Probing the functional constraints of influenza A virus NEP by deep mutational scanning
Qi Wen Teo,
Yiquan Wang,
Huibin Lv,
Michael S. Oade,
Kevin J. Mao,
Timothy J.C. Tan,
Yang Wei Huan,
Joel Rivera-Cardona,
Evan K. Shao,
Danbi Choi,
Chaoyang Wang,
Zahra Tavakoli Dargani,
Christopher B. Brooke,
Aartjan J.W. te Velthuis,
Nicholas C. Wu
Affiliations
Qi Wen Teo
Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA; Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
Yiquan Wang
Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
Huibin Lv
Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA; Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
Michael S. Oade
Department of Molecular Biology, Princeton University, Washington Road, Princeton, NJ 08544, USA
Kevin J. Mao
Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
Timothy J.C. Tan
Center for Biophysics and Quantitative Biology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
Yang Wei Huan
Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
Joel Rivera-Cardona
Department of Microbiology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
Evan K. Shao
Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
Danbi Choi
Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
Chaoyang Wang
Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
Zahra Tavakoli Dargani
Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
Christopher B. Brooke
Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA; Department of Microbiology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
Aartjan J.W. te Velthuis
Department of Molecular Biology, Princeton University, Washington Road, Princeton, NJ 08544, USA
Nicholas C. Wu
Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA; Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA; Center for Biophysics and Quantitative Biology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA; Carle Illinois College of Medicine, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA; