was found to play a critical role in joining the leader and body segments of sg RNAs in arteriviruses and coronaviruses (Pasternak et al., 2001
, 2003
; Zuniga et al., 2004
). The fusion of the sg RNA body to the leader sequence has been postulated to involve discontinuous extension during minus-strand RNA synthesis (Fig. 1b
) (Sawicki & Sawicki, 1995
). After attenuation of RNA synthesis at the body TRS, the nascent minus-strand is thought to translocate to the 5'-proximal region of the genomic template. Following TRS-guided base pairing with the template, RNA synthesis is resumed, adding the complement of the genomic leader sequence, thus completing the sg minus-strand. Positive-strand sg mRNAs are then transcribed from corresponding minus-strand sg RNA templates (Sawicki & Sawicki, 1995
).
|
Ten P-PMOs were designed to bind specifically to RNA sequences involved in different aspects of EAV amplification: genome replication, sg mRNA synthesis, and translation of genome and sg mRNAs. The 3'-terminal regions of the genome and antigenome were chosen as P-PMO target sites in an attempt to block replication, but only a moderate reduction of virus amplification was observed at relatively high oligomer concentrations. To assess whether the synthesis of sg mRNAs could be blocked exclusively, TRS motifs were targeted, but these P-PMOs were found to be ineffective at interfering with transcription. In contrast, all four of the antisense P-PMOs designed to base pair with sequences in the positive-strand genomic 5' UTR efficiently reduced virus amplification in a dose-responsive manner. At concentrations in the low micromolar range, some of these compounds completely, and others near-completely, inhibited virus amplification. In vitro translation assays indicated that these P-PMOs likely inhibited genome translation. Our data suggest that several of these compounds have antiviral potential at relatively low concentrations and also represent useful tools to study the molecular biology of arteriviruses.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RNA isolation and analysis.
Intracellular RNA from Vero-E6 cells was isolated at 36 h post-infection (p.i.), separated in denaturing agarose formaldehyde gels and analysed by hybridization to a 32P-labelled probe, which is complementary to the 3' end of the EAV genome and all sg mRNAs (5'-TTGGTTCCTGGGTGGCTAATAACTACTT-3') (van Marle et al., 1999). Dried gels were exposed to phosphorimager screens and scanned with a Personal Molecular Imager FX (Bio-Rad) after exposure. Band intensities were quantified with Quantity One v4.2.2 (Bio-Rad).
P-PMO design and synthesis.
Morpholino oligomers were designed to be complementary to target sequences in the EAV genome or antigenome. See Table 1, Fig. 1
and Results for P-PMO sequences and target locations. PMO is a single-stranded DNA analogue that has a morpholine ring in place of each riboside moiety and phosphorodiamidate intersubunit linkages instead of phosphorodiester linkage (Summerton & Weller, 1997
). The nucleoside bases attached to this backbone are the same as for DNA. In order to deliver the PMOs into cultured cells, an arginine-rich peptide NH2-RRRRRRRRRFF-CONH2, designated R9F2C (Moulton et al., 2004
), was covalently linked to the 5' end of each PMO. The performance of the R9F2C peptide varies with the amount of serum in the media (Neuman et al., 2004
; Moulton et al., 2004
). Hence, the 6 h P-PMO treatments were carried out in the absence of serum. All PMOs were synthesized at AVI BioPharma by methods described previously (Summerton & Weller, 1997
). The conjugation, purification and analysis of P-PMO compounds were carried out at AVI BioPharma according to methods described elsewhere (Moulton et al., 2004
).
|
Translation assays.
