RNA-dependent RNA polymerase activity associated with Eimeria necatrix virus particles containing either double-stranded or single-stranded RNA

Single-stranded (ss) RNA-containing and double-stranded (ds) RNA-containing virus particles of Eimeria necatrix were isolated by centrifugation through a CsCl gradient. RNA from the gradient fractions was identified as single-stranded or double-stranded by probing northern blots with digoxigenin-labeled riboprobes. These probes were generated with SP6 and T7 RNA polymerases from a partial cDNA clone derived from 5.6-kb viral dsRNA of E. necatrix. RNA-dependent RNA polymerase (RDRP) activity was identified in these CsCl-purified virus particles. The polymerase products of the ssRNA particles consisted of dsRNA indicating replicase activity, whereas the polymerase products of the dsRNA particles consisted of ssRNA indicating trancriptase activity. RNase treatment in high salt solution (0.3 M NaCI) of the pooled RDRP products revealed that the products consisted of both RNase-resistant dsRNA and RNase-sensitive ssRNA. These results show that both replicase and transcriptase activities were present in the purified virus. The digoxigenin-labeled products hybridized to both SP6 and T7 transcripts confirming the presence of both activities.