The complete 5284-nucleotide sequence ofthe double-stranded RNA genome of Leishmania RNA virus 1 (LRV1) was determined and contains three open reading frames (ORFs) on the plus (+) (mRNA) (3, 7). The 5.3-kilobase (kb) LRV1 dsRNA was purified by electrophoresis in 0.6% agarose/ TBE (0.09 M Tris borate/2 mM EDTA, pH 8.0) gels, electroelution, and ethanol precipitation. LRV1 Cloning. LRV1 cDNA clones were prepared from gel-purified RNA (5) or by PCR amplification. Their location within the LRV1 genome is shown in Fig. 1. First-strand cDNA was synthesized using mixed hexamer primers (LP series), oligo(dT) priming of Escherichia coli poly(A) polymerase-treated LRV1 RNA (U1 series); and oligonucleotides 87-19 and 87-20 (see Fig. 2) (LW or WAL series). Second strand was synthesized by the RNase H method (9), repaired, methylated, tinkered, and ligated into BamHI-digested pBS+ (LP series), EcoRI-digested AZAP (LJ series) or EcoRIdigested AZAPII (LW and WAL series) (10). Clones of 5' ends were obtained by PCR amplification of C-tailed (terminal deoxynucleotidyltransferase) first-strand cDNA synthesized\ using primers 90-122 and 87-19 for the (+)-and (-)-strand, respectively. Amplification with Bam-dG10 (CCGGATCCGGGGGGGGGG) and 90-145 or 89-195, respectively, used Replinase (DuPont/NEN) for 30 cycles of denaturation at 940C for 1 min, annealing at 450C for 1 mi, and extension at 720C for 2 min. The products were digested with BamHI and EcoRI (1P145 series), BamHI and Nhe I (1PN4 series), or BamHI and HindIll (1P195 series) and ligated into pBluescript II SK(-) (Stratagene) digested with the appropriate enzymes. For cloning the 3' ends, gel-purified dsRNA was C-tailed using poly(A) polymerase (BRL), cDNA was synthesized using Bam-dG10, and PCR amplification was carried out using 90-122 and 90-146 were used for first-strand cDNA synthesis and PCR amplification ofgel-purified LRV1 dsRNA for some internal LRV1 clones. Products were digested with EcoRI and ligated into EcoRI-digested pBluescript II SK(-) (PCR146-1 and P2R series).Sequence Analysis. Plasmid and PCR product DNA was sequenced by the dideoxy chain-termination method using Sequenase (United States Biochemical) (11) and sequence analysis was carried out using DNASTAR and PCFOLD (12) and CLUSTAL (13) software. Homology searches ofthe Swiss-Prot
Leishmania RNA virus-1 (LRV1) is a double-stranded RNA virus present in some Leishmania species. The virus genome consists of a 450-nucleotide, 5' untranslated region (UTR) followed by the coat gene and the RNA-dependent RNA polymerase (RDRP). It has been shown that the 5' end UTR of the genome promotes internal initiation of translation in an in-vitro assay, indicating the presence of an internal ribosomal entry site (IRES) element upstream of the coat gene. The nucleotide sequences of the 5' subterminal regions of six new isolates of LRV1, of different geographical origins, have now been determined. The RNA folding of the 5' subterminal region of LRV1 has been predicted, using a combination of thermodynamic parameters and folding constraints based on nucleotide substitutions. Furthermore, a putative pyrimidine-rich region (a feature unique to all IRES elements), which is complementary to the Leishmania 18S rRNA, has been identified. The significance and relevance of these findings in the context of the function of the 5' UTR of LRV1 as an IRES element are discussed.
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