High-temperature inducible cell-free transcription and replication of double-stranded RNAs within the parasitic protozoan Cryptosporidium parvum

Sporozoites of the protozoan parasite, Cryptosporidium parvum, were found to contain free, full-size plus strands transcribed from two extrachromosomal, cytoplasmic, virus-like double-stranded RNAs (dsRNAs). Cell-free transcription and replication of both dsRNAs were observed in crude sporozoite lysates. RNA polymerase activity was found to be dependent upon addition of Mg2+ or Mn2+, as well as the four ribonucleoside triphosphates, and was insensitive to inhibitors of cellular DNA-dependent RNA polymerase. Semiconservative transcription of the dsRNAs (plus strand synthesis) was observed at a wide range of temperatures, with an optimum of 50 degrees C. In contrast, replication (minus strand synthesis) was detected only at 50 and 60 degrees C.