Deletion or substitution of the aphthovirus 3' NCR abrogates infectivity and virus replication

M. Sáiz1,2, S. Gómez1,2, E. Martínez-Salas2 and F. Sobrino1,2

Centro de Investigación en Sanidad Animal, INIA, 28130 Valdeolmos, Madrid, Spain1
Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM), Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain2

Author for correspondence: Encarnación Martínez-Salas. Fax +34 91 3974799. e-mail emartinez{at}cbm.uam.es


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
The 3' noncoding region (NCR) of the genomic picornaviral RNA is believed to contain major cis-acting signals required for negative-strand RNA synthesis. The 3' NCR of foot-and-mouth disease virus (FMDV) was studied in the context of a full-length infectious clone in which the genetic element was deleted or exchanged for the equivalent region of a distantly related swine picornavirus, swine vesicular disease virus (SVDV). Deletion of the 3' NCR, while maintaining the intact poly(A) tail as well as its replacement for the SVDV counterpart, abrogated virus replication in susceptible cells as determined by infectivity and Northern blot assays. Nevertheless, the presence of the SVDV sequence allowed the synthesis of low amounts of chimeric viral RNA at extended times post-transfection as compared to RNAs harbouring the 3' NCR deletion. The failure to recover viable viruses or revertants after several passages on susceptible cells suggests that the presence of specific sequences contained within the FMDV 3' NCR is essential to complete a full replication cycle and that FMDV and SVDV 3' NCRs are not functionally interchangeable.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Foot-and-mouth disease virus (FMDV) is the causative agent of an acute systemic disease of cloven-hooved animals considered to be a major animal health problem world-wide (Kitching, 1999 ). FMDV is the prototypic member of the aphthoviruses, which belong to the family Picornaviridae (Rueckert, 1990 ). Swine vesicular disease is a highly contagious disease caused by the enterovirus swine vesicular disease virus (SVDV), which also belongs to the family Picornaviridae (Sutmoller, 1992 ). The clinical signs produced by SVDV are indistinguishable from those caused by FMDV in pigs. Both viruses share the ability to infect in vitro cultured IBRS-2 cells whereas only FMDV infects BHK-21 cells.

The FMDV genome consists of a single molecule of messenger-sense RNA of about 8500 nt containing a single ORF encoding the viral polyprotein, which is subsequently processed into the viral products (Ryan et al., 1989 ). The 5' end of the RNA is covalently linked to a small viral protein (VPg) (Sangar et al., 1977 ). The 5' noncoding region (NCR), about 1200 nt in length, is highly structured (Clarke et al., 1987 ) and contains several genetic elements necessary to control essential functions in the replication cycle. This region harbours an internal ribosome entry site (IRES) element that promotes the cap-independent translation initiation of the viral genome (Kühn et al., 1990 ; López de Quinto & Martínez-Salas, 1997 ). Immediately downstream of the stop codon at the end of the polyprotein ORF is a 3' NCR of about 90 nt followed by a genetically encoded poly(A) tract (Chatterjee et al., 1976 ).

The picornavirus 3' NCR has been predicted to contain stable secondary and tertiary structures by computer algorithms and biochemical structure probing (Pilipenko et al., 1992 ; Todd & Semler, 1996 ). A cis-acting determinant for replication is believed to reside in the 3' NCR where primary sequence and/or higher order motifs are presumably recognized by the replication complex in order to initiate the synthesis of the negative-strand RNA (Richards & Ehrenfeld, 1990 ; Rohll et al., 1995 ). Engineered disruption of the relevant structures predicted in this region results in an impairment of the replicative capacity of the viral genome (Rohll et al., 1995 ; Pierangeli et al., 1995 ).

Binding of viral proteins, or their precursors, to the 3' NCR has been reported for encephalomyocarditis virus (Cui et al., 1993 ) and poliovirus (PV) (Harris et al., 1994 ). In addition, picornavirus 3' NCR interacts with host proteins. A complex of proteins binds to human rhinovirus type 14 (HRV14) and PV1 3' NCRs and a deletion mutant in this region exhibited reduced binding capacity and a defective replication phenotype (Todd et al., 1995 ). Likewise, cellular factors interact with the 3' NCR of PV3, HRV14 and coxsackievirus B4 (CB4) and mutations reducing binding affinity had a deleterious effect on virus replication (Mellits et al., 1998 ). A nucleolar protein, nucleolin, has been shown to bind poliovirus 3' NCR. Relocalization of nucleolin induced by virus infection seems to play a role in the early stages of the virus replication cycle (Waggoner & Sarnow, 1998 ). The functional significance of those interactions remains to be determined, although some of them may play an important role in regulating the initiation of the negative-strand once the cis-elements have mediated the first recognition event or in modulating the specificity of this process.

Moreover, internal cis-acting replication elements (CREs) located in the picornavirus genome coding region are now being identified. This is the case with a hairpin structure in the HRV14 capsid protein precursor (McKnight & Lemon, 1998 ), in the VP2 coding sequence of two cardioviruses (Lobert et al., 1999 ) or a similar element located in the PV 2C coding region (Goodfellow et al., 2000 ). These findings contribute to highlight the complexity of a process in which different viral and cellular factors contribute to promote efficient initiation of negative-strand RNA synthesis (Agol et al., 1999 ).

Little is known about the role of the 3' NCR in aphthoviruses although some experimental evidence supports its relevance in the FMDV replication cycle. RNA transcripts spanning the 3'-terminal region of the FMDV genome, in both sense and antisense orientations, have been reported to inhibit infective particle formation following co-microinjection or co-transfection with viral RNA in BHK-21 cells (Gutiérrez et al., 1994 ). Inhibition of virus yield was also observed in FMDV-infected cells transiently expressing the interfering RNAs (Bigeriego et al., 1999 ).

In this report, we have examined the role of the 3' NCR of FMDV in the virus replication cycle by the deletion of this element and by its substitution with its counterpart of another swine picornavirus, SVDV, in a full-length infectious cDNA clone of FMDV. The effects of those alterations on replication and translation have been followed by infectivity and RNA analysis.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Construction of plasmids.
Plasmids are based on an FMDV O1K infectious full-length cDNA clone, pSP65FMDVpolyC (Falk et al., 1992 ; Zibert et al., 1990 ), which has an average poly(C) tract of 30 nt. Plasmid pDM, which contains unique StuI and EcoRV sites flanking the 3' NCR, was made by mutagenic PCR (Kunkel, 1985 ). In this clone, three nucleotide mismatches were artificially introduced to create the StuI and EcoRV sites at positions 9 and 86 from the stop codon, respectively (Fig. 1).



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Fig. 1. Schematic representation of different constructs based on an FMDV full-length cDNA clone. The restriction sites used for cloning are indicated. The sequence of the 3' region is shown above each plasmid. Artificially engineered residues are shown in bold letters. The stop codon is underlined and SVDV sequences are boxed.

 
To construct plasmid p{Delta}74, pDM was digested with StuI and EcoRV and the large fragment released was religated using T4 DNA ligase. The resulting clone has a deletion of most of the 3' NCR element (74 nt). Thus, only 15 virus residues remain between the stop codon and the poly(A) tract.

Prior to the construction of plasmid pCHIM, construct pTAG was made by recombinant PCR (Kunkel, 1985 ) using pDM as a template and primer pairs OL-1 (5' CGGACCGCAAATTGGCTCAGCGG 3') with ILAV (5' CCTCTGAGGGCCTAGGCGTCA 3') and IRAV (5' TGACGCCTAGGCCCTCAGAGG 3') with ILE-1 (5' TTTTTGGATATCAAGGAAGCGGGAAAAACCC 3'), respectively. The bold letters indicate nucleotides that were substituted in order to create the underlined AvrII site at nt -2 from the stop codon. A unique CelII site at position 635, relative to the first nucleotide of the 3D gene sequence (Fig. 1), was used for the 5' insertion of the PCR products. The final PCR product was digested with CelII and EcoRV (sites underlined in external primers OL-1 and ILE-1, respectively) and ligated into the pDM clone in which the fragment CelII–EcoRV had been previously excised. Thus, plasmid pTAG contains a unique AvrII site spanning the stop codon, which changes TAA in the original construct to TAG.

cDNA containing the 3' NCR of SVDV was amplified by RT–PCR from viral RNA of strain UK (Seechurn et al., 1990 ) using primers SVDCHIM-R (5' TTTTTTTTTTTTTTTGATATCCGCACCGAATACGG 3') and SVDCHIM-L (5' GGACTCCTTTTAACCTAGGAATTAGAGCACAATTAG 3'), which introduce the underlined EcoRV and AvrII sites, respectively. The RT–PCR product was then digested with AvrII and EcoRV and cloned into pTAG in which the fragment AvrII–EcoRV had been excised. The resulting plasmid, pCHIM, has the 3' NCR of SVDV inserted into the FMDV infectious clone and only a G residue remains between the stop codon and the aphthoviral sequence. In the 3' end, 6 nt corresponding to the FMDV 3' NCR remain preceding the poly(A) tract.

All plasmids were transformed into E. coli strain DH5{alpha} cells and sequenced along the 3' modified region using the fmol DNA cycle sequencing system (Promega). Prediction of secondary structures in the 3' NCR of the different constructs was performed using the M-Fold program of the GCG package. The average length of the poly(A) tract in all the constructs was 58 nt.

{blacksquare} In vitro RNA transcription.
Synthetic RNAs were obtained by transcribing HpaI-linearized plasmids with SP6 RNA polymerase (Promega), according to the manufacturer’s instructions. After transcription, the reaction mixture was incubated with RQ1 DNase (1 U/µg RNA) (Promega). RNAs were then phenol–chloroform-extracted and ethanol-precipitated. RNA integrity and concentration were determined by native agarose gel electrophoresis.

{blacksquare} Cells and transfections.
Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 2 to 5% foetal bovine serum and 1 mM HEPES pH 7·4. Semiconfluent (80%) BHK-21 or IBRS-2 (a swine-derived cell line) cell monolayers, split 1 day prior to transfection, were transfected with 3 to 5 µg of transcripts. Liposome-mediated transfection (Rose et al., 1991 ) was performed as described (López de Quinto & Martínez-Salas, 1997 ) and transfected cells were incubated at 37 °C or 34 °C until cytopathic effect (CPE) was observed. For those mutants that did not induce a visible CPE, new rounds of amplification were performed collecting separately the transfection medium and the monolayers lysed by three cycles of freeze–thawing. The lysate and the transfection medium were transferred independently onto fresh cell monolayers.

{blacksquare} Infection of suckling mice.
Virus used as the inoculum was produced by transfection of RNA onto BHK-21 cells followed by two rounds of amplification. Groups of 12 mice, 7-day-old, were inoculated intraperitoneally with 100 µl of a virus dilution in PBS. Dead animals were scored from 2 to 7 days after inoculation. LD50 corresponds to virus titre required to kill 50% of the inoculated animals.

{blacksquare} In vitro translation.
About 200 and 400 ng of each transcript was translated in 10 µl of rabbit reticulocyte lysate (Promega) in the presence of 10 µCi of [35S]methionine (10 mCi/ml). Reaction mixtures were incubated at 30 °C for 1 h. Aliquots of the translation products were loaded onto a 12% SDS–polyacrylamide gel. The gels were then dried and exposed to X-ray films (Agfa Curix RP2).

{blacksquare} Northern blot.
Extracts from transfected cells (about 1x106), frozen at different times post-transfection, were prepared in 260 µl of 0·5% NP-40, 120 mM NaCl and 50 mM Tris–HCl, pH 7·8. The lysate was then centrifuged at 12000 r.p.m. for 5 min and total cytoplasmic RNA was purified from the supernatant with TRI Reagent (Sigma).

Aliquots of RNAs (12·5% of each sample) were resolved through a formaldehyde gel and blotted onto a nylon Zeta probe membrane (Bio-Rad) using the vacublot XL system (Pharmacia). The probe was prepared by PCR using the 932 bp CelII–HpaI fragment from pSP65FMDVpolyC as a template (about 200 ng) and the antisense primer I-18 (5' GCCACCACGATGTCGTCTCC 3') in the presence of 50 µCi of [{alpha}-32P]dCTP (6000 Ci/mmol; Amersham). The probe was subsequently purified using a Nick column (Pharmacia). The resulting probe is a single-stranded DNA fragment of 392 nt complementary to nt 817–1207 of the 3Dpol gene. About 5x106 c.p.m. of the probe was added to the hybridization solution (50% formamide in 0·12 M Na2HPO4, pH 7·2, 0·25 M NaCl, 7% SDS) and incubated overnight at 42 °C. The membrane was then washed three times for 5 min at room temperature with 2x SSC/0·1% SDS; 0·5x SSC/0·1% SDS; 0·1x SSC/0·1% SDS, respectively, and exposed to X-ray film.

{blacksquare} RT–PCR analysis of viral RNA.
Serial dilutions of the total cytoplasmic RNA extracted as above were analysed by RT–PCR to determine the best signal to background ratio (data not shown). Basically, RNAs (1 µl of a 1/10 dilution) were heated at 93 °C for 2 min with 10 pmol of the antisense primer I-18 in a final volume of 10 µl, then cooled down at room temperature for 10 min prior to RT for 1 h in a 20 µl reaction containing 50 mM Tris–HCl, pH 8·3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 1 mM each dNTP, 40 U of RNasin RNase inhibitor (Promega) and 20 U of SuperScript II RNase H- reverse transcriptase (Gibco BRL).

For PCR amplification, 1/5 to 1/10 of the RT reaction was used in the 50 µl PCR mixture containing 10 mM Tris–HCl, pH 8·3, 50 mM KCl, 1·5 mM MgCl2, 0·2 mM each dNTP, 20 pmol each of the respective primers and 5 U of AmpliTaq DNA polymerase (Perkin Elmer). Samples were amplified in a UNO-thermoblock (Biometra) using a program that included an initial incubation at 94 °C for 10 min and 72 °C for 2 min (the enzyme was added at this point), followed by 30 cycles of denaturation at 94 °C for 1·5 min, annealing at 50 °C for 1 min and extension at 72 °C for 1·5 min. All the RT–PCR products were dependent on the presence of reverse transcriptase in the reaction mixture (data not shown), proving the specificity for RNA templates.

The primers used for the PCR analysis of viral RNA were D3-I(-) (5' GTACTGTGTGTAGTACTG 3'), complementary to nt 286–303 of the VP3 gene, and D3-V(+) (5' GACCCGAAGACGGCTGACC 3'), corresponding to nt 61–80 of the VP3 gene. The expected amplification product is a DNA fragment of 252 bp.

For positive-strand RNA amplification experiments D3-I was used as the antisense primer in the RT reaction at 42 °C.

Negative-strand RNA amplification was carried out using an external primer in the RT reaction, VP3NEST(+) (5' CCAACGTGCACGTCGCGGGT 3'), corresponding to the sequence identical to nt 617–637 of the VP2 gene, and cDNA synthesis was performed at 50 °C. Prior to PCR amplification with primers D3-I and D3-V, primer VP3NEST was removed by filtering the RT reaction through a Centricon YM-30 column (Millipore). Under these conditions, specific amplification dependent on the negative-strand was achieved for up to 1 ng of input RNA.

As a control for RNA extraction, an RT–PCR reaction using primers designed for amplification of cellular mRNA was performed using oligonucleotides g3pdh 3'(-) (5' AAGTTGTCATGGATGACCTTGGCCA 3') and g3pdh 5'(+) (5' CCATCACCATCTTCCAGGAGCGAG 3'), which amplify a 287 bp DNA fragment corresponding to nt 387–673 of human glyceraldehyde-3-phosphate dehydrogenase gene (accession no. X01111).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
RNAs harbouring a 3' NCR deletion are not infectious
To examine the role of the 3' NCR in the FMDV infectious cycle we constructed a derivative plasmid, pDM, from the FMDV O1K infectious full-length clone in which two unique restriction sites flanking the 3' NCR (StuI and EcoRV) were introduced (Fig. 1). RNAs derived from plasmid pDM are infectious at a level similar to that of the RNA derived from the wt construct (pSP65FMDVpolyC) (Table 1).


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Table 1. Infectivity of the different FMDV transcripts on BHK-21 and IBRS-2 cells and virulence of the recovered viruses in suckling mice

 
The viability of the viruses recovered from transfections with SP65FMDVpolyC and DM RNAs was analysed in suckling mice infected with second-passage viruses. The virulence observed for pDM-derived virus was similar to that obtained for the virus stock derived from the original clone (Table 1). This result proves the infection ability of viruses derived from pDM in an animal system.

To study the biological relevance of FMDV 3' NCR, we constructed the p{Delta}74 derivative (Fig. 1), harbouring a deletion of most of the 3' NCR. p{Delta}74 retains 15 virus residues between the stop codon and the poly(A) tract, which remains intact in all constructs. In vitro-transcribed RNAs harbouring the {Delta}74 mutation were transfected onto BHK-21 and IBRS-2 monolayers and cells were kept at 37 °C and monitored for CPE development. No CPE could be observed in these monolayers after 3 to 4 days while the positive control, DM RNA, consistently induced CPE about 19 h post-transfection.

In order to preserve cell viability for extended times following transfection and to give a better chance to slow-growing viruses to complete their life-cycle, transfected cells were incubated at 34 °C for up to 9 to 11 days. As BHK-21 cells exhibited an impaired ability to grow at 34 °C, these experiments were performed using IBRS-2 cells. During this time no CPE could be observed in cells transfected with {Delta}74 RNAs whereas transcripts derived from the positive control, pDM, induced CPE about 48 h post-transfection (Table 1).

No infectious virus could be recovered from cells transfected with {Delta}74 RNA, as indicated by the negative results of two additional rounds of amplification performed by transferring the transfection medium and the cell lysate onto fresh monolayers. Therefore, we conclude that deletion of the 3' NCR was deleterious for virus multiplication.

Exchange of the FMDV 3' NCR for its homologue from SVDV fails to produce infectious RNA
The use of chimeric viruses provides a useful tool to study the conservation of functional signals to initiate replication between related viruses. In order to study the possibility of a functional substitution of the 3' NCR of FMDV for that element of another picornavirus, we chose SVDV, a distantly related swine picornavirus, which has a similar growth capacity on IBRS-2 cells as FMDV and shares a 45% primary sequence homology in the 3' NCR. Prior to the construction of the FMDV–SVDV chimera, a new plasmid, pTAG, containing a unique AvrII site spanning the stop codon of the polyprotein which changes the codon from TAA to TAG was made (Fig. 1). Secondary structure prediction of the 3' NCR region in pTAG is identical to that of pDM, consisting of two main stem–loops that are highly conserved among all aphthovirus strains (data not shown). The intermediate construct pTAG allows the precise excision of the 3' NCR as an AvrII–EcoRV cassette and its replacement with a heterologous sequence bearing those restriction sites while keeping the 3Dpol sequence and the poly(A) tract intact. The infectivity on BHK-21 and IBRS-2 cells shown by transcripts produced from pTAG was similar to that obtained with pDM transcripts (Table 1), but the RNAs derived from the pTAG mutant caused smaller plaques. To construct pCHIM, the 3' NCR of SVDV RNA was amplified by RT–PCR and then ligated into pTAG (Fig. 1). Thus, pCHIM is a chimera of the FMDV full-length infectious clone in which the 3' NCR has been exchanged for that of SVDV. pCHIM contains a synonymous nucleotide substitution in the last codon of the 3D protein (Fig. 1). The predicted secondary structure for pCHIM shows no disruption of the secondary structure predicted for the SVDV RNA in that region (data not shown).

RNAs transcribed from pCHIM failed to induce visible CPE when transfected onto BHK-21 or IBRS-2 monolayers incubated at 37 °C (Table 1). Nevertheless, when cells transfected with CHIM RNA were incubated at 34 °C a diffuse cellular damage as detected by cell detachment was observed in IBRS-2 cells at extended times post-transfection (about 4 days). This effect was not visible in cells transfected with {Delta}74 RNA or in mock-transfected cells (data not shown). In no case were we able to recover infectious particles from cells transfected with CHIM RNAs, incubated at either 34 °C or 37 °C, after two additional passages on IBRS-2 cells using both the supernatant and the lysate from transfected cells harvested at different times post-transfection (from 3 to 9 days). These results indicate that the 3' NCRs of FMDV and SVDV are not functionally interchangeable.

Mutations in the 3' NCR have no effect on the in vitro translation efficiency of the FMDV RNA
To assess that the phenotype observed for mutant RNAs bearing extensive deletion or heterologous sequences in the 3' NCR was due to a major replication defect, we assayed the in vitro translation efficiency of the transcripts to rule out the possibility of significant translation dysfunction in these mutants (Fig. 2). Equal amounts of RNAs derived from constructs p{Delta}74, pCHIM and pTAG (Fig. 2b) were used to program in vitro translation reactions in a rabbit reticulocyte lysate system. The translation products are shown in Fig. 2(a). No significant differences in the viral protein patterns could be detected, suggesting that the multiplication defect associated with mutant RNAs was independent of the efficiency of translation.



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Fig. 2. Translation efficiency of transcripts in the rabbit reticulocyte lysate system. Lanes 1 and 2, 3 and 4, and 5 and 6 correspond to 200 ng and 400 ng of {Delta}74, CHIM and TAG RNAs, respectively. (a) SDS–PAGE and autoradiography of the in vitro translation products derived from RNA templates shown in (b). Protein molecular mass markers are indicated in kDa.

 
Analysis of RNA derived from primary transfections
The 3' NCR of different picornavirus RNAs has been shown to be involved in replicative events (Pierangeli et al., 1995 ; Richards & Ehrenfeld, 1990 ; Rohll et al., 1995 ). Accordingly, the lack of infectivity of {Delta}74 and CHIM RNAs could be due to a replication defect. In order to explore this possibility, the accumulation of positive-strand viral RNA in cells transfected with transcripts prepared from p{Delta}74, pCHIM and pTAG was assessed (Fig. 3). Full-length positive-strand RNA could be detected only in cells transfected with TAG RNA. Overexposure of the film did not yield full-length viral RNA bands in lanes corresponding to {Delta}74 and CHIM RNAs. Our failure to detect viral RNA in cells transfected with 3' mutant RNAs by Northern blot suggested major dysfunction defects for those mutants at the replication level.



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Fig. 3. Northern blot analysis of positive-strand viral RNA in IBRS-2 cells transfected with the transcripts indicated on the top of the panel or mock-transfected. Cells were incubated at 34 °C and harvested at the indicated times (in h) following transfection. The blot was probed with a {alpha}-32P-labelled single-stranded DNA fragment complementary to residues 817 to 1207 of the 3Dpol gene. The unspecific band (28S RNA), present also in mock-transfected cells, serves as a sample loading control.

 
In a further attempt to detect low levels of virus replication derived from those RNAs, we carried out an RT–PCR analysis of the RNAs extracted from transfected cells in order to specifically amplify viral positive- and negative-strand RNAs. Fig. 4(a) shows the results of the negative-strand amplification. Comparison of lanes corresponding to the three different transcripts indicates that neither {Delta}74 nor CHIM RNAs were able to reach replication levels comparable to the levels of infectious RNAs derived from pTAG, even at late times post-transfection (119 h). In contrast, a significant difference was observed between {Delta}74 and CHIM RNAs after 42 h post-transfection. In cells transfected with chimeric RNAs harbouring the 3' NCR of SVDV negative-strand RNA was detected at the 42 h time-point. However, at early times post-transfection (0·5 h), positive RT–PCR amplification was detected in both {Delta}74 and CHIM RNA lanes, presumably arising from remnants of transfection input RNA. The differences observed in synthesis of negative-strand RNA at 42 h post-transfection are unlikely to be due to incomplete elongation by aberrant initiation events of the negative-strand RNA as the primers used in the assay amplify an internal fragment of the genome (VP3).



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Fig. 4. RT–PCR analysis of transfected cells. IBRS-2 cells were incubated at 34 °C and harvested at the indicated times (in h) following transfection with the transcript indicated on the top of each panel. (a) Detection of negative-strand RNA following cDNA synthesis with primer VP3NEST. The PCR reaction was carried out using primers D3-I and D3-V as described in Methods. (b) Positive-strand RNA RT–PCR with primers D3-I and D3-V. (c) RT–PCR of cellular RNA with primers g3pdh3' and g3pdh5'. The expected sizes of the products are indicated with an arrow. M, size of markers (bp).

 
The results of the RT–PCR amplification of positive-strand RNA are shown in Fig. 4(b). The fact that positive-strand RNA remains detectable at times post-transfection as late as 119 h, even for genomes unable to replicate (as suggested in Fig. 4a) could be explained by the presence of low amounts of input RNA undetectable in the Northern blot assay. Nevertheless, slight differences in the band intensities were observed among the transcripts despite of the lack of linearity in the assay. The intensity of the bands from monolayers transfected with CHIM RNA was higher when compared to those transfected with {Delta}74 RNA, particularly at 42 h post-transfection. This is consistent with the detection of small amounts of negative-sense viral RNA in cells transfected with pCHIM transcripts (Fig. 4a).

Fig. 4(c) shows the results of the RT–PCR reactions with g3pdh primers as a control of sample RNA loading. The intensity of the bands with these primers did not correlate with the intensities of bands shown in Fig. 4(a) and (b), indicating that the differences observed in those reactions were not due to significant differences in RNA load.

Sequencing of the PCR products corresponding to positive-strand RNA amplification confirmed the presence of the {Delta}74-, SVDV 3' NCR- and TAG-specific sequences in viral RNAs from the respective transfections at 42, 90 and 119 h post-transfection.

The results obtained from cells transfected with mutant RNAs by RT–PCR analysis suggest that a certain advantage was conferred by the presence of the heterologous element from a distant picornavirus over the deletion of this region, even though none of the FMDV RNAs bearing the {Delta}74 deletion or the SVDV sequence in the 3' NCR were replication-competent at levels sufficient to make them viable and produce infectious viruses. However, we failed to detect the presence of viral proteins in cells transfected with {Delta}74 and CHIM transcripts in Western blot experiments (data not shown). Thus, CHIM RNA might replicate to a small extent but this is still below the threshold required for further rounds of virus multiplication.


