Fig. 6: Mutation of PB1-K578 affects the binding of free polymerase to NP and polymerase dimerization.

a, b Detection of NP binding to trimeric polymerase by co-affinity precipitation. Cells were transfected with PB1, PB2, HA-tagged PA, strep-tagged NP (a) and a firefly-encoding vRNA reporter (b) followed by strep-purification 24 h p.t. Co-precipitated proteins were detected by western blot. c Polymerase dimerization assessed by co-affinity precipitation. Cells were transfected with PB1, PB2 and both HA- and strep-tagged PA. 24 h p.t. the polymerase components bound to strep-tagged PA were purified. Co-precipitated proteins were detected by western blot. (Input Strep-PA signals derive from a blot analyzed in parallel). Quantification of the interaction is shown below the panels. Levels of HA-tagged PA were normalized to strep-tagged NP (a, b) or strep-tagged PA (c) and depicted below as the mean n-fold of WT (±SEM). The number of independent biological replicates (n) is provided in the figure. P values < 0.05 compared to WT from Dunnett’s multiple comparison one-way ANOVA are indicated. Source data are provided as a Source data file. d Model depicting the biological function of PB1-K578 ubiquitination during vRNA replication. Incoming vRNPs perform viral mRNA transcription for viral protein expression including the viral polymerase proteins and NP. A subpopulation of newly synthesized trimeric polymerase complexes acquires ubiquitination at PB1-K578 (cyan polymerase complexes with UB in yellow) which disrupts the interaction between PB1-K578 and the loop in the PB2-N1 domain, prevents early formation of the symmetric dimer and reduces the affinity to NP. The PB1-K578 ubiquitinated polymerase interacts with the vRNP-bound polymerase to form the ANP32A-stabilized asymmetric dimer for cRNA synthesis and encapsidation. For vRNA synthesis from the cRNA template, a non-ubiquitinated polymerase (violet polymerase complexes) interacts with the cRNP-associated polymerase under formation of the symmetric dimer. Ultimately, progeny vRNPs may perform secondary mRNA transcription, further rounds of vRNA replication or get exported out of the nucleus for progeny virion assembly. Substitution of the UB-acceptor site PB1-K578 with both alanine and arginine affects vRNA replication (right panel). While PB1-K578A retains the pool of free polymerases, PB1-K578 aborts progression of vRNA replication due to a depletion of free polymerases.