Early In Vitro Transcription Termination in Human H5 Influenza Viral RNA Synthesis

Abstract

Rapid diagnostic identification of the human 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 human 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 SP6 phage promoter systems. Gel analysis of RNA transcribed from plasmids containing the H5 sequence between a 5' SP6 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 SP6 RNA polymerase promoters to allow transcription in either direction with either polymerase. The T7 transcription product from purified, restricted plasmids in the vRNA direction only produced the 1,776-base full-length sequence and the 784-base fragment, instead of the three bands generated by the SP6 system, suggesting an early termination mechanism. Additionally, the T7 system produced a higher fraction of full-length vRNA transcripts than the SP6 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 magnesium 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 SP6 phage polymerase systems to minimize these early termination events during in vitro transcription of H5 influenza vRNA.

Experimental Pathways towards Developing a Rotavirus Reverse Genetics System: Synthetic Full Length Rotavirus ssRNAs Are Neither Infectious nor Translated in Permissive Cells

“…One band of each was consistent with the full length product; the other one was smaller. Another publication has found comparable results when in vitro transcribing the influenza H5N1 virus HA RNAs [33], where faster migrating bands were attributed to premature in vitro transcription termination due to a type II RNA hairpin loop terminator [33]. This is a plausible assumption for RV segments 3 and 6 as we know that RV ssRNA segments are highly structured [32].…”

“…Transcription products of segment 3 and 6 cDNA templates each migrated as double bands, one of the expected full length ssRNA and a second smaller transcript. The same dual product was seen for both segments when PCR amplicons were used as transcription templates (unpublished data), strongly suggesting that the shorter ssRNA had resulted from premature termination of transcription [33]. To ensure the bands were not gel-related artifacts, samples were separated by denaturing urea PAGE, and the same profile was seen (unpublished data).…”