Centro de Virología Animal (CEVAN), Serrano 669, Buenos Aires 1414, Argentina1
Instituto de Investigaciones Biotecnológicas de la Universidad de General San Martín (IIB-UNSAM), Av. Gral Paz s/n, INTI ed. 24, Provincia de Buenos Aires, Argentina2
Centro de Parásitos y Vectores (CEPAVE), La Plata, Buenos Aires, Argentina3
Laboratorio de Reproducción y Lactancia (LARLAC), Mendoza, Argentina4
Author for correspondence: Eduardo Scodeller (at CEVAN). Fax +54 1 856 4495. e-mail jltcevan{at}datamar.com.ar
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Abstract |
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Introduction |
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In order to gain a better understanding of the genomic organization and strategy of replication of TrV, its complete genomic nucleotide sequence was determined. We now report the full sequence of the TrV genome, which shows an organization resembling that of DCV, Plautia stali intestine virus (PSIV), Rhopalosiphum padi virus (RhPV), cricket paralysis virus (CrPV) and Himetobi P virus (HiPV).
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Methods |
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cDNA synthesis, cloning and sequencing.
Purified TrV RNA was used to synthesize cDNA with oligo(dT)1218 and random hexamer oligonucleotides as primers and a commercial kit (SuperScript Choice system for DNA synthesis; Life Technologies). The blunt-ended cDNA was ligated in the SmaI site of the plasmid vector pBluescript KS II (+) (Stratagene). The ligation mixture was used to transform E. coli DH5 cells. Identification and isolation of recombinant clones were carried out by following standard procedures (Sambrook et al., 1989
). Nine overlapping clones were obtained that covered most of the TrV genome except the 5' end. Both strands of each of the cDNA clones were sequenced completely. To obtain clones representing the 5' end of the TrV genome, 5' RACE was performed with a commercial kit (Life Technologies) following the manufacturers instructions. The 5'-terminal sequence was determined by comparison of the sequences of six clones. The results obtained by 5' RACE were further confirmed by primer extension. Primer extension was performed according to Sambrook et al. (1989)
with a 32P-labelled oligonucleotide complementary to nt 6887 of TrV RNA.
Nucleotide sequencing was performed by using the T7 Sequenase version 2.0 DNA sequencing kit (Amersham Life Science). Both strands of each cDNA clone were sequenced completely at least twice. The sequence of the 5' end of TrV genome was obtained by sequencing of six independent clones obtained by 5' RACE.
All computational sequence analyses were done by using the Lasergene package from DNAstar. Multiple alignments were performed by using CLUSTAL W (Thompson et al., 1994 ).
Protein sequencing.
Purified TrV virions were subjected to SDS10% PAGE (Laemmli, 1970 ), blotted onto a PVDF membrane (ProBlott) and stained with Coomassie brilliant blue according to the manufacturers instructions. Each stained protein band was excised and sequenced by the Edman degradation procedure.
Northern blot analysis.
Total RNA was extracted from the intestines of infected and non-infected T. infestans by using a Trizol reagent according to the manufacturers instructions. General procedures described by Sambrook et al. (1989) were used for Northern blot hybridization. Nitrocellulose membranes were probed with 32P-labelled PCR products amplified from all nine cDNA clones.
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Results and Discussion |
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Coding and non-coding regions of TrV genomic RNA
A computer-assisted analysis of the nucleotide sequence of TrV showed that the genomic RNA contains two large open reading frames (ORFs), nucleotides 5495936 (ORF1) and 61098715 (ORF2) (Fig. 1). The ORFs are located in different frames. ORF1 and ORF2 account for 88·6% of the TrV genome; the other 11·4% consists of untranslated regions. These include the 548 nt 5' untranslated region (UTR), a 172 nt intergenic region that separates ORF1 from ORF2 and a 295 nt 3' UTR [excluding the poly(A) tail]. The lengths of these three non-coding regions are in good agreement with those of other insect picorna-like viruses sequenced recently (Johnson & Christian, 1998
; Sasaki et al., 1998
; Moon et al., 1998
; Nakashima et al., 1999
).
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Alignments of the amino acid sequences of non-structural proteins
A computer analysis of the nucleotide sequence of the TrV genome revealed that the deduced amino acid sequence of ORF1 contained the core motifs of the picornavirus 2C RNA helicase, 3C cysteine protease and 3D RNA-dependent RNA polymerase (RdRp). These motifs are also conserved in the genomes of viruses in the families Comoviridae, Sequiviridae and Caliciviridae (Koonin & Dolja, 1993 ). The predicted amino acid sequences around these motifs in ORF1 were aligned with those of the insect picorna-like viruses DCV, PSIV, RhPV and HiPV. Fig. 2(a)
shows a multiple alignment of putative RNA helicase sequences of these viruses. The consensus sequence for RNA helicase, GX4GK (Gorbalenya et al., 1989
), which is proposed to be responsible for nucleotide binding, was found at amino acids 820826 of the deduced amino acid sequence of TrV.
