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Department of Virology II, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashi-murayama, Tokyo 208-0011, Japan
Correspondence
Tomoichiro Oka
oka-t{at}nih.go.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences reported in this paper are AY237420, AY237422, AY237423, AY646853, AY646854, AY237419, AY646855, DQ058829, AY646856, X86560, AY694184, AJ249939, AY603425, AF182760, NC_000940 and DQ125333.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Sapovirus (SaV) is a causative agent of gastroenteritis in humans and swine (Guntapong et al., 2004; Guo et al., 2001a; Hansman et al., 2004a, 2006; Johansson et al., 2005; Katayama et al., 2004; Nakata et al., 2000; Noel et al., 1997; Robinson et al., 2002; Vinjé et al., 2000) and is currently divided into five distinct genetic groups, genogroups I (GI)–GV, on the basis of the capsid protein gene (Farkas et al., 2004). The SaV strains that infect pigs belong to genogroup GIII and these strains are able to multiply in cultured cells (Chang et al., 2004; Flynn & Saif, 1988); however, the strains that infect humans, which belong to genogroups GI, GII, GIV and GV, have not yet been grown in cultured cells.
The SaV genome is a linear, polyadenylated, positive-sense, single-stranded RNA of about 7.5 kb with either two or three open reading frames (ORFs) (Clarke & Lambden, 2000). ORF1 encodes a 250 kDa polyprotein that contains amino acid motifs characteristic of caliciviruses, including 2C-like NTPase (NTPase), VPg, 3C-like protease (Pro), 3D-like RNA-dependent RNA polymerase (Pol) and capsid protein (VP1). The functional domains in the ORF1 polyprotein were predicted on the basis of the motifs found in the picornavirus polyprotein, and these domains are highly conserved among members of the family Caliciviridae (Green et al., 2000; Meyers et al., 2000). The functions of the proteins encoded by ORF2 and ORF3 have not yet been elucidated.
Proteolytic processing of the ORF1 polyprotein is a common feature of the caliciviruses (Green et al., 2000) and the cleavage sites have been mapped in detail in Rabbit hemorrhagic disease virus (RHDV) (König et al., 1998; Martin Alonso et al., 1996; Meyers et al., 2000; Wirblich et al., 1995, 1996), Feline calicivirus (FCV) (Sosnovtsev et al., 1998, 2002; Sosnovtseva et al., 1999) and norovirus (NoV) (Belliot et al., 2003; Blakeney et al., 2003; Hardy et al., 2002; Liu et al., 1996, 1999; Seah et al., 1999, 2003; Someya et al., 2000). The 3C-like protease of these viruses cleaves dipeptides containing either glutamic acid (E) or glutamine (Q) at the P1 position (i.e. the amino acid immediately upstream of the scissile bond) and those containing glycine (G), alanine (A), serine (S), threonine (T), aspartic acid (D) or asparagine (N) at the P1' position (i.e. the amino acid immediately downstream of the scissile bond). We have recently identified SaV ORF1 polyprotein-cleavage products of the GII Mc10 strain and these products are arranged in the following order: NH2–p11–p28–p35 (NTPase)–p32–p14 (VPg)–p70 (Pro–Pol)–p60 (VP1)–COOH (Oka et al., 2005b). Site-directed mutagenesis of the GDCG motif in the 3C-like protease fully abolished the proteolytic activity of this enzyme, thus demonstrating that the viral 3C-like protease was responsible for the cleavage (Oka et al., 2005b).
Our recent study using an Escherichia coli expression system also revealed that GII Mc10 3C-like protease cleaves the Q/G site in the rhinovirus 3C protease-recognition sequence (Oka et al., 2005a) in a manner similar to that of NoV Chiba virus 3C-like protease (Someya et al., 2000). Although the cleavage site between p14 (VPg) and p70 (Pro–Pol) of GII Mc10 was identified as E1055/A1056 by N-terminal amino acid sequencing (Oka et al., 2005a), the other cleavage sites are unknown. Our cleavage-products map indicated that the GII Mc10 ORF1 polyprotein should have at least six cleavage sites between six non-structural proteins and one structural protein (Oka et al., 2005b).
The aim of this study was to identify the remaining five cleavage sites of the Mc10 ORF1 polyprotein. Site-directed mutagenesis, an in vitro coupled transcription–translation system and N-terminal amino acid sequencing of E. coli-expressed recombinant proteins were used to identify all of the cleavage sites. In addition, the cleavage site between p14 and p70 was confirmed by site-directed mutagenesis. The dipeptide used for the cleavage sites was conserved among 16 SaV strains and was similar to those of other members of the family Caliciviridae.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The GII C12 strain was isolated from an infant with gastroenteritis in Sakai, Japan, in 2001 (Katayama et al., 2004). The GI NongKhai-50, GI Chanthaburi-74, GV NongKhai-24 (NK24) and GII SaKaeo-15 (SK15) strains were isolated from infants with gastroenteritis in NongKhai, Chanthaburi or SaKaeo, Thailand, between November 2002 and April 2003 (Guntapong et al., 2004). The GIV Ehime1107 and GIV Sw278 strains were isolated from an infant with gastroenteritis in Matsuyama, Japan, in 2002, and from an adult with gastroenteritis in Solna, Sweden, in 2003 (Hansman et al., 2005d). RNA extraction from the stool samples, cDNA synthesis and complete genome sequencing were performed as described previously (Katayama et al., 2002). The SaV sequences were classified phylogenetically according to the method of Farkas et al. (2004).
Full-length cDNA clones.
Plasmids containing a full-length Mc10 genome sequence with the T7 promoter, designated ‘pUC19/SaV Mc10 full-length’ and ‘pUC19/SaV Mc10 full-C1171A/ORF1’, of which the latter contains a 1169GDCG1172 to GDAG mutation in the protease, have been described previously (Oka et al., 2005b).
Site-directed mutagenesis.
Site-directed mutagenesis was performed by using the GeneTailor site-directed mutagenesis system (Invitrogen) with pUC19/SaV Mc10 full-length as a template. In brief, 100 ng plasmid was methylated in 16 µl reaction mixture according to the manufacturer's instructions and then PCR was performed in 100 µl reaction mixture containing 1 µl methylated DNA, 40 pmol each primer, KOD polymerase buffer, 0.2 mM each dNTP, 1 mM MgSO4 and 2 units KOD-Plus DNA polymerase (TOYOBO). The primers used for site-directed mutagenesis to generate the mutant full-length cDNA clones are represented in Table 1. After initial denaturation at 94 °C for 5 min, 20 cycles of amplification were performed. Each cycle consisted of denaturation at 94 °C for 30 s, primer annealing at 55 °C for 30 s and primer extension at 72 °C for 10 min, followed by a final extension at 72 °C for 15 min. E. coli DH5
-T1 cells (Invitrogen) were transformed with 2 µl PCR mixture, and the plasmids containing the mutation(s) in ORF1 were amplified. The resulting 17 full-length mutant cDNA clones were designated as follows: pUC19/SaV Mc10 full-ORF1-E69A (where E at amino acid residue 69 was changed to A), -EE6869AA (where E at amino acid position 68 was changed to A, and E at amino acid position 69 was changed to A), -Q112A, -Q325A, -E385A, -E430A, -EE429430AA, -Q666A, -EQ665666AA, -E940A, -EE939940AA, -EEE938939940AAA, -E1055A, -E10541055AA, -E1679A, -Q1690A and -E1722A. All of the full-length clones were verified by sequencing and no additional mutation was found.
