Importance of Arginine 20 of the Swine Vesicular D
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病菌学杂志 2005年第1期
Department of Exotic Disease, National Institute of Animal Health, Kodaira, Tokyo, Japan
Molecular Biology Division
Epidemiology Division, Institute for Animal Health, Pirbright, Woking, Surrey, United Kingdom
ABSTRACT
A major virulence determinant of swine vesicular disease virus (SVDV), an Enterovirus that causes an acute vesicular disease, has been mapped to residue 20 of the 2A protease. The SVDV 2A protease cleaves the 1D-2A junction in the viral polyprotein, induces cleavage of translation initiation factor eIF4GI, and stimulates the activity of enterovirus internal ribosome entry sites (IRESs). The 2A protease from an attenuated strain of SVDV (Ile at residue 20) is significantly defective at inducing cleavage of eIF4GI and the activation of IRES-dependent translation compared to the 2A protease from a pathogenic strain (J1/73, Arg at residue 20), but the two proteases have similar 1D-2A cleavage activities (Y. Sakoda, N. Ross-Smith, T. Inoue, and G. J. Belsham, J. Virol. 75:10643-10650, 2001). Residue 20 has now been modified to every possible amino acid, and the activities of each mutant 2A protease has been analyzed. Selected mutants were reconstructed into full-length SVDV cDNA, and viruses were rescued. The rate of virus growth in cultured swine kidney cells reflected the efficiency of 2A protease activity. In experimentally infected pigs, all four of the mutant viruses tested displayed much-reduced virulence compared to the J1/73 virus but a significant, albeit reduced, level of viral replication and excretion was detected. Direct sequencing of cDNA derived from samples taken early and late in infection indicated that a gradual selection-reversion to a more efficient protease occurred. The data indicated that extensive sequence change and selection may introduce a severe bottleneck in virus replication, leading to a decreased viral load and reduced or no clinical disease.
INTRODUCTION
Swine vesicular disease virus (SVDV) is an important member of the Enterovirus genus of the family Picornaviridae. This virus is closely related antigenically and genetically to human coxsackievirus B5 (17, 19, 39). Virulent strains of SVDV induce an acute vesicular disease in pigs (but not ruminants) (10, 29) that is clinically very similar to that induced by foot-and-mouth disease virus, a member of the Aphthovirus genus of the family Picornaviridae. Attenuated strains of SVDV have been isolated from apparently healthy pigs (25). Infectious cDNA clones have been generated for both pathogenic (J1/73, termed J1) and attenuated (H/3/76, termed 00) strains of SVDV (19, 21). Recent studies have mapped the key determinant of virulence within these strains to the 1D-2A coding region of the SVDV genome. It has been shown that the identity of residue 132 within 1D and particularly of residue 20 within the 2A protease can determine the pathogenicity of the virus (23, 24). Residue 20 of 2A is an arginine (R) in the virulent J1 strain but an isoleucine (I) in the attenuated 00 strain (19, 21).
In common with other picornaviruses, the positive-sense RNA genome of SVDV encodes a large polyprotein that is processed by internal viral proteases to the mature polypeptides. The enteroviruses encode two distinct proteases. The 2A protease is responsible for the primary cleavage at the 1D-2A junction, while the 3C protease (plus its precursor, 3CD) is required for all other cleavages within the polyprotein except for the 1AB cleavage that occurs following encapsidation of the genome. Both proteases are related to the trypsin-like family of serine proteases (6, 34), but each has a catalytic triad in which a cysteine (C) residue substitutes for the serine (S) nucleophile. The catalytic triad of the SVDV 2A protease comprises residues histidine 21, aspartate 39, and cysteine 110 (38). Thus, residue 20, a critical determinant of virus virulence, is adjacent to one component of the catalytic triad.
In addition to cleaving the 1D-2A junction, the SVDV 2A protease also induces the cleavage of translation initiation factor eIF4G (26, 35), a key component of the cap-binding complex, eIF4F (15). This complex also includes eIF4E (the cap-binding protein) and eIF4A (an RNA helicase). eIF4G makes multiple protein-protein interactions and acts as a bridge between capped mRNAs (bound by eIF4E) and eIF3 that is associated with the small ribosomal unit. Thus, eIF4G plays an important role in the initiation of cap-dependent protein synthesis on cellular mRNAs. The cleavage of eIF4G induced by the 2A protease is believed to be responsible for the inhibition of cellular protein synthesis in enterovirus-infected cells (7). The translation of picornavirus RNA is dependent on an internal ribosome entry site (IRES) located within the 5' untranslated region. Except for the hepatitis A virus IRES, the picornavirus IRES elements are still active when eIF4G has been cleaved by the 2A protease. Indeed, another function of the 2A protease is the stimulation of enterovirus IRES activity within cells in which eIF4G is cleaved (8, 18, 33). However, our studies suggest that the inhibition of cap-dependent translation (resulting from eIF4G cleavage) and the stimulation of IRES-directed translation by the 2A protease are independent processes (33).
We have demonstrated recently that the 2A protease from the attenuated strain (00) of SVDV was severely defective in inducing eIF4GI cleavage and its expression resulted in only modest stimulation of IRES activity (35). In contrast, the 2A protease from the virulent strain (J1) of SVDV efficiently induced eIF4GI cleavage and strongly stimulated IRES function. Both proteases efficiently cleaved the 1D-2A junction. To determine the effects of other amino acid substitutions at this position of the protease sequence, we have modified residue 20 of the SVDV strain J1 2A protease to each of the 20 amino acids and examined the three distinct properties of the mutant proteases generated. The different proteins display a spectrum of activities, with just one mutant being catalytically inactive. Some of the mutant protease sequences have been introduced into full-length cDNA clones of SVDV, and the effects of the mutations on the properties of the rescued viruses have been analyzed in cultured cells. Furthermore, four of the mutant viruses have also been compared to the wild-type (wt) J1 virus by experimental infection of pigs. Each of the mutant viruses displayed significantly reduced virulence, but viral replication and excretion occurred at a low level. Direct sequencing of viral cDNA derived at different times postinfection indicated that significant selection-reversion had occurred in vivo.
MATERIALS AND METHODS
Plasmid construction and mutagenesis. Plasmid pGEM3Z/J1 (35), which contains the coding region for a myc-tagged 1D-2A product derived from the virulent J1 strain of SVDV, was used as the backbone for all mutagenesis (Fig. 1). All DNA manipulations were performed essentially as described by the manufacturer or by using standard procedures (36). The SVDV cDNA, as an EcoRI-BamHI fragment (ca. 1,350 bp), was isolated from pGEM3Z/J1, ligated into similarly digested M13mp18 replicative-form (Rf) DNA, and transformed into Escherichia coli JM109. Phage from an individual plaque was amplified, and double-stranded Rf DNA was isolated. Mutagenesis with a Muta-GENE kit (Bio-Rad) was performed essentially as described by the manufacturer on the basis of the method of Kunkel (27). From the initial reaction, which used a degenerate pool of oligonucleotides (36-mers) in which the codon corresponding to residue 20 of 2A was changed to NNN (an equal mixture of all four bases at each position), mutations encoding 11 different amino acids plus the stop codons (TGA, designated , and TAG) were recovered. The sequence of the pool of degenerate primers was GCGCGCGTTGCGAGATGNNNATTCACCACTCTATAG. The residual mutations were generated by using the same mutagenesis procedure but with unique 36-mer primers that contained the required codons. The sequences of the particular codons used for each amino acid are shown in Table 1. Following determination of the 1D-2A coding sequence, preparations of double-stranded Rf DNA were made and digested with BamHI and EcoRI and the 1.35-kbp fragments were ligated into similarly digested pGEM3Z. All of the plasmids generated were then amplified individually to prepare DNA (with a QIAGEN midiprep kit) suitable for transfection into mammalian cells. The presence of the required mutations was confirmed with the BcaBEST dideoxy-sequencing kit (TAKARA), and several clones were also fully sequenced through the SVDV cDNA with a Beckman capillary sequencer (model CEQ2000XL). Control plasmid pGEM3Z/J1 (Fig. 1) has an in-frame deletion that removes the 1D-2A junction along with a critical N-terminal region of the 2A protease and lacks all 2A protease activity (35).
Protein expression analysis. In vitro transcription-and-translation (TNT) reactions were performed with the TNT rabbit reticulocyte system (Promega) with [35S]methionine essentially as described by the manufacturer. Aliquots (3 μl) were mixed with sample buffer containing sodium dodecyl sulfate (SDS) and dithiothreitol, boiled, and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) (28) and autoradiography. Quantitation of radioactivity was performed with a phosphorimager and Quantity One software (Bio-Rad).
Transient-expression assays within cells were performed as described previously (33). Briefly, plasmids (2.5 μg) were introduced with Lipofectin (8 μg; Life Technologies) into BHK cells (35-mm-diameter dishes) infected with recombinant vaccinia virus vTF7-3 (14), which expresses T7 RNA polymerase. After 20 h, cell extracts were prepared (400 μl). Aliquots (20 μl) were analyzed by SDS-PAGE and Western blotting with a monoclonal antibody (9E10; Roche) directed against the c-myc epitope tag on the N terminus of SVDV 1D (Fig. 1) or with sheep antibodies specific for the C terminus of eIF4GI as described previously (35). Detection was achieved with appropriate peroxidase-labeled secondary antibodies and chemiluminescence reagents (Amersham Pharmacia Biotech). To determine the ability of the mutant 2A proteases to activate IRES function, the test plasmids (0.5 μg) were cotransfected with dicistronic reporter plasmid pGEM-CAT/CB4/LUC (2 μg) containing the coxsackievirus B4 IRES (33) or with pC/SVDVJ(+)/L (2 μg), which has the SVDV J1 IRES (35), essentially as described above. Cell extracts were prepared, and the chloramphenicol acetyltransferase (CAT) and luciferase (LUC) expression levels were monitored by Western blot analysis as described previously (33). LUC enzyme activity, the indicator of IRES-directed translation, was quantitated with a LUC assay kit (Promega) and a Bio-orbit luminometer.
Reconstruction of full-length infectious cDNAs, virus rescue, and growth analysis. The mutant cDNA fragments (in the J1 background) encoding the single amino acid substitutions of residue 20 of the 2A protease gene were excised from the M13 Rf DNAs (described above) with SalI and BssHII (Fig. 1), and these fragments (about 740 bp) were inserted into a similarly digested transfer vector containing the SmaI (nucleotide [nt] 1755)-to-EcoRI (nt 5433) segment of H/3/76 (00) cDNA. From these transfer vectors, fragments were prepared with Bst1107I (nt 2233) and BssHII (nt 3368) and inserted into an infectious clone (pSVLS00) (22) of avirulent SVDV H/3/76 cut with the same enzymes (unique sites). Resulting chimeric plasmids containing the H/3/76 backbone with the SalI-BssHII fragment of the J1 strain modified at residue 20 of the 2A gene were selected, and infectious virus was rescued by transfection into COS-7 cells essentially as described previously (22). Infectious virus was amplified by two or three passages in the SKL (porcine kidney) cell line (37). The titer of each virus stock was determined by plaque assay on SKL cells. The sequences of the initial infectious plasmid clones and RNA purified from the virus stocks were confirmed by reverse transcription (RT)-PCR and nucleotide sequencing. Virus growth curves were obtained by infecting SKL cells with 0.1 PFU/cell and incubating them for up to 48 h. At each time point tested, the virus yield was determined by plaque assay on SKL cells as described previously (21, 22).
