Site entropy mapped to PB1 structure

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) 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 available H1N1 sequences. Of 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.