Generation of the short RNA transcript in Leishmaniavirus correlates with the growth of its parasite host, Leishmania

Leishmaniavirus 1 is a double-stranded RNA virus that infects the New World kinetoplastid parasites, Leishmania braziliensis, and Leishmania guyanensis. The isolated virus particles contain an RNA-dependent RNA polymerase which exhibits both transcriptase activity for genome-length plus-strand synthesis and replicase activity for genome-length minus-strand synthesis. Recently, we identified a 320 nucleotide short RNA transcript of Leishmaniavirus 1-4, derived from the 5' end of the viral plus-strand, which is generated by the virus capsid via site-specific cleavage of the full-length positive single-stranded RNA. We have hypothesized that this short RNA transcript functions to regulate the virus life cycle during the growth of its parasite host, Leishmania guyanensis. To address this hypothesis, we measured the relative amount of short RNA transcripts and the absolute number of viral genomes per infected cell from log through stationary phase of the parasite growth cycle. In vitro assays of the viral polymerase showed an overall increase in viral polymerase activity from log growth into stationary phase which mirrored an in vivo increase in the quantity of double-stranded genome as measured by agarose gel electrophoresis. We have developed competitive reverse transcription-polymerase chain reaction (RT-PCR) assays to measure the relative amounts of viral transcripts in infected cells as well as the number of viral genomes per infected cell. The results of these assays show that the amount of full-length virus transcripts peaks in the parasite stationary phase (132 transcripts per cell), and that the short transcript is most abundant in the early stationary phase cells (24 transcripts per cell).