Cap binding and cleavage activities are performed by the viral polymerase complex and depend on the interaction of the complex with the vRNA template. Polyadenylation of the viral mRNA transcripts occurs by reiterative copying of an oligo sequence adjacent to the 59 terminus of each vRNA. In addition, the viral polymerase complex generates full-length, uncapped copies of cRNA, which act as the replicative intermediate for production of progeny vRNAs that are assembled into new virions. While all three polymerase proteins are required for efficient replication and transcription in virus-infected cells, PB1 plays a central role in the formation of the structural backbone and catalytic activities of the RNA polymerase. It possesses four highly conserved regions of amino acids identified by comparative sequence analysis of all viral RNA-dependent DNA polymerases and RNA-dependent RNA polymerases. Together, these four conserved motifs form a large functional domain with at least one ‘invariant’ amino acid per motif. In a previous study, these ‘invariant’ amino acids were tested for their significance in polymerase activity in a minireplicon assay; additional mutations were also introduced into the influenza viral PB1 protein to resemble amino acid sequences found in the polymerases of other RNA viruses. Most of these mutations significantly reduced polymerase activity, demonstrating the critical role of the conserved motifs in PB1 for influenza virus transcription and replication. However, our inspection of influenza A virus PB1 sequences revealed a small number of PB1 proteins with non-consensus amino acids in the conserved motifs. Currently, it remains unknown whether these PB1 proteins support efficient viral replication and transcription of influenza viral RNAs. Here, we therefore tested selected PB1 variants for their replicative ability in minireplicon assays in cell culture. Our data show that these amino acid changes may significantly inactivate the polymerase, providing further support for the importance of these four conserved PB1 motifs for influenza polymerase activity. Based on analysis of currently available sequences in influenza databases, influenza Bortezomib viruses with non-consensus amino acids in the four conserved motifs of PB1 protein exist in nature, although rare in number. To assess the replicative ability of these PB1 proteins, we evaluated their transcription/replication in minireplicon assays and found that most of these PB1 mutations abolished polymerase functionality. Our data support the conclusions by Biswas and Nayak that the four conserved PB1 motifs are critical for replication and transcription. More importantly, our results raise the question of how viruses with these mutations exist in nature. The PB1 mutants tested exhibited similar polymerase activities in both the human and avian cells, arguing against host-specific effects. Therefore, a possible explanation for the existence of natural isolates with PB1 mutations that abrogate polymerase activity in our test system is the presence of compensatory mutations acquired by the respective viruses. Additionally, some of the PB1 mutants tested here were found in viruses not closely related to the model strain used. Hence, the respective mutations may not be active in the genetic background of BHG, but function in their authentic backgrounds. Additional experiments would be needed to address this issue.