Department of Biological Sciences, John Tabor Laboratories, University of Essex, Colchester CO4 3SQ, UK1
Author for correspondence: Glyn Stanway. Fax +44 1206 873416. e-mail stanwg{at}essex.ac.uk
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Abstract |
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Introduction |
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The picornavirus genome is 70009500 nucleotides long and is enclosed within a naked particle, made up of 60 copies of each of the capsid proteins. The genome encodes a single polyprotein, which undergoes a cleavage cascade performed by virus-encoded activities, to give the final virus proteins. The genomes of the various genera encode 10, 11 or 12 final proteins, but intermediates in the cascade can have a significant half-life and may have distinct functions (Rueckert, 1996 ). All picornaviruses share essentially the same genome organization. The capsid proteins (1A, 1B, 1C and 1D, commonly known as VP4, VP2, VP3 and VP1, respectively) are encoded towards the N terminus of the polyprotein and the non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D) are encoded downstream of these. VP4 and VP2 are assembled into the particle in the form of a precursor, VP0, the final step of assembly being the cleavage of this precursor, which appears to be required for virion infectivity and stability. However, in the human parechoviruses and in Aichi virus, this cleavage appears not to occur and consequently these particles have only three proteins, VP0, VP3 and VP1 (Hyypiä et al., 1992
; Stanway et al., 1994
; Yamashita et al., 1998
).
For most picornavirus proteins, there is clear homology between the corresponding protein-encoding region of different genera. One exception is L, the protein encoded at the extreme N terminus of the polyprotein in aphthoviruses, cardioviruses, ERBV and Aichi virus (Rueckert, 1996 ; Wutz et al., 1996
; Yamashita et al., 1998
). With the exception of aphthoviruses and ERBV, where they are homologues, picornavirus L proteins are structurally and functionally distinct (Rueckert, 1996
). The other variable picornavirus locus is the region encoding 2A. In rhinoviruses and enteroviruses, 2A is a trypsin-like, cysteine protease involved in polyprotein processing, while in cardioviruses, aphthoviruses, ERBV and PTV1, it is associated with an unusual processing activity, involving an AsnProGlyPro (NPGP) motif (Ryan & Flint, 1997
). The 2A proteins of parechoviruses, hepatoviruses and Aichi virus are not believed to be involved in processing (Schultheiss et al., 1995
; Jia et al., 1993
; Yamashita et al., 1998
). In addition, no similarity between the 2A protein of parechoviruses, hepatoviruses and Aichi virus has previously been noted.
We show here that the 2A proteins of parechoviruses are, in fact, homologous to the corresponding region of the polyprotein of the hepato-like virus, avian encephalomyelitis virus (AEV), and, to a lesser extent, to the 2A protein of Aichi virus (Marvil et al., 1999 ; Yamashita et al., 1998
). Moreover, these proteins are also related to a recently identified family of cellular proteins, possibly involved in the control of cell proliferation. These observations have important implications for an understanding of the replication of these viruses and of picornavirus evolution.
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Methods |
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Sequence comparisons.
Database searches were performed by using the EBI FASTA3 (http://www2.ebi.ac.uk/fasta3/) or BLAST2 (http://www2.ebi.ac.uk/blast2/) search facilities (Pearson & Lipman, 1988 ; Altschul et al., 1990
). A range of FASTA3 parameters was used to analyse the HPeV1 sequence and a match with AEV was found by using the blosum50 matrix, Ktup=2, GapPenOpen=-12 and GapPenExtend=-2. These parameters were used for all other searches. Clustal W (http://www2.ebi.ac.uk/clustalw/) was used to align multiple sequences (Thompson et al., 1994
).
Sequence analysis.
Sequences were analysed for the presence of likely transmembrane domains by using the program TMpred (http://www.ch.embnet.org/software/TMPRED form.html; Hofmann & Stoffel, 1993 ). The program ppsearch (http://www2.ebi.ac.uk/ppsearch/) was used to perform PROSITE pattern searches.
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Results and Discussion |
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It can be seen from the alignment of the homologous region of the three available HPeV sequences, AEV and Aichi virus, together with the cellular proteins, that there is conservation of all the sequence features described above (Fig. 1). In addition, the HPeV1/2 and Aichi 2A proteins are virtually co-N-terminal with H-rev107 and TIG3 and have a similar overall length. The HPeV1 position 24 region, hereafter termed the H-box, is seen in two forms among these proteins: HWA(I/L) in human and rat H-rev107, TIG3 and Aichi virus 2A; and H(Y/F)G(I/V) in HPeV1/2 and AEV 2A, together with LRAT. In addition, although there were no other absolutely conserved amino acids, the nature of some flanking residues is conserved and a GlyAsp (GD) dipeptide is seen in most of the proteins. The NCE region is less well conserved in Aichi virus 2A and only AsnCys (hereafter termed the NC motif) is seen. The residues flanking the Aichi virus H-box are also less closely related to those of the other proteins. However, even in this virus, the significance of the similarity is emphasized by the presence of the hydrophobic region downstream of the NC motif. Moreover, there is clear primary sequence identity between the Aichi virus 2A and all the cellular proteins in the NC motif region, where the common sequence NCXHFV is seen.
