AJ te Velthuis

The influenza virus polymerase is central to influenza virus evolution. Adaptive mutations within the polymerase are often a prerequisite for efficient spread of novel animal-derived viruses in human populations. The polymerase also determines fidelity and, therefore, the rate at which the virus will acquire mutations that lead to host range expansion, drug resistance, or antigenic drift. Despite its importance to viral replication and evolution, our understanding of the mutational effects and associated constraints on the influenza RNA-dependent RNA polymerase (RdRp) is relatively limited. We performed deep mutational scanning of the A/WSN/1933(H1N1) polymerase basic 1 (PB1), generating a library of 95.4% of amino acid substitutions at 757 sites. After accuracy filters, we were able to measure replicative fitness for 13,354 (84%) of all possible amino acid substitutions, and 13 were validated by results from pairwise competition assays. Functional and structural constraints were better revealed by individual sites involved in RNA or protein interactions than by major subdomains defined by sequence conservation. Mutational tolerance, as defined by site entropy, was correlated with evolutionary potential, as captured by diversity in the available H1N1 sequences. Of the 29 beneficial sites, many have either been identified in the natural evolution of PB1 or shown experimentally to have important impacts on replication and adaptation. Accessibility of amino acid substitutions by single nucleotide mutation was a key factor in determining whether mutations appeared in natural PB1 evolution. Our work provides a comprehensive map of mutational effects on a viral RdRp and a valuable resource for subsequent studies of influenza replication and evolution. IMPORTANCE The influenza virus polymerase is important for adaptation to new hosts and, as a determinant of mutation rate, for the process of adaptation itself. We performed a deep mutational scan of the polymerase basic 1 (PB1) protein to gain insights into the structural and functional constraints on the influenza RNA-dependent RNA polymerase. We find that PB1 is highly constrained at specific sites that are only moderately predicted by the global structure or larger domain. We identified a number of beneficial mutations, many of which have been shown to be functionally important or observed in influenza virus’ natural evolution. Overall, our atlas of PB1 mutations and their fitness impacts serves as an important resource for future studies of influenza replication and evolution.