Molecular characterization of human group C rotavirus genes 6, 7 and 9

Vivienne L. A. James1, Paul R. Lambden2, Yu Deng2, E. Owen Caul3 and Ian N. Clarke2

Public Health Laboratory1 and Department of Molecular Microbiology, University Medical School2, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, UK
Regional Virus Laboratory, Public Health Laboratory, Myrtle Road, Bristol BS2 8EL, UK3

Author for correspondence: Vivienne James. Present address: Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK. Fax +44 20 8205 1488. e-mail vjames{at}phls.nhs.uk


   Abstract
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Abstract
Introduction
Methods
Results and Discussion
References
 
Genes 6, 7 and 9 of human group C rotavirus ‘Bristol’ strain, encoding non-structural proteins (NSP) 3, 1 and 2, respectively, were cloned and sequenced. Human group C rotavirus genome segment 6 is 1350 bp and contains a single ORF of 1231 nucleotides (encoding 402 amino acids). Genome segment 7 is 1270 bp and encodes a protein of 394 amino acids and genome segment 9 is 1037 bp and encodes a 312 amino acid protein. The human group C rotavirus genes 6, 7 and 9 showed 78, 67 and 88% sequence identity, respectively, to the corresponding porcine group C rotavirus genes. The derived protein sequences were compared with those of the porcine ‘Cowden’ group C and mammalian group A rotavirus strains. The human group C rotavirus NSP1 protein sequence is one amino acid longer than the porcine group C equivalent. In common with group A and porcine group C rotaviruses, the human group C rotavirus NSP1 protein has a zinc finger motif. Human group C rotavirus NSP2 has two hydrophobic heptad repeat regions, a basic, RNA-binding domain and a basic, proline-rich region. Human group C rotavirus NSP3 has both single- and double-stranded RNA-binding domains and several hydrophobic heptad repeat regions, one of which forms a leucine zipper. This work completes the molecular characterization of the non-structural proteins of a human group C rotavirus. Phylogenetic analysis of all the non-structural genes of group A, B and C rotaviruses suggests that these viruses have diverged at a constant rate from a common ancestor.


   Introduction
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Abstract
Introduction
Methods
Results and Discussion
References
 
Rotaviruses are a genus of the family Reoviridae and are an important cause of gastroenteritis in humans and animals. Seven distinct rotavirus groups (A–G) have been described (Kapikian & Chanock, 1996 ), but only groups A, B and C have been identified in both humans and animals. Group A rotaviruses were recognized as human pathogens in 1973 (Bishop et al., 1973 ; Flewett et al., 1973 ). Group A rotaviruses are endemic and are the major cause of severe gastroenteritis in infants under 2 years of age. Human group B rotaviruses were originally described in 1983 (Hung et al., 1983 ) and have been responsible for several large outbreaks of severe gastroenteritis in adults in China. Although serological studies have suggested that infection with group B rotaviruses has occurred in other countries (Brown et al., 1987 ; Hung et al., 1987 ; Nakata et al., 1987 ), the virus was first reported outside China in 1999 (Krishnan et al., 1999 ). In this Indian study, group B rotaviruses were confirmed as the cause of five cases of severe adult diarrhoea in Calcutta during 1997–98. Group C rotavirus (Cowden strain) was first described in 1980 in pigs (Saif et al., 1980 ) and, in 1982, the first human group C rotavirus was described (Rodger et al., 1982 ). Sporadic cases and limited outbreaks, affecting both adults and children, have since been reported worldwide. Although serological evidence existed previously for group C rotavirus infections in cattle, the bovine ‘Shintoku’ strain was only isolated in 1991 (Tsunemitsu et al., 1991 ).