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TrV capsid protein location within ORF2
Four proteins have been detected in purified TrV particles by SDSPAGE. The three major components are VP1, VP2 and VP3 (with apparent molecular masses, based on their electrophoretic mobility, of 39, 37 and 33 kDa, respectively) with a minor component, VP0, of apparent molecular mass 45 kDa. Another low molecular mass protein, VP4, present in mammalian picornaviruses and also reported in other insect picorna-like viruses, could never be identified in TrV (Muscio et al., 1988 ). In order to determine the genetic order of the capsid proteins in ORF2, the N-terminal sequences of VP2 and VP3 of TrV were determined by Edman degradation. The N terminus of VP1 could not be determined because it was blocked. The N-terminal sequence of VP2 was SKPLTT, starting at nt 7045. The first three amino acids of VP2 in DCV, PSIV and TrV are identical, indicating a similar mechanism of proteolytic cleavage at this site of the polyprotein encoded by ORF2. The N terminus of VP3 was VGFASA, starting at nt 7900. This sequence showed no similarity to the N termini of VP3 of DCV or PSIV. However, in all three cases, the putative C-terminal residue of VP2 deduced from the sequence is Q. We could not obtain enough VP0 to perform sequencing reactions. Since VP2 and VP3 are located internally in ORF2, we assume that VP1 is encoded by the 5' end of the latter. This assumption is reinforced by the high degree of similarity of this region of ORF2 of TrV to VP1 of HiPV, PSIV, DCV and RhPV (data not shown).
Regarding the location of the putative (as yet unidentified) VP4 of TrV in ORF2, it is worth mentioning that the boundaries of the putative cleavage site of VP4/VP2 in PSIV (FGF/SKP) and DCV (LGF/SKP) are conserved in TrV (LGF/SKP) (Fig. 3). This might be an indication that proteolytic cleavage also occurs at this site in TrV. The boundaries of the VP1/VP4 cleavage site (Fig. 3
) were determined in PSIV by sequencing of VP0 and VP4. We could not find any region in ORF2 of TrV with similarity to this sequence. We therefore can not predict the existence of VP4 in TrV from these data.
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It has recently become clear that many small picorna-like viruses of insects are not like picornaviruses in their genomic organization. The first evidence was provided some years ago by Koonin & Gorbalenya (1992) . They analysed 1600 nt from the 3'-end sequence of CrPV published by King et al. (1987)
, which was postulated to be the coding sequence of the viral RdRp. However, Koonin & Gorbalenya (1992)
concluded that this region actually encoded capsid proteins, suggesting similarity in gene order between CrPV and the caliciviruses. More recently, the complete genome sequences of other insect picorna-like viruses have been published. Some of these viruses (DCV, PSIV and RhPV) have been found to have a novel genome organization. They contain two ORFs that are separated by an intervening sequence. The non-structural protein precursor is encoded in the 5'-proximal ORF and the capsid protein precursor is located in the 3'-proximal ORF. The initiation of translation of ORF2 occurs internally at an IRES sequence and the initiation codon is not an AUG. In contrast, sequence analysis of sacbrood virus of the honey bee (Ghosh et al., 1999
) and infectious flacherie virus of the silkworm (Isawa et al., 1998
) showed that their genomic organization resembles that of the picornaviruses of vertebrates. Acyrthosiphon pisum virus, although an insect picorna-like virus with calicivirus-like genome organization (van der Wilk et al., 1997
), was grouped away from the rest and appeared to be different from other insect viruses (Ghosh et al., 1999
). The TrV genome organization reported in this paper suggests that this virus belongs to the group formed by DCV, RhPV, PSIV and HiPV.
Relevance of the taxonomic classification of TrV
TrV is a pathogen of T. infestans, the insect vector of Trypanosoma cruzi, which causes Chagas disease in humans. Recent data from our laboratory support the use of TrV as a control agent for triatomines (O. Muscio, J. L. La Torre & E. A. Scodeller, unpublished results). However, there has been a general reluctance to use insect picornaviruses for biological control, because many members of the family Picornaviridae are mammalian pathogens.
The results presented in this paper indicate strongly that TrV may be included in this putative new family, which appears to be specific to insects. In addition, none of the mammalian viruses reported to date have this particular genome organization. Moreover, taking into account the feeding pattern of triatomines (Muscio et al., 1997 ), we can speculate that millions of humans have already been exposed to TrV. However, it was not possible to find anti-TrV antibodies in sera of Chagas disease patients living in endemic areas of very high incidence of triatomines, in spite of the fact that TrV was found in all wild T. infestans populations studied (Muscio et al., 1997
). Regarding the host range of TrV, preliminary results indicate that only members of the family Reduviidae are susceptible (O. Muscio, J. L. La Torre & E. A. Scodeller, unpublished results). All of these data reinforce the idea that TrV might be acceptable as a biological control agent for triatomines.
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Acknowledgments |
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Footnotes |
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References |
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Received 14 September 1999;
accepted 21 December 1999.