Analysis of virus replication and virulence in experimentally infected pigs. (i) Animals. Twenty Landrace crossbred Large White pigs (between 20 and 30 kg) that were seronegative for antibody to SVDV were housed in biosecure animal buildings and divided at random into five groups of four animals in individual boxes as follows: pigs UT23 to -26, box 1, inoculated with SVDV 2A-20 V; pigs UT27 to -30, box 2, inoculated with SVDV 2A-20 K; pigs UT31 to -34, box 3, inoculated with SVDV 2A-20 I; pigs UT35 to -38, box 4, inoculated with SVDV 2A-20 W; pigs UT39, -40, -43, and -44, box 5, inoculated with SVDV 2A-20 R (J1, wt).
Pigs were inoculated intradermally or subdermally in the heel pad of the left fore foot essentially as described previously for inoculation with foot-and-mouth disease virus (1, 2). The titers of the stock viruses were adjusted to 107.3 PFU (as measured in SLK cells) in tissue culture medium, and each inoculated animal received approximately 107 PFU in 0.5 ml. The animals were examined clinically for signs of swine vesicular disease, and rectal temperatures were recorded daily until 11 days after inoculation (dpi). On the basis of previous experience with swine vesicular disease, which can produce mild clinical symptoms, the clinical signs were scored as follows: a local lesion at the site of inoculation of less than 1.5 cm (small), 1 point; a local lesion at the site of inoculation of more than 1.5 cm (large), 2 points; small lesions on other feet, 3 points per foot; large lesions on other feet, 6 points per foot; lesions on or around the tongue, mouth, or snout, 3 points; lameness, 3 points. Each animal could have a maximum score of 26 points by this system.
(ii) Sample collection. Blood samples, together with nasal and rectal swabs, were taken immediately before inoculation (0 samples), 5 min after inoculation (blood only), and then at 1, 2, 3, 4, 7, 9, and 11 dpi, when the animals were killed. The blood samples were left to clot for 1 h at room temperature, and serum was separated and then stored at –80°C. For RNA extraction, an aliquot of serum was thawed, 2 volumes of serum was added to 3 volumes of lysis buffer (Roche Lysis Solution), and the mixture was stored at 4°C overnight before nucleic acid extraction and subsequent analysis were performed. Swabs were stored at –20°C, and then 1 ml of TRIzol Reagent (Life Technologies) was added to the tubes for RNA extraction. When the animals were killed at 11 dpi, selected samples, i.e., epithelium from the feet or tongue and liver from a few animals, were collected into RNAlater (Ambion, Austin, Tex.) and stored at –20°C prior to extraction.
(iii) qRT-PCRs. Real-time quantitative RT-PCRs (qRT-PCRs) were used to determine the amount of SVDV RNA in extracts of total nucleic acid from blood and swab samples, from virus inocula, and in total RNA from tissue samples by using the 2B-IR primer-probe set as described elsewhere (32). All quantitative measurements were based on standard dilution series derived with in vitro-transcribed SVDV RNA from a plasmid containing the SVDV IRES fragment (pGEM 3Z/J1 IRES) (35) and quantified by spectrophotometry. The stock RNA preparation contained 1010 SVDV genome equivalents per ml. This method of quantitation is influenced minimally by sample type and correlates well with infectivity (1-5, 31, 32).
(iv) Sequencing. All sequencing was performed on PCR products derived with the same cDNA or RNA samples as used for the quantitations described above. The sequencing was performed as consensus sequencing from samples amplified in a two-step nested PCR with primers flanking the 2A sequence and cycle sequencing with a Beckman capillary sequencer (model CEQ2000XL). Sequencing of individual cDNA products was performed in each direction and repeated multiple times to confirm the results.
(v) Assay for antibodies. Serum samples were tested by an enzyme-linked immunosorbent assay (ELISA) for the presence of antibodies to SVDV by the 5B7 monoclonal antibody blocking ELISA (9) and by virus neutralization assay (VNT) (16).
RESULTS
In vitro expression and processing by the SVDV 2A protease. Plasmid pGEM3Z/J1 (35) encodes a myc-tagged 1D-2A product derived from the pathogenic J1 strain of SVDV (Fig. 1). Mutagenesis was performed so that the codon for residue 20 of the 2A protease was changed to encode individually each of the possible 20 amino acids or a stop codon. Each of the mutants is identified by the single-letter code for the amino acid encoded at residue 20 (Table 1). As an initial screen of the properties of the mutated 2A proteases, each of the plasmid DNAs was used to program an in vitro TNT reaction with rabbit reticulocyte lysate and [35S]methionine. The samples were analyzed by SDS-PAGE and autoradiography (Fig. 2, top), and the products were quantitated with a phosphorimager. In these reactions, initially the 1D-2A precursor is generated, which is then cleaved through the action of the 2A protease to generate the products 1D and 2A. As expected, when a stop codon was introduced at the position of residue 20 within 2A, a stable product was generated corresponding to 1D fused to the first 19 amino acids of 2A (this fusion protein migrates only slightly more slowly than the mature 1D product). When a proline was introduced at residue 20, the 2A protease appeared to be completely inactive and a stable 1D-2A protein was made that was not processed during the course of the incubation. In all other cases, production of the full-length 1D-2A precursor was observed and processing occurred that was incomplete when the reaction was terminated. Similar results were obtained in three independent experiments, and the mean ratios of 1D-2A to 1D observed at a 2-h time point are presented in the bottom panel of Fig. 2. Apart from the P mutant, four other mutants also appeared significantly defective in their efficiency of processing of the 1D-2A junction, as judged by the high level of 1D-2A compared to that of the processed product 1D (Fig. 2, top and bottom). These defective mutants contained residues G, V, T, and D at position 20 of the protease. The D, G, and V mutants were the most defective of these, with little or no 2A product being detectable; although low levels of the 1D product were observed, these two products are clearly made in the same amounts but the sensitivity of detection depends on the methionine content and protein stability.
Intracellular expression and properties of the mutant 2A proteases. Further analysis of the properties of these mutant proteases was performed with transient expression assays of the plasmids within cells infected with recombinant vaccinia virus vTF7-3 (14), which expresses the T7 RNA polymerase. From extracts of transfected cells, the expression of the 1D-2A products was determined by Western blot analysis with an anti-myc tag antibody (the myc tag is located at the N terminus of the 1D sequence; Fig. 1). In our previous studies (35) with the 1D-2A region from the J1 and 00 strains of SVDV, we found that almost complete cleavage of the 1D-2A precursor occurred in this system and hence only the 1D species was detected by the anti-myc tag antibody. This was also the case for nearly all of the single amino acid mutants (Fig. 3). However, consistent with the in vitro TNT assay described above, the P mutant yielded only the unprocessed 1D-2A product. It is noteworthy that the yield of this product was also very high. The yields of the G, V, D, and stop codon () mutant products were also relatively high, whereas the level of products observed from the R, W, and H mutants was particularly low. On the basis of our previous studies (35), it seemed likely that most of the mutants (other than the P mutant) were inducing eIF4GI cleavage and thus inhibiting their own synthesis (to different degrees) since in these assays the 1D-2A precursors are expressed from monocistronic mRNAs lacking an IRES and hence are translated by a cap-dependent mechanism. To check this, the cleavage of translation initiation factor eIF4GI induced by the 2A protease was analyzed within the same cell extracts with an antibody directed against the C terminus of eIF4GI. Each of the mutant 2A proteases, other than the P mutant, induced eIF4GI cleavage; a subset of the data generated is shown in Fig. 4A. The nature of this assay and the fact that very low levels of 2A expression can induce eIF4GI cleavage make it difficult to determine any quantitative differences in the efficiency of eIF4GI cleavage in these assays. However, as discussed below, the higher level of expression of certain mutants, e.g., G, V, and D (Fig. 3), may be indicative of a slower rate of eIF4GI cleavage occurring within the cells.
IRES activation by the mutant 2A proteases. Plasmids encoding each of the mutant 2A proteases were cotransfected with plasmid pGEM-CAT/CB4/LUC into vTF7-3-infected BHK cells. This reporter plasmid expresses a dicistronic mRNA containing the IRES element from coxsackievirus B4 (33). In each experiment, the cell extracts (prepared after 20 h of incubation) were analyzed by SDS-PAGE and immunoblotting with antibodies directed against both the CAT and LUC proteins. Expression of the wt J1 strain 2A protease strongly stimulates CB4 IRES function in this assay (35), whereas the 2A protease from the 00 strain of SVDV has a significantly weaker effect. The analyses of a subset of extracts obtained in one experiment are shown in Fig. 4B and demonstrate the spectrum of observations made. Clear inhibition of CAT expression, reflecting the cleavage of eIF4GI induced by these plasmids (Fig. 4A) and the loss of cap-dependent protein synthesis, was observed for the M and J1 (R, wt) constructs, whereas the P and (stop codon) mutant proteins had little effect on the expression of CAT. In contrast, the M and R (J1, wt) constructs strongly stimulated LUC accumulation (reflecting IRES activity) whereas the P and mutant proteins had little or no effect on LUC expression (Fig. 4B). The ability of each of the 20 different mutant 2A proteases to activate the CB4 IRES, as indicated by the expression of LUC, is shown in Fig. 4C. The results presented are the means of three separate transfections and also closely reflect those obtained with a second dicistronic reporter plasmid, pC/SVDJ(+)/L (35), that contains the SVDV J1 IRES (data not shown). Neither the P mutant nor the pGEM3Z/J1 construct induced any increase in IRES activity. Expression of the D mutant resulted in about a twofold increase in LUC expression, while the G, V, and T mutants induced about a threefold increase in IRES function. Most of the other mutants (including the wt J1 sequence) induced about a five- to sixfold increase in IRES activity. However, mutants I, E, and K appeared slightly deficient in this activity (about a fourfold increase in activity) both in these assays and when assayed with the SVDV IRES construct (data not shown).
Analysis of 2A mutations within full-length infectious cDNA of SVDV. In order to examine the effects of the mutations at residue 20 of the 2A protease on the properties of SVDV, nine representative mutant cDNAs that had shown a range of different activities in the assays described above were reconstructed into the full-length infectious cDNA (pSVLS00) derived from the attenuated H/3/76 (00) strain (20). The individual mutant full-length cDNAs were sequenced to check for the presence of the expected mutations and then transfected into COS-7 cells to rescue the viruses. The mutant viruses that were generated were then amplified by infection of SKL cells. By 48 h postinfection, a complete cytopathic effect (CPE) was apparent in cells infected with the virus rescued from the pSVLS00 parental vector and with the full-length hybrid cDNA clones containing the L, W, and R mutations in the 2A protease. A lower level of CPE was observed in cells infected with the virus produced from the plasmids containing the K, E, and I sequences, but little or no CPE was apparent from the V, T, and D mutants (Table 2). All of these results appear consistent with the level of 2A protease activity detected with the in vitro assays described above (Fig. 2 and 4 and Table 2).
After further passage of the viruses, some CPE was evident from all of the viruses (although this was very weak for the D mutant) and the presence of SVDV RNA could be detected by RT-PCR (no signal was detected if the RT step was omitted; data not shown). The sequence of the 2A protease within the rescued viruses was determined following RT-PCR, and the presence of just the expected mutations was observed in each case (data not shown).
The growth properties of certain mutant viruses were compared (Fig. 5). The W and R mutants grew almost as well as the wt J1 virus in SKL cells, but the other mutant viruses (the K and I mutants and particularly the V mutant) were significantly slower at replicating. Furthermore, the W, R, and L mutants showed a large-plaque phenotype (like the J1 virus) whereas the V, T, and D mutant viruses produced only small plaques (like the attenuated 00 virus). The K, I, and E viruses produced an intermediate plaque size phenotype (Table 2). Thus, there was a good correlation between the replication rate of the rescued virus and the efficiency of the 2A protease activity scored with in vitro assays.
Analysis of experimental infection of pigs with mutant SVDV. In order to determine the effect of the 2A modifications on virulence, we selected four mutant viruses with single amino acid changes within this protease that displayed a range of in vitro activities and growth rates in tissue culture cells. Each of these selected viruses (W, K, I, and V) was compared to the wt J1 virus by experimental infection of pigs. It should be mentioned that previous work has shown that similar chimeric viruses that have an arginine (R) at position 20 of the 2A protease (as in the J1 wt virus) displayed a level of virulence comparable to that of the J1 wt virus (23).
(i) Quantitation of SVDV RNA in virus samples. The genome copy numbers in the virus inocula determined by qRT-PCR showed that the J1 (wt) inoculum (group 5) contained around 8.8 to 8.9 log10 genome copies per ml while the other four inocula (V, K, I, and W; groups 1 to 4) contained around 9.3 to 9.4 log10 copies of SVDV RNA per ml. Since all of the inocula had been adjusted to contain 107.3 PFU/ml, this indicated that the wt virus may have a slightly lower genome-to-PFU ratio (1.5 to 1.6 log10 copies per PFU) compared to the modified viruses (2.0 to 2.1 log10 copies per PFU). This slightly higher genome copy number in the modified viruses was also observed in the 5-min serum samples obtained from the inoculated pigs (see below), which for groups 2 to 4 (K, I, and W) contained approximately 0.5 log10 copies more than group 5 (J1 wt). The 5-min serum samples from group 1 inoculated pigs (V) contained approximately the same amount of genomes as those from the group 5 pigs. The reason for this is not known, but it may have resulted from a minor difference in the inoculations as these were done intradermally or subdermally in the heel pads and any virus in the circulation at 5 min after inoculation is derived from inoculated virus spilling into the local lymph vessels (4). The important point is that pigs inoculated with the mutant viruses all received at least as large a dose of virus as those that received the wt virus.
(ii) Clinical signs and evidence of virus replication in vivo. Small local vesicular lesions were observed in some of the group 5 (wt virus) pigs from 1 dpi and in some of the other groups on the following day. The lesions became relatively severe in the group 5 animals (maximum scores of 14, 20, 20, and 23 for the four pigs in the group), with severe lesions on most of the pigs' feet and on the snout, tongue, and lips of a single animal. In contrast, pigs in the other groups mainly had only local or minor lesions on additional feet and although a few of these animals had slightly more pronounced lesions (single animals with a score of 11 in groups 1 and 3 and two animals with scores of 7 and 8 in group 4, respectively), none of them were as severe as in the group 5 animals. No real lameness was observed, although the pigs in group 5 showed a slight "sticky gait" at 2 to 4 dpi and all of the pigs were eating normally and were not, apart from the lesions, much affected clinically by the infection. The average lesion scores for the groups are shown in Fig. 6A, and the maximum scores for the individual animals are shown in Table 3. The drop in scores seen toward the end of the observation period (Fig. 6A) was due to healing of the lesions.
The average temperatures of the group 1 to 4 pigs were in general normal, i.e., around 38°C, although the group 1 pigs had an average temperature of 39.1°C (a single animal had a temperature of 39.5°C) at 2 dpi. In contrast, group 5 pigs (with wt virus) had an increased temperature up to an average of 39.3°C at 2 to 7 dpi, with one animal having a temperature of 40.3°C on day 3 (data not shown).
As mentioned above, SVDV RNA could be observed in the sera of inoculated pigs, even in the samples taken 5 min after inoculation, at a level of around 3.4 to 4.2 log10 copies/ml (Fig. 6B). From this initial level of inoculated virus, the levels rose in the group 5 animals to 4.6 log10 copies/ml at day 1 and peaked at a level of 6.2 to 7.1 log10 copies/ml (average level of 6.7 log10 copies/ml) at 2 to 3 dpi (Fig. 6B and data not shown), after which the levels declined to 1.2 log10 copies/ml at day 7 and became undetectable from day 9. In contrast to this pattern, the levels of viral RNA in the serum of pigs in groups 1 to 4 initially decreased to around 1.5 to 2.4 log10 copies per ml at 1 dpi but then increased up to 3.2 to 6.2 log10 copies/ml (average levels around 3.3 to 5.3 log10 copies/ml, with the highest levels in groups 2 [K] and 4 [W]) at 2 to 3 dpi (Fig. 6B and data not shown). Thus, from day 1 to day 3, the level of viremia in the pigs infected with the mutant viruses was generally at least 100-fold lower than that observed in the animals that received the wt virus. Subsequently, the viremia slowly decreased and became undetectable on day 9 for groups 1 to 3 and on day 11 for group 4 (Fig. 6B). The decrease in virus RNA in the blood after day 3 correlated well with the onset of the serum antibody response that was detectable from day 4 (Fig. 6C). Antibodies to SVDV could be detected in all of the animals from 4 to 7 dpi by both ELISA and VNT. There was good agreement between the VNT (Fig. 6C) and ELISA (data not shown) results, with the highest titers being observed in animals in group 5 (wt virus).
(iii) Virus excretion from infected pigs. The SVDV RNA levels in nasal and rectal swabs are a measurement of virus excretion and are shown in Fig. 7. In the group 5 pigs, virus RNA (wt) could be detected in nasal swabs from day 1 and increased to levels just above 5 log10 copies/ml on day 3. There was a secondary peak on day 7 but only a low residual level of SVDV RNA at day 11. In contrast, viral RNA could not be detected in nasal swabs from pigs in groups 1 to 4 until 2 dpi. The amount of RNA peaked at levels of 3.2 to 6.3 log10 copies/ml at 7 dpi and then rapidly decreased to low levels by 11 dpi (Fig. 7A).
Analysis of the rectal swabs from the group 5 pigs (wt virus) detected viral RNA from day 1, and this increased to levels of around 3.7 log10 copies/ml on day 4 but then decreased slowly to only around 2 log10 copies/ml by day 11. In contrast, viral RNA could not be detected in rectal swabs from pigs in groups 1 to 4 until 4 dpi; excretion then peaked at a level of less than 3 log10 copies/ml at 7 to 9 dpi and then declined to less than 2 log10 copies/ml by 11 dpi (Fig. 7B).
In the tissue samples taken at the termination of the experiment at 11 dpi, around 4 to 11 log10 genome copies/g of tissue could be detected. The highest levels of viral RNA were detected in the epithelial samples from the feet (up to 11 log10 genome copies/g), although pigs that only had local lesions at the inoculation site had relatively low levels (data not shown). Tongue epithelium and liver samples from the pigs in group 5 (wt virus) had relatively low levels of viral RNA, up to 5.5 log10 copies/g (data not shown). There were no obvious differences in the levels of viral RNA between samples analyzed from the different groups, apart from the low levels detected in pigs that only had local lesions (as mentioned above); however, only 10 epithelial samples from the feet (data not shown) and a total of 18 samples were analyzed.
(iv) Sequence analysis of virus RNA from in vivo samples. Selected cDNAs used in the qRT-PCR studies described above were subjected to PCR amplification and direct sequencing of the SVDV 2A coding region. Samples selected for this analysis were serum samples collected at 3 dpi, nasal swabs collected at 7 dpi, and epithelial samples from the feet collected at 11 dpi, when the experiments were terminated. The results are shown in Table 3 and indicate that the significant late amplification and possibly residual virulence of the mutants (used for groups 1 to 4) were, at least in part, due to a gradual selection-reversion to a more efficient 2A protease. The modifications in the 2A sequence either generated a mutant form of the 2A protease with higher protease activity than the initial mutant or more generally changed to the wt amino acid (R) despite the fact that this involved up to three nucleotide changes. It is apparent that the generation of viruses within the infected animals containing the pathogenic wt sequence did not result in severe acute disease.
DISCUSSION
The major virulence determinant of SVDV for Japanese isolates J1/73 and H/3/76 has previously been mapped to residue 20 of the 2A protease (23). In this study, we have analyzed the impact of mutation of this residue on the activities of the protease and on the replication of the virus in tissue culture and during experimental infection of pigs.
The various mutant 2A proteases displayed a spectrum of activities. The P mutant was totally deficient in all three of the activities assayed. Presumably, the presence of this residue, adjacent to one component of the catalytic triad of the protease, causes some deformation of the polypeptide chain backbone that perturbs the geometry of the active site. All of the other mutants were functional, but the G, V, T, and D mutants were significantly defective in the 1D-2A cleavage assay and IRES activation. There is no obvious pattern to the nature of the residues that resulted in the production of either a highly active or a rather defective protease. For example, the A, L, and S mutant proteins are all highly active but the G, V, and T mutant proteins are all significantly defective; these amino acids are listed in order of having small aliphatic, hydrophobic, and hydroxyl group side chains in each group. The assay for the induction of eIF4GI cleavage was not very discriminating among the mutants, and all of the mutant proteins, except the P mutant protein, induced eIF4GI cleavage (data not shown). This presumably reflects the fact that even low levels of protease expression are sufficient to induce this cleavage; thus, presumably higher-level expression of a somewhat defective protease can still achieve the same effect. However, there is an indication that some of the mutant proteins induced eIF4GI cleavage more slowly. The effect of eIF4GI cleavage is to inhibit the initiation of cap-dependent protein synthesis (note that the 1D-2A proteins are expressed in this assay by this mechanism). Thus, if eIF4GI cleavage occurs more slowly, then it can be expected that cap-dependent expression of 1D-2A will be maintained longer (35). It is apparent that the expression level of certain mutations, e.g., P, G, V, and D, was significantly higher than that of other mutations, e.g., R, Y, W, and F. Thus, although the G, V, and D mutant proteins induced cleavage of eIF4GI, it is probable that the inactivation of the eIF4F complex happened relatively slowly in cells containing these proteins. Hence, these mutant proteins were synthesized longer and consequently accumulated to higher levels.
Certain other mutant proteins, namely, I, E, and K, appeared to be slightly defective in the IRES activation assay but did not appear to be significantly deficient in the in vitro 1D-2A cleavage assay. However, it was apparent that none of these mutant proteins were as effective as, for example, the R, Y, W, and F products, which were very active both in the IRES activation assays and in the TNT assay (Fig. 2 and 4).
It is interesting that the I mutant protein (in the J1 strain background) has a different phenotype than that which we observed for the 2A protease from the attenuated 00 strain of SVDV, which has an Ile at residue 20 (35). The 00 1D-2A protein is significantly deficient at inducing eIF4GI cleavage and about threefold less active in IRES activation than the J1 1D-2A protein, whereas the I mutant protein is not significantly defective in these activities. We were able to show that merely the change of I20 to R20 in the 00 1D-2A protein produced a protease that induced cleavage of eIF4GI and stimulated CB4 IRES activity about as efficiently as the J1 1D-2A protein does (35). However, there are six other amino acid differences between the SVDV 00 and J1 strains within the 1D-2A region (five differences in 1D and one in 2A). Of particular significance may be the nature of residue 132 within 1D, which is also linked to the virulence phenotype (23, 24). It is not entirely clear, however, how this residue (or any of the other different residues in 1D) could interact with residue 20 within 2A. It may be that although the 1D-2A junction is the primary cleavage site within the polyprotein that the two products can remain associated within the cell and perhaps this interaction modifies the activity of the protease. Recently, Foeger et al. (13) have shown an interaction between the 2A protease from human rhinovirus 2 and eIF4GI that is independent of the substrate-binding cleft of the protease. It was shown for this 2A protease that Leu17 (adjacent to catalytic residue His18, just as SVDV 2A protease residue 20 is adjacent to catalytic residue His21) is important in determining this interaction between the 2A protease and the N-terminal region of eIF4GI. Furthermore, mutation of Leu17 to Arg17 impaired this binding but did not affect 1D-2A processing activity by the protease (13). While this result has some similarity to the role of SVDV residue 20 in determining the activity of the 2A protease, there is also an important difference since in the SVDV 2A protease Arg20 is the wt residue. Thus, specific details of the interaction of the entero- and rhinovirus 2A proteases with eIF4GI still need to be determined.
Representatives of each of the different types of SVDV 2A mutant have been reconstructed into full-length infectious clones of SVDV, and the properties of the rescued mutant viruses have been examined in tissue culture (Fig. 5). Furthermore, the ability of some of these viruses to replicate and cause disease in pigs has also been examined. The ability of the viruses to replicate in tissue culture (Fig. 5) closely reflected the relative activity of the 2A protease in the in vitro assays (Fig. 2 and 4 and Table 2), and some of the mutants (e.g., W and R) replicate to a level similar to that of the wt J1 virus. It should be noted, however, that the virulent J1 strain and the attenuated 00 strain of SVDV also replicate with similar efficiency in a single-step growth curve (23) but the J1 virus does replicate to a higher level than the 00 strain in a low-multiplicity infection (T.I., unpublished results). However, only SVDV with the wt R residue at position 20 of the 2A protease causes severe disease in pigs (Fig. 6) (23, 24) but the W and K viruses induced the next highest level of viremia (Fig. 6B), which is in accord with their efficient growth in tissue culture cells (Fig. 5).
It was interesting that during the course of replication in tissue culture no evidence of sequence reversion was obtained. However, in the infected pigs the sequence encoding residue 20 of the protease was modified at a high frequency (Table 3). The changes that occurred all produced a 2A protease that functioned more efficiently than that found in the input virus. There was a strong bias toward reversion to the wt AGA codon encoding an R residue.
Picornaviruses lack proofreading-repair activities, and consequently the error rate during genome replication is high (approximately 10–4 substitutions per nucleotide copied, i.e., around one nucleotide misincorporation per genome per replication cycle). This inevitably leads to the generation of variant genomes (11). For example, live poliovirus type 3 vaccines contain a subset of viral genomes that contain the wt C at nt 472 in the 5' noncoding region, and it has been shown that the proportion of such genomes in the vaccine correlates with neurovirulence (12). In recipients of type 3 poliovirus vaccine, the reversion-selection to the wt nucleotide at nt 472 occurs by 3 to 6 days after vaccination, and by day 11, the type 3 virus also loses all or part of its temperature-sensitive growth phenotype because of another mutation-selection at amino acid residue 91 of VP3. Despite this relatively rapid reversion of the live vaccine to a more virulent phenotype, the frequency of vaccine-induced polio is very low (estimated at around 1:500,000 primary vaccines), suggesting that the immune system generally has time to prevent clinical disease, most likely by preventing or reducing viremia (30). Hence, it appears likely that our findings on pigs inoculated with mutant SVDVs are due to a combination of preexisting variant genomes in the inocula, initial local replication with further generation of mutants, selection for viral fitness, bottleneck transmission events, and the effects of the evolving immune response. In this light, the results obtained with the individual groups of pigs may be explained as follows. For the group 5 pigs (wt virus), most of the viral genomes encode a highly efficient protease (arginine at position 20) that is conserved because of positive selection for a rapidly replicating virus, with the result that the virus is able to spread relatively efficiently through the circulation and cause generalized lesions at other sites before the immune system is able to control the virus. For the other groups, the picture is more complex and we will discuss the findings separately for each of the groups and then try to tie the findings together.
Pigs in group 1 were inoculated with a mutant virus having a valine codon at position 20 of the 2A protease. According to the in vitro studies, valine at this position results in a rather inefficient protease (Fig. 2 and 4) and the virus grows relatively slowly in tissue culture (Fig. 5). Two pigs given this virus had no or minor local lesions, while the two other pigs had local lesions and lesions on one and three other feet, respectively. Interestingly, the pigs that developed secondary lesions had a significant early viremia with a majority of viral genomes (on the basis of consensus sequencing of viral RNA from serum at day 3) that contained a single nucleotide change resulting in a methionine codon; this produces a more efficient protease (Fig. 2 and 4 and Table 2). This was followed by two additional mutation-selection steps that generated the codon for the wt arginine. In contrast, the two other pigs in this group (having only minor or no local lesions) had a low or undetectable viremia but the major virus population that was present appeared to have changed all three nucleotides of codon 20 to encode an arginine residue. This finding indicates that an initial change at a single nucleotide can create a relatively efficient protease that allows additional replication and the further selection of efficient variants. However, the severe bottleneck caused by an initial selection for the wt sequence severely restricts early replication so that the immune system can control it, which prevents efficient spread through the circulation and thus development of secondary lesions.
Group 2 pigs were inoculated with a mutant with codon 20 of the protease changed to a lysine (K). This mutant protease was found to be slightly more efficient in vitro than the valine mutant, and the virus grew better in tissue culture (Fig. 2, 4, and 5). However, this codon only required a single nucleotide change to encode the wt arginine. This appeared to occur rapidly, as the consensus sequences obtained even at 3 dpi showed the wt AGA at position 20. Interestingly, although this change to an arginine only included selection-reversion at a single nucleotide position and the animals in this group had significant early viremia, this was reduced relatively early and excretion in nasal fluid and feces occurred relatively late compared to that in the other groups (Fig. 6 and 7). This early reduction in viremia and the late excretion correlated well with the fact that the pigs in this group only developed local, although rather severe, lesions that did not spread to their other feet.
Pigs in group 3 received the I mutant virus; the mutation resulted in a protease that has approximately the same efficiency in vitro as the lysine mutant discussed above, but the I mutant virus grew slightly more slowly in tissue culture than the K mutant virus did (Fig. 2, 4, and 5). In contrast to the lysine mutant, the isoleucine mutant needs to change two nucleotides to revert to the wt arginine. Of the four pigs in this group, three developed local lesions at the site of inoculation while a single pig developed both a local lesion and lesions on two of its other feet. Interestingly, this pig had the highest level of viremia in the group and consensus sequence analysis showed a single nucleotide change so that codon 20 encoded phenylalanine, which results in a highly efficient protease. In contrast, the virus from the other pigs in this group either initially retained the isoleucine at position 20 (albeit changing a single nucleotide [UT33, Table 3]) or changed at two nucleotide positions to the wt arginine codon (UT32, Table 3), indicating that a single change without a gain in protease efficiency or the severe bottleneck effect of an early two-nucleotide reversion-selection severely restricted the viral population's ability to cause generalized disease before the immune system took control.
Pigs in group 4 were inoculated with a mutant encoding tryptophan (W) at codon 20 of the 2A protease; this protease is almost as efficient as the wt, and the virus replicated with near wt efficiency in tissue culture (Fig. 2, 4, and 5). The pig with the greatest viral load at days 2, 3, and 7 (UT35, Table 3) had a majority of viral genomes that retained the tryptophan codon, and this caused local lesions and lesions on two of its other feet, while another pig (UT36, Table 3) initially had viral genomes that retained the tryptophan codon and had a high early viremia but then the majority of the population of viral genomes changed or reverted at two nucleotide positions to a wt arginine codon. However, this appeared to introduce a severe bottleneck and this pig had a lighter viral load at day 7 and only mild local lesions. The viral populations in the other two pigs in this group reverted early on at two nucleotide positions to the wt arginine codon, but this two-nucleotide reversion also appeared to introduce a severe bottleneck, as indicated by the reduced viral load and reduced clinical disease, although pig UT37 (Table 3) developed lesions on two of its other feet. However, the relatively severe generalized lesions on additional feet of this pig were most likely due to excessive abrasion of the foot epithelium caused by an unrelated bacterial joint infection leading to direct entry of excreted virus into damaged epithelium rather than from the circulation.
In conclusion, all four mutant viruses displayed much-reduced virulence compared to that of the wt J1/73 virus but a significant, albeit reduced, level of viral replication and excretion was observed. Direct sequencing of samples taken early and late in infection indicated that the significant replication and possibly residual virulence of these mutants were, at least in part, due to a gradual selection-reversion to either a more efficient protease or to the parental amino acid sequence despite the fact that this involved up to three nucleotide changes. It appeared that selection of viruses with the amino acid codon changed at one or two nucleotides from the wt sequence occurred rather easily but that three nucleotide changes involved selection of intermediate, slightly more efficient viruses, followed by further selection for viruses with the wt amino acid. Furthermore, the data indicated that extensive change and selection may introduce a severe bottleneck in virus replication, leading to a decreased viral load and reduced or no clinical disease. A single nucleotide change resulting in a relatively efficient protease, not necessarily having the wt arginine at residue 20, may be the most fit virus in terms of the level of viral replication and disease in this virus-host system. However, clearly an arginine at position 20 of the 2A protease is required for an efficiently replicating and fully virulent virus. Interestingly, it appeared that not only is an arginine required at this position, the codon selected was in each case AGA although five other arginine codons exist. The SVDV strains sequenced so far, except for the attenuated Japanese H/3/76 strain, all have an AGA codon at this position, and comparison of more than 40 human enterovirus B isolates (same virus species as SVDV) indicates that AGA is by far the preferred codon while AGG may also occur. CGC and CGG occur very seldom, and CGT and CGA did not occur (Nick Knowles, personal communication). It would be interesting to pinpoint the exact mechanism of this codon preference. Clearly, very subtle differences in the properties of the 2A protease can have a profound effect on the virulence of SVDV.
ACKNOWLEDGMENTS
This work was funded in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan. A.T.C. gratefully acknowledges the award of a studentship from the Biotechnology and Biological Sciences Research Council.
Jeanette Knight, Vidhi Aggarwal, and Ginette Wilsden are gratefully acknowledged for excellent technical assistance, and we thank Luke Fitzpatrick, Brian Taylor, Colin Randall, and Malcolm Turner for assistance with the handling and management of experimental animals.
T.I. and S.A. contributed equally to this work.
Present address: Danish Institute for Food and Veterinary Research, Department of Virology, Lindholm, DK-4771 Kalvehave, Denmark.
Present address: Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
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Molecular Biology Division
Epidemiology Division, Institute for Animal Health, Pirbright, Woking, Surrey, United Kingdom
ABSTRACT
A major virulence determinant of swine vesicular disease virus (SVDV), an Enterovirus that causes an acute vesicular disease, has been mapped to residue 20 of the 2A protease. The SVDV 2A protease cleaves the 1D-2A junction in the viral polyprotein, induces cleavage of translation initiation factor eIF4GI, and stimulates the activity of enterovirus internal ribosome entry sites (IRESs). The 2A protease from an attenuated strain of SVDV (Ile at residue 20) is significantly defective at inducing cleavage of eIF4GI and the activation of IRES-dependent translation compared to the 2A protease from a pathogenic strain (J1/73, Arg at residue 20), but the two proteases have similar 1D-2A cleavage activities (Y. Sakoda, N. Ross-Smith, T. Inoue, and G. J. Belsham, J. Virol. 75:10643-10650, 2001). Residue 20 has now been modified to every possible amino acid, and the activities of each mutant 2A protease has been analyzed. Selected mutants were reconstructed into full-length SVDV cDNA, and viruses were rescued. The rate of virus growth in cultured swine kidney cells reflected the efficiency of 2A protease activity. In experimentally infected pigs, all four of the mutant viruses tested displayed much-reduced virulence compared to the J1/73 virus but a significant, albeit reduced, level of viral replication and excretion was detected. Direct sequencing of cDNA derived from samples taken early and late in infection indicated that a gradual selection-reversion to a more efficient protease occurred. The data indicated that extensive sequence change and selection may introduce a severe bottleneck in virus replication, leading to a decreased viral load and reduced or no clinical disease.
INTRODUCTION
Swine vesicular disease virus (SVDV) is an important member of the Enterovirus genus of the family Picornaviridae. This virus is closely related antigenically and genetically to human coxsackievirus B5 (17, 19, 39). Virulent strains of SVDV induce an acute vesicular disease in pigs (but not ruminants) (10, 29) that is clinically very similar to that induced by foot-and-mouth disease virus, a member of the Aphthovirus genus of the family Picornaviridae. Attenuated strains of SVDV have been isolated from apparently healthy pigs (25). Infectious cDNA clones have been generated for both pathogenic (J1/73, termed J1) and attenuated (H/3/76, termed 00) strains of SVDV (19, 21). Recent studies have mapped the key determinant of virulence within these strains to the 1D-2A coding region of the SVDV genome. It has been shown that the identity of residue 132 within 1D and particularly of residue 20 within the 2A protease can determine the pathogenicity of the virus (23, 24). Residue 20 of 2A is an arginine (R) in the virulent J1 strain but an isoleucine (I) in the attenuated 00 strain (19, 21).
In common with other picornaviruses, the positive-sense RNA genome of SVDV encodes a large polyprotein that is processed by internal viral proteases to the mature polypeptides. The enteroviruses encode two distinct proteases. The 2A protease is responsible for the primary cleavage at the 1D-2A junction, while the 3C protease (plus its precursor, 3CD) is required for all other cleavages within the polyprotein except for the 1AB cleavage that occurs following encapsidation of the genome. Both proteases are related to the trypsin-like family of serine proteases (6, 34), but each has a catalytic triad in which a cysteine (C) residue substitutes for the serine (S) nucleophile. The catalytic triad of the SVDV 2A protease comprises residues histidine 21, aspartate 39, and cysteine 110 (38). Thus, residue 20, a critical determinant of virus virulence, is adjacent to one component of the catalytic triad.
In addition to cleaving the 1D-2A junction, the SVDV 2A protease also induces the cleavage of translation initiation factor eIF4G (26, 35), a key component of the cap-binding complex, eIF4F (15). This complex also includes eIF4E (the cap-binding protein) and eIF4A (an RNA helicase). eIF4G makes multiple protein-protein interactions and acts as a bridge between capped mRNAs (bound by eIF4E) and eIF3 that is associated with the small ribosomal unit. Thus, eIF4G plays an important role in the initiation of cap-dependent protein synthesis on cellular mRNAs. The cleavage of eIF4G induced by the 2A protease is believed to be responsible for the inhibition of cellular protein synthesis in enterovirus-infected cells (7). The translation of picornavirus RNA is dependent on an internal ribosome entry site (IRES) located within the 5' untranslated region. Except for the hepatitis A virus IRES, the picornavirus IRES elements are still active when eIF4G has been cleaved by the 2A protease. Indeed, another function of the 2A protease is the stimulation of enterovirus IRES activity within cells in which eIF4G is cleaved (8, 18, 33). However, our studies suggest that the inhibition of cap-dependent translation (resulting from eIF4G cleavage) and the stimulation of IRES-directed translation by the 2A protease are independent processes (33).
We have demonstrated recently that the 2A protease from the attenuated strain (00) of SVDV was severely defective in inducing eIF4GI cleavage and its expression resulted in only modest stimulation of IRES activity (35). In contrast, the 2A protease from the virulent strain (J1) of SVDV efficiently induced eIF4GI cleavage and strongly stimulated IRES function. Both proteases efficiently cleaved the 1D-2A junction. To determine the effects of other amino acid substitutions at this position of the protease sequence, we have modified residue 20 of the SVDV strain J1 2A protease to each of the 20 amino acids and examined the three distinct properties of the mutant proteases generated. The different proteins display a spectrum of activities, with just one mutant being catalytically inactive. Some of the mutant protease sequences have been introduced into full-length cDNA clones of SVDV, and the effects of the mutations on the properties of the rescued viruses have been analyzed in cultured cells. Furthermore, four of the mutant viruses have also been compared to the wild-type (wt) J1 virus by experimental infection of pigs. Each of the mutant viruses displayed significantly reduced virulence, but viral replication and excretion occurred at a low level. Direct sequencing of viral cDNA derived at different times postinfection indicated that significant selection-reversion had occurred in vivo.
MATERIALS AND METHODS
Plasmid construction and mutagenesis. Plasmid pGEM3Z/J1 (35), which contains the coding region for a myc-tagged 1D-2A product derived from the virulent J1 strain of SVDV, was used as the backbone for all mutagenesis (Fig. 1). All DNA manipulations were performed essentially as described by the manufacturer or by using standard procedures (36). The SVDV cDNA, as an EcoRI-BamHI fragment (ca. 1,350 bp), was isolated from pGEM3Z/J1, ligated into similarly digested M13mp18 replicative-form (Rf) DNA, and transformed into Escherichia coli JM109. Phage from an individual plaque was amplified, and double-stranded Rf DNA was isolated. Mutagenesis with a Muta-GENE kit (Bio-Rad) was performed essentially as described by the manufacturer on the basis of the method of Kunkel (27). From the initial reaction, which used a degenerate pool of oligonucleotides (36-mers) in which the codon corresponding to residue 20 of 2A was changed to NNN (an equal mixture of all four bases at each position), mutations encoding 11 different amino acids plus the stop codons (TGA, designated , and TAG) were recovered. The sequence of the pool of degenerate primers was GCGCGCGTTGCGAGATGNNNATTCACCACTCTATAG. The residual mutations were generated by using the same mutagenesis procedure but with unique 36-mer primers that contained the required codons. The sequences of the particular codons used for each amino acid are shown in Table 1. Following determination of the 1D-2A coding sequence, preparations of double-stranded Rf DNA were made and digested with BamHI and EcoRI and the 1.35-kbp fragments were ligated into similarly digested pGEM3Z. All of the plasmids generated were then amplified individually to prepare DNA (with a QIAGEN midiprep kit) suitable for transfection into mammalian cells. The presence of the required mutations was confirmed with the BcaBEST dideoxy-sequencing kit (TAKARA), and several clones were also fully sequenced through the SVDV cDNA with a Beckman capillary sequencer (model CEQ2000XL). Control plasmid pGEM3Z/J1 (Fig. 1) has an in-frame deletion that removes the 1D-2A junction along with a critical N-terminal region of the 2A protease and lacks all 2A protease activity (35).
Protein expression analysis. In vitro transcription-and-translation (TNT) reactions were performed with the TNT rabbit reticulocyte system (Promega) with [35S]methionine essentially as described by the manufacturer. Aliquots (3 μl) were mixed with sample buffer containing sodium dodecyl sulfate (SDS) and dithiothreitol, boiled, and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) (28) and autoradiography. Quantitation of radioactivity was performed with a phosphorimager and Quantity One software (Bio-Rad).
Transient-expression assays within cells were performed as described previously (33). Briefly, plasmids (2.5 μg) were introduced with Lipofectin (8 μg; Life Technologies) into BHK cells (35-mm-diameter dishes) infected with recombinant vaccinia virus vTF7-3 (14), which expresses T7 RNA polymerase. After 20 h, cell extracts were prepared (400 μl). Aliquots (20 μl) were analyzed by SDS-PAGE and Western blotting with a monoclonal antibody (9E10; Roche) directed against the c-myc epitope tag on the N terminus of SVDV 1D (Fig. 1) or with sheep antibodies specific for the C terminus of eIF4GI as described previously (35). Detection was achieved with appropriate peroxidase-labeled secondary antibodies and chemiluminescence reagents (Amersham Pharmacia Biotech). To determine the ability of the mutant 2A proteases to activate IRES function, the test plasmids (0.5 μg) were cotransfected with dicistronic reporter plasmid pGEM-CAT/CB4/LUC (2 μg) containing the coxsackievirus B4 IRES (33) or with pC/SVDVJ(+)/L (2 μg), which has the SVDV J1 IRES (35), essentially as described above. Cell extracts were prepared, and the chloramphenicol acetyltransferase (CAT) and luciferase (LUC) expression levels were monitored by Western blot analysis as described previously (33). LUC enzyme activity, the indicator of IRES-directed translation, was quantitated with a LUC assay kit (Promega) and a Bio-orbit luminometer.
Reconstruction of full-length infectious cDNAs, virus rescue, and growth analysis. The mutant cDNA fragments (in the J1 background) encoding the single amino acid substitutions of residue 20 of the 2A protease gene were excised from the M13 Rf DNAs (described above) with SalI and BssHII (Fig. 1), and these fragments (about 740 bp) were inserted into a similarly digested transfer vector containing the SmaI (nucleotide [nt] 1755)-to-EcoRI (nt 5433) segment of H/3/76 (00) cDNA. From these transfer vectors, fragments were prepared with Bst1107I (nt 2233) and BssHII (nt 3368) and inserted into an infectious clone (pSVLS00) (22) of avirulent SVDV H/3/76 cut with the same enzymes (unique sites). Resulting chimeric plasmids containing the H/3/76 backbone with the SalI-BssHII fragment of the J1 strain modified at residue 20 of the 2A gene were selected, and infectious virus was rescued by transfection into COS-7 cells essentially as described previously (22). Infectious virus was amplified by two or three passages in the SKL (porcine kidney) cell line (37). The titer of each virus stock was determined by plaque assay on SKL cells. The sequences of the initial infectious plasmid clones and RNA purified from the virus stocks were confirmed by reverse transcription (RT)-PCR and nucleotide sequencing. Virus growth curves were obtained by infecting SKL cells with 0.1 PFU/cell and incubating them for up to 48 h. At each time point tested, the virus yield was determined by plaque assay on SKL cells as described previously (21, 22).
Analysis of virus replication and virulence in experimentally infected pigs. (i) Animals. Twenty Landrace crossbred Large White pigs (between 20 and 30 kg) that were seronegative for antibody to SVDV were housed in biosecure animal buildings and divided at random into five groups of four animals in individual boxes as follows: pigs UT23 to -26, box 1, inoculated with SVDV 2A-20 V; pigs UT27 to -30, box 2, inoculated with SVDV 2A-20 K; pigs UT31 to -34, box 3, inoculated with SVDV 2A-20 I; pigs UT35 to -38, box 4, inoculated with SVDV 2A-20 W; pigs UT39, -40, -43, and -44, box 5, inoculated with SVDV 2A-20 R (J1, wt).
Pigs were inoculated intradermally or subdermally in the heel pad of the left fore foot essentially as described previously for inoculation with foot-and-mouth disease virus (1, 2). The titers of the stock viruses were adjusted to 107.3 PFU (as measured in SLK cells) in tissue culture medium, and each inoculated animal received approximately 107 PFU in 0.5 ml. The animals were examined clinically for signs of swine vesicular disease, and rectal temperatures were recorded daily until 11 days after inoculation (dpi). On the basis of previous experience with swine vesicular disease, which can produce mild clinical symptoms, the clinical signs were scored as follows: a local lesion at the site of inoculation of less than 1.5 cm (small), 1 point; a local lesion at the site of inoculation of more than 1.5 cm (large), 2 points; small lesions on other feet, 3 points per foot; large lesions on other feet, 6 points per foot; lesions on or around the tongue, mouth, or snout, 3 points; lameness, 3 points. Each animal could have a maximum score of 26 points by this system.
(ii) Sample collection. Blood samples, together with nasal and rectal swabs, were taken immediately before inoculation (0 samples), 5 min after inoculation (blood only), and then at 1, 2, 3, 4, 7, 9, and 11 dpi, when the animals were killed. The blood samples were left to clot for 1 h at room temperature, and serum was separated and then stored at –80°C. For RNA extraction, an aliquot of serum was thawed, 2 volumes of serum was added to 3 volumes of lysis buffer (Roche Lysis Solution), and the mixture was stored at 4°C overnight before nucleic acid extraction and subsequent analysis were performed. Swabs were stored at –20°C, and then 1 ml of TRIzol Reagent (Life Technologies) was added to the tubes for RNA extraction. When the animals were killed at 11 dpi, selected samples, i.e., epithelium from the feet or tongue and liver from a few animals, were collected into RNAlater (Ambion, Austin, Tex.) and stored at –20°C prior to extraction.
(iii) qRT-PCRs. Real-time quantitative RT-PCRs (qRT-PCRs) were used to determine the amount of SVDV RNA in extracts of total nucleic acid from blood and swab samples, from virus inocula, and in total RNA from tissue samples by using the 2B-IR primer-probe set as described elsewhere (32). All quantitative measurements were based on standard dilution series derived with in vitro-transcribed SVDV RNA from a plasmid containing the SVDV IRES fragment (pGEM 3Z/J1 IRES) (35) and quantified by spectrophotometry. The stock RNA preparation contained 1010 SVDV genome equivalents per ml. This method of quantitation is influenced minimally by sample type and correlates well with infectivity (1-5, 31, 32).
(iv) Sequencing. All sequencing was performed on PCR products derived with the same cDNA or RNA samples as used for the quantitations described above. The sequencing was performed as consensus sequencing from samples amplified in a two-step nested PCR with primers flanking the 2A sequence and cycle sequencing with a Beckman capillary sequencer (model CEQ2000XL). Sequencing of individual cDNA products was performed in each direction and repeated multiple times to confirm the results.
(v) Assay for antibodies. Serum samples were tested by an enzyme-linked immunosorbent assay (ELISA) for the presence of antibodies to SVDV by the 5B7 monoclonal antibody blocking ELISA (9) and by virus neutralization assay (VNT) (16).
RESULTS
In vitro expression and processing by the SVDV 2A protease. Plasmid pGEM3Z/J1 (35) encodes a myc-tagged 1D-2A product derived from the pathogenic J1 strain of SVDV (Fig. 1). Mutagenesis was performed so that the codon for residue 20 of the 2A protease was changed to encode individually each of the possible 20 amino acids or a stop codon. Each of the mutants is identified by the single-letter code for the amino acid encoded at residue 20 (Table 1). As an initial screen of the properties of the mutated 2A proteases, each of the plasmid DNAs was used to program an in vitro TNT reaction with rabbit reticulocyte lysate and [35S]methionine. The samples were analyzed by SDS-PAGE and autoradiography (Fig. 2, top), and the products were quantitated with a phosphorimager. In these reactions, initially the 1D-2A precursor is generated, which is then cleaved through the action of the 2A protease to generate the products 1D and 2A. As expected, when a stop codon was introduced at the position of residue 20 within 2A, a stable product was generated corresponding to 1D fused to the first 19 amino acids of 2A (this fusion protein migrates only slightly more slowly than the mature 1D product). When a proline was introduced at residue 20, the 2A protease appeared to be completely inactive and a stable 1D-2A protein was made that was not processed during the course of the incubation. In all other cases, production of the full-length 1D-2A precursor was observed and processing occurred that was incomplete when the reaction was terminated. Similar results were obtained in three independent experiments, and the mean ratios of 1D-2A to 1D observed at a 2-h time point are presented in the bottom panel of Fig. 2. Apart from the P mutant, four other mutants also appeared significantly defective in their efficiency of processing of the 1D-2A junction, as judged by the high level of 1D-2A compared to that of the processed product 1D (Fig. 2, top and bottom). These defective mutants contained residues G, V, T, and D at position 20 of the protease. The D, G, and V mutants were the most defective of these, with little or no 2A product being detectable; although low levels of the 1D product were observed, these two products are clearly made in the same amounts but the sensitivity of detection depends on the methionine content and protein stability.
Intracellular expression and properties of the mutant 2A proteases. Further analysis of the properties of these mutant proteases was performed with transient expression assays of the plasmids within cells infected with recombinant vaccinia virus vTF7-3 (14), which expresses the T7 RNA polymerase. From extracts of transfected cells, the expression of the 1D-2A products was determined by Western blot analysis with an anti-myc tag antibody (the myc tag is located at the N terminus of the 1D sequence; Fig. 1). In our previous studies (35) with the 1D-2A region from the J1 and 00 strains of SVDV, we found that almost complete cleavage of the 1D-2A precursor occurred in this system and hence only the 1D species was detected by the anti-myc tag antibody. This was also the case for nearly all of the single amino acid mutants (Fig. 3). However, consistent with the in vitro TNT assay described above, the P mutant yielded only the unprocessed 1D-2A product. It is noteworthy that the yield of this product was also very high. The yields of the G, V, D, and stop codon () mutant products were also relatively high, whereas the level of products observed from the R, W, and H mutants was particularly low. On the basis of our previous studies (35), it seemed likely that most of the mutants (other than the P mutant) were inducing eIF4GI cleavage and thus inhibiting their own synthesis (to different degrees) since in these assays the 1D-2A precursors are expressed from monocistronic mRNAs lacking an IRES and hence are translated by a cap-dependent mechanism. To check this, the cleavage of translation initiation factor eIF4GI induced by the 2A protease was analyzed within the same cell extracts with an antibody directed against the C terminus of eIF4GI. Each of the mutant 2A proteases, other than the P mutant, induced eIF4GI cleavage; a subset of the data generated is shown in Fig. 4A. The nature of this assay and the fact that very low levels of 2A expression can induce eIF4GI cleavage make it difficult to determine any quantitative differences in the efficiency of eIF4GI cleavage in these assays. However, as discussed below, the higher level of expression of certain mutants, e.g., G, V, and D (Fig. 3), may be indicative of a slower rate of eIF4GI cleavage occurring within the cells.
IRES activation by the mutant 2A proteases. Plasmids encoding each of the mutant 2A proteases were cotransfected with plasmid pGEM-CAT/CB4/LUC into vTF7-3-infected BHK cells. This reporter plasmid expresses a dicistronic mRNA containing the IRES element from coxsackievirus B4 (33). In each experiment, the cell extracts (prepared after 20 h of incubation) were analyzed by SDS-PAGE and immunoblotting with antibodies directed against both the CAT and LUC proteins. Expression of the wt J1 strain 2A protease strongly stimulates CB4 IRES function in this assay (35), whereas the 2A protease from the 00 strain of SVDV has a significantly weaker effect. The analyses of a subset of extracts obtained in one experiment are shown in Fig. 4B and demonstrate the spectrum of observations made. Clear inhibition of CAT expression, reflecting the cleavage of eIF4GI induced by these plasmids (Fig. 4A) and the loss of cap-dependent protein synthesis, was observed for the M and J1 (R, wt) constructs, whereas the P and (stop codon) mutant proteins had little effect on the expression of CAT. In contrast, the M and R (J1, wt) constructs strongly stimulated LUC accumulation (reflecting IRES activity) whereas the P and mutant proteins had little or no effect on LUC expression (Fig. 4B). The ability of each of the 20 different mutant 2A proteases to activate the CB4 IRES, as indicated by the expression of LUC, is shown in Fig. 4C. The results presented are the means of three separate transfections and also closely reflect those obtained with a second dicistronic reporter plasmid, pC/SVDJ(+)/L (35), that contains the SVDV J1 IRES (data not shown). Neither the P mutant nor the pGEM3Z/J1 construct induced any increase in IRES activity. Expression of the D mutant resulted in about a twofold increase in LUC expression, while the G, V, and T mutants induced about a threefold increase in IRES function. Most of the other mutants (including the wt J1 sequence) induced about a five- to sixfold increase in IRES activity. However, mutants I, E, and K appeared slightly deficient in this activity (about a fourfold increase in activity) both in these assays and when assayed with the SVDV IRES construct (data not shown).
Analysis of 2A mutations within full-length infectious cDNA of SVDV. In order to examine the effects of the mutations at residue 20 of the 2A protease on the properties of SVDV, nine representative mutant cDNAs that had shown a range of different activities in the assays described above were reconstructed into the full-length infectious cDNA (pSVLS00) derived from the attenuated H/3/76 (00) strain (20). The individual mutant full-length cDNAs were sequenced to check for the presence of the expected mutations and then transfected into COS-7 cells to rescue the viruses. The mutant viruses that were generated were then amplified by infection of SKL cells. By 48 h postinfection, a complete cytopathic effect (CPE) was apparent in cells infected with the virus rescued from the pSVLS00 parental vector and with the full-length hybrid cDNA clones containing the L, W, and R mutations in the 2A protease. A lower level of CPE was observed in cells infected with the virus produced from the plasmids containing the K, E, and I sequences, but little or no CPE was apparent from the V, T, and D mutants (Table 2). All of these results appear consistent with the level of 2A protease activity detected with the in vitro assays described above (Fig. 2 and 4 and Table 2).
After further passage of the viruses, some CPE was evident from all of the viruses (although this was very weak for the D mutant) and the presence of SVDV RNA could be detected by RT-PCR (no signal was detected if the RT step was omitted; data not shown). The sequence of the 2A protease within the rescued viruses was determined following RT-PCR, and the presence of just the expected mutations was observed in each case (data not shown).
