Segments of Influenza Virus Complementary RNA Synthesized In Vitro

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Abstract
In the presence of Mg2+ and a specific primer, ApG or GpG, the influenza WSN virion transcriptase synthesizes large, poly (A)-containing complementary(c)RNA. After removal of its poly (A) with RNase H in the presence of poly(dT), the in vitro cRNA distributed into 7 discrete bands during electrophoresis in acrylamide gels containig 6 M urea. The 8 known segments of virion (v) RNA also distributed into 7 bands under these conditions as 2, rather than the expected 3, large-sized segments were resolved. Each of the in vitro cRNA segments migrated slightly faster than the corresponding vRNA segment. To determine whether this difference in mobility reflects a difference in size between cRNA and vRNA, the double-stranded RNA formed by annealing labeled in vitro cRNA to unlabeled vRNA was subjected to various nuclease treatments and was analyzed by gel electrophoresis. Hybrids treated with RNase T2 or a combination of RNase T2 and RNase H migrated slightly faster than those treated only with RNase H, indicating that RNase T2 removed an RNA sequence other than poly(A), most probably a short sequence of vRNA not hydrogen bonded to cRNA. The in vitro cRNA segments are probably shorter than, and thus incomplete transcripts of, the corresponding vRNA segments. All 8 hybrids were resolved by gel electrophoresis, indicating that all 8 vRNA segments are transcribed into cRNA in vitro. The ApG primer probably initiates in vitro transcription exactly at the 3'' end of vRNA.