Identification of the minimal essential RNA sequences responsible for site-specific targeting of the Leishmania RNA virus 1-4 capsid endoribonuclease

The Leishmania RNA virus 1-4 capsid protein possesses an endoribonuclease a ctivity responsible for single-site-specific cleavage within the 450-nucleo tide 5' untranslated region of its own viral RNA transcript. To characteriz e the minimal essential RNA determinants required for site-specific cleavag e, mutated RNA transcripts were examined for susceptibility to cleavage by the virus capsid protein in an in vitro assay. Deletion analyses revealed t hat all determinants necessary for accurate cleavage are encoded in viral n ucleotides 249 to 342. Nuclease mapping and site-specific mutagenesis of th e minimal RNA sequence defined a stern-loop structure that is located 40 nu cleotides upstream from the cleavage site (nucleotide 320) and that is esse ntial for accurate RNA cleavage, Abrogation of cleavage by disruption of ba se pairing within the stem-loop was reversed through the introduction of co mplementary nucleotide substitutions that reestablished the structure. We a lso provide evidence that divalent cations, essential components of the cle avage reaction, stabilized the stem-loop structure in solution. That capsid -specific antiserum eliminated specific RNA cleavage provides further evide nce that the virus capsid gene encodes the essential endoribonuclease activ ity.