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(I)] were generated with the forward primer 5'-GGATCCTAATACGACTCACTATAGGGAACAGCCACCATGgcttccaagccattctacccaatagag-3', including the T7 promoter sequence (underlined) and a start codon (bold), and antisense primer 5'-T30TTAttctaagaacctaacggcccgg, including a stop codon (bold), and designated I-Prow, -Promut, -E69A, -EE6869AA, -Q112A, -Q325A, -E385A, -E430A, -EE429430AA, -Q666A, -EQ665666AA, -E940A, -EE939940AA, -EEE938939940AAA, -E1055A, -E10541055AA, -E1679A, -Q1690A and -E1722A. The truncated ORF1 templates [Fig. 1(II) and (III)] were similarly generated with the forward primers 5'-GGATCCTAATACGACTCACTATAGGGAACAGCCACCATGgccaaaggaaagaccaagcatggc-3' and 5'-GGATCCTAATACGACTCACTATAGGGAACAGCCACCATGgctcccacaccaattgttac-3', including the T7 promoter-encoding sequence (underlined) and a start codon (bold), and the antisense primer described above. The products were designated II-Prow, II-Promut, -E1055A, -EE10541055AA and III-Prow, -Promut, -E1679A, -Q1690A and -E1722A.
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) (Oka et al., 2005b). The complex was captured with protein A magnetic beads (New England Biolabs) as described previously (Oka et al., 2005b). The protein in the gel was blotted electrically onto a PVDF membrane (Immobilon-P; Millipore) and radiolabelled proteins were detected by a Bioimage Analyser BAS 2500 (Fujifilm).
Construction of E. coli expression plasmids.
The DNA fragment corresponding to the amino acid residues 926–1720 (nt 2789–5173) was amplified by PCR with 500 ng plasmid pUC19/SaV Mc10 full-ORF1-E1055A, which contains the nucleotide changes in the P1 position of the p14/p70 cleavage site, or UC19/SaV Mc10 full-ORF1-E1055A/C1171A, which contains an additional mutation in the GDCG motif, with a sense primer (5'-CAGGGGCCCCTGGGATCCcacaatgtttcatacctcgcc-3') including a BamHI site (underlined) and an antisense primer (5'-GCCGCTCGAGTCGACTCAGTGATGGTGATGGTGATGttcaaacactaatttggtggtctcttcactggggct-3'), including a 6xHis tag-encoding sequence (underlined), a stop codon (bold) and a SalI site (italic). The PCR products were purified and digested with BamHI and SalI (New England Biolabs) and cloned into the corresponding sites of the pGEX-4T-1 vector (Amersham Biosciences). DH5 cells (TOYOBO) were used for the transformation and propagation of the plasmids. The plasmids were designated pGEX-4T-1-
p32-p14-p70/E1055A and pGEX-4T-1-p32-p14-p70/E1055A/C1171A. The truncated ORF1 polyproteins were expressed as fusion proteins with glutathione S-transferase (GST) at the N terminus and 6xHis tag products at the C terminus.
Expression of recombinant proteins in E. coli.
E. coli BL21-CodonPlus-RIL cells (Stratagene) were transformed with the expression plasmid and incubated at 37 °C in Luria broth in the presence of 50 µg ampicillin ml–1 and 50 µg chloramphenicol ml–1 until the OD600 value reached 0.6–0.8. Expression was induced by addition of a final concentration of 1 mM IPTG followed by incubation at 37 °C for 3 h. The E. coli lysates or purified recombinant proteins were separated by SDS-PAGE and stained with GelCode blue staining reagent (Pierce) (Oka et al., 2005a). The recombinant proteins were purified by using TALON resin (BD Clontech) and subjected to N-terminal amino acid sequencing (APRO Science) (Oka et al., 2005a).
Nucleotide and amino acid sequence analyses.