The rotavirus genome consists of 11 segments of double-stranded (ds) RNA that encode six structural and five non-structural proteins. The different rotavirus groups can be identified by the characteristic profile of the dsRNA on SDS–PAGE and defined by terminal fingerprint analysis of the genome segments (Pedley et al., 1986 ). Sequence data are available for all eleven genome segments of the porcine group C rotavirus ‘Cowden’ strain, although many of these are incomplete and do not have defined 5' and 3' termini. Sequences are also available for genome segments 3–6, 8 and 10 of the bovine group C rotavirus ‘Shintoku’ strain. Genes corresponding to the structural proteins VP1 and VP2 and the non-structural proteins NSP1, 2 and 3 of human group C rotavirus have not been determined. The purpose of this work was to determine the coding assignments and sequences of the non-structural proteins NSP1, 2 and 3 of human group C rotavirus and to compare them with the corresponding porcine group C and mammalian group A rotavirus proteins.


   Methods
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Abstract
Introduction
Methods
Results and Discussion
References
 
{blacksquare} RNA extraction, cDNA synthesis and coding assignment.
Human group C rotavirus ‘Bristol’ strain has been described previously (Caul et al.,1990 ). Rotavirus dsRNA was extracted and purified from a faecal sample using the RNAzolB and Geneclean II procedures as described previously (Lambden & Clarke, 1995 ). Random-primed cDNA was synthesized and purified by Sephacryl S-400 spin-column chromatography as described previously (James et al., 1998 ).

M13 clones for each of the 11 gene segments of human group C rotavirus ‘Bristol’ strain have been constructed and stored as an ordered genomic library (Lambden et al., 1992 ). Briefly, the cloning method involved ligation of a single amino-linked modified oligonucleotide to the 3' termini of each dsRNA genome segment by using T4 RNA ligase. The tailed RNA was then converted to cDNA by using a complementary primer and reverse transcriptase. The resultant cDNA was annealed and repaired and then amplified by PCR by using a single complementary oligonucleotide primer. The PCR products were ligated into dephosphorylated, SmaI-cleaved M13 mp8 and transformed into E. coli JM101. This method increased the probability that the resultant clones would contain full-length inserts of the genes.

32P-labelled cDNA probes used for gene assignment studies were generated as described previously (Deng et al., 1995 ) and the coding assignments were determined by Northern blot analysis.

{blacksquare} Sequence analysis.
Sequences were compiled from M13 clones and by direct sequencing of RT–PCR amplicons. Recombinant M13 templates were prepared by standard techniques and sequenced initially with universal primer designed to hybridize 49 bp upstream of the SmaI cloning site by using the ABI PRISM terminator cycle sequencing kit (Applied Biosystems) and an Applied Biosystems model 373A automated sequencer. The complete sequences were determined by preparing primers and sequencing stepwise along the gene from each previous sequence. Internal primer sequences were designed approximately 150 bp upstream of the previously deduced sequence and resulted in read-lengths of 400–500 bp. The oligonucleotides were synthesized on a Millipore Expedite 8909 automated synthesizer by using {beta}-cyanoethyl phosphoramidite chemistry. Computer analyses of the sequence data were performed by using the Lasergene software (DNASTAR).


   Results and Discussion
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
Analysis of the M13 recombinants confirmed that they were full-length clones containing terminal sequences matching the group C rotavirus consensus 5' and 3' termini, GGC(A/T)4AA and TGTGGCT, respectively (Lambden & Clarke, 1995 ). More than 92% of the gene sequences were determined by direct sequencing of the RT–PCR amplicons and included the sequences of the entire ORFs. Northern blot analysis of group C rotavirus dsRNA with probes representing the full-length NSP1, NSP2 and NSP3 inserts confirmed the coding assignments of the clones for genes 6, 7 and 9 (data not shown).

