Molecular organization of Leishmania RNA virus 1.

Abstract

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

Tilting the balance between RNA interference and replication eradicates Leishmania RNA virus 1 and mitigates the inflammatory response

“…Previous siRNA studies in Leishmania analyzed RNAs using a tagged Argonaute inserted into an δago1 − knockout of Lbr M2903, which lacks LRV1 (9,29,38,39). Because the lines bearing LRV1 studied here had not been similarly modified, we sequenced total small RNAs (sRNAs) as an alternative.…”

“…This virus is a member of the Totiviridae family and consists of a single-segmented dsRNA genome that encodes only a capsid protein and an RNA-dependent RNA polymerase (RDRP) (8,9). It is most frequently found (as LRV1) in New World parasite species in the subgenus Viannia, such as Leishmania braziliensis (Lbr) and L. guyanensis (Lgy), which cause both cutaneous and mucocutaneous disease (6), but it has also been found sporadically in Old World subgenus Leishmania species (as LRV2) (10,11).…”

Type I interferons induced by endogenous or exogenous viral infections promote metastasis and relapse of leishmaniasis

“…Recently, we correlated the development of these more severe forms of leishmaniasis with the presence of Leishmania RNA virus (LRV1) within different species of Leishmania (3-6). Discovered in the 1980s (7,8), and since then for a long time neglected, Leishmaniavirus is a genus of double-stranded RNA viruses belonging to the Totiviridae family. Like most other viruses in this family, LRV1 is neither shed nor infectious, and thus can be seen as a persistent, endogenous viral element (9).…”

Successful transient introduction of leishmania RNA virus into a virally infected and an uninfected strain of Leishmania

“…In LRV1, the putative transcriptase and replicase were detected in virus-infected wholecell extracts from the promastigote stage of the parasite and in virus purified from both CsCl and sucrose gradients (8,9). More recently, the entire sequence for LRV1-1 was reported by Stuart and colleagues (10).…”