The PB1 N-Terminal Helix is Essential for Influenza Polymerase Catalysis

Abstract

Influenza A viruses (IAV), which are known to cause seasonal epidemic and occasional pandemics, rely on a heterotrimeric RNA-dependent RNA polymerase (RdRp) composed of the PB1, PB2, and PA subunits to replicate and transcribe its segmented RNA genome. The PB1 gene also encodes PB1-ΔN40, an N-terminally truncated isoform previously believed to be transcriptionally inactive due to its inability to interact with the PA subunit. However, recent findings from our lab show that PB1-ΔN40 is expressed earlier than full-length PB1 during infection and may exhibit transcriptional activity when bound with PB2 and PA, suggesting a previously unrecognized role in IAV viral infection cycle. To investigate the functional significance of PB1-ΔN40, we cloned, expressed, and purified RdRp complexes containing either full-length PB1 or PB1-ΔN40 from the H1N1 WSN strain. Using biochemical assays—including in vitro RNA replication and transcription—we then compared the activity and composition of these complexes. We found that PB1-ΔN40 was able to complex with PA and PB2, but that it had no replication or transcriptional activity in vitro, in line with the partial deletion of the PB1 active site. This work has thus provided critical insights into the mechanistic role of PB1-ΔN40 in IAV infection.