Gene segment 6
Genome segment 6, encoding the NSP3 (NS34) protein, is 1350 nucleotides long, the same length as the cognate bovine ‘Shintoku’ gene and two nucleotides longer than the genome sequence for the porcine ‘Cowden’ strain. The two extra nucleotides are at positions 1250 and 1251 in the human and bovine group C sequences in the 3' non-coding region. The NSP3 genome segment showed 78% identity to the porcine ‘Cowden’ equivalent and 79% identity to the bovine ‘Shintoku’ NSP3 gene. Similarly, the ‘Cowden’ and ‘Shintoku’ NSP3 genes share 78% sequence identity. The ATG translation initiation codon is at position 25, in a favourable context for translation initiation (Kozak, 1991 ). Computer analysis of the sequence revealed a single ORF of 1206 nucleotides and a 3' non-coding region of 120 nucleotides.

The amino acid sequence predicted a protein 402 amino acids long, acidic in nature (Fig. 1), with a predicted molecular mass of 45·3 kDa and a calculated isoelectric point of 4·85. Eight cysteine residues were conserved between the human, porcine and bovine NSP3 proteins at positions 60, 171, 200, 280, 284, 294, 353 and 369. Three major functional domains described for the group A rotavirus SA11F NSP3 protein (Mattion et al., 1992 ) were also present on the group C NSP3 protein. The regions are a basic region for single-stranded (ss) RNA binding, a heptad repeat region for oligomerization and a leucine zipper motif. The group A rotavirus NSP3 protein basic region extends from amino acids 81 to 150; this region is also charged in the group C rotaviruses, with a large number of lysine and arginine residues between amino acids 79 and 151. The region between amino acids 89 and 125 is conserved in the group C rotaviruses, with three amino acid changes between human and bovine strains and just two amino acid changes between the human and porcine strains. The consensus sequence for ssRNA-binding, (I/L)XXM(I/L)(S/T)XXG, seen in orbiviruses, reoviruses and group A rotaviruses (Rao et al., 1995 ) is found in this region and is located at amino acids 104–112 of NSP3 of SA11. In the human and bovine group C rotaviruses, the sequence 100LXXMLSXXG108 is conserved and is highlighted in Fig. 1. This is at variance with the ‘Cowden’ sequence, where the methionine residue is replaced by isoleucine (Qian et al., 1991 ).



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Fig. 1. Group C rotavirus NSP3 protein alignment. NSP3 protein sequences derived from the nucleotide sequences from human group C rotavirus, the porcine ‘Cowden’ strain and bovine ‘Shintoku’ strains were aligned using the CLUSTAL W program (Thompson et al., 1994 ). The sequences are numbered from the first amino acid residue. Identical amino acids are represented by dashes. The following functional domains are indicated: the ssRNA-binding consensus sequence (bold type), hydrophobic heptad repeats (lines above the sequence: asterisks indicate each seventh hydrophobic amino acid), the leucine zipper domains (shaded boxes with leucine/isoleucine residues underlined) and the dsRNA-binding domain (open box).

 
The group C rotaviruses also appear to have several regions of hydrophobic heptad repeats necessary for oligomerization of the NSP3 protein. The ‘Cowden’ strain has a leucine-rich region between amino acids 87 and 116, a short leucine zipper between amino acids 93 and 107 and another five hydrophobic heptad repeats between amino acids 95 and 123 (Mattion et al., 1992 ). In the human group C rotavirus, leucine at amino acid 87 is replaced by valine. Further heptad repeats are seen at amino acids 206–236, 286–312 and 323–351. These regions contain the amino acids proline and glycine, which would suggest the absence of an {alpha}-helical coil, although the heptad repeats might be sufficient to promote helix formation (Cohen & Parry, 1986 ). The last heptad repeat conforms to a leucine zipper of five repeats. Following the basic region, there is a short, highly acidic region extending from amino acids 153 to 171, which mirrors the structure found in the cognate group A rotavirus gene product (amino acids 151–169).

