In vivo deep mutational scanning and drug resistance profiling of Influenza A virus neuraminidase

Neuraminidase (NA) is an essential surface enzyme and drug target of influenza A virus. Its evolution can result in potential zoonotic transmission, seasonal epidemics, or the spreading of drug-resistant mutants. A comprehensive understanding of the mutational effect and drug resistance profiles of NA is essential for evaluating its evolutionary potentials. Here we established an in vivo high-throughput profiling system and quantified the replication capacity of NA mutants in mouse lung tissues at single-nucleotide resolution. The fitness of NA mutants generally correlated with natural mutation occurrence and was constrained by both the requirement to maintain protein stability and NA function. Applying the same system, we profiled the drug resistance to three most commonly used neuraminidase inhibitors (NAIs), including Zanamivir (ZA), Oseltamivir (OS) and Peramivir (PE). We confirmed reported drug-resistant mutations and validated new mutations. Notably, we identified a group of allosteric mutations that confer resistance to all three drugs, and may affect drug binding through interfering with the tetramerization of NA. Although the NAIs were widely used in clinic, the occurrence of NAI-resistant mutations in the past decade did not increase, possibly due to their fitness costs. In sum, we established an efficient deep mutational screening system in vivo. We provided a parallel characterization of both the fitness and drug resistance landscape of NA, which may guide the rational selection of anti-viral drugs for optimal therapeutic efficacy and the second generation NAI development.