1 CENTEX Shrimp, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
2 Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
3 CSIRO Livestock Industries, Long Pocket Laboratories, Indooroopilly, Queensland, Australia
Correspondence
Sarawut Jitrapakdee (at Department of Biochemistry)
scsji{at}mahidol.ac.th
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ABSTRACT |
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The nucleotide sequence reported in this paper has been submitted to GenBank with accession number AF540644.
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INTRODUCTION |
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Yellow head virus (YHV) is an enveloped, rod-shaped particle (approximately 40 nmx170 nm) with prominent surface projections (approximately 11 nm) and an inner helical nucleocapsid (Chantanachookin et al., 1993; Wang & Chang, 2000
; Loh et al., 1997
). Primarily based on the virion morphology and the presence of a single-stranded RNA genome (Wongteerasupaya et al., 1995
), YHV was previously reported as a rhabdovirus (Nadala et al., 1997
). However, it was subsequently demonstrated that the YHV genome is positive-sense RNA (Tang & Lightner, 1998
). Sequence analysis has also revealed that, like the closely related gill-associated virus (GAV) from Australia (Cowley et al., 1999
, 2000
), YHV contains a large replicase gene (ORF1b) that appears to be expressed as a polyprotein by ribosomal frame-shift at a slippery sequence upstream of a predicted pseudoknot structure (Sittidilokratna et al., 2002
). Considerations of sequence identity, genome organization and gene expression have indicated that GAV and YHV are related to coronaviruses, toroviruses and arteriviruses and are classified in new taxa (family Roniviridae, genus Okavirus) within the order Nidovirales (Cowley et al., 2000
; Sittidilokratna et al., 2002
; Cowley & Walker, 2002
).
Nadala et al. (1997) originally reported that YHV particles contain four structural proteins of approximately 170, 135, 67 and 22 kDa, of which the 135 kDa protein was glycosylated. However, Wang & Chang (2000)
subsequently reported only three major YHV proteins (110, 63 and 20 kDa), suggesting that the larger protein may be of cellular origin. In GAV only two genes (ORF2 and ORF3), located immediately downstream of the ORF1b gene, have been predicted to encode structural protein (Cowley et al., 2001
). ORF2 encodes a 22 kDa structural protein that appears to function as the nucleoprotein (J. A. Cowley and others, unpublished), and ORF3 encodes a polypeptide with the structural characteristics of a large glycoprotein with multiple membrane-spanning domains (Cowley & Walker, 2002
). In this paper, we report the nucleotide and deduced amino acid sequences of the YHV ORF3 region. We show that the region encodes the major viral structural glycoproteins (gp116 and gp64): these are synthesized as a polyprotein which undergoes post-translational proteolytic cleavage and glycosylation.
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METHODS |
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SDS-PAGE and N-terminal sequencing.
Protein samples (10 µg) were analysed by SDS-PAGE (Laemmli, 1970) under reducing conditions in 7·5 % or 15 % discontinuous gels. The fractionated proteins were transferred to a PVDF membrane in transfer buffer (10 mM CAPS/10 % methanol) using a semi-dry blotter (Hoeffer). Protein bands blotted onto the membrane were stained briefly in 0·3 % Coomassie brilliant blue R250. The protein bands were then excised and analysed by N-terminal sequencing. Edman degradation was done with an Applied Biosystems Sequencer at the Center for Genetic Engineering and Biotechnology, Bangkok, Thailand.
Polyclonal antibody production and immunoblot analysis.
Nitrocellulose-bound antigen was prepared as described by Diano et al. (1998). Briefly, 500 µg aliquots of total viral protein were blotted onto a nitrocellulose membrane (Amersham Pharmacia), excised and ground in 0·5 ml PBS (140 mM NaCl, 4 mM KCl, 2 mM KH2PO4, 8 mM Na2HPO4, pH 7·4) in liquid nitrogen. This antigen was emulsified with an equal volume of Freund's complete adjuvant (Sigma) and used to immunize two BALB/c mice by intraperitoneal injection. Mice were boosted at 1 week intervals with 100 µg of viral protein in Freund's incomplete adjuvant. The antisera were collected 1 week after the second boost and used for immunoblot analysis.
Proteins were transferred to a nitrocellulose membrane using a semi-dry electroblotting apparatus (Hoeffer). Following transfer, the membrane was blocked in 3 % BSA, 0·5 % Tween 20 in PBS for 3 h. The membrane was washed briefly in the same buffer without BSA and then reacted with a 1 : 10 000 dilution of mouse antiserum for 1 h. Goat anti-mouse polyclonal antibodies conjugated with alkaline phosphatase were then reacted for 2 h and immuno-reactive proteins were visualized by adding nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indoyl phosphate (BCIP).
Glycoprotein staining.
Glycoprotein detection was performed using the ECL glycoprotein detection system (Amersham Pharmacia). Proteins blotted onto PVDF membranes were oxidized with 10 mM sodium metaperiodate in 100 mM acetate buffer pH 5·5 at room temperature for 20 min in the dark. The membrane was then reacted with 0·35 mM biotin hydrazide in 100 mM acetate buffer pH 5·5 for 1 h, followed by incubation with streptavidin conjugated with alkaline phosphatase. The glycoprotein bands were visualized by adding NBT and BCIP. Thymol staining was done as described by Racusen (1979).
RT-PCR, cloning and sequencing.
YHV genomic RNA was extracted from purified virus using TRIzol Reagent (Invitrogen) and resuspended in DEPC-treated water. RT-PCR was performed in a total volume of 25 µl containing 200 ng genomic RNA, 0·4 mM of a forward primer (5'-GATCGGGGTACCTAAGCTTATGCTATCGACCTA-3') designed from the 3'-end of the ORF1b gene and an oligo(dT) primer (5'-TCTAGAGGATCCC-CGGTACCTTTTTTTTTTTTTTTTTTTT-3'). The SuperScript one-step RT-PCR system (Invitrogen) was used in the presence of 1 unit of Elongase (Invitrogen) according to the instruction manual. The RT-PCR profile consisted of an initial incubation at 50 °C for 30 min, 94 °C for 2 min followed by 35 cycles of amplification. Each cycle consisted of denaturation at 94 °C for 30 s and annealing/extension at 68 °C for 8 min. The RT-PCR product was either sequenced directly or cloned in pGEM-T Easy vector (Promega). The RT-PCR product was gel-purified using the QIAquick Gel Extraction kit (Qiagen) and directly sequenced using Big Dye reagent (ABI). Nucleotide sequences obtained from initial reactions were used to design new primers to generate overlapping sequences toward the 5'- and the 3'-ends of the fragment. Sequence chromatograms were then analysed and a consensus sequenced generated using SeqEd 1.0.3 (ABI).
Recombinant protein expression in E. coli.
The cDNA encoding full-length gp64 was generated by PCR using forward primer (YHV-D7) 5'-GCCTCTAGACATATGCTCGCTCCACGACAGGCACGTGTT-3' and reverse primer (YHV-D8) 5'-CATTGTGGATCCTCACTAGTGATGATGATGATGATGGGATCGTTTGGCTTTCGTTCTCATGGACGT-3'. The forward primer was designed from residues L1128 to V1135 in the ORF3 polyprotein and included an initiation codon and an NdeI restriction site (underlined). The reverse primer was designed from residues T1657 to S1666 and included stop codons (bold) and a BamHI restriction site (underlined). PCR was performed in a 25 µl reaction mixture containing 1x PCR buffer (10 mM Tris/HCl pH 8·3, 50 mM KCl, 1·5 mM MgCl2, 0·1 % Triton X-100), 0·2 mM of each dNTP, 1 ng oligo-primed cDNA, 0·25 µM of each primer and 2 units of Taq DNA polymerase (Perkin Elmer). The reaction mixture was subjected to 35 cycles of denaturation at 94 °C/30 s, annealing/extension at 68 °C/2 min, and followed by the final extension at 72 °C/10 min. The PCR products were cut with NdeI and EcoRI and ligated into the multiple cloning site of pET17b (Novagen). A transmembrane-deleted construct was generated by removal of 40 C-terminal residues (Y1627S1666) by PCR using primers YHV-D7 and YHV-D14 [5'-GAATTCTCACTAATCCCATGTCTTGCCGCCGAA; corresponding to residues F1620D1626 with stop codons (bold) and an EcoRI site extended at the 5'-end]. The PCR product was cloned into pDrive vector (Qiagen) and sequenced. The insert was digested with NdeI and EcoRI and cloned into the multiple cloning site of pET17b. The recombinant plasmids were then transformed into E. coli BL21(DE3) (Novagen). An overnight culture of BL21(DE3) was diluted 1 : 20 with fresh LB broth and grown at 37 °C to an OD600 of 0·50·6. The culture was then induced with 1 mM IPTG for 17 h. A 1 ml volume of the culture was pelleted, suspended in protein loading buffer, heated at 100 °C for 10 min and analysed by SDS-PAGE. The recombinant protein expressed in E. coli was confirmed by immunoblot analysis using gp64 polyclonal antiserum.
