Proteolytic Processing of Sapovirus ORF1 Polyprote
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病菌学杂志 2005年第12期
Department of Virology II, National Institute of Infectious Diseases
Department of Developmental Medical Sciences, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
ABSTRACT
The genome of Sapovirus (SaV), a causative agent of gastroenteritis in humans and swine, contains either two or three open reading frames (ORFs). Functional motifs characteristic to the 2C-like NTPase (NTPase), VPg, 3C-like protease (Pro), 3D-like RNA-dependent RNA polymerase (Pol), and capsid protein (VP1) are encoded in the ORF1 polyprotein, which is afterwards cleaved into the nonstructural and structural proteins. We recently determined the complete genome sequence of a novel human SaV strain, Mc10, which has two ORFs. To investigate the proteolytic cleavage of SaV ORF1 and the function of protease on the cleavage, both full-length and truncated forms of the ORF1 polyprotein either with or without mutation in 1171Cys to Ala of the GDCG motif were expressed in an in vitro coupled transcription-translation system. The translation products were analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by immunoprecipitation with region-specific antibodies. The ORF1 polyprotein was processed into at least 10 major proteins: p11, p28, p35, p32, p14, p70, p60, p66, p46, and p120. Seven of these products were arranged in the following order: NH2-p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. p66, p46 and p120 were precursors of p28-p35 (NTPase), p32-p14 (VPg), and p32-p14 (VPg)-p70 (Pro-Pol), respectively. Mutagenesis in the 3C-like protease motif fully abolished the proteolytic activity. The cleavage map of SaV ORF1 is similar to those of other heretofore known members of the family Caliciviridae, especially to rabbit hemorrhagic disease virus, a member of the genus Lagovirus.
INTRODUCTION
The family Caliciviridae contains four genera, Lagovirus, Vesivirus, Norovirus (NoV; formerly known as "Norwalk-like virus"), and Sapovirus (SaV, formerly known as "Sapporo-like virus"), in which rabbit hemorrhagic disease virus (RHDV), feline calicivirus (FCV), Norwalk virus, and Sapporo virus are assigned as the prototype strains (8, 19). SaV is associated with gastroenteritis in humans and swine (5, 9). Human SaV is predominantly isolated from infants and young children, though it is occasionally associated with outbreaks of gastroenteritis (10, 21, 22, 33). Phylogenetic analysis using SaV capsid protein VP1 revealed five genetic groups, genogroup I (GI) to GV (7). The human SaV are classified into GI, GII, GIV, and GV, whereas porcine SaV belongs to GIII (7, 24, 26). Although porcine SaV can grow in cultured cells (4, 9), neither cell culture nor animal models can support the replication of human SaV.
The SaV genome is a positive-sense, single-strand RNA molecule of approximately 7.5 kb that is polyadenylated at its 3' terminus. The SaV GI, GIV, and GV genomes are predicted to contain three main open reading frames (ORFs), whereas SaV GII and GIII have two ORFs (9, 16, 22, 23, 25). The SaV ORF1 encodes nonstructural proteins and the capsid protein VP1, while ORF2 and ORF3 encode proteins of yet unknown function. To date, seven complete genome sequences (accession numbers in parentheses) of SaV, GI Manchester (X86560), GI Dresden (AY694184), GII Bristol (AJ249939), and GII Mc10 (AY237420), GII Sakai C12 (AY603425), GIII PEC/Cowden (AF182760), and PEC/LL14 (AY425671) have been published.
The SaV ORF1 polyprotein contains amino acid motifs characteristic of caliciviruses (8, 36), including 2C-like NTPase (NTPase) (GXXGXGKS/T), VPg [KGK(N/T)K and (D/E)EY(D/E)E], 3C-like protease (Pro)(GDCG), 3D-like RNA-dependent RNA polymerase (Pol)(GLPSG and YGDD), and VP1 (PPG) (9, 28). The proteolytic processing of ORF1 by the virus-encoded protease has been reported in RHDV, FCV, and NoV, and detailed cleavage maps of ORF1 have been reported in these viruses (1, 20, 31, 36). A recent study with a full-length RNA transcript derived from the SaV GI Manchester strain indicated six major cleavage products in vitro (6), suggesting that SaV ORF1 is also cleaved into nonstructural and structural proteins by the virus-encoded protease. However, neither the cleavage map of the viral proteins nor the function of the virus-encoded protease involved in this cleavage has been elucidated yet.
In this study, the proteolytic processing of the ORF1 polyprotein of SaV Mc10, a novel human SaV GII strain, was analyzed by using an in vitro coupled transcription-translation system. We also evaluate the function of the virus-coded 3C-like protease on this proteolytic processing.
MATERIALS AND METHODS
Specimen. The Mc10 strain (Hu/SaV/Mc10/2000/Thailand) was isolated in an epidemiological screening of acute gastroenteritis patients in Thailand in June 2000 (10). The RNA extraction, reverse transcription-PCR, and complete nucleotide sequence analysis of the virus genome were performed as previously described (12).
Construction of plasmids containing the full-length genome. A plasmid harboring the entire Mc10 genome was constructed with two overlapping cDNA fragments utilizing a unique HindIII site at nucleotides (nt) 3599 to 3604. The 5' fragment corresponding to nt 1 to 3735 was amplified with a sense primer (5'-GCGGGATCCTAATACGACTCACTATAGGgtgattggttagtatggcttccaagccattctacccaatag-3') including a BamHI site (underlined) and a T7 RNA polymerase promoter sequence (bold) and an antisense primer (5'-TTGGGCCATGCAGGTGAGCG-3'). The 3' fragment corresponding to nt 2188 to 7458 was amplified with the sense primer (5'-TCCACCTCCCACATACAGTG-3') and antisense primer (5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGAGCTCAGATCTT29ccaagaaagcacggctgc-3') including a poly(A) tail, a BglII site (underlined), and a SacI site (double underlined). The 5' fragments were digested with BamHI and HindIII, and the 3' fragments were digested with HindIII and SacI (New England Biolabs, Beverly, MA). pUC19 vector (Toyobo, Tokyo) was digested with BamHI and SacI (New England Biolabs). These three fragments were purified and ligated to create a plasmid containing a full-length Mc10 genome sequence with the T7 promoter, which was designated as pUC19/SaV Mc10 full-length. The 3' end of this clone contained a poly(A) tract flanking a unique BglII site which was used to linearize the plasmid for runoff transcription. The complete nucleotide sequence of the insert was determined to confirm the original sequence.
Preparation of region-specific antibodies. A panel of antibodies specific to the ORF1 polyprotein was prepared. DNA fragments corresponding to regions A to J (Fig. 1A) were amplified with pUC19/SaV Mc10 full-length as a template by using the primers listed in Table 1. These regions were first cloned into Gateway pDONR201 vector (Invitrogen, Carlsbad, CA) and then transferred to Gateway pDEST 17 vector (Invitrogen) according to the manufacturer's protocol. All plasmids were verified by sequence analysis. Escherichia coli BL21-AI (Invitrogen) was transformed with plasmids A to J and incubated at 37°C in Luria broth (Invitrogen) containing 50 μg/ml of ampicillin until the optical density at 600 nm reached 0.6 to 0.8. The expression was induced in a final concentration of 0.2% (wt/vol) arabinose followed by incubation at 37°C for 3 h. The cultured E. coli cells were centrifuged at 10,000 x g for 10 min at 4°C and resuspended in equilibration/wash buffer (50 mM sodium phosphate, 300 mM NaCl, pH 7; BD Clontech, Palo Alto, CA) supplemented with 8 M urea. The cell suspension was frozen at –80°C, thawed once, rotated at room temperature for 2 h, and centrifuged at 10,000 x g for 10 min at room temperature. The His6-tagged recombinant protein in the supernatant was absorbed with TALON resin (BD Clontech) and eluted with the elution buffer (50 mM sodium phosphate, 300 mM NaCl, 150 mM imidazole, pH 7; BD Clontech) supplemented with 8 M urea. The purified recombinant proteins were dialyzed against phosphate-buffered saline (PBS) containing 4 M urea (pH 7.4). The protein concentration was measured with a protein assay kit (Bio-Rad, Hercules, CA). Five hundred micrograms of each recombinant protein was used to subcutaneously immunize New Zealand White rabbits as described previously (13). The immunoglobulin G (IgG) fraction was purified from serum with rProtein A Sepharose (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's instructions and dialyzed against PBS (pH 7.4). The specificity of the purified IgG fraction was examined by Western blot analysis using the respective recombinant protein.
Site-directed mutagenesis of the full-length cDNA clone. Site-directed mutagenesis was performed with pUC19/SaV Mc10 full-length as a template, using the GeneTailor site-directed mutagenesis system (Invitrogen) according to the manufacturer's instructions. The sense primer, 5'-CCAACAAAGCGTGGGGACGCGGGCACACCC-3', contains nucleotide changes (underlined) converting TGT (1171Cys) to GCG (1171Ala) in the GDCG motif. As the antisense primer,5'-GTCCCCACGCTTTGTTGGGTATCCATTTATGATGCG-3' was pre-pared. The nucleotide sequence of the full-length mutant clone was confirmed and designated as pUC19/SaV Mc10 full-C1171A/ORF1.
In vitro transcription-translation assay. In vitro protein synthesis was performed using TNT T7 Quick for PCR DNA (Promega, Madison, WI). The primers used to generate the templates for the T7 polymerase coupled transcription-translation assay in rabbit reticulocytes are represented in Table 2, and the translation products are depicted in Fig. 1B. PCR was performed with 500 ng of pUC19/SaV Mc10 full-length, or pUC19/SaV Mc10 full-C1171A/ORF1 in 100 μl of the reaction mixture containing 40 pmol of each primer, KOD polymerase buffer, 0.2 mM each deoxynucleoside triphosphate (dNTP), 1 mM MgSO4, and 2 units of KOD-Plus-DNA polymerase (Toyobo). After initial denaturation at 94°C for 5 min, 25 cycles consisting of denaturation at 94°C for 30 s, primer annealing at 55°C for 30 s, and primer extension at 72°C for the appropriate period were performed, followed by a final extension at 72°C for 15 min. Five microliters of the PCR mixture was added to 50 μl of the TNT reaction mixture containing 40 μl of TNT master mixture (Promega) and 40 μCi of Redivue Pro-mix L-35S in vitro cell labeling mix (Amersham Biosciences). The TNT reaction was performed at 30°C for 1.5 h, and 2 μl of the solution was mixed with 20 μl of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (62.5 mM Tris-HCl [pH 6.8], 5% [wt/vol] sucrose, 2% [wt/vol] SDS, 0.002% [wt/vol] bromophenol blue with 5% [vol/vol] 2-mercaptoethanol), heated at 95°C for 5 min, and loaded onto 5 to 20% Tris-Gly polyacrylamide gel (D.R.C., Tokyo). The proteins in the gel were blotted onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA) using a semidry electroblotting apparatus. The radiolabeled proteins were detected by a Bioimage Analyzer BAS 2500 (Fuji Film, Tokyo).
Immunoprecipitation. For radioimmunoprecipitation, 10 μl of the TNT translation reaction mixture was diluted with 80 μl of radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP40, 1 mM EDTA; Upstate Biotechnology Inc., Lake Placid, NY) and incubated with 5 μg of region-specific antibodies. After incubation for 1 h on ice, 25 μl of a suspension of protein A magnetic beads (New England Biolabs) and 900 μl of RIPA buffer were added. The mixture was gently rotated at 4°C for 1 h and then washed three times with 1 ml of RIPA lysis buffer. The immunoprecipitated proteins were resuspended in 20 μl of SDS-PAGE sample buffer and heated at 95°C for 5 min prior to analysis with 5 to 20% Tris-Gly polyacrylamide gel. The proteins were blotted onto an Immobilon-P polyvinylidene difluoride membrane. Immunoprecipitated radioactive proteins were detected with a Bioimage Analyzer BAS 2500 (Fuji Film).
Nucleotide sequence analysis. Nucleotide sequence analysis was performed with the Big Dye Terminator version 3.1 Cycle Sequencing Ready Reaction kit (Applied Biosystems, Tokyo) and an automated sequencer, the 3100 Avanti genetic analyzer (Applied Biosystems). Nucleotide sequences were assembled with Sequencher version 4.1 (Gene Codes Corporation, Ann Arbor, MI). Nucleotide and amino acid sequences were analyzed with GENETYX Mac version 11.2.2 (Genetyx Corporation, Tokyo).
Nucleotide sequence accession number. The GenBank accession number of the SaV Mc10 genome sequence is AY237420.