To test the effect of various P-PMOs on translation, cell-free translation assays were carried out using the previously described pDualLuc-scan1 reporter gene construct for EAV genome translation (van den Born et al., 2005). In vitro transcribed, capped bicistronic DualLuc-scan1 transcripts were in vitro translated in the presence of various P-PMOs using the Rabbit Reticulocyte Lysate system according to the manufacturer's protocol (Promega). Luciferase expression was measured using a luminometer (Turner Designs TD-20/20) and the Dual-Luciferase Reporter assay (Promega) on the basis of enzymic activity.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To assess whether using P-PMOs could reduce the synthesis of sg mRNAs, several TRS motifs were targeted (Fig. 1b). PMOTRS interactions were expected to interfere with body TRS-to-leader TRS base pairing, which is a crucial step in the production of sg RNAs (van Marle et al., 1999
; Pasternak et al., 2001
). The leader TRS of EAV is predicted to be located in the 20 nt loop of the so-called leader TRS hairpin (LTH; Fig. 1
). One P-PMO was complementary to the entire LTH loop (TRS1-as), whereas another P-PMO spanned both the leader TRS and the downstream replicase translation initiation codon (TRS1+AUG-as). A P-PMO complementary to TRS1-as (TRS1-s) was an appropriate control in these transcription-inhibition studies, as it was designed to target the complement of the leader TRS which is not involved in TRS base pairing. In addition, the RNA7 body TRS was targeted with two P-PMOs designed to interfere specifically with sg RNA7 synthesis. One P-PMO (TRS7-as) was complementary to the RNA7 body TRS in the genomic template and was designed to interfere with the attenuation step that has been postulated to precede the discontinuous step during sg minus-strand RNA synthesis. The other (TRS7-s) was designed to anneal to the RNA7 body TRS complement at the 3' end of the nascent minus-strand that, following attenuation, is presumed to be translocated to and base pair with the leader TRS.
Four of the P-PMOs were designed against targets located in the genomic 5' UTR. In addition to the 5'HP-as, TRS1-as and TRS1+AUG-as compounds, described above, a P-PMO with a sequence antisense to the 5'-terminal 20 nt (5'TERM-as) was designed to interfere with pre-initiation of translation of all sg mRNAs and genomic mRNA. Although P-PMO TRS1+AUG-as (Fig. 1a) was designed to interfere with genome translation (as well as sg mRNA synthesis) it is the only one of the four with little likelihood of interfering with sg mRNA translation, as all sg mRNAs lack most of the 16 contiguous nucleotides immediately downstream of the leader TRS that are part of the TRS1+AUG-as target sequence (den Boon et al., 1996
). In contrast, translation of all sg mRNAs and genomic RNA could be affected by 5'TERM-as and 5'HP-as as they target sequences in the common leader. A nonsense P-PMO (SCR) was also synthesized as a control for off-target effects of the P-PMO chemistry. P-PMO design details are summarized in Table 1
.
P-PMOs display low cytotoxicity and have EAV-specific antiviral activity
A cytotoxicity assay was performed in which each P-PMO was tested on Vero-E6 cells at concentrations of 0, 5, 10, 20, 40 or 80 µM in the culture medium. Cells were cultured with P-PMOs for 6 h in the absence of serum to allow P-PMO uptake, after which P-PMO-free medium containing 8 % FCS was given. Cell proliferation was monitored by microscopy after 3 days of incubation. At the highest concentration (80 µM) some of the P-PMOs clearly affected cell growth, as was evident from the reduced cell density compared with untreated cells. At 40 µM no morphological growth anomalies were apparent (data not shown), but as a precaution the P-PMO concentration never exceeded 20 µM in any subsequent experiment.
To assess P-PMO specificity and cytotoxicity, the effect of each compound on EAV amplification was compared to its effect on the amplification of the (unrelated) togavirus SinV. Cells were post-treated with 20 µM of each P-PMO, following inoculation with EAV or SinV under identical cell culture conditions, and virus yields were measured at 36 or 24 h p.i., respectively. SinV titres in the untreated control reached approximately 106 p.f.u. ml1 and were not significantly altered by P-PMO treatment (Fig. 2). Remarkably, several P-PMOs reduced the EAV titre by several orders of magnitude or even to undetectable levels (Fig. 2
). At 20 µM, control P-PMO SCR induced an approximately threefold reduction in EAV titre, an effect that was not observed with SinV. However, the much stronger anti-EAV effect observed with most EAV-targeted P-PMOs was clearly specific and not due to cytotoxicity or a generic barrier to cellular entry or egress of virus, as such non-specific effects would likely have caused an inhibition of SinV amplification as well.