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
The precise mechanisms involved in initiating and regulating replicative events in infected cells remain elusive in picornaviruses. However, the relevance of the 3' NCR in initiation of negative-strand RNA synthesis has been well established as an important cis-acting signal mediating the process. Extensive mutagenesis analysis of this region has shown primary sequence and secondary and tertiary order motifs essential for preserving a wt replication phenotype. However, the extent of conservation of those signals among different picornaviruses is difficult to predict, as results inferred for different viruses may be in apparent contradiction. Binding of several viral and host proteins to the 3' NCR has also been reported. Some of these interactions may play a role in replication.

Previous work on picornaviruses has shown that PV and HRV14 RNAs harbouring complete deletions of the 3' NCR are infectious. The recovered viruses demonstrated a defective growth phenotype and an RNA replication defect, suggesting that the presence of the 3' NCR may not be an absolute requirement for genome replication in these viruses (Todd et al., 1997 ). In contrast, individual excision of the two stem–loops of PV3 3' NCR had been previously reported to abolish replication (Rohll et al., 1995 ).

We have examined the role of the 3' NCR in FMDV, an aphthovirus for which there is no available information regarding replication signals. Our results with the 3' NCR mutants show that {Delta}74 RNAs were not infectious when transfected into susceptible cells as no CPE could be observed and we were unable to recover infectious virus after two rounds of amplification.

The functional inter-exchange of PV 3' NCR for that of CB4 or HRV14 has been reported (Rohll et al., 1995 ). When the donor sequences came from hepatitis A virus (HAV) or bovine enterovirus, the replication levels were significantly lower than those for the PV3, CB4 or HRV14 recombinants exhibiting the wt replication phenotype. In contrast, Pierangeli et al. (1995) reported the deleterious effect on replication of the substitution of PV 3' NCR with the equivalent sequences of HAV RNA.

Since the {Delta}74 deletion, spanning most of the FMDV 3' NCR and including the two predicted stem–loops, resulted in an impairment of infectivity, we studied the functional substitution of the element with that of the distantly related enterovirus SVDV. A chimeric construct, pCHIM (Fig. 1), did not induce visible CPE in BHK-21 or IBRS-2 cells at 3 to 4 days post-transfection, as compared to DM RNAs, which induced CPE about 19 h post-transfection. We were not able to recover infectious virus in this case, similar to the situation seen with the {Delta}74 mutant.

In order to hypothesize replication defects in {Delta}74 and CHIM RNAs, it was necessary to rule out the possibility of major translation defects. In recent reports the highly conserved 98 nt X region of the 3' NCR of hepatitis C virus has been shown to enhance translation (Ito et al., 1998 ; Ito & Lai, 1999 ). When the in vitro translation efficiency of the different FMDV transcripts in a rabbit reticulocyte lysate system was compared, no relevant differences could be detected between infectious and uninfectious RNAs (Fig. 2). This result suggests that the inability of {Delta}74 and CHIM RNAs to direct RNA replication efficiently was unrelated to their translation initiation efficiency.

In further experiments we used IBRS-2 cells that were able to grow at lower temperatures (34 °C) and may have provided trans-acting factors specifically required for the replication competence of the FMDV–SVDV chimera.

The accumulation of positive-sense FMDV RNA, monitored by Northern blot, indicated that only TAG transcripts were able to replicate successfully, while RNAs derived from p{Delta}74 and pCHIM did not accumulate at levels detectable by Northern blot.

In our search for RNA synthesized de novo in cells transfected with {Delta}74 and CHIM RNAs, we carried out an RT–PCR analysis to detect lower amounts of RNA. In the negative-strand amplification experiments we detected viral RNA at 42 h post-transfection in cells transfected with CHIM RNA, but not in monolayers transfected with {Delta}74 RNA (Fig. 4a). Positive-strand amplification revealed slight differences in the intensity of the bands corresponding to {Delta}74 and CHIM RNA-transfected cells respectively, at different times post-transfection.

