Influenza virus mRNA synthesis by the RNA-dependent RNA polymerase involves binding and cleavage of capped cellular mRNA by the PB2 and PA subunits, respectively, and extension of viral mRNA by PB1. However, the mechanism for such a dynamic process is unclear. Using high-throughput mutagenesis and sequencing analysis, we have not only generated a comprehensive functional map for the microdomains of individual subunits, but have also revealed the PA linker to be critical for polymerase activity. This PA linker binds to PB1 and also forms ionic interactions with the PA C-terminal channel. Nearly all mutants with five amino acid insertions in the linker were non-viable. Our model further suggests that the PA linker may play an important role in the conformational changes that occur between stages that favor capped mRNA binding and cleavage and those associated with viral mRNA synthesis.

IMPORTANCE:

The RNA-dependent RNA polymerase of influenza virus consists of the PB1, PB2 and PA subunits. By combining genome-wide mutagenesis analysis with the recently discovered crystal structure of the influenza polymerase heterotrimer, we generated a comprehensive functional map of the entire influenza polymerase complex. We identified the microdomains of individual subunits, including the catalytic domains, the interaction interfaces between subunits, and nine linkers interconnecting different domains. Interestingly, we found that mutants with five amino acid insertions in individual linkers were non-viable, suggesting the critical roles these linkers play in coordinating spatial relationships between the subunits. We furthermore identified an extended PA linker that binds to PB1 and also forms ionic interactions with the PA C-terminal channel