Mechanism of co-transcriptional cap snatching by influenza polymerase
Summary
This Nature paper defines, at near-atomic resolution, how the influenza virus RNA-dependent RNA polymerase (FluPol) engages host RNA polymerase II (Pol II) and the elongation factor DSIF to perform co-transcriptional “cap snatching” — the process by which the virus steals 5′ capped fragments from nascent host transcripts to prime viral mRNA synthesis. Using biochemical assays, cell-based minigenome tests and two cryo-EM structures (pre- and post-cleavage), the authors show that FluPol recognises a phosphorylated Pol II–DSIF early elongation complex, positions its PA endonuclease near the Pol II RNA exit channel and binds the capped RNA in PB2, then cleaves a 10–15 nt primer which is steered into FluPol’s polymerase active site for viral transcription.
Key Points
- FluPol preferentially binds a Pol II–DSIF elongation complex whose CTD is phosphorylated at serine-5; CTD phosphorylation is the main determinant for stable recruitment.
- DSIF enhances FluPol endonuclease cleavage of Pol II-bound capped RNA; the PA endonuclease docks near the Pol II RNA exit channel and interfaces with DSIF (KOWx-4).
- Cryo-EM structures capture a pre-cleavage complex with continuous density from the cap in PB2 to the Pol II active site, and a post-cleavage complex where the snatched primer’s 3′ end is directed toward FluPol’s PB1 active site.
- Mutations at two newly identified interfaces (PA–DSIF and PB2–Pol II) reduce FluPol activity in cells; some PB2 mutations specifically impair viral transcription and reduce viral viability.
- After cleavage, FluPol adopts a pre-initiation-like conformation that readies the snatched primer for annealing to the viral template with minimal further rearrangement.
- The window for cap snatching is the early elongation/paused Pol II state (Pol II–DSIF / PEC) and is incompatible with later activated elongation complexes (EC*), linking cap snatching tightly to transcription phase.
- Structures and conserved interfaces provide targets for future in silico screening, though small-molecule disruption of protein–protein interfaces remains challenging.
Content summary
Rotsch et al. reconstituted mammalian Pol II elongation complexes (with cap(1)-RNA and CAK-phosphorylated CTD), added human DSIF and recombinant influenza A/H7N9 FluPol (PA E119D mutant to stabilise complexes), and used SEC, fluorescence endonuclease assays and cryo-EM to map interactions. SEC and cleavage assays show CTD phosphorylation strongly promotes FluPol recruitment and stimulates cleavage; DSIF further boosts cleavage and primer usability for extension. Two cryo-EM maps (pre- and post-cleavage at ~3 Å focused resolution) reveal FluPol binding adjacent to the Pol II RNA exit channel: PA contacts DSIF KOWx-4 and PB2 inserts at the RPB1/RPB3/RPB11 dock, with visible density for serine-5 phosphorylated CTD. Post-cleavage structure shows the 3′ end of the cleaved primer guided into the polymerase active site, with the priming loop poised—explaining efficient transition to initiation. Mutational analyses (in vitro and cell-based) validate functional importance of the interfaces, and reverse-genetics viruses carrying specific PB2 mutations show reduced fitness.
Context and relevance
This work settles a longstanding mechanistic question about when and how influenza performs cap snatching in the host nucleus. By connecting FluPol activity to defined Pol II transcriptional states (early elongation/pausing), it links viral transcription initiation to host transcription dynamics and capping enzymes (notably CMTR1). The structures explain why cap(1) methylation and CTD phosphorylation matter, why DSIF stimulates primer capture, and why cap snatching is restricted to a specific transcriptional window — insights relevant for antiviral strategy design and for understanding host–pathogen interplay at the transcriptional level.
Author style
Punchy: this is a clear, high‑impact structural and functional dissection of influenza cap snatching. The data are comprehensive — biochemistry, cryo‑EM, cell assays and virology — and the conserved interfaces make the findings broadly relevant across influenza strains.
Why should I read this?
Look — if you care about how influenza hijacks host transcription (and you probably should), this paper gives you the blueprint. It shows exactly where FluPol grabs the nascent cap, how DSIF and Pol II phosphorylation bait the trap, and which bits of the polymerase you’d have to mess with to stop it. Big for mechanism, and handy for anyone hunting antiviral targets.