Leishmania RNA Virus 1-Mediated Cap-Independent Translation

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Abstract
Recently, a group of related Leishmania RNA viruses (Leishmania RNA virus 1 [LRV1]) has been isolated from Leishmania guyanensis and L. brasiliensis. These viruses persist in the cytoplasm and contain double-stranded RNA genomes. Miniexon sequences are absent from the 5' end of the viral RNA, and the 5' end of the viral RNA lacks a cap structure, suggesting that LRV1 has evolved a cap-independent mechanism of translation. Cap-independent translation of picornavirus genomic RNA requires a cis element, within the 5' untranslated region (UTR), referred to as an internal ribosome entry site (IRES). In order to find out if the 5' UTR of LRV1 possessed IRES activity, we modified a Leishmania expression vector, pX63NEO-GUS, so that it would produce a dicistronic transcript in which the neomycin phosphotransferase gene was separated from the downstream beta-glucuronidase (GUS) gene by the LRV1 5' UTR. High levels of GUS activity were detected in L. major stably transformed with this plasmid. Elimination of the first 120 nucleotides of the viral 5' UTR lowered GUS activity 10-fold. Furthermore, when the entire 5' UTR was eliminated, GUS activity was undetectable. These results, together with the absence of trans-spliced GUS transcripts, are consistent with the hypothesis that the 5' UTR of LRV1 functions as an IRES element. The ability to couple expression of genes via an IRES element should prove useful in genetic experiments with Leishmania spp.