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RESULTS |
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Each of the three YHV structural proteins (gp116, gp64 and p20) was subjected to N-terminal sequence analysis. The N-terminal sequences for glycoproteins gp116 and gp64 were determined to be T-I-L-S-G-I-P-E-K-D- and L-A-P-R-Q-A-R-V-X-G- (X, uncertain residue), respectively. No sequence was obtained for protein p20, which appeared to be blocked at the N terminus.
Nucleotide sequence and deduced amino acid sequence of ORF3
A region of the YHV genome extending from the 3'-poly(A) tail to a locus at the 3'-end of the ORF1b gene was amplified by RT-PCR. Analysis of the amplified product by agarose gel electrophoresis revealed a single band of approximately 6·0 kbp. By comparison with the known complete sequence of GAV (Cowley et al., 2000; Cowley & Walker, 2002
) and available partial sequence data on the YHV genome (N. Sittidilokratna and others, unpublished data), the size of the amplified product was consistent with the expected size of 3'-end of YHV genome (Fig. 2
). Nucleotide sequence analysis revealed that the region contained two long open reading frames in the same sense (+) as ORF1b. The largest open reading frame (ORF3) commenced 848 nucleotides downstream of the ORF1b termination codon and comprised 4998 nucleotides. The complete nucleotide sequence and deduced amino acid sequence of YHV ORF3 is shown in Fig. 3
. ORF3 encodes a polypeptide of 1666 amino acids with a predicted molecular mass of 185 713 Da and a pI of 6·68. Alignment with the N-terminal sequences of mature gp116 and gp64 identified perfect identity with residues T229E236 and L1128V1135 respectively of the encoded polypeptide. The data indicated that gp116 and gp64 are derived by post-translational proteolysis of the ORF3 polyprotein. Based on the identified N-terminal sequences and presumed sites of proteolysis, the calculated molecular mass for unglycosylated gp116 was 101 734 Da and gp64 was 58 599 Da. The predicted size of these products was consistent with evidence of post-translational glycosylation of each protein (Fig. 1B, C
).
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DISCUSSION |
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Nadala et al. (1997) reported previously that YHV contains four major structural proteins (molecular masses 170, 135, 67 and 22 kDa) of which only the 135 kDa protein was reported to be glycosylated. Wang & Chang (2000)
reported only three structural proteins (molecular masses 110, 63 and 20 kDa). Our results are in close agreement with this more recent report and we demonstrate that each of the larger structural proteins is glycosylated. Small differences in the molecular mass of the structural proteins could well be due to differences in the conditions of electrophoresis or to variations in the pattern of glycosylation of different YHV isolates.
N-terminal sequence analysis of gp116 and gp64 allowed precise identification of sequences encoding these proteins within the 1666 amino acid ORF3 polyprotein. The size of the YHV polyprotein is similar to those of the glycoproteins of vertebrates coronaviruses including avian infectious bronchitis virus (1160 amino acids) (Binns et al., 1985), feline infectious peritonitis virus (1452 amino acids) (de Groot et al., 1987a
), murine hepatitis virus (1376 amino acids) (Spann et al., 1988
), porcine epidemic diarrhoea virus (Duarte & Laude, 1994
) (1383 amino acids) and human respiratory coronavirus (1353 amino acids) (Mounir & Talbot, 1993
). However, the predicted structure of the ORF3 polyprotein is more complex, comprising six putative transmembrane regions generating three ectodomains and two intra-virion endodomains. By contrast, coronaviruses contain a single transmembrane domain located near the C terminus of the spike polyprotein.