|
|
The P-PMOs could be divided into three groups based on their ability to reduce the number of eGFP-positive cells. The first group consisted of TRS1-s and SCR, both of which had almost no effect on EAVGFP amplification when compared to the untreated control cells. At the highest P-PMO concentration used (20 µM), a relatively small reduction of about 25 % in the number of eGFP-positive cells was observed (Fig. 3b). A second P-PMO group, exhibiting intermediate efficacy, consisted of 5'HP-s, TRS7-as, TRS7-s, 3'TERM-as and 5'TERM-s, which, at relatively high compound concentrations, protected the majority (5095 %) of cells (Fig. 3b
). The third group comprises the four most active P-PMOs. Three of these compounds (5'HP-as, TRS1+AUG-as and 5'TERM-as) when present at 10 µM, and the fourth (TRS1-as) at 20 µM, were able to apparently protect all cells from virus infection (Fig. 3b
). In summary, most of the P-PMOs generated a substantial degree of inhibition of virus amplification when administered to cells immediately after virus infection.
Several P-PMOs inhibit virus amplification in a dose-dependent manner
To quantify the relationship between dose and efficacy of the various P-PMOs, cells were post-treated at varying P-PMO concentrations for 6 h following EAVGFP infection. Virus titres in cell culture media harvested at 36 h p.i. were determined by plaque assays. Not unexpectedly, the relative titre reductions observed with the different P-PMO dose levels (Fig. 3c) were in agreement with the relative number of infected cells determined by eGFP autofluorescence (Fig. 3b
). The group of four P-PMOs with the highest inhibition efficiency (5'HP-as, TRS1+AUG-as, 5'TERM-as, and to a lesser extent TRS1-as) generated EAVGFP titre reductions of four logs or greater, from a peak titre of 2·0x105 p.f.u. ml1 in untreated cells, to 30 p.f.u. ml1 or below in treated cells. These four compounds are all complementary to sequences in the genomic 5' UTR, indicating that this region is the most effectual target area for this class of compounds, at least in EAV. It is noteworthy that the small amount of residual virus from the inoculum that is usually detectable in low dilutions of culture medium, was not recovered in plaque assays (Fig. 3c
; see also Fig. 2
). The most likely explanation is that even residual amounts of highly efficacious P-PMOs prevented detectable plaque formation. Therefore, the P-PMO concentration at which virus particle production was completely prevented could not be exactly determined.
Some of the P-PMOs that do not have target sequences in the 5' UTR were able to inhibit virus amplification in a dose-dependent manner, but significant effects were observed only at relatively high concentrations (10 µM). TRS1-s, which targets the leader TRS complement, was completely ineffective (Fig. 3b and c
). Moderate effects of up to one log-reduction were observed for 5'HP-s and TRS7-s (Fig. 3c
), both of which target the minus-strand. The result obtained with 5'HP-s was particularly interesting because it targeted the complement of the conserved hairpin B in the 5' UTR (Fig. 1a
), suggesting that this sequence (and/or putative structure), located in the 3' end of the antigenome, plays a role in genome replication. Fairly strong inhibitory effects of approximately two log-reduction were obtained with 3'TERM-as, 5'TERM-s and TRS7-as. The former two target the 3' terminus of the genome and antigenome, respectively, and these results suggest that they interfere with the interaction between EAV RNA and the replicase complex.