RESULTS AND DISCUSSION
Functional motifs in Mc10 ORF1. The complete nucleotide sequence analysis demonstrated that the Mc10 genome consists of 7,458 nt, excluding the 3' poly(A) tail, and has two ORFs. ORF1 consists of 6,837 nucleotides (nt 14 to 6850) and encodes a protein of 2,278 amino acids (aa) with an estimated molecular mass of 250 kDa. ORF2 consists of 501 nucleotides (nt 6850 to 7350) and encodes a protein of 167 aa that overlaps the 3' end of ORF1 by 1 nt. An amino acid homology search revealed that the Mc10 ORF1 polyprotein contains conserved sequence motifs characteristic of calicivirus, which include NTPase (481GPPGIGKT488), VPg (942KGKTK946 and 964DEYDE968), Pro (1169GDCG1172), Pol (1504GLPSG1508 and 1552YGDD1555), and VP1 (1856PPG1858) (Fig. 1A). These motifs are also found in other SaV strains and allowed us to predict several cleavage sites of putative mature nonstructural and structural proteins, which in turn served as templates to generate region-specific antibodies.
Generation of region-specific antibodies. To generate a panel of antibodies specific to the ORF1 polyprotein, 10 protein fragments (Fig. 1A, proteins A to J) were expressed in E. coli BL21-AI cells as N-terminal His6-tagged fusion proteins, as described in the Materials and Methods. We selected these regions based on the characteristic amino acid motifs and multiple alignments of the available SaV ORF1 aa. Because the residues Q (or E)/G, A, S, T, D, and N have been identified as the cleavage sites in other caliciviruses (1, 20, 31), the boundaries of some of these regions were selected between the highly conserved dipeptides among SaV strains (data not shown). SDS-PAGE analysis of the arabinose-induced proteins in E. coli demonstrated that all of these proteins were efficiently expressed with the expected sizes, except for region I. Inefficient expression of a corresponding region in E. coli was reported in RHDV and FCV (31, 36), and a common highly hydrophobic domain observed in these viruses may be the reason. Most of the proteins were expressed as insoluble forms, and they were solubilized with 8 M urea. Under this condition, the proteins A to H, but not J, were efficiently extracted and purified by affinity chromatography using His6 tag.
We expressed eight viral proteins, A to H (Fig. 1A), which correspond to 83% of the ORF1 polyprotein, and region-specific antibodies were prepared.
Proteolytic processing of ORF1 polyprotein in vitro. Proteolytic processing of Mc10 ORF1 was investigated with an in vitro translation using a rabbit reticulocyte lysate. In order to produce a full-length ORF1 fragment, an experiment using runoff RNA products from the pUC19/SaV Mc10 full-length clone was originally performed. However, this was unsuccessful due to the low translation level (data not shown). Therefore, a DNA fragment containing the entire ORF1 was amplified by PCR with a set of primers, which included a T7 promoter sequence in the sense primer (Table 2). The ORF1 polyprotein was then expressed by using an in vitro coupled transcription-translation system in the presence of 35S-labeled methionine and cysteine (Fig. 1B, construct I). SDS-PAGE of the radiolabeled proteins followed by image analysis showed that at least seven proteins, p28, p32, p35, p46, p60, p66, and p120, were generated, demonstrating that ORF1 polyprotein "I-Prow" was translated and proteolytically cleaved (Fig. 2A, lane I-Prow). A time course analysis revealed that there was no accumulation of the 250-kDa primary translation product even at the early stage of the incubation, indicating that the processing of ORF1 occurred cotranslationally or rapidly after translation, as observed in RHDV, FCV, and NoV (1, 2, 11, 15, 20, 32).
To determine whether the cleavage is dependent on the virus-encoded 3C-like protease, a DNA fragment containing a C1171A mutation was prepared and used for an in vitro transcription-translation. As shown in the lane I-Promut (Fig. 2A), only a single major band of 250 kDa was observed. Thus, the viral protease is responsible for the processing, and the Cys residue in the GDCG motif is critical for the proteolytic activity, as seen in other caliciviruses (1-3, 15, 27, 32).
Immunoprecipitation with region-specific antibodies. To identify the cleavage products, an in vitro reaction mixture of I-Prow protein was subjected to the immunoprecipitation with region-specific antibodies (anti-A to -H antibodies). As shown in Fig. 2B, lane A, three major proteins of 11, 28, and 66 kDa were precipitated with anti-A antibody (Fig. 2B, lane A), whereas p11 was not identified as a specific cleavage product in the direct SDS-PAGE analysis due to an abundance of cellular proteins (Fig. 2A and 2B, lane I-Prow). Anti-B antibody precipitated 35-kDa proteins (Fig. 2B, lane B). Anti-C antibody precipitated 46-kDa proteins (Fig. 2B, lane C). Three major proteins of 14, 46, and 120 kDa were precipitated with anti-D antibody (Fig. 2B, lane D), whereas p14 was not detectable in the direct SDS-PAGE analysis due to an abundance of cellular proteins (Fig. 2B, lane I-Prow). Neither anti-E nor -F antibodies precipitated a specific cleavage product (Fig. 2B, lanes E and F), although these antibodies reacted with I-Promut and with proteins E and F expressed in E. coli (data not shown). Both anti-G and -H antibodies precipitated 60-kDa proteins (Fig. 2B, lanes G and H).
The above results indicated the following: (i) p11, p28, p35, and p66 were products derived from the N-terminal region of the ORF1; (ii) p14 and p46 were products derived from the central region of the ORF1; (iii) p46 was either unprocessed or stable p14-p32, since this protein was precipitated with both anti-C and -D antibodies; and (iv) p60 was a product derived from the C-terminal region of ORF1, since this protein was precipitated with both anti-G and -H antibodies. The cleavage products of ORF1, except p32 and p120, were thus identified by the region-specific antibodies. Furthermore, two additional products, p11 and p14, were found which were not detected by direct SDS-PAGE analysis.
Proteolytic processing of N- and C-terminally truncated translation products. To further analyze the cleavage products, five truncated templates—one C-terminally truncated, three N-terminally truncated, and one both C- and N-terminally truncated—were generated and expressed by an in vitro coupled transcription-translation system (Fig. 1B, II to VI). The expressed regions were selected according to the cleavage products sizes. We used identical boundaries to the regions to prepare the region-specific antibodies. The expression was carried out with both wild-type (designated as Prow) and C1171A mutant (designated as Promut) templates, and the 35S-labeled products were analyzed by direct SDS-PAGE (Fig. 3) and immunoprecipitation with region-specific antibodies (Fig. 4). When mutant proteins I- to VI-Promut were expressed, only single major protein bands of p250, p190, p180, p145, p130, or p70, which corresponded to their respective estimate sizes, were observed (Fig. 3, I- to VI-Promut). Similarly, VI-Prow was not further processed (Fig. 3, VI-Prow). In contrast, I- to V-Prow were extensively processed and the following cleavage patterns were obtained. (i) II-Prow was processed into p11, p28, p35, p32, p14, p66, p46, and p120, which were identical to I-Prow, except for p60, confirming that p60 is located in the C-terminal ORF1 (Fig. 3, II-Prow; Fig. 4, II Prow). (ii) III-Prow was cleaved into p32, p14, p70, p46, p120, and p60 (Fig. 3, III-Prow; Fig. 4, III-Prow). (iii) IV-Prow was cleaved into p14, p70, p84, and p60 (Fig. 3, IV-Prow; Fig. 4, IV-Prow); and (iv) V-Prow was processed into p70 and p60 (Fig. 3, V-Prow). p66 was identified as an unprocessed or stable p28-p35, since this protein was detected in additional N-terminally truncated Prow forms II' (corresponding to aa 70 to 1720) but not in II" (corresponding to aa 326 to 1720), and since the p66 that appeared in the cleavage products of Prow forms II' could be precipitated with anti-A antibody (data not shown).
Since p32 and p120 were found in I- to III-Prow but not in IV- or V-Prow, p32 and p120 are derived from the central region of ORF1 (Fig. 3, I- to V-Prow). Although p70 was difficult to detect in the I- and II-Prow processed products due to the nonspecific or the inappropriate internal translation protein(s) seen in I- and II-Promut (Fig. 3, I- and II-Promut), this protein appeared in the cleavage products of III-, IV-, and V- Prow (Fig. 3, III-, IV-, and V-Prow), and was identical in size to the VI-Prow and VI-Promut products (Fig. 3, VI-Prow and VI-Promut). p70 in III- to VI-Prow and VI-Promut was immunoprecipitated with both anti-E and -F antibodies (Fig. 4, III- to VI-Prow, and VI-Promut). Although the identification of p70 in I- and II-Prow by immunoprecipitation was not clear (Fig. 4, I- and II-Promut), we concluded that p70 was the cleavage product derived from the central region of ORF1. In addition, we observed p84 and assigned it to p14-p70 based on its molecular size and immunoreactivity to anti-D, -E, and -F antibodies. We did not further analyze this product in this study, because this protein was detected only in IV-Prow and is likely to be a construct-dependent unprocessed or stable product.
Cleavage map of SaV ORF1. The ORF1 cleavage map was generated using full-length and/or truncated forms of the ORF1 polyprotein. We concluded that Mc10 ORF1 polyprotein was processed into at least 10 major proteins—p11, p28, p35, p32, p66, p14, p46, p70, p120 and p60—by an in vitro coupled transcription-translation system. Seven of these products were arranged in the order NH2-p11-p28-p35-p32-p14-p70-p60-COOH, and the amino acid motifs indicated that p35, p14, p70, and p60 correspond to NTPase, VPg, Pro-Pol, and VP1, respectively (Fig. 1C). p66, p46, and p120 were identified as p28-p35 (NTPase), p32-p14 (VPg), and p32-p14 (VPg)-p70 (Pro-Pol).
The cleavage map and product sizes are strikingly similar to those of RHDV, except in the case of Pro-Pol, which is processed into Pro and Pol, and the RHDV polyprotein is arranged in the order NH2-p16-p23-p37 (NTPase)-p29-p13 (VPg)-p15 (Pro)-p58 (Pol)-p60 (VP1)-COOH (36).
Although p70 (Pro-Pol) was not further cleaved under the conditions used in this study, it should be noted that Pro-Pol has also been identified as a stable product in RHDV, FCV, and NoV in in vitro translation systems (1, 18, 32, 36). Extension of the incubation time from 1.5 h to 24 h or the presence of a canine pancreatic microsomal membrane fraction had no effect (data not shown). Further cleavage of Pro-Pol to Pro and Pol has been shown in RHDV and NoV, but not in FCV, when the Pro-Pol-containing region was expressed in mammalian cells (14, 20, 27, 31, 32), and a similar event was observed in RHDV, FCV, and NoV in E. coli (17, 29, 30, 32, 34, 35). These observations indicated that the cleavage of Pro-Pol to Pro and Pol is dependent on the expression system.
In this study, SaV ORF1 cleavage was investigated for the first time with full-length and N- and C-terminally truncated constructs of the ORF1 polyprotein either with or without mutation in 1171Cys to Ala of the GDCG motif derived from the GII Mc10 strain. After the in vitro transcription-translation reaction, the cleavage products were analyzed with region-specific antibodies against a panel of viral protein fragments. When both the N- and C-terminally truncated ORF1 constructs were expressed, the cleavage products of the expected size were generated from each truncated form of ORF1, indicating that our prediction of the boundaries of each product was almost accurate except for the N-terminal region. Our preliminary data showed that SaV Pro-containing region expressed in E. coli has cleavage activity between Q/G within the rhinovirus 3C-like protease recognition sequence as reported in Chiba virus (29; Oka et al. unpublished observation). This result supports the possibility that SaV protease has a similar dipeptide recognition pattern (cleaves after Q or E) to those of other caliciviral proteases. At the beginning of our study, four SaV full-length genome sequences including Mc10 were available, but now, seven strains of SaV full genome sequences which belong to GI, GII, and GIII, respectively, are available. Multiple alignments of the available SaV ORF1 amino acid sequence revealed the presence of the conserved dipeptide-QG, -QA, -EG, and -EA, which are likely used as cleavage sites. From these data, we speculated that selected sites, E940/A941 and E1055/A1056 in the central region, are likely the cleavage sites, but in the case of N- and C-terminal regions, several candidate sites exist close to the selected sites described in this study (data not shown); therefore, we did not conclude any real cleavage sites in this study. The identification of each cleavage site by another approach, i.e., N-terminal amino acid sequencing and/or site-directed mutagenesis of the candidate cleavage sites, is now under investigation.
In conclusion, we determined the SaV ORF1 cleavage map and showed that the viral 3C-like protease was shown to be responsible for this proteolytic processing, and the proteolytic activity was fully abolished by replacing 1171Cys within the GDCG motif of the 3C-like protease.
