Status lab-led Areas Influenza Genome Regulation and Replication
One heterotrimeric polymerase performs two incompatible reactions on the same eight templates. Messenger RNA synthesis requires the enzyme to stay bound in cis to the 5 prime template end, which is also what forces the stuttering that builds the poly(A) tail, while genome synthesis is primer independent and requires the enzyme to read through that same end. Perez 2010 states the paradox directly and records that the models then available did not reconcile it. The field had correlates for the transition, including stabilisation of the complementary RNA intermediate, nucleotide concentration and the soluble pools of nucleoprotein and polymerase, but no molecular event that reassigned the enzyme.
Deep sequencing of the sub-40 nucleotide RNA fraction of infected lung epithelial cells found a discrete species of 22 to 27 nucleotides matching the 5 prime terminus of each of the eight genomic segments, distributed as a terminal hotspot rather than as breakdown product, produced by H1N1, H3N2 and H5N1 and across human, canine, murine and avian systems, and not induced by an unrelated virus or by type I interferon (Perez 2010). Locked nucleic acid inhibition directed at one segment's species depleted that segment's genomic RNA while sparing messenger and complementary RNA and the other seven segments. Perez 2012 then placed the species on the complementary RNA intermediate as template, showed it stays nuclear and is largely excluded from virions, mapped binding to the PB1 and PA heterodimer with the basic residue R566 of PA most important, and reproduced the effect in a cell-free reaction with purified trimer where synthetic small viral RNA promoted full-length synthesis even with its 3 prime hydroxyl blocked, which excludes priming, with the first 13 nucleotides sufficient. A recombinant virus unable to generate the species from the neuraminidase segment lost genome synthesis for that segment alone while retaining neuraminidase messenger RNA. Perez 2012 states in its own discussion that this is the first example of a small RNA controlling RNA-dependent RNA polymerase activity, and that priority claim belongs to the paper rather than to this document.
What is now known is that the influenza polymerase carries a ligand, made as a product of copying its own genome, whose presence shifts its output toward full-length genome synthesis for the segment it came from. What is not known is how the species is made, whether it causes the transition rather than accompanying it, or whether each segment truly carries its own loaded replicase and whether that is how the eight segments are held in balance. No polymerase occupancy measurement and no structure of the complex exists in either paper, and Perez 2012 cautions that the promiscuity of a short synthetic mimetic in vitro may not reflect what the virus uses in a cell. The line did not continue as a dedicated program. Its clearest later appearance is as a distinct band alongside mini-viral RNA in Nilsson-Payant 2021.
Substantiated by - Influenza A virus-generated small RNAs regulate the switch from transcription to replication, identifies the species, establishes that it is made across subtypes and hosts, and shows segment-specific loss of genome synthesis when it is blocked - A Small-RNA Enhancer of Viral Polymerase Activity, establishes the template, the polymerase binding site and a non-priming mechanism in a reconstituted reaction, and carries the priority claim
Influenza A virus small viral RNAs are shown to be synthesized from the complementary RNA intermediate, to load into the RNA binding cleft of the polymerase PA subunit, and to act there as segment-specific allosteric enhancers of full-length genome synthesis.
Influenza A virus produces a family of 22 to 27 nucleotide small viral RNAs corresponding to the 5 prime end of each genomic segment, which accumulate as the polymerase shifts toward genome synthesis and whose inhibition selectively depletes genomic RNA without comparably affecting messenger or complementary RNA.