The surface-exposed PA-loop of the influenza A virus polymerase is required for viral genome replication.

The heterotrimeric influenza A virus RNA-dependent RNA polymerase complex, composed of PB1, PB2, and PA subunits, is responsible for transcribing and replicating the viral RNA genome. The N-terminal endonuclease domain of the PA subunit performs endonucleolytic cleavage of capped host RNAs to generate capped RNA primers for viral transcription. A surface-exposed flexible loop (PA(51-72)-loop) in the PA endonuclease domain has been shown to be dispensable for endonuclease activity. Interestingly, the PA(51-72)-loop was found to form different intramolecular interactions depending on the conformational arrangement of the polymerase. In this study, we show that a PA subunit lacking the PA(51-72)-loop assembles into a heterotrimeric polymerase with PB1 and PB2. We demonstrate that in a cellular context, the PA(51-72)-loop is required for RNA replication but not transcription by the viral polymerase. In agreement, recombinant viral polymerase lacking the PA(51-72)-loop is able to carry out cap-dependent transcription but is inhibited in de novo replication initiation in vitro. Furthermore, viral RNA (vRNA) synthesis is also restricted during ApG-primed extension, indicating that the PA(51-72)-loop is required not only for replication initiation but also for elongation on a cRNA template. We propose that the PA(51-72)-loop plays a role in the stabilization of the replicase conformation of the polymerase. Together, these results further our understanding of influenza virus RNA genome replication in general and highlight a role of the PA endonuclease domain in polymerase function in particular. IMPORTANCE Influenza A viruses are a major global health threat, not only causing significant morbidity and mortality every year but also having the potential to cause severe pandemic outbreaks like the 1918 influenza pandemic. The viral polymerase is a protein complex which is responsible for transcription and replication of the viral genome and therefore is an attractive target for antiviral drug development. For that purpose it is important to understand the mechanisms of how the virus replicates its genome and how the viral polymerase works on a molecular level. In this report, we characterize the role of the flexible surface-exposed PA(51-72)-loop in polymerase function and offer new insights into the replication mechanism of influenza A viruses.