Institute of Molecular Agrobiology, 1 Research Link, The National University of Singapore, Singapore 1176041
Clinical Microbiology and Public Health Laboratory and Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 2QW, UK2
Xinjiang August 1st Agricultural University, Xinjiang, People's Republic of China3
Author for correspondence: Liu Ding Xiang.Fax +65 8727007. e-mail liudx{at}ima.org.sg
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Abstract |
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Introduction |
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Recently, the properties and functions of the non-structural proteins of GARs have begun to be elucidated. The NSP1 sequence is highly variable among GAR strains, especially those from different host species. However, NSP1 contains a cysteine-rich region in its N terminus that is conserved among GARs and group C rotaviruses (GCRs), suggesting the presence of a zinc finger motif and its essential role for the function of this protein. There is evidence suggesting that part or almost all of NSP1 of GAR is not essential for virus replication (Hua & Patton, 1994 ; Taniguchi et al., 1996
), as mutant viruses carrying genes encoding C-terminally truncated NSP1 proteins can grow in cell cultures. To date, non-defective C-terminal deletion of a large region has only been detected in NSP1 from GARs. NSP5 is a serine- and threonine-rich protein and has been shown to be an O-glycosylated (Gonzalez & Burrone, 1991
) and phosphorylated protein (Welch et al., 1989
; Afrikanova et al., 1996
; Poncet et al., 1997
). It is highly conserved among different GAR strains. NSP1, as well as NSP2 and NSP3, are RNA-binding proteins associated with replicative intermediates (Hua & Patton, 1994
). NSP5 was shown to be a kinase that autophosphorylates (Blackhall et al., 1997
). However, their roles in virus replication remain to be clarified.
Group B rotavirus (GBR) infections in humans have been reported (Hung et al., 1984 ; Bai & Shen, 1985
; Eiden et al., 1985
) and the viruses have been isolated from a variety of animal species. To our knowledge, only one strain was adapted to grow in cell culture (Sanekata et al., 1996
) and a few can be passaged in young animals (Vonderfecht et al., 1984
; Wang et al., 1989
). This is an obstacle in studying the molecular biology of GBRs. Currently, identification of genes of GBRs depends largely on sequence comparison with GAR strains and between GBR strains, RNARNA hybridization assays and analysis of proteins translated in vitro. The assignment of gene 6 (or 7) to NSP1 and gene 11 to NSP5 was based on the similarity of deduced amino acid sequences of GBRs to those of GARs; its validity needs to be confirmed. In contrast to the NSP1 genes of GARs and GCRs, the equivalent gene of GBRs contains two ORFs instead of one, although ORF1 has not been shown to be translatable even in in vitro translation systems (Eiden & Allen, 1992
). The ORF2-encoded protein shares the highest similarity with NSP1 among viral proteins of GARs. The deduced amino acid sequences encoded by ORFs 1 and 2 appear not to contain a zinc finger motif(s), commonly present in NSP1 of GARs and GCRs and assumed to be essential for the function of the protein. This raises an immediate question about the properties and function of the NSP1 gene of GBRs. NSP5 of GBRs has not been shown to be a phosphoprotein. However, its deduced amino acid sequence shows that it is a serine/threonine-rich protein, typical of NSP5 of GARs, despite it being only distantly related.
In this report, we have determined the complete sequences of genes 6 and 11 of an ovine GBR isolate, KB63. Sequence comparisons showed that they are cognate genes of GAR and GBR genes encoding NSP1 and NSP5; however, they share low similarity even with the equivalent genes of GBRs. Sequence analysis showed that gene 6 contains three overlapping ORFs. Of them, only ORFs 1 and 2 were translatable in a cell-free translation system. As ORFs 2 and 3 may be derived from a single ORF corresponding to ORF2 of the NSP1 gene of other GBR strains, the product encoded by ORF2 represents a C-terminally truncated form of the GBR NSP1 protein. Furthermore, as KB63 can be passaged further in young goats, this truncated protein may be non-defective. We also demonstrate that in vitro expression of gene 11 resulted in the phosphorylation of the in vitro-translated polypeptides. In view of the difficulty of adapting GBRs in a cell culture system and the relative rarity of the GBR isolates reported so far, this study has added useful information to our current understanding of the sequence diversity and functions of the non-structural proteins encoded by GBR genes 6 and 11.
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Methods |
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Extraction and PAGE of genomic dsRNA.
A partially purified virus suspension (0·5 ml) was used for extraction of dsRNA (Rodger & Holmes, 1979 ). The genomic segments were separated on 10% polyacrylamide gels and visualized by silver staining (Follett et al., 1984
). Viral RNA was further purified by using silica particles (Geneclean II) as described previously (Boom et al., 1990
) for amplification of the viral genome.
Amplification of viral genes by RTPCR.
A sequence-independent strategy developed by Lambden et al. (1992) was used to amplify the full-length dsRNA fragments of KB63. Briefly, 3'-blocked (amino) primer 1 (5' CCCGTCGACGAATTCTTT 3'NH2) was ligated to both 3' ends of dsRNA segments by using T4 RNA ligase (Life Technologies) according to the manufacturer's instructions. Unligated primer 1 was removed by chromatography on a Sephacryl S-400 (Promega) spin column. Primer 1-tailed dsRNA was denatured by heating to 90 °C for 5 min and cooled rapidly on ice in the presence of primer 2 (5' AAAGAATTCGTCGACGGG), the reverse complement of primer 1. Synthesis of cDNA was performed in a 50 µl reaction mixture containing 50 ng primer 1-tailed, primer 2-annealed dsRNA, 50 mM Tris-HCl (pH 8·3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0·5 mM each dNTP, 40 units RNasin (Promega) and 500 units Moloney murine leukaemia virus reverse transcriptase (Superscript, Life Technologies). After incubation at 37 °C for 1 h, the remaining RNA was hydrolysed by addition of 5 µl 1 M NaOH at 65 °C for 1 h followed by neutralization with 5 µl 1 M HCl and 5 µl 1 M TrisHCl (pH 7·5). The resulting plus and minus cDNA strands were annealed at 65 °C overnight and purified on a Sephacryl S-400 spin column.
The annealed strands were elongated with Taq DNA polymerase (Promega) at 72 °C for 5 min and 2 µl of the repaired cDNA and 100 ng primer 1 were used for PCR amplification. The PCR amplification was performed in a 50 µl reaction mixture containing 10 mM TrisHCl (pH 9·0 at 25 °C), 50 mM KCl, 1·5 mM MgCl2, 0·1% Triton X-100, 0·2 mM each dNTP and 1 unit Taq DNA polymerase. The PCR consisted of 35 cycles of denaturation at 94 °C for 20 s, annealing at 50 °C for 20 s and extension at 72 °C for 4 min (the extension time on the final cycle was increased to 10 min). After the sequences were obtained for both genes, RTPCR was also performed, as described above except that the extension time was decreased to 2 min, with primers specific for genes 6 and 11 of KB63. The location and orientation of the specific oligonucleotides used are shown in Fig. 2.
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DNA sequencing and analysis.
Dideoxynucleotide chain-termination sequencing with a Sequenase version 2.0 kit (United States Biochemical) or automated sequencing of both strands was carried out with SP6 and T7 primers. Direct sequencing of PCR products was carried out with primers specific for genes 6 and 11 of KB63 as described previously (Shen et al., 1994 ). Sequence data from gel reading was assembled by using the Staden sequence analysis program (Staden, 1982
, 1984
). Further analyses were carried out with the GCG suite of programs.
Northern blot hybridization.
Northern blot hybridization was performed by using either 32P-labelled probes prepared by reverse transcription of dsRNA of KB63 as described above or DIG-labelled probes prepared by transcription of plasmids pCR42 and pCR39, which contain full-length cDNAs of genes 6 and 11, respectively. DIG-labelled probes were prepared by using the DIG RNA labelling kit (Boehringer Mannheim) according to the manufacturer's protocol. After PAGE, dsRNA was transferred to nylon membrane (Hybond-N+, Amersham) and Northern blotting was carried out as described previously (Lambden et al., 1992 ).