Nucleotide sequence analysis was performed with a BigDye Terminator (version 3.1) cycle sequencing ready reaction kit (Applied Biosystems) and an automated sequencer, the 3100 Avanti genetic analyser (Applied Biosystems). Nucleotide sequences were assembled with the program SEQUENCHER version 4.2.2 (Gene Codes Corporation). Nucleotide and amino acid sequences were analysed with GENETYX Mac software, version 12.2.6 (Genetyx Corporation).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The other potential cleavage sites were selected on the basis of our findings that SaV 3C-like protease cleaves next to E or Q residues. An amino acid alignment with seven SaV strains, i.e. GI Manchester (GenBank accession no. X86560
[GenBank]
), GI Dresden (AY694184
[GenBank]
), GII Bristol (AJ249939
[GenBank]
), GII Mc10 (AY237420
[GenBank]
), GII C12 (AY603425
[GenBank]
), GIII PEC (AF182760
[GenBank]
) and GIII PEC LL14 (NC_000940
[GenBank]
), allowed us to identify six potential cleavage sites (E69/G70, Q325/G326, Q666/G667, E940/A941, E1679/G1680 and E1722/G1723) (Fig. 1). In addition, we tested four additional dipeptides (Q112/G113, E385/A386, E430/G431 and Q1690/A1691) (Table 1), because these sites were conserved among five human SaV strains: GI Manchester, GI Dresden, GII Bristol, GII Mc10 and GII C12. Although our strategy was to alter the E or Q residues at position P1 to an A residue, five of 11 potential cleavage sites, including E1055/A1056, had amino acid(s) that were able to create a potential novel cleavage site immediately upstream of the P1 position (i.e. 68EE69, 429EE430, 665EQ666, 938EEE940 and 1054EE1055) (Table 1). Therefore, mutant clones containing either double or triple substitutions were prepared. To this end, we constructed 17 full-length mutant cDNA clones for the 11 potential cleavage sites, as shown in Table 1 and Fig. 1.
Cleavage of the ORF1 polyprotein in an in vitro coupled transcription–translation system
To identify the potential cleavage sites in the ORF1 polyprotein, 17 linear template DNAs containing the T7 promoter were amplified by PCR as described in Methods; these sites corresponded to entire ORF1 regions and were designated I-E69A, -EE6869AA, -Q112A, -Q325A, -E385A, -E430A, -EE429430AA, -Q666A, -EQ665666AA, -E940A, -EE939940AA, -EEE938939940AAA, -E1055A, -E10541055AA, -E1679A, -Q1690A and -E1722A. Then, in vitro coupled transcription–translation was performed and the expressed proteins were analysed by SDS-PAGE or immunoprecipitation. Two DNA templates, I-Prow, which encodes the wild-type protease, and I-Promut, which encodes the mutant protease, were used as the positive and negative controls for proteolytic processing. I-Prow produced at least nine proteins, i.e. p11, p14, p28, p32, p35, p46, p60, p66 and p120 (Fig. 2, lanes 10 and 20), whereas I-Promut produced a major 250 kDa product in SDS-PAGE (Fig. 2, lanes 11 and 21) (Oka et al., 2005b). To detect p11 and p14, immunoprecipitation with anti-A and anti-D region-specific antibodies was performed (Fig. 1). The detection of p70 (Pro–Pol) was difficult when the entire ORF1 region was expressed, as described previously (Oka et al., 2005b). A 
100 kDa product clearly visible in Fig. 2 was present in all samples analysed, including the I-Promut sample. This suggested that it was probably an artefact of the expression system, an internal initiation product or a terminally truncated protein, and is not discussed further in this study. As shown in Fig. 2, 10 constructs – I-E69A, -EE6869AA, -Q325A, -Q666A, -EQ665666AA, -EE939940AA, -EEE938939940AAA, -E1055A, -E10541055AA and -E1722A – demonstrated cleavage patterns different from those of I-Prow, demonstrating clearly that the proteolytic processing of the ORF1 polyprotein was blocked in these constructs, as described in the following sections.