P-PMOs targeting the EAV 5' UTR can markedly interfere with translation
To investigate the effect of the above-identified highly active compounds on EAV genome translation, a cell-free translation assay was performed using the previously described pDualLuc-scan1 construct (van den Born et al., 2005). Briefly, the T7 promoter of this vector directs the synthesis of a bicistronic mRNA harbouring the EAV 5' UTR fused to the firefly luciferase (Fluc) reporter gene followed by an Encephalomyocarditis virus (EMCV) IRES-driven Renilla luciferase (Rluc) gene, which serves as an internal standard in the experiments. In vitro transcribed, capped DualLuc-scan1 mRNA was added to rabbit reticulocyte lysate and the translation of the two reporter genes was measured. The expression of both Fluc and Rluc was analysed in the presence of the P-PMOs targeting the 5' UTR (5'TERM-as, 5'HP-as, TRS1+AUG-as and TRS1-as), SCR and 3'TERM-as. The latter compound is complementary to the 3'-terminal region of the EAV genome that is also present in DualLuc-scan1 mRNA (Fig. 4
a). Unless mRNA circularization is required for efficient translation in vitro, 3'TERM-as would not be expected to have activity in this system. At the highest P-PMO concentration used in this assay (1 µM), expression from the Rluc control cistron was not affected and could be used to normalize the Fluc activity (Fig. 4b
). At nanomolar concentrations all P-PMOs targeting the 5' UTR generated dose-dependent reduction of Fluc expression, with a 50 % inhibition concentration (IC50) in the 10100 nM range. Both controls, the SCR and 3'TERM-as P-PMOs, did not show nearly as high a reduction in translation, with IC50 values of approximately 1000 nM (Fig. 4c
).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The 5' UTR of the EAV genome was found to contain the most sensitive targets for inhibition with P-PMOs (Table 1 and Fig. 1
). All four P-PMOs directed to the 5' UTR were able to profoundly inhibit EAV amplification at low micromolar concentrations (Fig. 3b and c
), with the P-PMO targeting the 5' genomic terminus (5'TERM-as) consistently generating the strongest effect. For arteriviruses and coronaviruses, the genomic 5' UTR provides an appealing target area, because all viral mRNA share a large part of this region, the common leader sequence, due to the unique mechanism of sg RNA synthesis. Consequently, P-PMOs 5'TERM-as and 5'HP-as may inhibit the translation of replicase-coding EAV genomic RNA as well as all six sg mRNAs. In vitro translation assays indicated that their antiviral effect was exerted, at least partly, by inhibition of translation (Fig. 4
). A cell culture-based translation assay was also attempted, using transient expression of the luciferase reporters driven by the cytomegalovirus promoter that is present, in addition to the T7 promoter, in pDualLuc-scan1. However, for unexplained reasons, P-PMO treatment equally affected the expression of both Fluc and the internal standard Rluc in a sequence-specific and dose-dependent manner (data not shown). This suggested a problem with plasmid DNA transfection or reporter mRNA synthesis or stability in the presence of P-PMO and, consequently, we had to refrain from using this assay.
Our results indicate that genome replication can also be reduced by targeting the genome termini (P-PMOs: 3'TERM-as and 5'TERM-s). Since translation assays indicated that the mode of action of 3'TERM-as was unlikely to be through interference with translation (Fig. 4), we assumed that it exerted its inhibitory effect by acting on particular events during RNA synthesis. Rather unexpectedly, TRS7-as also significantly suppressed virus amplification (Fig. 3c
), despite its target sequence being located approximately 450 nt from the genomic 3' end. Whether the P-PMO duplexing to this target affected the initiation of replication or perhaps blocked the processing RdRp complex is unclear.
No effect on sg mRNA synthesis was observed when leader or body TRS motifs were targeted. A likely explanation is that the target sequences for these P-PMOs were not accessible due to RNA structural or intracellular ultrastructural constraints. Genome replication and sg mRNA synthesis are thought to occur in association with dedicated double membrane vesicles that are believed to provide a suitable platform for viral RNA synthesis (Pedersen et al., 1999). This protective microenvironment may physically prevent P-PMOs from entering replication complexes. Incoming viral genomes, which are translated in the cytoplasm in a similar manner to cellular mRNAs, are likely to be more accessible and thus susceptible to antisense P-PMO hybridization. Another possibility is that virus-encoded enzymes such as the nsp10 helicase can displace the P-PMO during sg RNA synthesis prior to the interaction between the leader TRS and the body TRS.