Construction of plasmids.
Plasmids pCR42 and pCR39, which contain the full-length cDNAs of genes 6 and 11, respectively, were obtained by ligating RTPCR fragments directly into TA vector pCR II as described above.
For in vitro expression, full-length cDNAs of genes 6 and 11 were transferred into plasmid pKT0 (Liu et al., 1994 ), generating plasmids pKT42 (containing gene 6) and pKT39 (containing gene 11). This was achieved by cloning EcoRV/BamHI-digested cDNA fragments containing genes 6 and 11 into PvuII/BamHI-digested plasmid pKT0.
Plasmid pKT42, in which most of the ORF1 region was deleted, was constructed by inserting a PvuII/BamHI-digested cDNA fragment covering the gene 6 sequence from nucleotide 216 to the 3' terminus into PvuII/BamHI-digested pKT0.
Transcription and translation in vitro.
Plasmids were linearized with appropriate restriction enzymes, extracted with phenolchloroform and precipitated with ethanol. Transcription was performed by using T7 RNA polymerase (Promega) as described by Sambrook et al. (1989) . Translation of in vitro-synthesized transcripts was carried out in rabbit reticulocyte lysates (RRL, Promega) or wheatgerm extracts (WGE, Promega) according to the manufacturer's instructions. After incubation at 30 °C for 1 h, aliquots of [35S]methionine-labelled polypeptides were analysed by SDSPAGE and detected by autoradiography.
Dephosphorylation of polypeptides expressed in vitro.
[35S]Methionine-labelled proteins synthesized in vitro were dephosphorylated by using calf intestinal alkaline phosphatase (CIP, Promega) in a 20 µl reaction mixture containing 50 mM TrisHCl (pH 9·3), 10 mM MgCl2, 1 mM ZnCl2 and 10 mM spermidine. After 1 h incubation at 37 °C, the samples were mixed with an equal volume of 2x loading buffer (Laemmli, 1970 ) and analysed on 15% SDSpolyacrylamide gels.
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Results |
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Sequence analysis of KB63 gene 6
The nucleotide sequence of gene 6 was deduced initially from the insert contained within plasmid pCR42 and was confirmed in two ways. The sequences of the 5' and 3' ends were confirmed by sequencing three other copies of the gene identified during the initial screening of insert-containing plasmids. An internal 998 bp fragment was amplified directly from viral RNA by RTPCR by using three oligonucleotide primers (SS-1, SS-2 and SS-5) complementary to the sequence initially determined from pCR42 (Fig. 2). RTPCR with these primers generated fragments of the expected sizes that were subsequently sequenced.
Data generated from these analyses showed that gene 6 of KB63 contained 1275 nucleotides and three long overlapping ORFs (ORFs 13) with the potential to encode polypeptides of 101, 175 and 146 amino acids, respectively (Fig. 2). These three ORFs were flanked by untranslated regions of 42 nucleotides at the 5' end and 60 nucleotides at the 3' end (Fig. 2
). A 95 nucleotide overlap was present between ORFs 1 and 2 and there was a 7 nucleotide overlap between ORFs 2 and 3. The AUG start codon for ORF2 was flanked by a sequence (GAAATGG) associated with efficient initiation (Kozak, 1981
). The sequences flanking the start codons for ORF1 and ORF3 contained pyrimidines in the -3 and +4 positions, respectively, and thus would be expected to direct translation less efficiently than that of ORF2.