The envelope spikes of vertebrate coronaviruses and toroviruses comprise a large (180 kDa) glycoprotein that is often cleaved by a cellular protease to yield two similarly sized subunits (S1 and S2) which remain non-covalently associated in virions (Sturman et al., 1985
; Cavanagh, 1995
; Snijder & Horzinek, 1995
). In YHV, gp64 and gp116 are also generated by proteolysis of a
180 kDa polyprotein. Although each is rich in cysteine residues, SDS-PAGE under non-reducing conditions indicated that gp116 and gp64 are not covalently associated. It is possible that, like the S1 and S2 subunits of coronavirus surface glycoprotein, gp64 and gp116 are associated non-covalently to form the peplomers evident on the virion surface. In coronaviruses, the S2 subunit appears to form the membrane-bound stalk while the S1 subunit forms the globular head of the spike (de Groot et al., 1987b
) which interacts with the host cell receptor (Kubo et al., 1994
; Suzuki & Taguchi, 1996
). The S2 subunit contains several functional domains including a membrane anchor, six strictly conserved cysteine residues and a leucine zipper motif (Britton, 1991
) which has been shown to mediate the oligomerization of the subunits and induce cell fusion (Grosse & Siddell, 1994
; Luo et al., 1999
). The S2 ectodomain also contains two large amphipathic
-helices with a heptad repeat that have been proposed to mediate coiled-coil interchain interactions (de Groot et al., 1987b
). As reported for GAV (Cowley & Walker, 2002
), examination of YHV gp64 and gp116 sequences revealed no heptad repeats or significant amphipathic
-helices, suggesting an absence of coiled-coil structures. Further work is required to determine whether the processed gp116 and gp64 are associated to form the envelope spikes.
Sequence analysis was facilitated by use of an oligo(dT) primer to amplify the 3'-terminal region of the YHV genome. This approach was adopted in the expectation that the YHV genome would be polyadenylated a characteristic of the viruses in the order Nidovirales. The sequence data showed that the ORF3 gene, encoding the YHV structural glycoproteins, is located downstream of the ORF1b gene that encodes replication enzymes and has features characteristic of nidoviruses (Sittidilokratna et al., 2002). In GAV, ORF2 (located in the genome between ORF1b and ORF3) appears to encode a 22 kDa nucleoprotein (J. A. Cowley and others, unpublished) and this corresponds to the major YHV virion protein p20. Clearly, GAV and YHV are closely related in sequence and share a common gene organization which appears unique amongst nidoviruses in that the nucleoprotein gene (ORF2) is upstream of the structural glycoprotein gene (ORF3) and there is no discrete gene encoding the integral membrane (M) protein (Cowley & Walker, 2002
). However, it is evident from the location of proteolytic cleavage sites in the YHV ORF3 polyprotein that the N-terminal fragment has not yet been identified in virions or infected cells. If no further cleavage occurs, this 227 amino acid (25·4 kDa) fragment will be of similar size to the integral membrane proteins of coronaviruses and toroviruses (225262 amino acids) which occur abundantly in virions and appear to have a role in intracellular budding (Rottier, 1995
). The YHV ORF3 N-terminal fragment also contains three membrane-spanning domains characteristic of the M proteins but the predicted membrane topology is in the reverse orientation, with an N-terminal cytoplasmic tail and the C terminus oriented external to the membrane. Also, as p20 appears to be the nucleoprotein encoded in ORF2 (J. A. Cowley and others, unpublished), there is no evidence that YHV has a major virion component corresponding to the coronavirus M protein. The use of antibodies against suitable peptides in the N-terminal domain should assist identification and characterization of the N-terminal fragment of the ORF3 polyprotein.
The molecular masses of gp116 and gp64 calculated from the nucleotide sequence are smaller (15 kDa and
4 kDa, respectively) than those estimated by SDS-PAGE for the mature forms of the proteins. The size differences can be explained in part by glycosylation as revealed by the detection of carbohydrate (Fig. 1
) and the presence of putative N- and O-linked glycosylation sites in each protein. Although the expression level of recombinant gp64 was relatively poor, it was readily detected using polyclonal antibodies raised against the native form. The size of recombinant gp64 was consistent with the molecular mass calculated from deduced amino acid sequence. Poor expression of gp64 in E. coli suggests that the protein is unstable and may not be properly folded when expressed independently of gp116. An attempt to express gp64 as the fusion protein with glutathione S-transferase failed to improve the yield but expression was improved after removal of the C-terminal transmembrane domain (Fig. 6C
). Similar results were obtained when forms of gp116 with and without transmembrane domain were expressed in E. coli (data not shown). Expression of these proteins in insect cells, which may present an intracellular environment similar to shrimp cells, could offer a more useful approach to investigations of the processing and assembly of the glycoproteins encoded in YHV ORF3.
Overall, these data indicate that, despite defining similarities in the ORF1b region, YHV, like GAV, has a genome organization and glycoprotein expression strategy that are fundamentally different from vertebrate nidoviruses. The unique features of the viral structural glycoproteins support the classification of YHV with GAV as new taxa (family Roniviridae, genus Okavirus) within the Nidovirales (Cowley et al., 2000; Sittidilokratna et al., 2002
; Cowley & Walker, 2002
).
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ACKNOWLEDGEMENTS |
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Received 4 September 2002;
accepted 26 November 2002.