Early in vitro transcription termination in XXXX H5 influenza viral RNA synthesis.

@entity1 @entity8949 @entity44038 @entity1760
Rapid diagnostic identification of the @entity1 H5 influenza virus is a strategic cornerstone for outbreak prevention. We recently reported a method for direct detection of viral RNA from a highly pathogenic @entity1 H5 influenza strain (A/Hanoi/30408/2005(H5N1)), which necessarily was transcribed in vitro from non-viral sources. This article provides an in-depth analysis of the reaction conditions for in vitro transcription (IVT) of full-length influenza H5 RNA, which is needed for diagnostic RNA production, for the T7 and @entity8949 phage promoter systems. Gel analysis of RNA transcribed from plasmids containing the H5 sequence between a 5' @entity8949 promoter and 3' restriction site (BsmBI) showed that three sequence-verified bands at 1,776, 784, and 591 bases were consistently produced, whereas only one 1,776-base band was expected. These fragments were not observed in H1 or H3 influenza RNA transcribed under similar conditions. A reverse complement of the sequence produced only a single band at 1,776 bases, which suggested either self-cleavage or early termination. Aliquots of the IVT reaction were quenched with EDTA to track the generation of the bands over time, which maintained a constant concentration ratio. The H5 sequence was cloned with T7 and @entity8949 RNA polymerase promoters to allow transcription in either direction with either polymerase. The T7 transcription product from purified, restricted plasmids in the @entity44038 direction only produced the 1,776-base full-length sequence and the 784-base fragment, instead of the three bands generated by the @entity8949 system, suggesting an early termination mechanism. Additionally, the T7 system produced a higher fraction of full-length @entity44038 transcripts than the @entity8949 system did under similar reaction conditions. By sequencing we identified a type II RNA hairpin loop terminator, which forms in a transcription direction-dependent fashion. Variation of the @entity1760 concentration produced the greatest impact on termination profiles, where some reaction mixtures were unable to produce full-length transcripts. Optimized conditions are presented for the T7 and @entity8949 phage polymerase systems to minimize these early termination events during in vitro transcription of H5 influenza @entity44038 .