In all our experiments a post-treatment procedure was performed, although it has been found to be less effective compared with pre-treatment (Kinney et al., 2005). However, we encountered several undesired side effects when using a P-PMO pre-treatment, probably due to the positively charged arginine-rich peptide conjugate. Firstly, to a certain extent, pre-treatment with P-PMOs protected cells against virus infection, an observation that was made for both EAV and SinV. This non-specific effect was also noted in a previous study (Kinney et al., 2005
). Secondly, in some cases high P-PMO concentrations actually increased virus titres as determined by plaque assays. For example, at both 4 and 10 µM of 5'TERM-as there were no eGFP-positive cells detected, but the amount of residual virus from the inoculum recovered from the 10 µM sample was approximately ten times higher than in the 4 µM sample (data not shown). This suggested that virus particles from the inoculum may adhere to the outside of cells pre-treated with certain P-PMO concentrations more than they do to non-treated cells. Finally, in cell culture-based translation studies, the efficiency of cationic-lipid mediated plasmid DNA transfection appeared to be influenced by P-PMO (data not shown). P-PMOs have been shown to accumulate in cellular membranes (Moulton et al., 2003
, 2004
). This accumulation of charged molecules may interfere with virus entry (and release) as well as cationic-lipid uptake. A post-infection P-PMO treatment strategy can be recommended to avoid these problems, and was done for all the experiments reported here.
We consistently observed that for any individual cell P-PMO treatment using our eGFP-expressing EAVGFP recombinant generally resulted in an all or nothing outcome of infection. Either eGFP expression was completely absent, indicating that EAVGFP was not able to replicate, or a bright fluorescence was observed, indicating that a vigorous virus infection was in progress. This phenomenon could be explained by low-level EAVGFP replication in the presence of P-PMO, slowly generating more and more viral RNA. At some point, the number of viral targets could reach a threshold level over which the number of P-PMO molecules becomes inconsequential, allowing the virus to replicate robustly.
P-PMOs appear to have some potential as antisense therapeutics in vivo. They behave with predictability and potency, and displayed little non-specificity in most of our systems. The ability of a post-infection P-PMO treatment to significantly reduce the degree of viral amplification further supports the idea that this class of compounds may be suitable for therapeutic development. Delivery of PMOs into cells can clearly be enhanced by using peptide conjugation, which may prove to be an important factor in future drug design. One obstacle may be that serum reduces the cellular uptake of the type of arginine-rich peptide used in this study (Neuman et al., 2004). However, new types of peptide conjugates may be able to overcome this problem (Deas et al., 2005
).
Arteriviruses and coronaviruses are a major economic problem in the swine and cattle breeding industry, and coronaviruses also cause a considerable amount of human disease, such as severe acute respiratory syndrome and a substantial percentage of cases of the common cold syndrome (Saif et al., 1988; Albina, 1997
; Makela et al., 1998
; Cook, 2002
; Peiris et al., 2003
). The arterivirus EAV has proven to be a safe and convenient tool to study various aspects of nidovirus molecular biology, in particular viral RNA synthesis and replicase function. Continued in vitro studies and in vivo approaches with various types of P-PMOs will be required to assess the potential of these compounds to protect against and/or treat diseases caused by nidoviruses. In any case, it is clear from this study that these compounds represent useful reagents for exploring various aspects of molecular virology, especially translation mechanics.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Boguslavsky, D., Ierusalimsky, V., Malyshev, A., Balaban, P. & Belyavsky, A. (2003). Selective blockade of gene expression in a single identified snail neuron. Neuroscience 119, 1518.[CrossRef][Medline]
Cook, J. (2002). In Poultry Diseases, 5th edn, pp. 298306. Edited by F. Jordan, M. Pattison, D. Alezander & T. Faragher. London: W. B. Saunders.