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; Fig. 1). If the cleavage site was abolished, then these two products (i.e. p11 and p28) would be expected to disappear, whereas p39, corresponding to p11–p28, would appear. In vitro coupled transcription–translation with I-E69A as the template resulted in the accumulation of p39 (Fig. 2, lane 1), which was immunoprecipitated with anti-A antibody (Fig. 3a, lane 1), indicating clearly that p39 was indeed p11–p28. The construct I-EE6869AA had a cleavage pattern identical to that of I-E69A (Fig. 2, lanes 1 and 2), demonstrating that the cleavage occurred between E69 and G70 and that no alternative cleavage occurred between E68 and A69. The loss of p66, which corresponds to p28–p35, was also observed in both the I-E69A and I-EE6869AA translation products (Fig. 2, lanes 1 and 2; Fig. 3a, lane 1). I-Q112A had little effect on the proteolytic processing (Fig. 2, lane 3), demonstrating that Q112/G113 is not a cleavage site between p11 and p28. Based on these findings, we concluded that the cleavage site between p11 and p28 is E69/G70.
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; Fig. 1). The translation products from the I-E385A, I-E430A and I-EE429430AA constructs were similar to those of I-Prow (Fig. 2, lanes 5, 6, 7, 10 and 20), indicating that E385/A386 and E430/G431 are not cleavage sites between p28 and p35. In contrast, the cleavage products from I-Q325A showed a loss of p28 and p35 and an accumulation of p66 (Fig. 2, lane 4; Fig. 3a, lane 2). Therefore, we concluded that the cleavage site between p28 and p35 is Q325/G326.
Cleavage site between p35 and p32
One site, Q666/G667, was tested for the putative cleavage site between p35 and p32 (Table 1; Fig. 1). The translation products from I-Q666A showed a loss of p35 and p32; in addition, a loss of p66, p46 and p120 was observed, as well as an accumulation of p67, p81 and p150 (Fig. 2, lane 8). The antibodies raised against fragment C immunoprecipitated p67, p81 and p150 (Fig. 3b, lane 1), demonstrating that these products corresponded to p35–p32, p35–p32–p14 and p35–p32–p14–p70, respectively. I-EQ665666AA had a cleavage pattern identical to that of I-Q666A (Fig. 2, lanes 8 and 9), indicating that alternative cleavage did not occur between E665 and A666. Based on these results, we concluded that the cleavage site between p28 and p35 is Q666/G667.
Cleavage sites between p32 and p14
E940/A941 was predicted as the putative cleavage site between p32 and p14 (Table 1; Fig. 1) (Oka et al., 2005b). The cleavage products from I-EE939940AA and I-EEE938939940AAA showed a loss of p32 and p14 (Fig. 2, lanes 13 and 14). In contrast, the translation products from I-E940A were similar to those of I-Prow (Fig. 2, lanes 12, 10 and 20), indicating that the newly created 939EA940 was utilized as the alternative cleavage site. These results suggested that the cleavage site between p32 and p14 was E940/A941; however, this interpretation of the results was inconclusive and, therefore, N-terminal amino acid sequencing analysis was carried out. pGEX 4T-1-p32-p14-p70/E1055A, a plasmid encoding p32–p14–p70 (aa 926–1720) with a mutation at 1055E/A1056 in the protease, was expressed as an N-terminal GST and C-terminal 6xHis tag fusion protein in E. coli and used to analyse the N terminus of p14, because our previous study indicated that the cleavage between p14 and p70 occurred efficiently when p14–p70 (aa 941–1720) was expressed as an N-terminal GST and C-terminal 6xHis tag fusion recombinant protein in E. coli (Oka et al., 2005a). Three major products of approximately 110, 84 and 26 kDa were visualized when the total lysate was analysed by SDS-PAGE (Fig. 4a, lane 1). These products were considered to be GST–p32–p14–p70–6xHis, p14–p70–6xHis and GST–p32, respectively, on the basis of their molecular sizes and their affinity to TALON resin (data not shown). N-terminal amino acid sequencing of the purified 84 kDa protein revealed the sequence AKGKT, which corresponds to aa 941–945 of the Mc10 ORF1 polyprotein. pGEX-4T-1-p32-p14-p70/E1055A/C1171A produced a major product of 110 kDa (Fig. 4a, lane 2), demonstrating clearly that the proteolytic processing was dependent on the 3C-like protease, as described previously (Oka et al., 2005a, b). We therefore concluded that the cleavage site between p32 and p14 is E940/A941.
