Nathalie Lejal

The influenza A virus hijacks host cell machinery by “snatching” molecular caps from cellular RNAs to prime its own transcription. While this process is typically associated with the beginning of genes, this study reveals that the viral RNA polymerase exerts a far-reaching influence on host gene expression. Using genome-wide mapping and comparative pathway analyses, we show that viral polymerase (FluPol) recruitment is decoupled from general host transcriptional activity; instead, FluPol is markedly enriched at a distinct subset of promoters and enhancers associated with defense-related pathways. Beyond the initiation site, FluPol remains associated with host transcripts throughout elongation, extending into downstream-of-gene (DoG) regions. It further accumulates at gene ends, potentially contributing to widespread defects in transcription termination. By remaining linked to host transcripts across the entire gene body, FluPol could extend its opportunity for cap-snatching and impair the recycling of cellular transcription machinery. Together, these findings demonstrate how FluPol accumulation across key cellular defense loci spans from initiation through to termination, driving host transcriptional attenuation.

Influenza virus transcription is catalyzed by the viral RNA-polymerase (FluPol) through a cap-snatching activity. The snatching of the cap of cellular mRNA by FluPol is preceded by its binding to the flexible C-terminal domain (CTD) of the RPB1 subunit of RNA-polymerase II (Pol II). To better understand how FluPol brings the 3′-end of the genomic RNAs in close proximity to the host-derived primer, we hypothesized that FluPol may recognize additional Pol II subunits/domains to ensure cap-snatching. Using binary complementation assays between the Pol II and influenza A FluPol subunits and their structural domains, we revealed an interaction between the N-third domain of PB2 and RPB4. This interaction was confirmed by a co-immunoprecipitation assay and was found to occur with the homologous domains of influenza B and C FluPols. The N-half domain of RPB4 was found to be critical in this interaction. Punctual mutants generated at conserved positions between influenza A, B, and C FluPols in the N-third domain of PB2 exhibited strong transcriptional activity defects. These results suggest that FluPol interacts with several domains of Pol II (the CTD to bind Pol II), initiating host transcription and a second transcription on RPB4 to locate FluPol at the proximity of the 5′-end of nascent host mRNA.

Influenza virus transcription is catalyzed by the viral RNA-polymerase (FluPol) through a cap-snatching activity. The snatching of the cap of cellular mRNA by FluPol is preceded by its binding to the flexible C-terminal domain (CTD) of the RPB1 subunit of RNA-polymerase II (Pol II). To better understand how FluPol brings the 3’-end of the genomic RNAs in close proximity to the host-derived primer, we hypothesized that FluPol may recognize additional Pol II subunits/domains to ensure cap-snatching. Using binary complementation assays between the Pol II and FluPol subunits and their structural domains, we revealed an interaction between the N-third domain of PB2 and RPB4. This interaction was confirmed by a co-immunoprecipitation assay and found to occur with the homologous domains of influenza B and C FluPols. Residues [1-72] of RPB4 were found critical in this interaction. Numerous punctual mutants generated at conserved positions between influenza A, B and C FluPols in the N-third domain of PB2 exhibited strong transcriptional activity defect. These results suggest that FluPol interacts with several domains/subunits of Pol II, the CTD to bind Pol II initiating host transcription and a second on RPB4 to locate FluPol at the proximity of the 5’-end of nascent host mRNA.