Besides ssRNA binding, the NSP3 proteins of the group C rotaviruses have been shown to bind dsRNA (Langland et al., 1994 ). The group C rotavirus NSP3 proteins are 87–89 amino acids longer than their group A counterparts. The carboxy-terminal region of the group C NSP3 protein contains the dsRNA-binding domain, with a consensus sequence LX39(G/A)XGXSKKXAKXXAAXX(A/I)LXXL (Langland et al., 1994 ). This consensus region is also present in human group C rotavirus (amino acids 340–400).

Gene segment 7
The non-structural protein NSP1 (NS53) is encoded by gene 7 in group C rotaviruses. The gene sequence is 1270 nucleotides long. Computer analysis revealed a single ORF of 1182 nucleotides with a 5' non-coding region of 37 bp and a 3' non-coding region of 48 bp. The nucleotide sequence GTCAACATGG in the region of the putative ATG codon (underlined), with A at -3 and G at +4, constitutes a strong signal for initiation of translation (Kozak, 1991 ). The human group C rotavirus gene 7 sequence is 35 nucleotides longer than the porcine equivalent (Bremont et al., 1993 ), with an extra 31 nucleotides at the 5' terminus, one extra base at the 3' terminus and a novel extra codon at positions 135–137. The sequence has 67% identity to the equivalent ‘Cowden’ sequence.

The predicted protein sequence contains 394 amino acid residues, with a molecular mass of 46·6 kDa (Fig. 2). It is a basic protein with a calculated isoelectric point of 8·9. Human group C rotavirus NSP1 has 61% amino acid sequence identity to the equivalent ‘Cowden’ sequence, which contains 393 amino acids. This is consistent with other reports that suggest that NSP1 is the most variable rotavirus protein and that diversity in sequences is evident between strains from different species (Hua et al., 1993 ; Dunn et al., 1994 ). Studies examining reassortants between rotavirus strains have implicated NSP1 in host-range selection (Graham et al., 1987 ; Palombo & Bishop, 1994 ) and pathogenic phenotype (Broome et al., 1993 ).



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Fig. 2. Group C rotavirus NSP1 protein sequence alignment. NSP1 protein sequences derived from the nucleotide sequences from human group C rotavirus and the porcine ‘Cowden’ strain were aligned using CLUSTAL W. The sequences are numbered from the first amino acid residue. Identical amino acids are represented by dashes. The zinc finger motifs are underlined. *, Gap introduced to maximize the alignment.

 
The group C rotavirus NSP1 proteins (393–394 amino acids) are much smaller than the corresponding group A rotavirus NSP1 proteins, which are 486–495 amino acids in length. No function has been found for the C-terminal 300 amino acids of the group A rotaviruses (Hua et al., 1994 ). Group A rotavirus mutants lacking either the central or carboxy half of the protein were viable and therefore this region is not required for virus replication in vitro (Tian et al., 1993 ; Hua & Patton,1994 ). The functional regions of the group A rotaviruses, demonstrated by deletion mapping (Hua et al., 1994 ), are the RNA-binding domain and the intracellular localization domain. The RNA-binding domain is cysteine rich and forms a zinc finger (Bremont et al., 1987 ; Mitchell & Both, 1990 ; Hua et al., 1993 , 1994 ; Xu et al., 1994 ). Analysis of the human group C NSP1 amino acid sequence shows the zinc finger motif, CX2CX3CX4CX2CX3HXCX2CX4C, between amino acids 40 and 69. An identical motif is also found in the ‘Cowden’ strain NSP1. Both the human and porcine group C sequences have only four residues before the final cysteine of the motif at position 69, compared with the group A NSP1 proteins, which have five amino acids at this position. This region is not required for growth in cell culture, as viable mutants lacking the zinc finger motif coding region have been isolated (Taniguchi et al., 1996a ). Additionally, low frequency reassortment occurs between strains lacking the cysteine-rich region and heterologous virus: thus, this region is not essential for genome segment reassortment, although the intact NSP1 enhances the efficiency of genome segment assortment (Okada et al., 1999 ). A second motif of HX2(C/H)X6CX2C has been noted in a few group A rotavirus strains (Bremont et al., 1987 ; Taniguchi et al., 1996b ). A corresponding sequence, HX2CHX4C, at amino acids 271–280 is conserved between the NSP1 of human and porcine group C rotaviruses.