Deas, T. S., Binduga-Gajewska, I., Tilgner, M. & 7 other authors (2005). Inhibition of flavivirus infections by antisense oligomers specifically suppressing viral translation and RNA replication. J Virol 79, 45994609.
den Boon, J. A., Kleijnen, M. F., Spaan, W. J. M. & Snijder, E. J. (1996). Equine arteritis virus subgenomic mRNA synthesis: analysis of leader-body junctions and replicative-form RNAs. J Virol 70, 42914298.[Abstract]
González, J. M., Gomez-Puertas, P., Cavanagh, D., Gorbalenya, A. E. & Enjuanes, L. (2003). A comparative sequence analysis to revise the current taxonomy of the family Coronaviridae. Arch Virol 148, 22072235.[CrossRef][Medline]
Heasman, J. (2002). Morpholino oligos: making sense of antisense? Dev Biol 243, 209214.[CrossRef][Medline]
Jubin, R., Vantuno, N. E., Kieft, J. S., Murray, M. G., Doudna, J. A., Lau, J. Y. & Baroudy, B. M. (2000). Hepatitis C virus internal ribosome entry site (IRES) stem loop IIId contains a phylogenetically conserved GGG triplet essential for translation and IRES folding. J Virol 74, 1043010437.
Kinney, R. M., Huang, C. Y., Rose, B. C., Kroeker, A. D., Dreher, T. W., Iversen, P. L. & Stein, D. A. (2005). Inhibition of dengue virus serotypes 1 to 4 in vero cell cultures with morpholino oligomers. J Virol 79, 51165128.
Liu, Y., Sinha, S. & Owens, G. (2003). A transforming growth factor-control element required for SM
-actin expression in vivo also partially mediates GKLF-dependent transcriptional repression. J Biol Chem 278, 4800448011.
Makela, M. J., Puhakka, T., Ruuskanen, O., Leinonen, M., Saikku, P., Kimpimaki, M., Blomqvist, S., Hyypia, T. & Arstila, P. (1998). Viruses and bacteria in the etiology of the common cold. J Clin Microbiol 36, 539542.
McCaffrey, A. P., Meuse, L., Karimi, M., Contag, C. H. & Kay, M. A. (2003). A potent and specific morpholino antisense inhibitor of hepatitis C translation in mice. Hepatology 38, 503508.[CrossRef][Medline]
Molenkamp, R., Greve, S., Spaan, W. J. M. & Snijder, E. J. (2000a). Efficient homologous RNA recombination and requirement for an open reading frame during replication of equine arteritis virus defective interfering RNAs. J Virol 74, 90629070.
Molenkamp, R., Rozier, B. C., Greve, S., Spaan, W. J. M. & Snijder, E. J. (2000b). Isolation and characterization of an arterivirus defective interfering RNA genome. J Virol 74, 31563165.
Molenkamp, R., van Tol, H., Rozier, B. C., van der Meer, Y., Spaan, W. J. M. & Snijder, E. J. (2000c). The arterivirus replicase is the only viral protein required for genome replication and subgenomic mRNA transcription. J Gen Virol 81, 24912496.
Moulton, H. M., Hase, M. C., Smith, K. M. & Iversen, P. L. (2003). HIV Tat peptide enhances cellular delivery of antisense morpholino oligomers. Antisense Nucleic Acid Drug Dev 13, 3143.[CrossRef][Medline]
Moulton, H. M., Nelson, M. H., Hatlevig, S. A., Reddy, M. T. & Iversen, P. L. (2004). Cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides. Bioconjug Chem 15, 290299.[CrossRef][Medline]
Muto, E., Tabata, Y., Taneda, T., Aoki, Y., Muto, A., Arai, K. & Watanabe, S. (2004). Identification and characterization of Veph, a novel gene encoding a PH domain-containing protein expressed in the developing central nervous system of vertebrates. Biochimie 86, 523531.[CrossRef][Medline]
Nasevicius, A. & Ekker, S. C. (2000). Effective targeted gene knockdown in zebrafish. Nat Genet 26, 216220.[CrossRef][Medline]
Neuman, B. W., Stein, D. A., Kroeker, A. D., Paulino, A. D., Moulton, H. M., Iversen, P. L. & Buchmeier, M. J. (2004). Antisense morpholino-oligomers directed against the 5' end of the genome inhibit coronavirus proliferation and growth. J Virol 78, 58915899.
