Functional and genetic constraints on influenza virus replication and fidelity

The rapid evolution of influenza viruses has led to reduced vaccine efficacy, episodic drug resistance, and the emergence of novel seasonal and pandemic strains. The influenza virus polymerase complex is central to the evolution of influenza A viruses (IAV), as it is a major factor in adaptation to new hosts, and its replicative fidelity determines the rate at which the virus will acquire mutations that lead to host range expansion, drug resistance, or antigenic drift. The long-term goal of this project is to elucidate the mechanisms through which novel viral variants are generated, which is critical to understanding how viruses emerge and spread. The objective of this project is define how competing selective forces drive the evolution of the IAV polymerase. The central hypothesis is that there is an inherent conflict between replication speed and fidelity, and that the evolution of the IAV polymerase is highly constrained by the high mutation rate of PB1, its RNA-dependent RNA polymerase (RdRp). This project will apply phylogenetic analysis, mutation rate assays, deep mutational scanning (DMS), molecular dynamic modeling, and in vitro polymerase assays to define the structural and functional constrains on the IAV polymerase. The feasibility of this approach is supported by preliminary data, which show that: (i) phylogenetic approaches can define the mutational pathway taken by the IAV polymerase as it adapts to human hosts; (ii) deep mutational scanning (DMS) can systematically define the impact of amino acid substitutions on polymerase function; (iii) a novel fluctuation test provides precise measurements of mutation rates for each nucleotide substitution class; (iv) the combination of molecular dynamic modeling and biochemistry can define functionally important interactions within the polymerase heterotrimer. Detailed analyses of the IAV polymerase will be accomplished in three aims. (Aim 1) Define the trade-off between replicative speed and fidelity over 50 years of H3N2 evolution. Competition assays and fluctuation tests will be used to determine the functional impact of adaptive mutations that have arisen in the natural evolution of PB1. (Aim 2) Measure the structural and functional impacts of all amino acid mutations in the influenza virus RdRp. DMS of the PB1 protein with serial passage in the absence and presence of mutagenic nucleosides will be used to evaluate the impact of each mutation on viral replication and mutation rates. (Aim 3) Define how amino acid interactions within the polymerase complex affect replication and fidelity. Dynamic modeling and in vitro assays will be used to mechanistically interrogate how co-selected mutations in PB2, PB1 and PA determine mutation rate. This work is innovative, because it uniquely combines a range of complementary approaches to test a novel hypothesis regarding the evolution of viral polymerases. The proposed research is significant, because it will define how selection on replication rate and fidelity shape the short- and long-term evolution of influenza viruses.