Gene segment 9
Gene segment 9 encodes the non-structural protein NSP2 (NS35). The nucleotide sequence is 1037 bp long and shares 88% identity (between nucleotides 21 and 1015) to the gene 9 sequence of the porcine ‘Cowden’ strain (Bremont et al., 1993 ). The sequence contains a single ORF of 936 nucleotides with a 5' untranslated leader sequence of 42 bp and an initiator codon in a favourable context for translation initiation (Kozak, 1991 ). The predicted protein sequence of 312 amino acids has a predicted molecular mass of 35·9 kDa and has 93·9% identity to the ‘Cowden’ NSP2 protein (Fig. 3).



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Fig. 3. Rotavirus NSP2 protein alignments. NSP3 protein sequences derived from the nucleotide sequences from human group C rotavirus, the porcine ‘Cowden’ strain and group A porcine (OSU) and human (DS1) strains were aligned using CLUSTAL W. The sequences are numbered from the first amino acid residue. Identical amino acids are represented by dots. The following features are indicated: basic amino acids (shaded and boxed), conserved regions (shaded) and hydrophobic heptad repeats ({blacktriangledown}). -, Gap introduced to maximize the alignment.

 
The NSP2 proteins have been shown to bind ssRNA in a sequence-independent manner, with binding confined to oligomerized states of NSP2 (Kattoura et al., 1992 ). Patton et al. (1993) postulated that the conserved and highly basic region from amino acids 205 to 241 of group A rotaviruses was the RNA-binding domain. This region is also highly basic in the group C rotaviruses and most of the basic amino acids are conserved. Preceding this basic region are two short regions of hydrophobic heptad repeats (amino acids 124–138 and 157–171) that are conserved between all the sequences and could form the oligomerization domain.

The first seven amino acids are absolutely conserved between all group A and group C rotavirus NSP2 proteins. Only one cysteine residue is absolutely conserved between the group A and group C NSP2 proteins, at position 6. Truncated NSP2 loses its ability to bind RNA when more than four amino acids are absent from the termini (Kattoura et al., 1992 ): therefore the conserved cysteine at position 6 could be involved in stabilizing the oligomerized NSP2 proteins.

Two other regions are well conserved between the group A and group C rotavirus NSP2 proteins. The first is a basic region (amino acids 47–60) with a run of three proline residues with the consensus sequence IXYGXAPPPXF(K/N/R)(K/N/R)R. Proline-rich domains have been identified in several transcription regulators that both activate and repress gene transcription (Hanna-Rose & Hansen, 1996 ). NSP2 localizes in the viroplasm (Petrie et al., 1984 ) and NSP2 has been shown in vivo to form part of a viral enzyme complex with replicase activity (Aponte et al., 1996 ). Proline-rich activation motifs are frequently associated with serine and threonine residues (Hanna-Rose & Hansen, 1996 ). NSP5, which also localizes in the viroplasm, interacts with NSP2 (Poncet et al., 1997 ) and has a high serine and threonine content. The interaction between NSP2 and NSP5 could regulate rotavirus RNA synthesis.

Between amino acid residues 109 and 139, a second, conserved, basic region is located followed by the first of the hydrophobic heptad repeats with consensus sequence (V/I)RHLENLX2RX5D(V/I)LX5LX5(L/M)I. NSP2 has been shown to form a complex with VP1, the putative RNA polymerase, in infected cells (Kattoura et al., 1994 ). Furthermore, NSP2 has an affinity for dsRNA (Kattoura et al., 1992 ) and associates with all 11 dsRNA segments, although Aponte et al. (1996) showed that, in the replicase complexes, some regions of the RNAs were single stranded, supporting the theory of a regulatory role in RNA replication.

