Laboratory of Insect Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei Province 430072, China
Correspondence
Yuanyang Hu
yyhu{at}whu.edu.cn
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ABSTRACT |
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INTRODUCTION |
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A number of small RNA-containing insect viruses have been named picorna-like, based on similarities to mammalian picornaviruses in composition, size of capsid proteins and other biophysical properties. To date, the complete nucleotide sequences of 13 picorna-like viruses from various species of insects have been reported, which have revealed differences in their genomic organization. Sacbrood virus (SBV; Ghosh et al., 1999), IFV (Isawa et al., 1998
) and PnPV (Wu et al., 2002
) have a genomic organization that resembles that of typical mammalian picornaviruses, containing a unique large open reading frame (ORF) with the capsid proteins located at the 5' terminus. For Drosophila C virus (DCV; Johnson & Christian, 1998
), Rhopalosiphum padi virus (RhPV; Moon et al., 1998
), Plautia stali intestine virus (PSIV; Sasaki et al., 1998
), Himetobi P virus (HiPV; Nakashima et al., 1999
), Triatoma virus (TrV; Czibener et al., 2000
), Black queen-cell virus (BQCV; Leat et al., 2000
), Acute bee paralysis virus (ABPV; Govan et al., 2000
), Cricket paralysis virus (CrPV; Wilson et al., 2000
) and Aphid lethal paralysis virus (ALPV; van Munster et al., 2002
), the nucleotide sequences indicated that two ORFs and the structural proteins are located in the 3'-terminal region. The viruses DCV, PSIV, RhPV, CrPV, TrV, HiPV, BQCV and ABPV have been assigned to a new genus named Cricket paralysis-like virus (Christian et al., 2000
) and more recently named Cripavirus (family Dicistroviridae; Mayo, 2002
). Although there are some sequence similarities amongst these insect viruses and mammalian picornaviruses, some fundamental differences are also present. The genome of dicistroviruses contains two ORFs that are separated by an intergenic region. The 5' ORF encodes the non-structural proteins and the downstream ORF encodes the capsid proteins. Since EoPV is the first characterized picorna-like virus infecting wild insects in China, it is of interest to determine its genomic organization.
We report here the complete nucleotide sequence of EoPV. The EoPV genome contains a single large ORF encoding the capsid proteins at the 5' terminus of the genome with the non-structural proteins at the 3' end of the genome. This organization is similar to typical mammalian picornaviruses. Phylogenetic analysis of the RNA-dependent RNA polymerase (RdRp) showed that EoPV, PnPV, SBV and IFV belong to a new group of insect-infecting ssRNA viruses that are distinct from the dicistroviruses.
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METHODS |
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SDS-PAGE.
The molecular masses of the EoPV capsid proteins were determined by SDS-PAGE. Purified EoPV virons were separated on 16 % SDS-polyacrylamide gels. After electrophoresis, the gels were stained with Coomassie brilliant blue R-250. The molecular masses of the proteins were determined by comparison with standard proteins of a broad-range molecular mass marker (Bio-Rad) using a UV scanning system (UVP).
RNA isolation and cDNA synthesis.
EoPV genomic RNA was extracted from purified viral particles using TRIzol reagent (Gibco-BRL), following the manufacturer's instructions. cDNA fragments complementary to EoPV RNA were synthesized using Superscript II reverse transcriptase (Invitrogen). First-strand synthesis was conducted with oligo(dT)1218 as a primer, based on the genomic characteristics of picornaviruses. The cDNA fragments were inserted into the SmaI site of pUC18. The ligation mixtures were transformed into Escherichia coli DH5 cells. The remainder of the genome was cloned by a similar process, using primers designed according to the sequences toward the end of each individual clone using the Primer Premier 5 program (Premier Biosoft Int.). Overlapping clones were obtained by this strategy, which covered the entire genome except for its 5' end. cDNA from the 5' end of the EoPV genome was synthesized using the 5' rapid amplification of cDNA ends (RACE) technique (Invitrogen). The tailing reaction in the RACE procedure was performed with TdT and dCTP. PCR fragments were cloned into the PMD-18 Vector (Takara).
Nucleotide sequencing.
Both strands of three individual cDNA clones were sequenced by the dideoxynucleotide chain-termination method using the Applied Biosystems Sequencer model 377, with universal sequencing and walking primers.
Sequence and phylogenetic analysis.
The DNA sequences of the EoPV genome were analysed and translated into amino acid sequences using the BioEdit program. Nucleotide and amino acid sequence data were further analysed against all sequences in the GenBank using the BLAST (Altschul et al., 1990) and FASTA (Pearson & Lipman, 1988
) programs. Multiple alignments were performed using CLUSTAL W (Thompson et al., 1994
) and phylogenetic analysis was conducted using the neighbour-joining method as implemented in the CLUSTAL W program.
The virus sequences and accession numbers used in this paper are as follows: ALPV (AF536531), Human poliovirus (PV, V01149), Foot-and-mouth disease virus (FMDV, AF189157), Encephalomyocarditis virus (EMCV, X87335), Hepatitis A virus (HAV, M14707), DCV (AF014388), HiPV (AB017037), TrV (AF178440), RhPV (AF022937), PSIV (AB006531), BQCV (AF183905), ABPV (AF150629), CrPV (AF218039), IFV (AB000906), SBV (AF092924), PnPV (AF323747) and Acyrthosiphon pisum virus (APV, AF024514).
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RESULTS |
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Capsid proteins
The molecular masses of the EoPV capsid proteins obtained from purified EoPV virons were analysed by 16 % SDS-PAGE (Fig. 1). Only two capsid protein bands with molecular masses of 31·5 (CP1) and 28·8 kDa (CP2) were observed. However, the relative amount of CP2 was approximately 2·5 times greater than that of CP1, as determined by scanning with a UV system.
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The C-terminal region (aa 26302876) of the EoPV polyprotein was similar to the sequences of the RdRp proteins of the Picornaviridae, Sequiviridae, Comoviridae and insect picorna-like viruses. All eight conserved motifs in the RdRp of positive-strand RNA viruses (Koonin, 1991) were also identified in this region (Fig. 3C
). An acid motif V (SGX3TX3N), core motif VI (YGDD) for nucleotide binding and motif VII (FLKR) for catalytic function were located at aa 27662775, 28052808 and 28422845, respectively. The highest sequence identity was 91 % between the putative RdRp and the PnPV polyprotein. The deduced amino acid sequence of the conserved domain of the EoPV RdRp was compared with all sequences in the protein sequence databases using the BLASTP program. The overall identities with insect picorna-like viruses were: PnPV (91 %), SBV (26 %), IFV (25 %), TrV (24 %), PSIV and HiPV (22 %), RhPV (24 %), ALPV (27 %), DCV (39 %) and CrPV (24 %). EoPV shared low identities (<22 %) with other insect picorna-like viruses and mammalian picornaviruses.
Phylogenetic analysis
The highly conserved fragments of the RdRp proteins containing motifs I to VIII (300 aa) of the picornaviruses and picorna-like viruses (Koonin & Dolja, 1993
) were used in a phylogenetic analysis. The neighbour-joining tree method was used and the results reflected the current systematic assignment of the viruses. As shown in Fig. 4
, EoPV was most closely related to PnPV, followed by SBV and IFV. These four viruses seemed to belong to one cluster, while another cluster (the dicistroviruses), containing PSIV, RhPV and DCV, appeared to be more distantly related. The mammalian picornaviruses EMCV, FMDV and PV were quite distinct from the insect picorna-like viruses and formed a separate cluster.