ACKNOWLEDGMENTS
This work was supported in part by a grant for Research on Emerging and Re-emerging Infectious Diseases from the Ministry of Health, Labor and Welfare of Japan. G. Hansman obtained a Ph.D. scholarship from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Department of Developmental Medical Sciences, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
ABSTRACT
The genome of Sapovirus (SaV), a causative agent of gastroenteritis in humans and swine, contains either two or three open reading frames (ORFs). Functional motifs characteristic to the 2C-like NTPase (NTPase), VPg, 3C-like protease (Pro), 3D-like RNA-dependent RNA polymerase (Pol), and capsid protein (VP1) are encoded in the ORF1 polyprotein, which is afterwards cleaved into the nonstructural and structural proteins. We recently determined the complete genome sequence of a novel human SaV strain, Mc10, which has two ORFs. To investigate the proteolytic cleavage of SaV ORF1 and the function of protease on the cleavage, both full-length and truncated forms of the ORF1 polyprotein either with or without mutation in 1171Cys to Ala of the GDCG motif were expressed in an in vitro coupled transcription-translation system. The translation products were analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by immunoprecipitation with region-specific antibodies. The ORF1 polyprotein was processed into at least 10 major proteins: p11, p28, p35, p32, p14, p70, p60, p66, p46, and p120. Seven of these products were arranged in the following order: NH2-p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. p66, p46 and p120 were precursors of p28-p35 (NTPase), p32-p14 (VPg), and p32-p14 (VPg)-p70 (Pro-Pol), respectively. Mutagenesis in the 3C-like protease motif fully abolished the proteolytic activity. The cleavage map of SaV ORF1 is similar to those of other heretofore known members of the family Caliciviridae, especially to rabbit hemorrhagic disease virus, a member of the genus Lagovirus.
INTRODUCTION
The family Caliciviridae contains four genera, Lagovirus, Vesivirus, Norovirus (NoV; formerly known as "Norwalk-like virus"), and Sapovirus (SaV, formerly known as "Sapporo-like virus"), in which rabbit hemorrhagic disease virus (RHDV), feline calicivirus (FCV), Norwalk virus, and Sapporo virus are assigned as the prototype strains (8, 19). SaV is associated with gastroenteritis in humans and swine (5, 9). Human SaV is predominantly isolated from infants and young children, though it is occasionally associated with outbreaks of gastroenteritis (10, 21, 22, 33). Phylogenetic analysis using SaV capsid protein VP1 revealed five genetic groups, genogroup I (GI) to GV (7). The human SaV are classified into GI, GII, GIV, and GV, whereas porcine SaV belongs to GIII (7, 24, 26). Although porcine SaV can grow in cultured cells (4, 9), neither cell culture nor animal models can support the replication of human SaV.
The SaV genome is a positive-sense, single-strand RNA molecule of approximately 7.5 kb that is polyadenylated at its 3' terminus. The SaV GI, GIV, and GV genomes are predicted to contain three main open reading frames (ORFs), whereas SaV GII and GIII have two ORFs (9, 16, 22, 23, 25). The SaV ORF1 encodes nonstructural proteins and the capsid protein VP1, while ORF2 and ORF3 encode proteins of yet unknown function. To date, seven complete genome sequences (accession numbers in parentheses) of SaV, GI Manchester (X86560), GI Dresden (AY694184), GII Bristol (AJ249939), and GII Mc10 (AY237420), GII Sakai C12 (AY603425), GIII PEC/Cowden (AF182760), and PEC/LL14 (AY425671) have been published.
The SaV ORF1 polyprotein contains amino acid motifs characteristic of caliciviruses (8, 36), including 2C-like NTPase (NTPase) (GXXGXGKS/T), VPg [KGK(N/T)K and (D/E)EY(D/E)E], 3C-like protease (Pro)(GDCG), 3D-like RNA-dependent RNA polymerase (Pol)(GLPSG and YGDD), and VP1 (PPG) (9, 28). The proteolytic processing of ORF1 by the virus-encoded protease has been reported in RHDV, FCV, and NoV, and detailed cleavage maps of ORF1 have been reported in these viruses (1, 20, 31, 36). A recent study with a full-length RNA transcript derived from the SaV GI Manchester strain indicated six major cleavage products in vitro (6), suggesting that SaV ORF1 is also cleaved into nonstructural and structural proteins by the virus-encoded protease. However, neither the cleavage map of the viral proteins nor the function of the virus-encoded protease involved in this cleavage has been elucidated yet.
In this study, the proteolytic processing of the ORF1 polyprotein of SaV Mc10, a novel human SaV GII strain, was analyzed by using an in vitro coupled transcription-translation system. We also evaluate the function of the virus-coded 3C-like protease on this proteolytic processing.
MATERIALS AND METHODS
Specimen. The Mc10 strain (Hu/SaV/Mc10/2000/Thailand) was isolated in an epidemiological screening of acute gastroenteritis patients in Thailand in June 2000 (10). The RNA extraction, reverse transcription-PCR, and complete nucleotide sequence analysis of the virus genome were performed as previously described (12).
Construction of plasmids containing the full-length genome. A plasmid harboring the entire Mc10 genome was constructed with two overlapping cDNA fragments utilizing a unique HindIII site at nucleotides (nt) 3599 to 3604. The 5' fragment corresponding to nt 1 to 3735 was amplified with a sense primer (5'-GCGGGATCCTAATACGACTCACTATAGGgtgattggttagtatggcttccaagccattctacccaatag-3') including a BamHI site (underlined) and a T7 RNA polymerase promoter sequence (bold) and an antisense primer (5'-TTGGGCCATGCAGGTGAGCG-3'). The 3' fragment corresponding to nt 2188 to 7458 was amplified with the sense primer (5'-TCCACCTCCCACATACAGTG-3') and antisense primer (5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGAGCTCAGATCTT29ccaagaaagcacggctgc-3') including a poly(A) tail, a BglII site (underlined), and a SacI site (double underlined). The 5' fragments were digested with BamHI and HindIII, and the 3' fragments were digested with HindIII and SacI (New England Biolabs, Beverly, MA). pUC19 vector (Toyobo, Tokyo) was digested with BamHI and SacI (New England Biolabs). These three fragments were purified and ligated to create a plasmid containing a full-length Mc10 genome sequence with the T7 promoter, which was designated as pUC19/SaV Mc10 full-length. The 3' end of this clone contained a poly(A) tract flanking a unique BglII site which was used to linearize the plasmid for runoff transcription. The complete nucleotide sequence of the insert was determined to confirm the original sequence.
Preparation of region-specific antibodies. A panel of antibodies specific to the ORF1 polyprotein was prepared. DNA fragments corresponding to regions A to J (Fig. 1A) were amplified with pUC19/SaV Mc10 full-length as a template by using the primers listed in Table 1. These regions were first cloned into Gateway pDONR201 vector (Invitrogen, Carlsbad, CA) and then transferred to Gateway pDEST 17 vector (Invitrogen) according to the manufacturer's protocol. All plasmids were verified by sequence analysis. Escherichia coli BL21-AI (Invitrogen) was transformed with plasmids A to J and incubated at 37°C in Luria broth (Invitrogen) containing 50 μg/ml of ampicillin until the optical density at 600 nm reached 0.6 to 0.8. The expression was induced in a final concentration of 0.2% (wt/vol) arabinose followed by incubation at 37°C for 3 h. The cultured E. coli cells were centrifuged at 10,000 x g for 10 min at 4°C and resuspended in equilibration/wash buffer (50 mM sodium phosphate, 300 mM NaCl, pH 7; BD Clontech, Palo Alto, CA) supplemented with 8 M urea. The cell suspension was frozen at –80°C, thawed once, rotated at room temperature for 2 h, and centrifuged at 10,000 x g for 10 min at room temperature. The His6-tagged recombinant protein in the supernatant was absorbed with TALON resin (BD Clontech) and eluted with the elution buffer (50 mM sodium phosphate, 300 mM NaCl, 150 mM imidazole, pH 7; BD Clontech) supplemented with 8 M urea. The purified recombinant proteins were dialyzed against phosphate-buffered saline (PBS) containing 4 M urea (pH 7.4). The protein concentration was measured with a protein assay kit (Bio-Rad, Hercules, CA). Five hundred micrograms of each recombinant protein was used to subcutaneously immunize New Zealand White rabbits as described previously (13). The immunoglobulin G (IgG) fraction was purified from serum with rProtein A Sepharose (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's instructions and dialyzed against PBS (pH 7.4). The specificity of the purified IgG fraction was examined by Western blot analysis using the respective recombinant protein.
Site-directed mutagenesis of the full-length cDNA clone. Site-directed mutagenesis was performed with pUC19/SaV Mc10 full-length as a template, using the GeneTailor site-directed mutagenesis system (Invitrogen) according to the manufacturer's instructions. The sense primer, 5'-CCAACAAAGCGTGGGGACGCGGGCACACCC-3', contains nucleotide changes (underlined) converting TGT (1171Cys) to GCG (1171Ala) in the GDCG motif. As the antisense primer,5'-GTCCCCACGCTTTGTTGGGTATCCATTTATGATGCG-3' was pre-pared. The nucleotide sequence of the full-length mutant clone was confirmed and designated as pUC19/SaV Mc10 full-C1171A/ORF1.
In vitro transcription-translation assay. In vitro protein synthesis was performed using TNT T7 Quick for PCR DNA (Promega, Madison, WI). The primers used to generate the templates for the T7 polymerase coupled transcription-translation assay in rabbit reticulocytes are represented in Table 2, and the translation products are depicted in Fig. 1B. PCR was performed with 500 ng of pUC19/SaV Mc10 full-length, or pUC19/SaV Mc10 full-C1171A/ORF1 in 100 μl of the reaction mixture containing 40 pmol of each primer, KOD polymerase buffer, 0.2 mM each deoxynucleoside triphosphate (dNTP), 1 mM MgSO4, and 2 units of KOD-Plus-DNA polymerase (Toyobo). After initial denaturation at 94°C for 5 min, 25 cycles consisting of denaturation at 94°C for 30 s, primer annealing at 55°C for 30 s, and primer extension at 72°C for the appropriate period were performed, followed by a final extension at 72°C for 15 min. Five microliters of the PCR mixture was added to 50 μl of the TNT reaction mixture containing 40 μl of TNT master mixture (Promega) and 40 μCi of Redivue Pro-mix L-35S in vitro cell labeling mix (Amersham Biosciences). The TNT reaction was performed at 30°C for 1.5 h, and 2 μl of the solution was mixed with 20 μl of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (62.5 mM Tris-HCl [pH 6.8], 5% [wt/vol] sucrose, 2% [wt/vol] SDS, 0.002% [wt/vol] bromophenol blue with 5% [vol/vol] 2-mercaptoethanol), heated at 95°C for 5 min, and loaded onto 5 to 20% Tris-Gly polyacrylamide gel (D.R.C., Tokyo). The proteins in the gel were blotted onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA) using a semidry electroblotting apparatus. The radiolabeled proteins were detected by a Bioimage Analyzer BAS 2500 (Fuji Film, Tokyo).
Immunoprecipitation. For radioimmunoprecipitation, 10 μl of the TNT translation reaction mixture was diluted with 80 μl of radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP40, 1 mM EDTA; Upstate Biotechnology Inc., Lake Placid, NY) and incubated with 5 μg of region-specific antibodies. After incubation for 1 h on ice, 25 μl of a suspension of protein A magnetic beads (New England Biolabs) and 900 μl of RIPA buffer were added. The mixture was gently rotated at 4°C for 1 h and then washed three times with 1 ml of RIPA lysis buffer. The immunoprecipitated proteins were resuspended in 20 μl of SDS-PAGE sample buffer and heated at 95°C for 5 min prior to analysis with 5 to 20% Tris-Gly polyacrylamide gel. The proteins were blotted onto an Immobilon-P polyvinylidene difluoride membrane. Immunoprecipitated radioactive proteins were detected with a Bioimage Analyzer BAS 2500 (Fuji Film).
Nucleotide sequence analysis. Nucleotide sequence analysis was performed with the Big Dye Terminator version 3.1 Cycle Sequencing Ready Reaction kit (Applied Biosystems, Tokyo) and an automated sequencer, the 3100 Avanti genetic analyzer (Applied Biosystems). Nucleotide sequences were assembled with Sequencher version 4.1 (Gene Codes Corporation, Ann Arbor, MI). Nucleotide and amino acid sequences were analyzed with GENETYX Mac version 11.2.2 (Genetyx Corporation, Tokyo).
Nucleotide sequence accession number. The GenBank accession number of the SaV Mc10 genome sequence is AY237420.
