Replication-competent foot-and-mouth disease virus RNAs lacking capsid coding sequences

Gerald M. McInerney1, Andrew M. Q. King1, Natalie Ross-Smith1 and Graham J. Belsham1

BBSRC Institute for Animal Health, Pirbright, Woking, Surrey GU24 0NF, UK1

Author for correspondence: Graham Belsham. Fax +44 1483 232448. e-mail graham.belsham{at}bbsrc.ac.uk


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RNA transcripts were prepared from plasmids encoding an infectious cDNA of foot-and-mouth disease virus (FMDV) or derivatives in which the leader (Lab and Lb) and capsid protein coding sequences were deleted or replaced by sequences encoding chloramphenicol acetyltransferase (CAT). The transcripts were electroporated into BHK cells and the expression of CAT and the FMDV 3C protease was monitored. Detection of CAT and 3C was dependent on the ability of the transcript to replicate. All of the Lb coding sequence and 94% of P1 (the capsid protein precursor) coding sequence could be deleted without any apparent effect on the ability of the RNA to replicate. Thus, no cis-acting replication element is present within this region of the FMDV genome. Trans-encapsidation of these FMDV replicons was very inefficient, which may explain the lack of production of defective-interfering particles in FMDV-infected cells.


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Foot-and-mouth disease virus (FMDV) is a member of the Aphthovirus genus within the family Picornaviridae. It causes an economically important disease of cloven-hoofed mammals. The FMDV genome is a single-stranded, positive-sense RNA of approximately 8 kb linked at its 5' end to VPg (3B) and polyadenylated at the 3' terminus. Translation of the single open reading frame produces a polyprotein that is processed proteolytically to the mature viral proteins (reviewed in Belsham, 1993 ). The primary cleavage products are L, P1–2A, P2 and P3 (see Fig. 1). The leader (L) protease is produced in two forms, Lab and Lb, due to the use of two different initiation codons. The P1–2A precursor yields the structural proteins, while P2 and P3 contain proteins required for genome replication, including 3Dpol (the RNA polymerase), and 3Cpro, required for protein processing.



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Fig. 1. Structure of plasmids encoding FMDV RNAs used in this study. The infectious cDNA, pT7S3, encoding FMDV has been published previously (Ellard et al., 1999 ). Modifications to produce the other plasmids are described in the text. The restriction enzyme sites required for these modifications are indicated. CCn, Poly(C) tract.

 
Replication of poliovirus, the prototype picornavirus, is dependent on sequences within the 5' and 3' untranslated regions (UTRs) of the genome (Andino et al., 1990 ; Borman et al., 1994 ; Pilipenko et al., 1996 ; Sarnow, 1989 ). More surprisingly, the replication of RNA from human rhinovirus 14 (HRV-14) (McKnight & Lemon, 1996 , 1998 ) and cardioviruses (Lobert et al., 1999 ) has also been demonstrated to require elements within the capsid coding region (P1). These sequences, termed cis-acting replication elements (CREs), act at the level of the RNA. Recently, a similar CRE has been located within the P2 coding sequence of poliovirus (Goodfellow et al., 2000 ).

Little work has been done on the replication of FMDV RNA, which is distinctive in having a long poly(C) tract within the 5' UTR and three copies of the 3B coding sequence. Defective genomes of FMDV have been generated during serial passage, but the deletions were restricted to the L coding region (Charpentier et al., 1996 ). The absence of capsid defective interfering (DI) genomes, as seen in poliovirus infections (Cole et al., 1971 ), suggests a role for P1 sequences in replication, either as protein or as a CRE. To determine whether the FMDV P1 sequences of FMDV are necessary for replication, FMDV RNAs lacking capsid sequences have been produced and analysed.

pT7S3{Delta}LP1 (Fig. 1) was constructed by digesting pT7S3 (Ellard et al., 1999 ), an infectious cDNA of FMDV O1K, with SunI and re-ligating the 9935 bp fragment. This introduced a 1152 bp deletion from 50 nt upstream of the 3' end of the L coding sequence to midway through the 1C coding sequence. This construct encodes a truncated L protein fused to the C-terminal portion (50·4%) of the P1 region.

Plasmids containing the chloramphenicol acetyltransferase (CAT) sequence in place of Lb and regions of P1 were also produced. pT7Rep (Fig. 1), with a deletion within the capsid sequences similar in size to pT7S3{Delta}LP1, was constructed in several steps. Using pT7S3 as template, a PCR with the primers 5' dCACTTTGTACTGCTGGTGACAGGC 3' (within the FMDV IRES) and 5' dTCTTACGTACATTTTCCCTGTG 3' produced a 270 bp fragment, with a SnaBI restriction site (underlined) introduced adjacent to the Lb initiation codon (complement in bold). This fragment was inserted into EcoRV-digested pBluescript II SK (+) (Stratagene) to produce pBSger1. A PCR was used to create a 665 bp fragment containing the CAT sequence flanked by SnaBI and EcoRV sites at the 5' and 3' termini, respectively. The primers were 5' dGCGCTACGTAATGGAGAAAAAAA 3' (GER3) and 5' dAAAAGATATCAACGCCCCGCCCTGC 3', with pKSGSCC (Drew & Belsham, 1994 ) as the template. This fragment was cloned into EcoRV-digested pGEM-5Zf+ (Promega) to produce pGEMger2. A 715 bp SnaBI–SacI fragment from pGEMger2 was ligated into a 3165 bp SnaBI–SacI fragment of pBSger1 to produce pBSger3. Finally, an 886 bp KpnI–EcoRV fragment from pBSger3 was ligated into a fragment from pT7S3 generated by digestion with Bsu36I, blunt-ending with the Klenow fragment of DNA polymerase I and digestion with KpnI. This produced pT7Rep (Fig. 1), containing the sequence of FMDV strain O1K, with the CAT sequence situated in-frame, in place of Lb and the 5'-proximal 59·6% of the P1 region. This construct was described briefly by Belsham et al. (2000) . Digesting pT7Rep with SalI and re-ligating the 9692 bp fragment produced pT7Rep{Delta}Sal (Fig. 1), which encodes a polyprotein with an inactive 3Dpol lacking 76 residues. A second CAT-containing plasmid was constructed, pT7Rep2 (Fig. 1), which lacked further P1 coding sequence. A PCR was performed with primers GER3 (as above) and 5' dAAAACCCGGGTCGCCCCGCCCTGC 3' and pKSGSCC as the template. This produced a fragment containing the CAT sequence flanked by SnaBI (as in pT7Rep) and XmaI (in italics) sites. Following digestion with these enzymes, the fragment was ligated into similarly digested pT7Rep to produce pT7Rep2, which lacks the coding sequence for Lb and 94% of P1.

The replication of the RNAs was determined indirectly by assaying protein production. This approach has significant advantages over determining RNA production, since there is no pre-existing virus-encoded protein present within the cells and none is introduced by the electroporation of the RNA into cells. Each plasmid was linearized with HpaI [immediately 3' of the poly(A) tail] and RNA transcripts were prepared by using T7 RNA polymerase (Ambion T7 MEGAscript). The transcripts were analysed on agarose gels to confirm their yield and integrity and were introduced into baby hamster kidney (BHK) cells by electroporation, as described previously (Belsham et al., 2000 ). Cell lysates were prepared at 2, 4, 6 and 8 h post-electroporation and analysed for expression of CAT by using a CAT ELISA (Boehringer) and for production of FMDV 3C by immunoblotting with MAb IG1 (kindly provided by E. Brocci, Brescia), as described previously (Belsham et al., 2000 ).

Cells electroporated with RNA derived from the 3Dpol-defective plasmid pT7Rep{Delta}Sal did not express detectable CAT at any time (Fig. 2). However, CAT expression was readily demonstrated in cells that received RNAs derived from pT7Rep and pT7Rep2 from 4 h post-treatment. The level of CAT declined gradually from 4 to 8 h post-electroporation (Fig. 2). The decrease was particularly marked in cells that received pT7Rep-derived RNA, and no CAT could be detected in these cells after 24 h (data not shown). The absence of CAT expression from the replication-defective RNA showed that the CAT produced from pT7Rep and pT7Rep2 RNAs was not the result of translation of input RNA alone and that these RNAs must have been replicating. This was confirmed by RNA dot-blot analysis (data not shown). Extensive cytopathic effect, similar to that seen following virus infection, was observed in cells treated with pT7Rep RNA, and this increased until approximately 50% of cells had lysed (as judged by trypan blue exclusion). This may explain the reduction in CAT signal in these cells at late times post-electroporation. The CAT expression from pT7Rep2 RNA was approximately twice that from pT7Rep RNA. Transient expression analysis of the plasmid DNAs in BHK cells (infected with the recombinant vaccinia virus vTF7-3, which expresses the T7 RNA polymerase; Fuerst et al., 1986 ) showed CAT expression from pT7Rep2 approximately double that from pT7Rep (data not shown). It seems likely that the different levels of signal reflect differences in the stability of the distinct CAT–{Delta}P1–2A fusion proteins and/or their recognition in the assay. The kinetics of CAT protein accumulation were similar in both cases and this is probably indicative of a similar rate of replication by the two RNAs. The fact that replication occurred in a substantial fraction of cells treated with the replicon RNAs, and that it occurred so rapidly, was of some surprise, as the shortened poly(C) tract encoded by the infectious cDNA plasmid and non-wt terminal RNA sequences had been expected to impair initial replication.



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Fig. 2. Time-course of CAT expression in cells electroporated with FMDV replicon RNAs. BHK cells were electroporated with RNA transcripts derived from pT7Rep (filled bars), pT7Rep2 (open bars) or pT7Rep{Delta}Sal (hatched bars) or with no transcripts (mock, cross-hatched bars). At the times indicated, cell extracts were prepared and analysed for CAT expression by ELISA.

 
The production of CAT could only allow monitoring of the replication of replicons containing this sequence. To compare their replication with full-length FMDV RNA and other replicons lacking CAT, we examined the expression of the FMDV 3C protease. Extracts of cells electroporated with RNA derived from each of the plasmids shown in Fig. 1 were analysed by immunoblotting for FMDV 3C and for cellular actin (Fig. 3). Significant accumulation of 3C was observed by 4 h post-electroporation in cells treated with RNA derived from pT7Rep, pT7Rep2, pT7S3 and pT7S3{Delta}LP1 (note that the 3CD precursor of 3C was also detected by the anti-3C antibody, plus a background protein that migrates between these species that is present in uninfected BHK cells; see Belsham et al., 2000 ). In each case, the level of 3C then declined (in accordance with the decline in the expression of CAT; Fig. 2). In part, this loss of protein could be accounted for by loss of cells, as indicated by the loss of actin (Fig. 3 D–F). No 3C expression was detected in cells that received the pT7Rep{Delta}Sal RNA, demonstrating that detectable expression of 3C, like CAT, required RNA replication.



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Fig. 3. Time-course of FMDV 3C expression in cells electroporated with FMDV replicon RNAs. BHK cells were electroporated with RNA transcripts derived from pT7Rep or pT7Rep2 (A, D), pT7S3 or pT7Rep{Delta}Sal (B, E) or pT7S3{Delta}LP1 (C, F) or with no transcripts (lanes labelled ‘Mock’) as shown and cell extracts were prepared at the indicated times (h post-treatment). Aliquots were analysed by SDS–PAGE and immunoblotting for the presence of FMDV 3C (and its precursor 3CD) (A–C) and cellular actin (D–F). Detection onto X-ray film was achieved with chemiluminescence reagents (Pierce).

 
The time of onset of 3C detection was similar for each replication-competent RNA, suggesting no major differences in their rates of replication. The production of infectious virus by cells electroporated with pT7S3-derived RNA was almost complete by 8 h, and most cells that had taken up RNA had lysed by this time (data not shown).

This study describes the replication of FMDV genomes carrying various deletions, the largest encompassing the complete Lb coding region and the 5'-proximal 94% of the P1 coding region. Although replication-competent FMDV RNAs lacking the L region have been reported previously (Piccone et al., 1995 ; Charpentier et al., 1996 ), the RNAs described here are the first FMDV RNAs missing capsid coding sequences that have been shown to replicate. Analysis of the FMDV type O1K RNA sequence using MFOLD from the GCG package predicted a stable secondary structure of approximately 80 nt within the coding sequence for the C-terminal region of 1C (VP3). This region was predicted to adopt a very limited range of structures and resembled the CREs described in rhino- and cardioviruses (McKnight & Lemon, 1998 ; Lobert et al., 1999 ). This sequence was deleted within pT7Rep2, but was present within pT7Rep. Since both of these RNAs replicated, this motif cannot act as a CRE. The CREs described initially are within the 1D (VP1) coding region of HRV-14 (McKnight & Lemon, 1998 ) and the 1B (VP2) coding region of the cardioviruses (Lobert et al., 1999 ). No CRE is present within most of the P1 region of poliovirus, since replicons lacking these sequences have been produced (Barclay et al., 1998 ). However, a CRE has recently been identified in the poliovirus P2 coding sequence (Goodfellow et al., 2000 ). If a CRE exists within the FMDV non-structural protein coding sequences, it would not have been identified in our study.

The pT7Rep and pT7Rep2 RNAs were encapsidated in trans within FMDV capsid proteins when electroporated cells were subsequently infected (10 p.f.u. per cell, 2 h post-electroporation) with homologous wt virus (data not shown). The relative efficiencies of encapsidation of replicons and wt genomes were calculated by transferring supernatants from such cells to fresh cells and comparing the numbers of cells that expressed CAT (detected by a CAT staining kit, Boehringer) with the number of wt virus plaques. The apparent efficiency of encapsidation of the replicons was found to be extremely low (at best, 1 in 36000 for pT7Rep and 1 in 20000 for pT7Rep2) and these efficiencies were reduced further when heterotypic FMDV serotypes were used as helper (data not shown). This efficiency is much lower than the encapsidation rates reported for poliovirus replicons (Barclay et al., 1998 ). The reasons for this difference are unknown, but may reflect a strong preference during encapsidation for FMDV capsid proteins provided in cis. This is consistent with the fact that the only defective genomes of FMDV have deletions in L rather than within the capsid coding region (Charpentier et al., 1996 ). There may be selection against packaging of the FMDV–CAT replicon RNAs due to their reduced size compared with the intact genome (the pT7Rep transcript is 86% of wt length and pT7Rep2 is 77% of wt length), but encapsidation was observed of a poliovirus replicon only 75% of the wt length (Barclay et al., 1998 ). The demonstration of encapsidation at any level indicates that no RNA-based encapsidation signal exists within the Lb region or the 5'-proximal 94% of the P1 region of FMDV O1K RNA.


   Acknowledgments
 
We thank E. Brocci (Brescia) for the anti-FMDV 3C MAb. G.M.M. gratefully acknowledges a studentship from the Institute for Animal Health and helpful advice from Dr Fiona Ellard.


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Received 18 January 2000; accepted 4 April 2000.