Sequence comparison (Table 1) revealed nucleotide similarity between KB63 gene 6 and ADRV gene 6 as well as IDIR gene 7 (54·2% and 54·7% identity, respectively), which have been proposed to encode the NSP1 genes of GBRs (Eiden, 1994
; E. R. Mackow, unpublished results, GenBank accession no. M91435). As mentioned above, KB63 gene 6 contains three ORFs, while the NSP1 genes of both ADRV and IDIR contain only two. Deduced amino acid sequences of ORFs 13 of KB63 were compared and aligned with the deduced amino acid sequences of ORF1 (Table 1
and Fig. 3
a) and ORF2 of the ADRV and IDIR NSP1 genes. Amino acid identity ranged from 34·9 to 48·0%, with polypeptide 2 showing the most conservation and polypeptide 3 the least (Table 1
). These results confirm the findings of the nucleotide comparisons and indicate that KB63 gene 6 ORF1 is homologous to ORF1 of other GBRs, while ORF3 was probably derived from ORF2 by a frame shift event. A single base insertion (or two-base deletion) upstream of the ORF2 stop codon would lead to the formation of a single ORF encoding a polypeptide just one (or two) amino acid(s) shorter than the equivalent product of IDIR (Fig. 3b
). The low level of identity between these sequences made it difficult to define the position of any deletion or insertion. It might have occurred between nucleotides 740 and 750, since an insertion or a deletion in this region would maintain CXXC upstream and RWXXXGXGX downstream, which are conserved among the three strains (Fig. 3b
). The sequence of this region was reconfirmed four times from independently amplified RTPCR products to reduce the likelihood that the proposed frame shift was the result of an artifact introduced during RTPCR or sequencing.
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In the RRL system, no product could be detected when ORF1 RNA was translated (Fig. 5, lane 2). In vitro translation of the full-length RNA (lane 3), ORF2/3 RNA (lane 4) and ORF2 RNA (lane 5) all resulted in the detection of two proteins with apparent molecular masses of 23 and 25 kDa. These results suggest that both the 23 and 25 kDa products may be derived from ORF2, although their apparent molecular masses are higher than the 20 kDa calculated from the amino acid sequence. The 23 kDa protein may represent a premature termination product of ORF2, although post-translational modification of the polypeptide cannot be excluded.
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In vitro expression and dephosphorylation of KB63 gene 11 products
In vitro transcription of KB63 gene 11 was performed by using plasmid pKT39 digested with BamHI. The RNA was translated in the RRL system and the translated products were analysed on 15% SDSpolyacrylamide gels. Four major bands, with apparent molecular masses of 19, 21, 23 and 25 kDa, were produced (Fig. 6, lane 2). Since the apparent molecular masses of three of the major bands were higher than the calculated molecular mass of 18·7 kDa, pKT39 was linearized with SspI, which cuts at nucleotide 483 (133 bases upstream the stop codon), and transcribed and translated in vitro. The result (lane 4) suggested that only the 25 kDa species was the full-length product, while the 19, 21 and 23 kDa bands may be premature termination products. In vitro translation of gene 11 was also carried out in the WGE system and similar results were obtained (data not shown).
Interestingly, at least three faint bands were present above the major 25 kDa band, one above the 19 kDa and one above the 21 kDa proteins. NSP5 of GARs has been reported to be phosphorylated both in intact cells and in a cell-free system (Afrikanova et al., 1996 ; Blackhall et al., 1997
; Poncet et al., 1997
) and the gene product itself has been proposed to be a kinase which autophosphorylates. To determine whether these faint, higher molecular mass bands were the result of phosphorylation, CIP was used to dephosphorylate the in vitro-translated products. Treatment of in vitro-synthesized proteins with CIP resulted in the disappearance of the three faint bands above the 25 kDa species (Fig. 6
, lanes 1 and 2). A similar effect was also observed when the truncated products were treated with CIP (lanes 3 and 4). These results indicate that the in vitro-translated products of this gene were indeed phosphorylated.
