Influenza A virus encodes conserved promoter sequences. Using minimal replication assays-transfections with viral polymerase, nucleoprotein, and a genomic template-these sequences were identified as 13nt at the 5' end of the genomic RNA (U13) and 12nt at the 3' end (U12). Other than the fourth 3' nucleotide, the U12 and U13 sequences are identical between all eight RNA molecules of the segmented influenza A genome. However, individual segments can exhibit different dynamics during infection. Influenza NS2, which modulates transcription and replication differentially between genomic segments, may provide an explanation. Here, we assess how internal sequences of two genomic segments, HA and PB1, contribute to NS2-dependent replication and map such interactions down to individual nucleotides in PB1. We find that the expression of NS2 significantly alters sequence requirements for efficient replication beyond the identical U12 and U13 sequences, providing a potential mechanism for segment-specific replication dynamics across the influenza genome.