Panavas, T., Pogany, J. & Nagy, P. D. (2002). Analysis of minimal promoter sequences for plus-strand synthesis by the Cucumber necrosis virus RNA-dependent RNA polymerase. Virology 296, 263274.[CrossRef][Medline]
Pasternak, A. O., van den Born, E., Spaan, W. J. M. & Snijder, E. J. (2001). Sequence requirements for RNA strand transfer during nidovirus discontinuous subgenomic RNA synthesis. EMBO J 20, 72207228.[CrossRef][Medline]
Pasternak, A. O., van den Born, E., Spaan, W. J. M. & Snijder, E. J. (2003). The stability of the duplex between sense and antisense transcription-regulating sequences is a crucial factor in arterivirus subgenomic mRNA synthesis. J Virol 77, 11751183.[CrossRef]
Pedersen, K. W., van der Meer, Y., Roos, N. & Snijder, E. J. (1999). Open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex. J Virol 73, 20162026.
Peiris, J. S., Lai, S. T., Poon, L. L. & 13 other authors (2003). Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361, 13191325.[CrossRef][Medline]
Saif, L. J., Redman, D. R., Brock, K. V., Kohler, E. M. & Heckert, R. A. (1988). Winter dysentery in adult dairy cattle: detection of coronavirus in the faeces. Vet Rec 123, 300301.[Medline]
Sawicki, S. G. & Sawicki, D. L. (1995). Coronaviruses use discontinuous extension for synthesis of subgenome-length negative strands. Adv Exp Med Biol 380, 499506.[Medline]
Snijder, E. J. & Meulenberg, J. J. M. (2001). In Fields Virology, 4th edn, pp. 12051220. Edited by D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott Williams & Wilkins.
Summerton, J. (1999). Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochim Biophys Acta 1489, 141158.[Medline]
Summerton, J. & Weller, D. (1997). Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev 7, 187195.[Medline]
Tijms, M. A., van Dinten, L. C., Gorbalenya, A. E. & Snijder, E. J. (2001). A zinc finger-containing papain-like protease couples subgenomic mRNA synthesis to genome translation in a positive-stranded RNA virus. Proc Natl Acad Sci U S A 98, 18891894.
Tortorici, M. A., Broering, T. J., Nibert, M. L. & Patton, J. T. (2003). Template recognition and formation of initiation complexes by the replicase of a segmented double-stranded RNA virus. J Biol Chem 278, 3267332682.
van den Born, E., Gultyaev, A. P. & Snijder, E. J. (2004). Secondary structure and function of the 5'-proximal region of the equine arteritis virus RNA genome. RNA 10, 424437.
van den Born, E., Posthuma, C. C., Gultyaev, A. P. & Snijder, E. J. (2005). Discontinuous subgenomic RNA synthesis in arteriviruses is guided by an RNA hairpin structure located in the genomic leader region. J Virol 79, 63126324.
van Marle, G., Dobbe, J. C., Gultyaev, A. P., Luytjes, W., Spaan, W. J. M. & Snijder, E. J. (1999). Arterivirus discontinuous mRNA transcription is guided by base pairing between sense and antisense transcription-regulating sequences. Proc Natl Acad Sci U S A 96, 1205612061.
Zuniga, S., Sola, I., Alonso, S. & Enjuanes, L. (2004). Sequence motifs involved in the regulation of discontinuous coronavirus subgenomic RNA synthesis. J Virol 78, 980994.
Received 1 May 2005;
accepted 22 July 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |