1 Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Cantoblanco, 28049 Madrid, Spain
2 Centro de Investigación en Sanidad Animal, INIA, Valdeolmos, 28130 Madrid, Spain
Correspondence
Francisco Sobrino
fsobrino{at}cbm.uam.es
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ABSTRACT |
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Introduction |
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Sense and antisense viral sequences have been shown to block specific steps in virus infection (reviewed by Agrawal, 1992; Bischofberger & Wagner, 1992
; Cohen et al., 1991
). This observation has led to the use of synthetic oligonucleotides and RNA transcripts to interfere with virus multiplication (Day et al., 1991
; Hanecak et al., 1996
; Ramírez et al., 1995
; Yao et al., 1996
). Antiviral strategies based on the induction of RNA-mediated resistance derived from the expression of non-coding sequences have potential advantages, such as the lack of harmful effects in cells. In fact, endogenous antisense RNAs have been identified as a physiological mechanism of gene regulation (Hildebrandt & Nellen, 1992
; Kolb et al., 2001
; Mizuno et al., 1984
). In general, factors that influence the efficiency of virus interference by RNA transcripts inside cells are poorly understood (reviewed by Branch, 1998
; Erickson & Izant, 1992
; Hélène & Toulmé, 1990
). However, several examples of inhibition of animal and plant viruses by constitutive antisense RNA expression have been reported (Day et al., 1991
; Ramírez et al., 1995
; Sczakiel & Pawlita, 1991
), including the development of transgenic animals expressing viral transcripts; these animals show a diminished susceptibility to infection by different viruses (Mizutani et al., 1993
).
The FMDV genome consists of a positive-stranded RNA molecule of about 8500 nt, which contains two long non-coding regions (NCRs) flanking a unique open reading frame (ORF). Replication and translation (Sangar, 1979) occur in the cytoplasm of infected cells (Arlinghaus & Polatnick, 1969
). Cap-independent translation initiation of FMDV RNA (Belsham & Brangwyng, 1990
; Kühn et al., 1990
; Martínez-Salas et al., 1993
) starts at two AUG codons (Beck et al., 1983
; Cao et al., 1995
; López de Quinto & Martínez-Salas, 1999
), following ribosome recognition of the upstream internal ribosome entry site (IRES). The IRES element is a cis-acting, highly structured 465 nt region that mediates RNAprotein interactions essential for ribosome recognition (reviewed by Martínez-Salas et al., 2001
), including the eukaryotic initiation factor eIF4G (López de Quinto & Martínez-Salas, 2000
; López de Quinto et al., 2001
), a key factor to initiate translation.
A highly ordered structure is also predicted at the 3'NCR, which contains a genome-encoded poly(A) tail. The 3'NCR is essential for FMDV replication in cell culture (Sáiz et al., 2001) and according to what is known for other picornaviruses, this region is likely to be involved in the interaction with cellular and/or viral proteins during RNA replication (Mellits et al., 1998
; Todd & Semler, 1996
).
We have shown previously that RNA molecules containing the 3'NCR in both antisense and sense orientations were able to transiently inhibit the infectivity of homologous (type C) FMDV RNA following co-transfection in BHK-21 cells. Likewise, antisense, but not sense, transcripts from the 5' region, including the proximal part of the IRES element and the functional initiator AUG codons, were also inhibitory (Gutiérrez et al., 1994). These inhibitions, which reached values up to 90 %, were dose-dependent and FMDV-specific and also affected heterologous FMDV RNAs of serotypes O and A (Bigeriego et al., 1999
).
RNA-dependent inhibition of viruses that do not present a nuclear stage is complex, probably due to cytoplasm compartmentalization of the replication process and the difficulties in attaining effective concentrations of the interfering molecule in adequate locations (reviewed by Hélène & Toulmé, 1990). Therefore, the efficiency of inhibition exerted by the 3' and 5'NCRs can depend greatly on the delivery system used. Preliminary results indicated that transient expression of type C antisense transcripts targeted to the 5' and 3'NCRs exerted significant inhibitions of homologous FMDV replication (Bigeriego et al., 1999
). Here, we report that stable expression of antisense FMDV 5'NCR transcripts and to a lesser extent, of antisense 3'NCR transcripts alone or in combination with antisense 5'NCR transcripts, confers high levels of inhibition against FMDV in BHK-21 cells. The resistance observed is FMDV-specific, extends to the seven virus serotypes and does not affect other related picornaviruses.
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Methods |
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Construction of plasmids.
RNA sequences containing FMDV 5' or 3'NCRs were used to prepare cDNAs prior to their insertion into plasmid pRSV/L (de Wet et al., 1987). In this vector, FMDV RNA transcripts in either the sense (S) or the antisense (AS) orientation were expressed under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter. Plasmids pR5'S, pR5'AS, pR3'S and pR3'AS contain cDNAs obtained by PCR from plasmids p-5'1 and p-3'1, described previously (Gutiérrez et al., 1994
), using the following pairs of primers: 5'S, 5'-CATAAGCTTAGCTTCTACCCCTG-3' (sense) and 5'-TAGCCCGG GAATTCCATTTTTCC-3' (antisense); 5'AS, 5'-AATCCCGGGAGCTTCTACCCCTG-3' (sense) and 5'-GCGAAGCTTAATTCCATTTTTCC-3' (antisense); 3'S, 5'-GGC AAGCTTAGCGACAAAGGTTTTG-3' (sense) and 5'-ATACCCGG GGGATTAAGGAAGCGGG-3' (antisense); and 3'AS, 5'-ATACCCGGGAGCGACAAAGGTTTTG-3' (sense) and 5'-GGCAAGCTTGGATTAAGGAAGCGGG-3' (antisense). Letters in bold indicate nucleotides inserted to create an HindIII site, letters in italics indicate nucleotides inserted to create a SmaI site and letters underlined correspond to FMDV NCR sequences. PCR products were digested with HindIII/SmaI and ligated into pRSV/L, in which the fragment HindIIISmaI containing the luciferase ORF had been excised previously. In these constructs, the polyadenylation signal from SV40 remained intact. FMDV RNA fragments (397 nt) transcribed from plasmids pR3'S and pR3'AS spanned the last 302 nt of the viral ORF (corresponding to the C terminus of the 3D polymerase gene) and the 95 nt of the 3'NCR (Fig. 1
A). FMDV RNA fragments (156 nt) transcribed from plasmids pR5'S and pR5'AS comprised the last 63 nt of the IRES element and the first 93 nt of the viral ORF, including the two functional initiator AUG codons (Fig. 1A
). Restriction enzyme analysis and DNA sequencing verified the insert orientation in the recombinant plasmids.
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Stable cell transformation.
FMDV plasmids (5 µg) were co-transfected with pBSpacp (pPur) (de la Luna et al., 1988
) at a molar ratio of 3:1 in BHK-21 cells (grown in 60 mm diameter dish) by means of conventional calcium phosphate precipitate (Martínez-Salas & Domingo, 1995
). The plasmid pPur encodes puromycin acetyl-transferase, whose expression leads to resistance to the antibiotic puromycin. Cells resistant to puromycin (purR) (10 µg ml-1) were selected. Then, both independent purR clones and uncloned populations (UPs) were expanded in the presence of puromycin (2·5 µg ml-1) to avoid loss of integrated DNAs (de la Luna & Ortín, 1992
). The efficiency of cell transformation with pPur was about 0·1 %, in agreement with previous BHK-21 cell transformations (Martínez-Salas & Domingo, 1995
). Cell stocks were kept frozen in liquid nitrogen for further analysis.
Analysis of FMDV RNA expression.
Cell monolayers seeded on 60 mm diameter dishes (containing about 4x106 cells) were lysed in 200 µl 0·5 % NP-40, 120 mM NaCl and 50 mM Tris/HCl, pH 7·8. Cellular extracts were centrifuged for 5 min at 12 000 r.p.m. at 4 °C. Cytoplasmic RNA was then extracted from the supernatants as described (Chomczynski & Sacchi, 1987). To eliminate traces of DNA, samples were incubated for 1 h at 37 °C with DNase RQ1 (2 units) and were used for the detection of viral RNA. RT-PCR amplification of 5' and 3' FMDV NCR sequences was performed as described (Rodríguez et al., 1992
), using serial dilutions of cytoplasmic RNA (starting with an RNA aliquot corresponding to about 105 cells). For retrotranscription, the primers used for plasmid constructions, described above, were employed. First, reverse transcription reactions were allowed to proceed in the presence of the antisense primer. Then, the corresponding sense primer was added and the mixture was amplified by PCR. DNA products were separated on a 2 % agarose gel and visualized by ethidium bromide staining. As an internal control, amplification of mRNA from the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (GenBank accession number X0111) was carried out in the same set of RNA samples using the primers antisense (5'-AAGTTGTCATGGATGACCTTGGCCA-3') and sense (5'-CCATCACCATCTTC CAGGAGCGAG-3'). The DNA product amplified (287 bp) corresponded to nt 387673 of the GAPDH gene.
For Northern blot analysis, cytoplasmic RNA (10 µg) was electrophoresed on a denaturing agarose (1·5 %)fomaldehyde (7 %) gel. RNAs obtained in vitro from plasmids p-5'1 and p-3'1 (linearized with XbaI) using T7 RNA polymerase were used as molecular mass markers. The gel was transferred to a nylon Zeta probe membrane (Bio-Rad) (Sáiz et al., 2001), which was hybridized with a mixture of 5x106 c.p.m. of each of 5'S and 3'S FMDV radioactive RNA probes. 5'S and 3'S 32P-labelled probes were obtained from SacI-linearized plasmids p-5'1 and p-3'1, respectively, by in vitro transcription with SP6 RNA polymerase in the presence of 50 µCi [
-32P]CTP (5x105 Bq) (Ramos & Martínez-Salas, 1999
). The stronger signal detected in the clones expressing 3'AS relative to those clones expressing 5'AS, may be the consequence of differences in the specific activity of the probe and/or on RNA stability. A DNA fragment was obtained by reverse transcription of cytoplasmic RNA with the GAPDH antisense primer followed by PCR amplification after the addition of the GAPDH sense primer. Then, the antisense primer was used to obtain single-stranded, labelled DNA in the presence of 50 µCi [
-32P]dCTP. This fragment (containing 5x106 c.p.m.) was used to probe GAPDH RNA sequences.
Virus yield inhibition assay.
To assess the capacity of FMDV to grow in BHK-21 cells expressing different FMDV transcripts, monolayers (about 80 % confluent) of transiently transfected cells at 24 h post-transfection, or stably transformed cell lines and clones, were grown in triplicate in 60 mm diameter dishes. Cells were infected with FMDV C-S8 at an m.o.i. of 0·1 p.f.u. per cell. After 1 h of adsorption, the inoculum was removed and cells were washed twice with DMEM. The infection was then allowed to proceed in DMEM supplemented with 4 % FBS. Samples of supernatant were taken at different times (h) post-infection (p.i.) and the virus titre (p.f.u. ml-1) was determined three times on BHK-21 cells, as described (Bigeriego et al., 1999; Gutiérrez et al., 1993
). The percentage of virus yield inhibition (PI) was calculated as the mean of three independent determinations relative to the p.f.u. recovered in control assays (cells transfected with pRSV/L for transient expressions and clones or UPs transformed with pPur alone for stably transformed cells).
Plaque reduction (PR) assay.
The determination of PR was performed using triplicate monolayers of transformed cells (4x1066x106) and clones that were grown in 60 mm diameter dishes (about 80 % confluent), infected with 50100 p.f.u. of the virus of interest. After 1 h of adsorption at 37 °C, cells were washed twice with PBS and overlaid with DMEM containing 0·7 % agar, as reported previously (Gutiérrez et al., 1994). At 36 h p.i., cells were fixed and stained with crystal violet (0·25 % crystal violet, 10 % ethanol and 2 % formaldehyde in PBS) for 2 h and the number of p.f.u. was scored. The percentage of PR was calculated as the mean of three independent experiments relative to the p.f.u. recovered in control assays (cells transfected with pRSV/L for transient expressions and clones or UPs transformed with pPur alone for stably transformed cells).
Cell detachment assay.
The effect of expression of viral transcripts on cytopathic effect (CPE) (cell detachment) caused by FMDV infection was assessed using a colorimetric procedure (Lewis et al., 1989; Ramírez et al., 1995
). Briefly, 104 cells were plated on 60 mm diameter dishes and infected 24 h later with FMDV C-S8 at an m.o.i. of 0·1 p.f.u. per cell. After adsorption, cells were washed and the infection was allowed to proceed for 5 days in DMEM supplemented with 5 % FCS. The surviving cells were washed three times with PBS and stained with crystal violet for 2 h. Dishes were washed extensively with tap water and air dried. The dye was then eluted with 1 ml per dish of 50 % ethanol and 0·5 % NaCl. Absorbance measurements were recorded at 570 nm, in the linear range of a Spectronic 1001, Bausch and Lomb spectrophotometer. Absorbance values (0·100·15) given by plastic dishes incubated for 6 days with DMEM alone (negative controls) were substracted from all values. The percentage of cell detachment was calculated relative to the values obtained in plates in which cells were maintained in the absence of FMDV, which was set as 100 %.
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Results |
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To study the effect of the expression of FMDV transcripts on susceptibility to virus infection, transfected cells (at 24 h post-transfection) were infected with FMDV C-S8 at an m.o.i. of 0·1 p.f.u. per cell. A transient inhibition of virus yield (PI) was observed in cells transfected with FMDV-interfering transcripts, being higher with transcript 5'AS, with PI around 5066 % at 3240 h post-transfection. No significant PI values were observed at 60 h post-transfection, a time in which a reduction in the level of transcript expression was detected by RT-PCR (data not shown). Mock transfections or cells transfected with plasmid pR5'S did not show a reduction in virus yield at any time-point assayed (Fig. 1C). Significant percentages of PR were also observed relative to the p.f.u. recovered in monolayers of cells transfected with pRSV/L. Inhibition of PR was higher for 5'AS and extended at least to 48 h p.i. (data not shown).
Selection of BHK-21 cell clones stably expressing FMDV transcripts
The above results prompted us to explore the antiviral potential of FMDV transcripts 5'S, 5'AS and 3'AS under conditions of continuous supply of these interfering molecules within cells. To this end, the selectable marker plasmid pPur alone or in combination with the pRSV derivatives expressing FMDV transcripts were transfected into BHK-21 cells. Cell lines were selected for their capacity to grow in the presence of puromycin (purR) from cells transfected with pPur or co-transfected with this plasmid and pR5'S, pR5'AS, pR3'AS or pR5'AS+pR3'AS. The effect of transgene expression on cell survival was estimated from the number and size (diameter) of purR clones that expressed the corresponding FMDV transcript. FMDV transcript expression was detected in 78100 % of the clones analysed from different transfections, with various colony sizes (Table 1).
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All purR clones were stable, keeping the expression of the corresponding FMDV transcript(s) for at least 40 serial passages, including several freezethaw cycles. In all cases, no differences in the ability to produce a cell monolayer were observed among the stable clones, with the exception of those derived from pR5'S, which exhibited a slower growth capacity.
Stable expression of NCR transcripts confers specific resistance against FMDV in BHK-21 cells
The antiviral effect of FMDV transcript expression was first studied by determining the distribution frequency of percentages of PR relative to the titre recovered from clones transfected with plasmid pPur with no insert (Fig. 2). The frequency of clones showing a PR above 60 % was significantly higher for pR5'AS clones than for pR3'AS and pR5'AS+pR3'AS clones. No significant PR was observed in six clones derived from pR5'S and in mock-transfected BHK-21 cells (Fig. 2
and Table 2
). The specificity of the PR observed was confirmed by the lack of significant PR values obtained when a related picornavirus, EMCV, was included in the assay (Table 2
).
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The inhibition on virus yield in clones expressing FMDV transcripts was compared with the extent of CPE in infected monolayers. While untransfected cells and clones derived from pPur and pPur+pR5'S showed an extensive CPE within less than 1 day, CPE was observed from days 4 to 7 p.i. in clones expressing transcripts 5'AS, 3'AS and 5'AS+3'AS (Table 2). As expected, the percentage of cell survival, estimated from the number of attached cells at day 6 p.i., was significantly higher in these clones showing delayed CPE (Table 2
).
Inhibition of FMDV infection correlates with the expression of NCR transcripts
The level of expression of FMDV transcripts in clones expressing 5'AS, 3'AS and 5'AS+3'AS was determined in confluent monolayers (at 24 h post-seeding) by Northern blot analysis of cytoplasmic RNA, using transcript-specific, 32P-labelled RNA probes of sense polarity, as described in Methods. Expression of the engineered transcript(s) was found in all clones analysed, including those expressing 5'AS+3'AS (Fig. 4). A stronger signal was always detected in clones expressing 3'AS than in those expressing 5'AS.
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These results indicate that constitutive expression of 5'AS NCR transcripts confers heterotypic, FMDV-specific protection against virus infection.
