Characterization of double-stranded RNA satellites associated with the Trichomonas vaginalis virus

Three small and distinct satellite double-stranded RNAs (dsRNAs) denoted s1, s1', and s2 were recently described for a Trichomonas vaginalis isolate harboring a dsRNA virus. Since characterization of these satellite dsRNAs might provide insight into the virus replication cycle and virus-host interactions, full-length cDNAs to s1 and s1' dsRNAs were synthesized and sequenced. s1 dsRNA has 688 bp, and s1' dsRNA has 616 bp. A 228-bp open reading frame that begins at nucleotide 37 was detected on a putative sense strand of s1. All satellite RNAs were found associated with RNA-dependent RNA polymerase (RDRP) activity that banded on CsCl gradients. Within carrier trichomonads, satellite RNAs synthesized single-stranded replicative intermediates. An in vitro assay was established to assess replication of satellite RNAs. Transcripts generated from s1 cDNA, for example, served as a template for the viral RDRP. These templates had a polarity similar to that of the replicative intermediate found in the satellite-harboring parasites. Importantly, the recognition of s1 RNA was shown to be specific, since unrelated RNAs did not serve as templates for RDRP under the same experimental conditions. The data indicate that the cDNA of s1 has a specific and essential sequence needed for recognition by the viral RDRP and for subsequent RNA synthesis. Both s1 and s1' have conserved domains, albeit of unproven function, but which may be required for replication.