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Abstract: The RNA polymerase of influenza A virus is a heterotrimeric complex of PB1, PB2 and PA subunits that is required for transcription and replication of the viral genome. Here, we demonstrate a differential requirement of the PA subunit for binding to the vRNA and cRNA promoters — specifically, PA is more important for binding to the cRNA than the vRNA promoter. Furthermore, five point mutations were identified in the L163–I178 region of PA, which resulted in an inhibition of polymerase activity when provided with a cRNA compared to vRNA promoter. Cross-linking studies suggested that this inhibition was due to a reduction in promoter binding of the mutant polymerases to the cRNA promoter. We conclude that the L163–I178 region of PA is directly or indirectly involved in cRNA promoter binding and suggest a novel function for PA in modulating promoter binding. [Copyright &y& Elsevier]