Phylogenetic analyses
Genomic reassortment during co-infection in vivo and in vitro with distinct strains of group A rotaviruses is well documented (Ramig, 1997 ). Evolution of the different rotavirus serogroups could have arisen because of reassortment events and divergent evolution. Alternatively, the different rotavirus serogroups may have arisen from a common ancestor, in which case all genes would display equal divergence. To test these hypotheses, unrooted phylogenetic trees were constructed for groups A, B and C rotavirus NSP1, 2, 3, 4 and 5 gene sequences. Multiple alignments were performed with CLUSTAL X (Thompson et al., 1997 ) and unrooted trees were generated by using the neighbour-joining method (Saitou & Nei, 1987 ). Trees were subjected to a bootstrap analysis (Felsenstein, 1985 ) using 1000 data sets and output as a graphic representation by using DRAWTREE in the PHYLIP package (Felsenstein, 1993 ).

Non-structural proteins have been considered to be more useful than structural proteins in determining the ancestral relationships of different viruses (Clewley, 1998 ). Expression of the non-structural proteins is confined within the host cell and non-structural proteins are not subject to the conformational structural constraints that affect the structural proteins, suggesting they would be subject to less evolutionary pressure than the structural proteins. However, Xu et al. (1994) demonstrated that divergence of gene 5 of group A rotaviruses encoding NSP1 exceeded that of the divergence of the major neutralizing antigen, VP7. Phylogenetic analysis of NSP3 proteins (Rao et al., 1995 ) revealed that the rodent group B rotavirus (IDIR), porcine group C rotavirus (Cowden) and various mammalian group A rotavirus strains belonged to three different groups with a distinct ancestral origin. In the study presented here, the phylogenetic trees for each of the five genes encoding the non-structural proteins were essentially similar (for NSP3 comparison see Fig. 4), with three major branches, of similar length, for the three serogroups analysed. This branching pattern suggests that the groups may have diverged at the same time, possibly from a common ancestor, rather than as a result of reassortment of the genes. No reassortants have been detected between strains of different rotavirus groups (A–G) (Taniguchi & Urasawa, 1995 ) and divergence of the rotavirus serogroups is so great that reassortment between viruses from different serogroups does not appear to occur (Yolken et al., 1988 ). Comparison of the group C branch of the phylogenetic trees suggests that the human, porcine and bovine group C rotaviruses also developed from a common ancestor at a later time. It may still be possible for reassortment to occur between group C rotaviruses from different animal species in vitro, although there is no evidence for naturally occurring reassortment (Grice et al., 1994 ; Jiang et al., 1996 ).



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Fig. 4. Unrooted phylogenetic tree of the NSP3 gene (also representative of the NSP 1, 2, 4 and 5 genes) showing the relationship between the mammalian rotavirus groups. Trees were subjected to a bootstrap analysis using 1000 data sets. Shaded ellipses have been added to highlight the individual groups. Accession numbers (in parentheses) for rotaviruses are as follows: RRV (X81426), IS2 (X76645), OSU (X81431), GO (X81430), WA (X81434), ST3 (X81436), RF (Z21639), NCDV (X81429), IDIR (L09722), ADRV (M91436), ‘Bristol’ (AJ132203), ‘Cowden’ (M69115) and ‘Shintoku’ (L12390).

 

   Acknowledgments
 
We thank Dr M. Pickett for the construction of the oligonucleotide primers. V.L.A.J. was funded for this work by the Public Health Laboratory Service.


   Footnotes
 
The EMBL accession numbers for the sequences of the human group C rotavirus ‘Bristol’ genes 6, 7 and 9 are AJ132203AJ132205.


   References
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Abstract
Introduction
Methods
Results and Discussion
References
 
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Received 29 March 1999; accepted 4 August 1999.