Therefore, we conclude that the deletion of the 3' NCR had a deleterious effect on FMDV replication. Its presence seems to be a strict requirement for the initial rounds of replication as no viable viruses, including revertants or pseudorevertants containing additional mutations, were recovered. The SVDV 3' NCR, replacing the FMDV homologue, was unable to provide the same function in cis. The extent of identity of both elements at the primary sequence level is 45% and the secondary structure predicted for SVDV 3' NCR does not resemble that of FMDV 3' NCR. The secondary structure was maintained intact in the pCHIM construct (data not shown). FMDV and SVDV cause indistinguishable symptomatic diseases in pigs and productively infect IBRS-2 cultured cells. However, their replicative signals, at least at the 3' NCR level, seem to be different. The lack of infectivity of CHIM transcripts on IBRS-2 cells is unlikely to be due to the absence of specific host factors for SVDV replication. Interestingly though, the presence of the SVDV 3' NCR conferred to the RNA a slight advantage in early replication events as compared to RNAs harbouring complete 3' NCR deletions.

Our results for FMDV may be in apparent contradiction to data obtained using entero- and rhinoviruses. Although the main replication mechanisms have been assumed to be common for all picornaviruses, some group-dependent strategies cannot be ruled out.

Novel internal domains essential for replication are being discovered in the coding regions of the picornaviral genome. All these CREs resemble each other functionally but differ in sequence, structure and location. The CREs in rhino- and cardioviruses are not functionally exchangeable while the CRE of Mengo virus can replace that of Theiler’s virus (Lobert et al., 1999 ). Interestingly, a lethal 3' NCR mutation could be partly rescued by a compensating mutation within the CRE of HRV14 (McKnight & Lemon, 1998 ).

The ability of an FMDV replicon lacking the Lb and most of the P1 (capsid protein precursor) coding sequences to replicate suggests that no CRE is contained in that region of the genome (McInerney et al., 2000 ). An additional distinguishing feature among enteroviruses, rhinoviruses and aphthoviruses is the different type of IRES contained within their 5' end regions differing in sequence and structure.

The possible interaction of the 5' and 3' NCRs during translation and replication of picornavirus RNA is currently being investigated. Inter-regulation of both processes seems to be mediated by the formation of specific RNA–protein complexes in the 5' end clover leaf structure (Gamarnik & Andino, 1998 ). Additionally, recent data favour a model where binding of cellular proteins may establish a physical link between the 5' and 3' NCRs (Herold & Andino, 2000a ). Several IRESs, including those of different virus families, have been exchanged without drastic consequences for virus replication (Alexander et al., 1994 ; Rohll et al., 1994 ; Lu & Wimmer, 1996 ). However, some requirements in terms of functional compatibility of sequences present in the 5' end, 3' end and CREs may exist and therefore make it difficult to predict the viability of modified RNAs bearing engineered mutations and replacements in those regions. Thus, disruption of signals strictly required for efficient replication, present in the 3' NCR, may cause major growing defects leading to non-viable phenotypes as well as revertants and pseudorevertants carrying compensatory mutations in the 3' NCR or other regions involved in the regulation of the process.

The essential role played by internal sequences of the 3' NCR has been recently highlighted in a model of initiation of negative-strand synthesis (Paul et al., 2000 ), in which uridylylated VPg was transferred to the 3' NCR and then transferred back to the poly(A) tract. The fact that replicative forms of PV RNA can be isolated by poly(A) tract selection (Herold & Andino, 2000 b ) strongly supports this model. Consistent with the biological relevance of signals within the 3' NCR, the data shown here for FMDV indicate that specific sequences within this region are absolutely required to initiate replication, since neither deletion nor SVDV-substituted 3' NCR mutants could replicate efficiently in transfected cells. What is noteworthy is that all the mutants shared the same poly(A) tails and the last four residues, irrespective of their replication competence, indicating that either a structural element essential for FMDV RNA synthesis is missing or disrupted in p{Delta}74 and pCHIM or that more than four residues in the FMDV 3' NCR are required to initiate replication.

Our data on FMDV 3' NCR add new information to the poorly characterized regulation of aphthoviruses replication. The experimental approach using derivatives from an infectious FMDV cDNA clone allows the study of infectivity both in vitro on cultured cell lines and in vivo in suckling mice. We are currently focussed on the study of the minimal requirements needed at the level of primary and secondary structures for replication competence in FMDV RNA.


   Acknowledgments
 
We are grateful to Ewald Beck who kindly provided plasmid pSP65FMDVpolyC containing the full-length sequence of FMDV O1K. We thank J. I. Núñez for his experimental help with mice. This work was supported by CICYT (grant BIO 99-0833-C02-01), Comunidad de Madrid (grant 08.2/0024/1997) and DGES (grant PM 98.0122). M. Sáiz was a postdoctoral fellow from the Spanish Ministry of Education and Culture.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 27 July 2000; accepted 11 September 2000.