The growth properties of certain mutant viruses were compared (Fig. 5). The W and R mutants grew almost as well as the wt J1 virus in SKL cells, but the other mutant viruses (the K and I mutants and particularly the V mutant) were significantly slower at replicating. Furthermore, the W, R, and L mutants showed a large-plaque phenotype (like the J1 virus) whereas the V, T, and D mutant viruses produced only small plaques (like the attenuated 00 virus). The K, I, and E viruses produced an intermediate plaque size phenotype (Table 2). Thus, there was a good correlation between the replication rate of the rescued virus and the efficiency of the 2A protease activity scored with in vitro assays.
Analysis of experimental infection of pigs with mutant SVDV. In order to determine the effect of the 2A modifications on virulence, we selected four mutant viruses with single amino acid changes within this protease that displayed a range of in vitro activities and growth rates in tissue culture cells. Each of these selected viruses (W, K, I, and V) was compared to the wt J1 virus by experimental infection of pigs. It should be mentioned that previous work has shown that similar chimeric viruses that have an arginine (R) at position 20 of the 2A protease (as in the J1 wt virus) displayed a level of virulence comparable to that of the J1 wt virus (23).
(i) Quantitation of SVDV RNA in virus samples. The genome copy numbers in the virus inocula determined by qRT-PCR showed that the J1 (wt) inoculum (group 5) contained around 8.8 to 8.9 log10 genome copies per ml while the other four inocula (V, K, I, and W; groups 1 to 4) contained around 9.3 to 9.4 log10 copies of SVDV RNA per ml. Since all of the inocula had been adjusted to contain 107.3 PFU/ml, this indicated that the wt virus may have a slightly lower genome-to-PFU ratio (1.5 to 1.6 log10 copies per PFU) compared to the modified viruses (2.0 to 2.1 log10 copies per PFU). This slightly higher genome copy number in the modified viruses was also observed in the 5-min serum samples obtained from the inoculated pigs (see below), which for groups 2 to 4 (K, I, and W) contained approximately 0.5 log10 copies more than group 5 (J1 wt). The 5-min serum samples from group 1 inoculated pigs (V) contained approximately the same amount of genomes as those from the group 5 pigs. The reason for this is not known, but it may have resulted from a minor difference in the inoculations as these were done intradermally or subdermally in the heel pads and any virus in the circulation at 5 min after inoculation is derived from inoculated virus spilling into the local lymph vessels (4). The important point is that pigs inoculated with the mutant viruses all received at least as large a dose of virus as those that received the wt virus.
(ii) Clinical signs and evidence of virus replication in vivo. Small local vesicular lesions were observed in some of the group 5 (wt virus) pigs from 1 dpi and in some of the other groups on the following day. The lesions became relatively severe in the group 5 animals (maximum scores of 14, 20, 20, and 23 for the four pigs in the group), with severe lesions on most of the pigs' feet and on the snout, tongue, and lips of a single animal. In contrast, pigs in the other groups mainly had only local or minor lesions on additional feet and although a few of these animals had slightly more pronounced lesions (single animals with a score of 11 in groups 1 and 3 and two animals with scores of 7 and 8 in group 4, respectively), none of them were as severe as in the group 5 animals. No real lameness was observed, although the pigs in group 5 showed a slight "sticky gait" at 2 to 4 dpi and all of the pigs were eating normally and were not, apart from the lesions, much affected clinically by the infection. The average lesion scores for the groups are shown in Fig. 6A, and the maximum scores for the individual animals are shown in Table 3. The drop in scores seen toward the end of the observation period (Fig. 6A) was due to healing of the lesions.
The average temperatures of the group 1 to 4 pigs were in general normal, i.e., around 38°C, although the group 1 pigs had an average temperature of 39.1°C (a single animal had a temperature of 39.5°C) at 2 dpi. In contrast, group 5 pigs (with wt virus) had an increased temperature up to an average of 39.3°C at 2 to 7 dpi, with one animal having a temperature of 40.3°C on day 3 (data not shown).
As mentioned above, SVDV RNA could be observed in the sera of inoculated pigs, even in the samples taken 5 min after inoculation, at a level of around 3.4 to 4.2 log10 copies/ml (Fig. 6B). From this initial level of inoculated virus, the levels rose in the group 5 animals to 4.6 log10 copies/ml at day 1 and peaked at a level of 6.2 to 7.1 log10 copies/ml (average level of 6.7 log10 copies/ml) at 2 to 3 dpi (Fig. 6B and data not shown), after which the levels declined to 1.2 log10 copies/ml at day 7 and became undetectable from day 9. In contrast to this pattern, the levels of viral RNA in the serum of pigs in groups 1 to 4 initially decreased to around 1.5 to 2.4 log10 copies per ml at 1 dpi but then increased up to 3.2 to 6.2 log10 copies/ml (average levels around 3.3 to 5.3 log10 copies/ml, with the highest levels in groups 2 [K] and 4 [W]) at 2 to 3 dpi (Fig. 6B and data not shown). Thus, from day 1 to day 3, the level of viremia in the pigs infected with the mutant viruses was generally at least 100-fold lower than that observed in the animals that received the wt virus. Subsequently, the viremia slowly decreased and became undetectable on day 9 for groups 1 to 3 and on day 11 for group 4 (Fig. 6B). The decrease in virus RNA in the blood after day 3 correlated well with the onset of the serum antibody response that was detectable from day 4 (Fig. 6C). Antibodies to SVDV could be detected in all of the animals from 4 to 7 dpi by both ELISA and VNT. There was good agreement between the VNT (Fig. 6C) and ELISA (data not shown) results, with the highest titers being observed in animals in group 5 (wt virus).
(iii) Virus excretion from infected pigs. The SVDV RNA levels in nasal and rectal swabs are a measurement of virus excretion and are shown in Fig. 7. In the group 5 pigs, virus RNA (wt) could be detected in nasal swabs from day 1 and increased to levels just above 5 log10 copies/ml on day 3. There was a secondary peak on day 7 but only a low residual level of SVDV RNA at day 11. In contrast, viral RNA could not be detected in nasal swabs from pigs in groups 1 to 4 until 2 dpi. The amount of RNA peaked at levels of 3.2 to 6.3 log10 copies/ml at 7 dpi and then rapidly decreased to low levels by 11 dpi (Fig. 7A).
Analysis of the rectal swabs from the group 5 pigs (wt virus) detected viral RNA from day 1, and this increased to levels of around 3.7 log10 copies/ml on day 4 but then decreased slowly to only around 2 log10 copies/ml by day 11. In contrast, viral RNA could not be detected in rectal swabs from pigs in groups 1 to 4 until 4 dpi; excretion then peaked at a level of less than 3 log10 copies/ml at 7 to 9 dpi and then declined to less than 2 log10 copies/ml by 11 dpi (Fig. 7B).
In the tissue samples taken at the termination of the experiment at 11 dpi, around 4 to 11 log10 genome copies/g of tissue could be detected. The highest levels of viral RNA were detected in the epithelial samples from the feet (up to 11 log10 genome copies/g), although pigs that only had local lesions at the inoculation site had relatively low levels (data not shown). Tongue epithelium and liver samples from the pigs in group 5 (wt virus) had relatively low levels of viral RNA, up to 5.5 log10 copies/g (data not shown). There were no obvious differences in the levels of viral RNA between samples analyzed from the different groups, apart from the low levels detected in pigs that only had local lesions (as mentioned above); however, only 10 epithelial samples from the feet (data not shown) and a total of 18 samples were analyzed.
(iv) Sequence analysis of virus RNA from in vivo samples. Selected cDNAs used in the qRT-PCR studies described above were subjected to PCR amplification and direct sequencing of the SVDV 2A coding region. Samples selected for this analysis were serum samples collected at 3 dpi, nasal swabs collected at 7 dpi, and epithelial samples from the feet collected at 11 dpi, when the experiments were terminated. The results are shown in Table 3 and indicate that the significant late amplification and possibly residual virulence of the mutants (used for groups 1 to 4) were, at least in part, due to a gradual selection-reversion to a more efficient 2A protease. The modifications in the 2A sequence either generated a mutant form of the 2A protease with higher protease activity than the initial mutant or more generally changed to the wt amino acid (R) despite the fact that this involved up to three nucleotide changes. It is apparent that the generation of viruses within the infected animals containing the pathogenic wt sequence did not result in severe acute disease.
DISCUSSION
The major virulence determinant of SVDV for Japanese isolates J1/73 and H/3/76 has previously been mapped to residue 20 of the 2A protease (23). In this study, we have analyzed the impact of mutation of this residue on the activities of the protease and on the replication of the virus in tissue culture and during experimental infection of pigs.
The various mutant 2A proteases displayed a spectrum of activities. The P mutant was totally deficient in all three of the activities assayed. Presumably, the presence of this residue, adjacent to one component of the catalytic triad of the protease, causes some deformation of the polypeptide chain backbone that perturbs the geometry of the active site. All of the other mutants were functional, but the G, V, T, and D mutants were significantly defective in the 1D-2A cleavage assay and IRES activation. There is no obvious pattern to the nature of the residues that resulted in the production of either a highly active or a rather defective protease. For example, the A, L, and S mutant proteins are all highly active but the G, V, and T mutant proteins are all significantly defective; these amino acids are listed in order of having small aliphatic, hydrophobic, and hydroxyl group side chains in each group. The assay for the induction of eIF4GI cleavage was not very discriminating among the mutants, and all of the mutant proteins, except the P mutant protein, induced eIF4GI cleavage (data not shown). This presumably reflects the fact that even low levels of protease expression are sufficient to induce this cleavage; thus, presumably higher-level expression of a somewhat defective protease can still achieve the same effect. However, there is an indication that some of the mutant proteins induced eIF4GI cleavage more slowly. The effect of eIF4GI cleavage is to inhibit the initiation of cap-dependent protein synthesis (note that the 1D-2A proteins are expressed in this assay by this mechanism). Thus, if eIF4GI cleavage occurs more slowly, then it can be expected that cap-dependent expression of 1D-2A will be maintained longer (35). It is apparent that the expression level of certain mutations, e.g., P, G, V, and D, was significantly higher than that of other mutations, e.g., R, Y, W, and F. Thus, although the G, V, and D mutant proteins induced cleavage of eIF4GI, it is probable that the inactivation of the eIF4F complex happened relatively slowly in cells containing these proteins. Hence, these mutant proteins were synthesized longer and consequently accumulated to higher levels.
Certain other mutant proteins, namely, I, E, and K, appeared to be slightly defective in the IRES activation assay but did not appear to be significantly deficient in the in vitro 1D-2A cleavage assay. However, it was apparent that none of these mutant proteins were as effective as, for example, the R, Y, W, and F products, which were very active both in the IRES activation assays and in the TNT assay (Fig. 2 and 4).