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Discussion |
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BHK-21 cell lines and clones, which stably expressed transcripts 3'AS, 5'AS, 5'AS+3'AS or 5'S (negative control), were obtained using purR as a marker for selection. Expression of individual FMDV transcripts did not affect cloning efficiency, as estimated by the number of clones selected relative to the number of clones obtained from cells expressing puromycin alone, except for transcript 5'AS from which a lower number of clones was recovered. In general, purR clones could be efficiently expanded while maintaining detectable levels of the specific FMDV transcript expression (Table 1). The higher levels of PR in homologous infections with C-S8 virus were observed among pR5'AS clones, with an average of 79±16 %, and a clear increase in the frequency of clones showing PR values over 60 % (Fig. 2
). Likewise, a delay in the visualization of extensive CPE, from 4 to 7 days, as well as a higher percentage of cell survival, at 7 days p.i., was observed (Table 2
).
The data reported here show that the 156 nt transcript complementary to the FMDV translation initiation region in the viral RNA elicits an effective antiviral activity when stably expressed in FMDV-susceptible cells. The targeted region in the viral RNA is essential during the life cycle of the virus. The proximal part of the FMDV IRES is involved in the interaction with several eIFs, including eIF4G (López de Quinto & Martínez-Salas, 2000). Remarkably, expression of the sense transcript, 5'S, does not confer significant antiviral activity (Fig. 2
and Table 2
), despite being highly structured (López de Quinto & Martínez-Salas, 1999
; López de Quinto et al., 2001
). Therefore, the possibility of a double-stranded, RNA-mediated antiviral response is unlikely. On the other hand, transcript 5'S does not seem to compete efficiently for any of the RNA-binding proteins essential for IRES activity. Another possibility to explain the efficient antiviral response induced by the 5'AS transcript involves the formation of RNA hybrids, which would either block translation initiation or induce double-stranded, RNase-specific cleavage. This would be consistent with the antiviral response exerted by 5'AS being highly selective, as evidenced by its lack of effect on EMCV infection.
Our results indicate that the levels of transcript expression within each group of 5'AS and 3'AS clones, estimated by a Northern blot assay, correlate with the extent of the inhibition to FMDV infection (Table 2 and Fig. 4
). Unlike what was observed in previous co-transfection experiments with viral RNA (Bigeriego et al., 1999
; Branch, 1998
), the combined expression of transcript 5'AS and 3'AS did not result in an increase of the inhibition of FMDV infection relative to that obtained when transcript 3'AS was expressed individually. Thus, clones pR5'AS+pR3'AS showed PR and PI values that were, in general, lower than those of pR5'AS (Table 2
and Fig. 2
). Further work is required to understand the lack of additive effects in the inhibitions observed in clones expressing the two transcripts, which could be related to interactions between the 3' end viral RNA sequences from transcript 3'AS and the sequences spanned by transcript 5'AS. The cloning efficiency of pR5'AS- or pR5'AS+pR3'AS-transfected cells was lower than that obtained for the remaining transcripts (Table 1
), indicating that high levels of expression of transcript 5'AS might negatively affect cell viability.
In agreement with previous data in co-transfection experiments with viral RNA (Bigeriego et al., 1999; Gutiérrez et al., 1994
), virus inhibitions observed in clones stably expressing the interfering transcripts are specific for FMDV and do not extend to a related picornavirus EMCV, which shows nucleotide sequence similarities of 19 (5'AS) and 23 (3'AS) % (Bigeriego et al., 1999
and references therein; GenBank accession numbers NC001479 and AJ133357). The level of nucleotide sequence divergence at the NCRs between FMDV serotypes is around 20 % (Bigeriego et al., 1999
), while the equivalent figure for the VP1 gene, which encodes a capsid protein contributing to important antigenic sites of the virus, is 40 % (Domingo et al., 1990
). This sequence conservation in the NCR transcripts permits the induction of cross-resistance to heterologous FMDV infection, extending to isolates from the seven different FMDV serotypes, in clones expressing transcript 3'AS or 5'AS. This result may represent a significant advantage over the lack of cross-protection among animals immunized with FMDVs of different serotypes (reviewed by Domingo et al., 1990
). The stability of the PI (Fig. 5B
) in viruses recovered from clones expressing transcripts 3'AS or 5'AS upon six serial passages in these cells suggests a low frequency in the selection of mutants resistant to the inhibition exerted by these transcripts.
The results reported here encourage the possibility of producing transgenic animals expressing 5'AS sequences as a new antiviral approach. However, further work is required to asses whether high levels of expression of interfering transcripts, particularly of 5'AS, are compatible with cell viability.
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ACKNOWLEDGEMENTS |
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Received 28 June 2002;
accepted 10 October 2002.