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; Fig. 1) (Oka et al., 2005a). This conclusion was confirmed by site-directed mutagenesis. The translation products from I-E1055A and II-E1055A prevented proteolytic cleavage between p14 and p70 (Fig. 2, lane 15; Fig. 4b, lane 1). The constructs I- and II-EE10541055AA, which contain a double mutation, had a cleavage pattern identical to that of the constructs I- and II-E1055A (Fig. 2, lanes 16 and 17; Fig. 4b, lanes 1 and 2), indicating that no alternative cleavage occurred between E1054 and A1055.
Cleavage site between p70 and p60
Three potential cleavage sites, E1679/G1680, Q1690/A1691 and E1722/G1723, were predicted between p70 and p60 (Table 1 and Fig. 1). The cleavage products from I-E1679A and I-Q1690A were identical to those of I-Prow (Fig. 2, lanes 17 and 18), indicating that E1679/G1680 and Q1690/A1691 are not cleavage sites between p70 and p60. In contrast, the cleavage products from the I-E1722A construct showed a loss of p120 and p60 and an accumulation of p180 (Fig. 2, lane 19). Antibodies raised against the H fragment (aa 1951–2278) (Oka et al., 2005b) immunoprecipitated p180 from the I-E1722A construct (data not shown), demonstrating that p180 corresponds to p120–p60. To further confirm the cleavage between E1722 and G1723, III-Prow was expressed (Fig. 1). We observed two proteins, p70 and p60, as described previously (Fig. 4c, lane 4) (Oka et al., 2005b). In contrast, III-Promut produced a single major band, p130 (Fig. 4c, lane 5). III-E1722A produced a p130 band, as did III-Promut (Fig. 4c, lanes 3 and 5). In contrast, the cleavage patterns from III-E1679A and III-Q1690A were identical to those of III-Prow (Fig. 4c, lanes 1, 2 and 4). Therefore, we concluded that the cleavage site between p70 and p60 is E1722/G1723.
Cleavage sites of the ORF1 polyprotein
The cleavage sites of the SaV GII Mc10 ORF1 polyprotein were identified as E69/G70, Q325/G326, Q666/G667, E940/A941, E1055/A1056 and E1722/G1723 (Table 2). Although seven full-length SaV genome sequences, including Mc10, were available at the beginning of this study, we had recently determined nine human full-length SaV genome sequences: the GI Mc114, N21, Nongkhai50, Chantaburi74, GIIMc2, SK15, GIVEhime1107, Sw278 and GV NK24 strains. The cleavage sites in ORF1 were highly conserved among these 16 SaV strains and either E or Q was found at the P1 position, whereas A, G or S was found at the P1' position (Table 3). The dipeptide sequences at the cleavage sites were similar to those of other caliciviruses, namely, E or Q at the P1 position and A, G, S, T, D or N at the P1' position (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Multiple alignment of 16 SaV ORF1 amino acid sequences revealed that the amino acids for the cleavage sites (P1/P1') are either E or Q at the P1 position and G, A or S at the P1' position (Table 3), which have also been observed in other members of the family Caliciviridae, i.e. RHDV, FCV and NoV (Table 2) (Belliot et al., 2003; Liu et al., 1996, 1999; Sosnovtsev et al., 2002; Wirblich et al., 1995, 1996). Site-directed mutagenesis demonstrated clearly that the P1 amino acid plays a critical role in SaV Mc10 ORF1 proteolytic processing, as observed similarly in RHDV (Martin Alonso et al., 1996; Meyers et al., 2000; Wirblich et al., 1995), FCV (Sosnovtsev et al., 1998, 2002) and NoV (Belliot et al., 2003; Blakeney et al., 2003; Hardy et al., 2002; Liu et al., 1996; Seah et al., 1999, 2003). In addition, our studies also revealed that additional factors are important for the recognition of the cleavage site by an SaV 3C-like protease, because (i) although the dipeptide sequences corresponding to 1679EG1680 in the Mc10 sequence were conserved among the 16 strains, they were never utilized as a cleavage site, and (ii) the newly generated dipeptide sequences, 68EA69, 665EA666, 938EA939 and 1054EA1055, which are positioned immediately