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DISCUSSION |
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Typical mammalian picornaviruses have three major capsid proteins (2441 kDa) and a small protein (VP4, 5·513·5 kDa). Some insect picorna-like viruses, such as CrPV, IFV and PnPV, obey this rule. However, other insect picorna-like viruses only have three major capsid proteins, for example ALPV, RhPV, SBV, APV and HiPV. It is interesting to note that EoPV had only two detectable capsid proteins of 31·5 kDa (CP1) and 28·8 kDa (CP2) on 16 % SDS-polyacrylamide gels. The relative amount of CP2 was 2·5 times greater than that of CP1. Among the insect picorna-like viral agents, EoPV resembles PnPV in both genome length and genome organization and shows an overall nucleotide sequence identity of 81·24 % and amino acid sequence identity of 87·37 % based on analysis using the DNAMAN program. It is possible that the structural proteins might be encoded in the same region and order. Pair alignment of EoPV and PnPV showed that aa 3211192 of the EoPV polyprotein had 89·79 % identity with aa 3201191 of PnPV, which encodes CP1 to CP4 of PnPV. The 31·5 kDa capsid protein of EoPV is similar to CP1 of PnPV in size; the 28·8 kDa capsid protein resembles CP2 and CP3 of PnPV in size, but EoPV does not appear to have the third capsid protein nor the small capsid protein (VP4 or CP4). Considering the relationship between EoPV and PnPV, it is possible that the structural proteins are encoded in the same order. The region where CP1 and CP3 are identified is located at a similar distance from the polyprotein terminus in both viruses. Because aa 3311192 of the EoPV polyprotein shares 89·79 % identity with the capsid protein of PnPV and has conserved sequences analogous to the cleavage sites of the PnPV capsid proteins, it is possible that the 28·8 kDa band contained two capsid proteins with the same molecular mass, which is consistent with theoretical deductions.
It is known that most cleavage reactions catalysed by the picornavirus 3C protease occur within a small subset of dipeptides consisting of Q-T, -S, -T, -V and -M (Hellen et al., 1989). The putative cleavage sites of CP1/CP4 and CP2/CP3 are present in the polyproteins of SBV, IFV and dicistroviruses (van Munster et al., 2002
; Liljas et al., 2002
), but absent in the polyproteins of PnPV and EoPV.
At the N terminus of IFV, SBV, apthoviruses and FMDV, a (sometimes putative) leader protein (L) precedes the first structural protein (Isawa et al., 1998; Ghosh et al., 1999
). The L protein of FMDV and apthoviruses can cleave its own C terminus, releasing it from VP4. A conserved cysteinetryptophan amino acid pair and a histidine residue have been found to be essential for the apthovirus L protease activity (Gorbalenya et al., 1991
; Piccione et al., 1995
; Roberts & Belsham, 1995
). Since the coding region for the structural proteins of PnPV starts at aa 320, Wu et al. (2002)
suggested that the PnPV genome may encode a leader polypeptide of 36·7 kDa prior to the structural protein-coding region. If this is true, we would also expect such a protein in EoPV, since the structures of these viral genomes are similar within this region. However, the conserved cysteinetryptophan amino acid pair and a histidine residue was not found in the putative L protein of EoPV, PnPV or IFV, nor in the L protein of SBV (Isawa et al., 1998
; Ghosh et al., 1999
), which suggests that these L proteins may not be proteases.
The conserved motif NPGP, which defines the 2A/2B junction in IFV, PnPV, cardioviruses and apthoviruses (Hahn & Palmenberg, 1996; Donnelly et al., 1997
), is also present in EoPV. Interestingly, similar to PnPV, there are two such motifs found within the EoPV polyprotein at aa 572575 and 11891192. The first sequence is located inside the structural protein-coding region. The second motif is located near the C terminus of the EoPV structural protein-coding region, which suggests that it might also be functionally active. Recently, in a study of the cleavage mechanism of the aphthovirus 2A/2B polyprotein, it was proposed that the activity of this site depends not on a proteolytic reaction, but on a novel translational effect that involves a putative ribosomal skip from one codon to the next without the formation of a peptide bond (Donnelly et al., 2001a
). Some 2A-like sequences have already been identified at various locations within the ORFs of insect virus polyproteins (reviewed by Donnelly et al., 2001b
). In IFV, a presumably functional 2A-like sequence was found near the downstream end of the structural protein-coding region, which probably functions as it does in mammalian picornaviruses (Isawa et al., 1998
). In DCV, ABPV and CrPV there are conserved 2A-like sequences in the N-terminal region of the replicative ORF1 (Johnson & Christian, 1998
; Govan et al., 2000
; Wilson et al., 2000
). The second EoPV 2A-like sequence (aa 11721191) has a similar location to the 2A-like motif in IFV and PnPV and it also appears to be functionally active.
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Received 9 September 2003;
accepted 22 December 2003.