The other characteristic feature of the virus and cellular proteins is the long (1824 amino acids) hydrophobic region. According to the predictive program TMpred, this is highly likely to be a transmembrane domain (Hofmann & Stoffel, 1993 ). Deletion of this region has been shown to reduce the activity of H-rev107 severely, suggesting that it plays a key role in the function of this protein (Sers et al., 1997
). In all the proteins, except LRAT, this putative transmembrane domain is followed immediately by a basic amino acid. A further notable similarity is that seen between the H-rev107 proteins and HPeV 2A, where the motif ALTXKAXXXKXXL (positions 4757 in HPeV1) can be seen in analogous positions.
In terms of overall amino acid identity, the H-rev107 and TIG3 proteins are closely related, while the other cellular protein, LRAT, is relatively distant from these. This is also evident from the alignment, which shows that LRAT has a longer N-terminal region and two relative insertions compared with the other cellular proteins (Fig. 1). If these are removed from comparisons, LRAT clearly clusters with the cellular proteins, but remains a distant member (Fig. 2
). Among the virus proteins, as expected, those from the human parechoviruses form a tight cluster.
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From its earliest isolation, it was observed that HPeV1 causes a distinctive cellular pathology, involving characteristic nuclear changes (Wigand & Sabin, 1961 ; Shaver et al., 1961
; Jamison, 1974
). The report that H-rev107 associates with the nuclear membrane, together with the relationships described here, raises the intriguing possibility that this pathology results from the action of the 2A protein (Sers et al., 1997
).
It is assumed that many virus proteins originated by acquisition from the host genome. In the case of picornaviruses, the structural similarity of 3C (and that of the related entero/rhinovirus 2A protein) to cellular trypsin-like proteases is cited as an example (Gorbalenya, 1992 ). The papain-like, aphthovirus L protein may also have arisen from capture of a host RNA. The presence in coxsackievirus A9 of an ArgGlyAsp (RGD)-containing, C-terminal VP1 extension (relative to other enteroviruses), with similarity to transforming growth factor
1, is a more speculative example of a region of a picornavirus protein with a putative cellular origin (Chang et al., 1989
, 1992
; Hughes et al., 1995
). Apart from these cases, the observation reported here, that some picornavirus 2A proteins have cellular homologues, is the clearest evidence in picornaviruses for such a mechanism.
The demonstration that the 2A proteins of three picornaviruses are related reduces the apparent diversity of this locus and, as shown in Fig. 3, picornaviruses fall essentially into four groups. These are viruses with: a trypsin-like protease (rhinoviruses/enteroviruses); an NPGP motif involved in 2A/2B cleavage (cardioviruses/aphthoviruses); a 2A unrelated to known proteins (hepatoviruses); or an H-box/NC protein (parechoviruses/Aichi virus/AEV). Although the C terminus of the cardiovirus 2A is homologous to the aphthovirus protein, it has an additional 130 N-terminal amino acids. The occurrence of an H-box/NC protein at the same locus in three diverse picornavirus groups suggests that it was acquired by a single event in a common ancestor and that it has been maintained through subsequent divergence. Since AEV is related to hepatoviruses, which lack a corresponding protein, it is likely that hepatoviruses lost this protein after divergence from the AEV lineage. This may have taken place through drift, or through deletion and subsequent capture of another protein-encoding sequence at this locus. In view of the distinct nature of the 2A proteins of AEV and hepatoviruses, it may be preferable to consider them as members of separate genera, notwithstanding the fact that they are more closely related to one another than to other picornaviruses in other parts of the genome (Marvil et al., 1999
). Recently, a parechovirus-like virus has been isolated from rodents (Nicklasson et al., 1998
, 1999
). It will be interesting to ascertain whether this virus has an H-box/NC protein and, if so, how closely related it is to that of HPeVs.
Relatively diverse picornaviruses have H-box/NC proteins and it is possible that they occur in other viruses. It was therefore interesting to find similar features in one genetic group of the caliciviruses (group II), containing Norwalk, Southampton and Lordsdale viruses, by comparison with H-rev107 sequences (Lambden et al., 1993 ; Dingle et al., 1995
; Hardy & Estes, 1996
). These viruses each have sequences reminiscent of the H-box and also possess an NC motif (Fig. 4
). Caliciviruses have a similar relative arrangement of non-structural proteins to picornaviruses, which can be summarized: NTP-binding protein (possibly helicase); 3C-like protease; polymerase (2C; 3C; 3D in picornaviruses). The calicivirus H-box/NC protein is upstream of the NTP-binding protein and the relative positions of the motifs are similar to those of the picornavirus protein (Fig. 4
). The calicivirus protein is distinguished from the picornavirus 2A by the absence of a significant transmembrane domain, suggesting that it may function in a different manner. However, it seems that H-box/NC proteins may occur in several RNA viruses, suggesting that they may contribute an important, but as yet unknown function to the virus life-cycle.
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Acknowledgments |
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References |
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Received 1 June 1999;
accepted 20 September 1999.