Mixed populations of viruses may have been sampled in which a virus with a mutant, nonfunctional PB1 protein was ‘rescued’ by a coinfecting virus with a functional PB1 gene/protein. Finally, we cannot rule out the possibility that erroneous sequences were submitted to influenza sequence databases. Interestingly, mutation K480R in motif IV of the PB1 subunit consistently demonstrated slightly increased polymerase activity in H5N1 and H1N1 polymerase complexes relative to wild-type. The amino acid alteration at this position is considered conservative since lysine and arginine are basic amino acids that share similar chemical structures. As this mutation has been found in several early pandemic 2009 virus isolates, we used reverse genetics to introduce the K480R mutation into the PB1 subunit of the A/California/04/09 virus strain to assess viral growth characteristics. Although the K480R mutation yielded increased amounts of replication products and slightly increased replicative ability in the minireplicon assays, the mutation did not affect virus titers in cell culture. We have considered two likely possibilities to explain the discrepancy in replication efficiency results between the in vitro assays and growth curve analysis. While the K480R mutation reproducibly enhanced transcription/replication in two different polymerase backgrounds and in two different cell lines, the observed increase may not be MLN4924 sufficient to translate into a detectable rise in virus titer of the CA04 strain. Alternatively, the mutation in PB1 may increase transcription/replication, but concomitantly interferes with another step in the viral life cycle. Structural data of the PB1 protein, along with the rest of the heterotrimeric polymerase, may help in the interpretation of mutational analysis of these four conserved motifs. To date, there are no crystal structures available of the central region of the PB1 subunit in which the consensus motifs are found. However, based on secondary structure predictions, the ‘invariant’ amino acids are frequently located within or proximal to turn structures that may be crucial for their proper positioning in processes such as cation binding and template specificity. Considering the ‘invariant’ amino acids are embedded in blocks of conserved residues, it is conceivable that the four motifs work cooperatively to form a well-defined functional unit constituting the polymerase module. Therefore, intermotif distances may also be critical for polymerase functionality by providing the correct folding and three-dimensional structure of the protein. potential secondary structures of the four conserved PB1 motifs that contribute to influenza polymerase activity. Despite lack of functional information on the individual conserved PB1 motifs of the influenza virus, sequence comparison and structural analysis of other RNA and DNA dependent viruses suggest motif III as the core of the transcriptase-replicase activity. This motif is defined by a centrally located SDD consensus sequence for influenza viruses. Common among all RNA-dependent RNA polymerases, the two ‘invariant’ aspartic acid residues are flanked by hydrophobic residues and located in the turn of a beta-turn-beta supersecondary structure. Consequently, motif III has been implicated in several functions including metal and phosphate binding, template recognition, and other catalytic functions.