RESULTS AND DISCUSSION
Functional motifs in Mc10 ORF1. The complete nucleotide sequence analysis demonstrated that the Mc10 genome consists of 7,458 nt, excluding the 3' poly(A) tail, and has two ORFs. ORF1 consists of 6,837 nucleotides (nt 14 to 6850) and encodes a protein of 2,278 amino acids (aa) with an estimated molecular mass of 250 kDa. ORF2 consists of 501 nucleotides (nt 6850 to 7350) and encodes a protein of 167 aa that overlaps the 3' end of ORF1 by 1 nt. An amino acid homology search revealed that the Mc10 ORF1 polyprotein contains conserved sequence motifs characteristic of calicivirus, which include NTPase (481GPPGIGKT488), VPg (942KGKTK946 and 964DEYDE968), Pro (1169GDCG1172), Pol (1504GLPSG1508 and 1552YGDD1555), and VP1 (1856PPG1858) (Fig. 1A). These motifs are also found in other SaV strains and allowed us to predict several cleavage sites of putative mature nonstructural and structural proteins, which in turn served as templates to generate region-specific antibodies.
Generation of region-specific antibodies. To generate a panel of antibodies specific to the ORF1 polyprotein, 10 protein fragments (Fig. 1A, proteins A to J) were expressed in E. coli BL21-AI cells as N-terminal His6-tagged fusion proteins, as described in the Materials and Methods. We selected these regions based on the characteristic amino acid motifs and multiple alignments of the available SaV ORF1 aa. Because the residues Q (or E)/G, A, S, T, D, and N have been identified as the cleavage sites in other caliciviruses (1, 20, 31), the boundaries of some of these regions were selected between the highly conserved dipeptides among SaV strains (data not shown). SDS-PAGE analysis of the arabinose-induced proteins in E. coli demonstrated that all of these proteins were efficiently expressed with the expected sizes, except for region I. Inefficient expression of a corresponding region in E. coli was reported in RHDV and FCV (31, 36), and a common highly hydrophobic domain observed in these viruses may be the reason. Most of the proteins were expressed as insoluble forms, and they were solubilized with 8 M urea. Under this condition, the proteins A to H, but not J, were efficiently extracted and purified by affinity chromatography using His6 tag.
We expressed eight viral proteins, A to H (Fig. 1A), which correspond to 83% of the ORF1 polyprotein, and region-specific antibodies were prepared.
Proteolytic processing of ORF1 polyprotein in vitro. Proteolytic processing of Mc10 ORF1 was investigated with an in vitro translation using a rabbit reticulocyte lysate. In order to produce a full-length ORF1 fragment, an experiment using runoff RNA products from the pUC19/SaV Mc10 full-length clone was originally performed. However, this was unsuccessful due to the low translation level (data not shown). Therefore, a DNA fragment containing the entire ORF1 was amplified by PCR with a set of primers, which included a T7 promoter sequence in the sense primer (Table 2). The ORF1 polyprotein was then expressed by using an in vitro coupled transcription-translation system in the presence of 35S-labeled methionine and cysteine (Fig. 1B, construct I). SDS-PAGE of the radiolabeled proteins followed by image analysis showed that at least seven proteins, p28, p32, p35, p46, p60, p66, and p120, were generated, demonstrating that ORF1 polyprotein "I-Prow" was translated and proteolytically cleaved (Fig. 2A, lane I-Prow). A time course analysis revealed that there was no accumulation of the 250-kDa primary translation product even at the early stage of the incubation, indicating that the processing of ORF1 occurred cotranslationally or rapidly after translation, as observed in RHDV, FCV, and NoV (1, 2, 11, 15, 20, 32).
To determine whether the cleavage is dependent on the virus-encoded 3C-like protease, a DNA fragment containing a C1171A mutation was prepared and used for an in vitro transcription-translation. As shown in the lane I-Promut (Fig. 2A), only a single major band of 250 kDa was observed. Thus, the viral protease is responsible for the processing, and the Cys residue in the GDCG motif is critical for the proteolytic activity, as seen in other caliciviruses (1-3, 15, 27, 32).
Immunoprecipitation with region-specific antibodies. To identify the cleavage products, an in vitro reaction mixture of I-Prow protein was subjected to the immunoprecipitation with region-specific antibodies (anti-A to -H antibodies). As shown in Fig. 2B, lane A, three major proteins of 11, 28, and 66 kDa were precipitated with anti-A antibody (Fig. 2B, lane A), whereas p11 was not identified as a specific cleavage product in the direct SDS-PAGE analysis due to an abundance of cellular proteins (Fig. 2A and 2B, lane I-Prow). Anti-B antibody precipitated 35-kDa proteins (Fig. 2B, lane B). Anti-C antibody precipitated 46-kDa proteins (Fig. 2B, lane C). Three major proteins of 14, 46, and 120 kDa were precipitated with anti-D antibody (Fig. 2B, lane D), whereas p14 was not detectable in the direct SDS-PAGE analysis due to an abundance of cellular proteins (Fig. 2B, lane I-Prow). Neither anti-E nor -F antibodies precipitated a specific cleavage product (Fig. 2B, lanes E and F), although these antibodies reacted with I-Promut and with proteins E and F expressed in E. coli (data not shown). Both anti-G and -H antibodies precipitated 60-kDa proteins (Fig. 2B, lanes G and H).
The above results indicated the following: (i) p11, p28, p35, and p66 were products derived from the N-terminal region of the ORF1; (ii) p14 and p46 were products derived from the central region of the ORF1; (iii) p46 was either unprocessed or stable p14-p32, since this protein was precipitated with both anti-C and -D antibodies; and (iv) p60 was a product derived from the C-terminal region of ORF1, since this protein was precipitated with both anti-G and -H antibodies. The cleavage products of ORF1, except p32 and p120, were thus identified by the region-specific antibodies. Furthermore, two additional products, p11 and p14, were found which were not detected by direct SDS-PAGE analysis.