upstream of the identified cleavage sites, were not recognized as alternative cleavage sites, with the exception of 939EA940. The role of the amino acid sequence surrounding cleavage sites has been reported in other caliciviruses (Belliot et al., 2003; Hardy et al., 2002; Wirblich et al., 1995). Hardy et al. (2002) noted the importance of phenylalanine (F) at the P4 position to achieve efficient cleavage between p48 (N-terminal protein) and p41 (NTPase) of the NoV Norwalk virus. Interestingly, Y and F were found at the P4 position in NoVs MD145 (p37/p40) and Southampton viruses (p45/p40), respectively (Table 2). Similarly, an F residue was found at the P4 position in three of five cleavage sites in the ORF1 and one in the ORF2 of FCV (Table 2). In contrast, the amino acid at the P4 position of the RHDV ORF1 polyprotein was not conserved (Table 2). As shown in Tables 2 and 3, either F or Y is found at P4 in five of six cleavage sites in the Mc10 ORF1. In a future study, it would be interesting to evaluate whether these amino acids play crucial roles in proteolytic processing, because these P4 amino acids (F or Y) were completely conserved (except for the p32/p14 cleavage site) among the 16 SaV strains (data not shown).
In addition, it appears that proteolytic processing is regulated by other factors, because (i) the construct E1722A (Fig. 2, lane 19), which contains mutations far from the original cleavage sites, Q666/G667 and E1055/A1056, led to a decreased production of p46, and (ii) the mutant constructs E430A and E429430AA seem to have decreased the proteolytic activity between p28 and p35 and were likely to have caused the accumulation of p66 (Fig. 2, lanes 6 and 7). The mutations E430A and E429430AA were positioned in the helix domain of p35 (NTPase) (data not shown). Paul et al. (1994) also reported that the proteolytic processing of the poliovirus polyprotein was affected when an amino acid substitution was introduced in the 2C NTPase N-terminal helix region. Sosnovtsev et al. (1998, 2002) demonstrated in the mutagenesis of an FCV infectious cDNA clone that the proteolytic processing of the virus-encoded polyprotein is critical for the growth of the virus. The ORF1 polyprotein of SaV and RHDV encodes both the non-structural proteins and the major structural protein (VP1), and Parra et al. (1993) reported that the purified RHDV virion had only an MEG sequence in its N terminus and suggested that the RHDV virion was derived from subgenomic RNA. In contrast, Sibilia et al. (1995) reported that both RHDV VP1 translated from the subgenomic RNA and that cleaved from ORF1 polyprotein led to the assembly of virus-like particles (VLPs) that were antigenically similar to purified viruses. Although the N terminus of the VP1 of the native SaV virion has not yet been determined, the expression of the putative VP1 with either MEG or MEA at its N terminus has been shown to form VLPs in SaV GI, GII, GIII and GV strains in insect or mammalian cells (Chen et al., 2004; Guo et al., 2001b; Hansman et al., 2005a, b, c; Jiang et al., 1999; Numata et al., 1997; Oka et al., 2006). Because the cleavage site, E1722/G1723, is in the proximity of the putative capsid start codon of the subgenomic RNA, it is of interest to determine whether the VP1 produced from the SaV ORF1 polyprotein would be able to form VLPs.
In conclusion, we defined the cleavage sites of the ORF1 polyprotein of the SaV GII Mc10 strain by expressing mutant constructs in an in vitro coupled transcription–translation system and by N-terminal amino acid sequencing of the E. coli-expressed recombinant proteins. Our study demonstrated that the cleavage sites were highly conserved among genetically and antigenically different SaV strains, and therefore have important role(s) in SaV replication.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 28 December 2005;
accepted 26 June 2006.
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