It is interesting that the I mutant protein (in the J1 strain background) has a different phenotype than that which we observed for the 2A protease from the attenuated 00 strain of SVDV, which has an Ile at residue 20 (35). The 00 1D-2A protein is significantly deficient at inducing eIF4GI cleavage and about threefold less active in IRES activation than the J1 1D-2A protein, whereas the I mutant protein is not significantly defective in these activities. We were able to show that merely the change of I20 to R20 in the 00 1D-2A protein produced a protease that induced cleavage of eIF4GI and stimulated CB4 IRES activity about as efficiently as the J1 1D-2A protein does (35). However, there are six other amino acid differences between the SVDV 00 and J1 strains within the 1D-2A region (five differences in 1D and one in 2A). Of particular significance may be the nature of residue 132 within 1D, which is also linked to the virulence phenotype (23, 24). It is not entirely clear, however, how this residue (or any of the other different residues in 1D) could interact with residue 20 within 2A. It may be that although the 1D-2A junction is the primary cleavage site within the polyprotein that the two products can remain associated within the cell and perhaps this interaction modifies the activity of the protease. Recently, Foeger et al. (13) have shown an interaction between the 2A protease from human rhinovirus 2 and eIF4GI that is independent of the substrate-binding cleft of the protease. It was shown for this 2A protease that Leu17 (adjacent to catalytic residue His18, just as SVDV 2A protease residue 20 is adjacent to catalytic residue His21) is important in determining this interaction between the 2A protease and the N-terminal region of eIF4GI. Furthermore, mutation of Leu17 to Arg17 impaired this binding but did not affect 1D-2A processing activity by the protease (13). While this result has some similarity to the role of SVDV residue 20 in determining the activity of the 2A protease, there is also an important difference since in the SVDV 2A protease Arg20 is the wt residue. Thus, specific details of the interaction of the entero- and rhinovirus 2A proteases with eIF4GI still need to be determined.
Representatives of each of the different types of SVDV 2A mutant have been reconstructed into full-length infectious clones of SVDV, and the properties of the rescued mutant viruses have been examined in tissue culture (Fig. 5). Furthermore, the ability of some of these viruses to replicate and cause disease in pigs has also been examined. The ability of the viruses to replicate in tissue culture (Fig. 5) closely reflected the relative activity of the 2A protease in the in vitro assays (Fig. 2 and 4 and Table 2), and some of the mutants (e.g., W and R) replicate to a level similar to that of the wt J1 virus. It should be noted, however, that the virulent J1 strain and the attenuated 00 strain of SVDV also replicate with similar efficiency in a single-step growth curve (23) but the J1 virus does replicate to a higher level than the 00 strain in a low-multiplicity infection (T.I., unpublished results). However, only SVDV with the wt R residue at position 20 of the 2A protease causes severe disease in pigs (Fig. 6) (23, 24) but the W and K viruses induced the next highest level of viremia (Fig. 6B), which is in accord with their efficient growth in tissue culture cells (Fig. 5).
It was interesting that during the course of replication in tissue culture no evidence of sequence reversion was obtained. However, in the infected pigs the sequence encoding residue 20 of the protease was modified at a high frequency (Table 3). The changes that occurred all produced a 2A protease that functioned more efficiently than that found in the input virus. There was a strong bias toward reversion to the wt AGA codon encoding an R residue.
Picornaviruses lack proofreading-repair activities, and consequently the error rate during genome replication is high (approximately 10–4 substitutions per nucleotide copied, i.e., around one nucleotide misincorporation per genome per replication cycle). This inevitably leads to the generation of variant genomes (11). For example, live poliovirus type 3 vaccines contain a subset of viral genomes that contain the wt C at nt 472 in the 5' noncoding region, and it has been shown that the proportion of such genomes in the vaccine correlates with neurovirulence (12). In recipients of type 3 poliovirus vaccine, the reversion-selection to the wt nucleotide at nt 472 occurs by 3 to 6 days after vaccination, and by day 11, the type 3 virus also loses all or part of its temperature-sensitive growth phenotype because of another mutation-selection at amino acid residue 91 of VP3. Despite this relatively rapid reversion of the live vaccine to a more virulent phenotype, the frequency of vaccine-induced polio is very low (estimated at around 1:500,000 primary vaccines), suggesting that the immune system generally has time to prevent clinical disease, most likely by preventing or reducing viremia (30). Hence, it appears likely that our findings on pigs inoculated with mutant SVDVs are due to a combination of preexisting variant genomes in the inocula, initial local replication with further generation of mutants, selection for viral fitness, bottleneck transmission events, and the effects of the evolving immune response. In this light, the results obtained with the individual groups of pigs may be explained as follows. For the group 5 pigs (wt virus), most of the viral genomes encode a highly efficient protease (arginine at position 20) that is conserved because of positive selection for a rapidly replicating virus, with the result that the virus is able to spread relatively efficiently through the circulation and cause generalized lesions at other sites before the immune system is able to control the virus. For the other groups, the picture is more complex and we will discuss the findings separately for each of the groups and then try to tie the findings together.
Pigs in group 1 were inoculated with a mutant virus having a valine codon at position 20 of the 2A protease. According to the in vitro studies, valine at this position results in a rather inefficient protease (Fig. 2 and 4) and the virus grows relatively slowly in tissue culture (Fig. 5). Two pigs given this virus had no or minor local lesions, while the two other pigs had local lesions and lesions on one and three other feet, respectively. Interestingly, the pigs that developed secondary lesions had a significant early viremia with a majority of viral genomes (on the basis of consensus sequencing of viral RNA from serum at day 3) that contained a single nucleotide change resulting in a methionine codon; this produces a more efficient protease (Fig. 2 and 4 and Table 2). This was followed by two additional mutation-selection steps that generated the codon for the wt arginine. In contrast, the two other pigs in this group (having only minor or no local lesions) had a low or undetectable viremia but the major virus population that was present appeared to have changed all three nucleotides of codon 20 to encode an arginine residue. This finding indicates that an initial change at a single nucleotide can create a relatively efficient protease that allows additional replication and the further selection of efficient variants. However, the severe bottleneck caused by an initial selection for the wt sequence severely restricts early replication so that the immune system can control it, which prevents efficient spread through the circulation and thus development of secondary lesions.
Group 2 pigs were inoculated with a mutant with codon 20 of the protease changed to a lysine (K). This mutant protease was found to be slightly more efficient in vitro than the valine mutant, and the virus grew better in tissue culture (Fig. 2, 4, and 5). However, this codon only required a single nucleotide change to encode the wt arginine. This appeared to occur rapidly, as the consensus sequences obtained even at 3 dpi showed the wt AGA at position 20. Interestingly, although this change to an arginine only included selection-reversion at a single nucleotide position and the animals in this group had significant early viremia, this was reduced relatively early and excretion in nasal fluid and feces occurred relatively late compared to that in the other groups (Fig. 6 and 7). This early reduction in viremia and the late excretion correlated well with the fact that the pigs in this group only developed local, although rather severe, lesions that did not spread to their other feet.
Pigs in group 3 received the I mutant virus; the mutation resulted in a protease that has approximately the same efficiency in vitro as the lysine mutant discussed above, but the I mutant virus grew slightly more slowly in tissue culture than the K mutant virus did (Fig. 2, 4, and 5). In contrast to the lysine mutant, the isoleucine mutant needs to change two nucleotides to revert to the wt arginine. Of the four pigs in this group, three developed local lesions at the site of inoculation while a single pig developed both a local lesion and lesions on two of its other feet. Interestingly, this pig had the highest level of viremia in the group and consensus sequence analysis showed a single nucleotide change so that codon 20 encoded phenylalanine, which results in a highly efficient protease. In contrast, the virus from the other pigs in this group either initially retained the isoleucine at position 20 (albeit changing a single nucleotide [UT33, Table 3]) or changed at two nucleotide positions to the wt arginine codon (UT32, Table 3), indicating that a single change without a gain in protease efficiency or the severe bottleneck effect of an early two-nucleotide reversion-selection severely restricted the viral population's ability to cause generalized disease before the immune system took control.
Pigs in group 4 were inoculated with a mutant encoding tryptophan (W) at codon 20 of the 2A protease; this protease is almost as efficient as the wt, and the virus replicated with near wt efficiency in tissue culture (Fig. 2, 4, and 5). The pig with the greatest viral load at days 2, 3, and 7 (UT35, Table 3) had a majority of viral genomes that retained the tryptophan codon, and this caused local lesions and lesions on two of its other feet, while another pig (UT36, Table 3) initially had viral genomes that retained the tryptophan codon and had a high early viremia but then the majority of the population of viral genomes changed or reverted at two nucleotide positions to a wt arginine codon. However, this appeared to introduce a severe bottleneck and this pig had a lighter viral load at day 7 and only mild local lesions. The viral populations in the other two pigs in this group reverted early on at two nucleotide positions to the wt arginine codon, but this two-nucleotide reversion also appeared to introduce a severe bottleneck, as indicated by the reduced viral load and reduced clinical disease, although pig UT37 (Table 3) developed lesions on two of its other feet. However, the relatively severe generalized lesions on additional feet of this pig were most likely due to excessive abrasion of the foot epithelium caused by an unrelated bacterial joint infection leading to direct entry of excreted virus into damaged epithelium rather than from the circulation.
In conclusion, all four mutant viruses displayed much-reduced virulence compared to that of the wt J1/73 virus but a significant, albeit reduced, level of viral replication and excretion was observed. Direct sequencing of samples taken early and late in infection indicated that the significant replication and possibly residual virulence of these mutants were, at least in part, due to a gradual selection-reversion to either a more efficient protease or to the parental amino acid sequence despite the fact that this involved up to three nucleotide changes. It appeared that selection of viruses with the amino acid codon changed at one or two nucleotides from the wt sequence occurred rather easily but that three nucleotide changes involved selection of intermediate, slightly more efficient viruses, followed by further selection for viruses with the wt amino acid. Furthermore, the data indicated that extensive change and selection may introduce a severe bottleneck in virus replication, leading to a decreased viral load and reduced or no clinical disease. A single nucleotide change resulting in a relatively efficient protease, not necessarily having the wt arginine at residue 20, may be the most fit virus in terms of the level of viral replication and disease in this virus-host system. However, clearly an arginine at position 20 of the 2A protease is required for an efficiently replicating and fully virulent virus. Interestingly, it appeared that not only is an arginine required at this position, the codon selected was in each case AGA although five other arginine codons exist. The SVDV strains sequenced so far, except for the attenuated Japanese H/3/76 strain, all have an AGA codon at this position, and comparison of more than 40 human enterovirus B isolates (same virus species as SVDV) indicates that AGA is by far the preferred codon while AGG may also occur. CGC and CGG occur very seldom, and CGT and CGA did not occur (Nick Knowles, personal communication). It would be interesting to pinpoint the exact mechanism of this codon preference. Clearly, very subtle differences in the properties of the 2A protease can have a profound effect on the virulence of SVDV.
ACKNOWLEDGMENTS
This work was funded in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan. A.T.C. gratefully acknowledges the award of a studentship from the Biotechnology and Biological Sciences Research Council.
Jeanette Knight, Vidhi Aggarwal, and Ginette Wilsden are gratefully acknowledged for excellent technical assistance, and we thank Luke Fitzpatrick, Brian Taylor, Colin Randall, and Malcolm Turner for assistance with the handling and management of experimental animals.
T.I. and S.A. contributed equally to this work.
Present address: Danish Institute for Food and Veterinary Research, Department of Virology, Lindholm, DK-4771 Kalvehave, Denmark.
Present address: Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
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