Proteolytic processing of N- and C-terminally truncated translation products. To further analyze the cleavage products, five truncated templates—one C-terminally truncated, three N-terminally truncated, and one both C- and N-terminally truncated—were generated and expressed by an in vitro coupled transcription-translation system (Fig. 1B, II to VI). The expressed regions were selected according to the cleavage products sizes. We used identical boundaries to the regions to prepare the region-specific antibodies. The expression was carried out with both wild-type (designated as Prow) and C1171A mutant (designated as Promut) templates, and the 35S-labeled products were analyzed by direct SDS-PAGE (Fig. 3) and immunoprecipitation with region-specific antibodies (Fig. 4). When mutant proteins I- to VI-Promut were expressed, only single major protein bands of p250, p190, p180, p145, p130, or p70, which corresponded to their respective estimate sizes, were observed (Fig. 3, I- to VI-Promut). Similarly, VI-Prow was not further processed (Fig. 3, VI-Prow). In contrast, I- to V-Prow were extensively processed and the following cleavage patterns were obtained. (i) II-Prow was processed into p11, p28, p35, p32, p14, p66, p46, and p120, which were identical to I-Prow, except for p60, confirming that p60 is located in the C-terminal ORF1 (Fig. 3, II-Prow; Fig. 4, II Prow). (ii) III-Prow was cleaved into p32, p14, p70, p46, p120, and p60 (Fig. 3, III-Prow; Fig. 4, III-Prow). (iii) IV-Prow was cleaved into p14, p70, p84, and p60 (Fig. 3, IV-Prow; Fig. 4, IV-Prow); and (iv) V-Prow was processed into p70 and p60 (Fig. 3, V-Prow). p66 was identified as an unprocessed or stable p28-p35, since this protein was detected in additional N-terminally truncated Prow forms II' (corresponding to aa 70 to 1720) but not in II" (corresponding to aa 326 to 1720), and since the p66 that appeared in the cleavage products of Prow forms II' could be precipitated with anti-A antibody (data not shown).
Since p32 and p120 were found in I- to III-Prow but not in IV- or V-Prow, p32 and p120 are derived from the central region of ORF1 (Fig. 3, I- to V-Prow). Although p70 was difficult to detect in the I- and II-Prow processed products due to the nonspecific or the inappropriate internal translation protein(s) seen in I- and II-Promut (Fig. 3, I- and II-Promut), this protein appeared in the cleavage products of III-, IV-, and V- Prow (Fig. 3, III-, IV-, and V-Prow), and was identical in size to the VI-Prow and VI-Promut products (Fig. 3, VI-Prow and VI-Promut). p70 in III- to VI-Prow and VI-Promut was immunoprecipitated with both anti-E and -F antibodies (Fig. 4, III- to VI-Prow, and VI-Promut). Although the identification of p70 in I- and II-Prow by immunoprecipitation was not clear (Fig. 4, I- and II-Promut), we concluded that p70 was the cleavage product derived from the central region of ORF1. In addition, we observed p84 and assigned it to p14-p70 based on its molecular size and immunoreactivity to anti-D, -E, and -F antibodies. We did not further analyze this product in this study, because this protein was detected only in IV-Prow and is likely to be a construct-dependent unprocessed or stable product.
Cleavage map of SaV ORF1. The ORF1 cleavage map was generated using full-length and/or truncated forms of the ORF1 polyprotein. We concluded that Mc10 ORF1 polyprotein was processed into at least 10 major proteins—p11, p28, p35, p32, p66, p14, p46, p70, p120 and p60—by an in vitro coupled transcription-translation system. Seven of these products were arranged in the order NH2-p11-p28-p35-p32-p14-p70-p60-COOH, and the amino acid motifs indicated that p35, p14, p70, and p60 correspond to NTPase, VPg, Pro-Pol, and VP1, respectively (Fig. 1C). p66, p46, and p120 were identified as p28-p35 (NTPase), p32-p14 (VPg), and p32-p14 (VPg)-p70 (Pro-Pol).
The cleavage map and product sizes are strikingly similar to those of RHDV, except in the case of Pro-Pol, which is processed into Pro and Pol, and the RHDV polyprotein is arranged in the order NH2-p16-p23-p37 (NTPase)-p29-p13 (VPg)-p15 (Pro)-p58 (Pol)-p60 (VP1)-COOH (36).
Although p70 (Pro-Pol) was not further cleaved under the conditions used in this study, it should be noted that Pro-Pol has also been identified as a stable product in RHDV, FCV, and NoV in in vitro translation systems (1, 18, 32, 36). Extension of the incubation time from 1.5 h to 24 h or the presence of a canine pancreatic microsomal membrane fraction had no effect (data not shown). Further cleavage of Pro-Pol to Pro and Pol has been shown in RHDV and NoV, but not in FCV, when the Pro-Pol-containing region was expressed in mammalian cells (14, 20, 27, 31, 32), and a similar event was observed in RHDV, FCV, and NoV in E. coli (17, 29, 30, 32, 34, 35). These observations indicated that the cleavage of Pro-Pol to Pro and Pol is dependent on the expression system.
In this study, SaV ORF1 cleavage was investigated for the first time with full-length and N- and C-terminally truncated constructs of the ORF1 polyprotein either with or without mutation in 1171Cys to Ala of the GDCG motif derived from the GII Mc10 strain. After the in vitro transcription-translation reaction, the cleavage products were analyzed with region-specific antibodies against a panel of viral protein fragments. When both the N- and C-terminally truncated ORF1 constructs were expressed, the cleavage products of the expected size were generated from each truncated form of ORF1, indicating that our prediction of the boundaries of each product was almost accurate except for the N-terminal region. Our preliminary data showed that SaV Pro-containing region expressed in E. coli has cleavage activity between Q/G within the rhinovirus 3C-like protease recognition sequence as reported in Chiba virus (29; Oka et al. unpublished observation). This result supports the possibility that SaV protease has a similar dipeptide recognition pattern (cleaves after Q or E) to those of other caliciviral proteases. At the beginning of our study, four SaV full-length genome sequences including Mc10 were available, but now, seven strains of SaV full genome sequences which belong to GI, GII, and GIII, respectively, are available. Multiple alignments of the available SaV ORF1 amino acid sequence revealed the presence of the conserved dipeptide-QG, -QA, -EG, and -EA, which are likely used as cleavage sites. From these data, we speculated that selected sites, E940/A941 and E1055/A1056 in the central region, are likely the cleavage sites, but in the case of N- and C-terminal regions, several candidate sites exist close to the selected sites described in this study (data not shown); therefore, we did not conclude any real cleavage sites in this study. The identification of each cleavage site by another approach, i.e., N-terminal amino acid sequencing and/or site-directed mutagenesis of the candidate cleavage sites, is now under investigation.
In conclusion, we determined the SaV ORF1 cleavage map and showed that the viral 3C-like protease was shown to be responsible for this proteolytic processing, and the proteolytic activity was fully abolished by replacing 1171Cys within the GDCG motif of the 3C-like protease.
ACKNOWLEDGMENTS
This work was supported in part by a grant for Research on Emerging and Re-emerging Infectious Diseases from the Ministry of Health, Labor and Welfare of Japan. G. Hansman obtained a Ph.D. scholarship from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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