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Discussion |
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Alignment of the putative gene 6 sequence with its homologues in other GBRs revealed significant diversity. Amino acid identity with ADRV and IDIR was only 34·9 and 48·0%, respectively, while the equivalent value between ADRV and IDIR was more than 63·5% (NSP1 encoded by ORF2). These values fall well within the range of identities (24·697·0%) found when the NSP1 proteins of GARs were compared and suggest that the sequence diversity that characterizes this protein in GARs is duplicated in GBRs. One significant discrepancy between the NSP1 gene of GBRs and those of GARs and GCRs is the absence of the conserved zinc finger motif located in the N-terminal region of the protein in the latter two groups of viruses. Its absence from the sequence of ADRV and IDIR is supported by the more divergent sequence of KB63 reported here and raises questions about the functional relatedness of the NSP1 proteins of these three groups of viruses. Another interesting result yielded by the sequence of KB63 gene 6 is the presence of three overlapping ORFs. Bicistronic genomic segments have been reported in reoviruses, GARs and the two GBR strains sequenced to date. However, while expression of two ORFs has been documented for the reovirus S1 gene and rotavirus (SA11) NSP5 gene, only one ORF (ORF2) has been shown to be expressed from the GBR NSP1 gene (Ernst & Shatkin, 1985 ; Belli & Samuel, 1991
; Mattion et al., 1991
). We have demonstrated that the first two of the three ORFs of KB63 gene 6 were expressed in the WGE translation system, supporting its designation as a bicistronic genome segment.
On the basis of the sequence analysis, we propose that ORF3 may have resulted from a single base deletion (or two-base insertion) near the 3' end of ORF2. No expression from this ORF could be detected in either the RRL or WGE system, although adaptation of the virus to growth in cell culture or generation of specific antiserum would be required to confirm that it is not expressed in vivo. This apparent C-terminal deletion of the original ORF2 is clearly not a cell culture artifact, since KB63 was isolated from and passaged in young goats prior to analysis. Such C-terminal deletions of gene 6 ORF2 have not been reported previously for GBRs but are corroborated by similar findings in viable GARs. Three non-defective mutants encoding partially or almost-totally C-terminally truncated NSP1 have been isolated (Hua & Patton, 1994 ; Taniguchi et al., 1996
). These mutants appear to have emerged as a result of a point mutation, a deletion and a mutation accompanied by a gene rearrangement. The results presented in this study suggest that, as in the case of GARs, a large part of GBR NSP1 is non-essential for virus replication. In addition, the demonstration that the C-terminal portion of NSP1 is non-essential for replication provides some phenotypic support for the classification of gene 6 ORF2 as NSP1, which to date has been based on sequence comparison alone (Eiden, 1994
).
Gene 11 of KB63 shows a high degree of sequence diversity at the amino acid level when compared with those of other sequenced GBRs (54·1% vs 79·4% identity). This observation is somewhat surprising, given the high degree of conservation of reported NSP5 sequences from GARs, which exceeds 78% identity (Blackhall et al., 1997 ). This low level of absolute amino acid identity is all the more interesting since analysis of the amino acids within the protein shows high levels of serine and threonine, typical of NSP5 characterized from GARs and GBRs. Thus, KB63 NSP5 may play a structurally distinct role in rotavirus replication. The diversity of KB63 NSP5 also enables us to identify three conserved and two variable regions that may be useful for future determination of functional domains.
Recent studies have demonstrated that NSP5 from GAR strains SA11 and RF is phosphorylated in vitro and in cell culture and suggested that it functions as a serine/threonine kinase that can autophosphorylate in vitro (Afrikanova et al., 1996 ; Poncet et al., 1997
; Blackhall et al., 1997
). In addition, this protein is located in viroplasms and appears to be associated with NSP2, suggesting that it might play a role in the formation of the virus replication machinery. We report that KB63 NSP5 is phosphorylated in a phenotypically similar manner to its homologues amongst GARs, despite its low level of absolute amino acid identity. Once again, this observation provides phenotypic support for the designation of this gene as NSP5. Investigation of whether this protein is also present within viroplasms will have to await adaptation of the virus to cell culture and generation of other reagents.
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Acknowledgments |
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Footnotes |
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References |
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Received 25 January 1999;
accepted 12 April 1999.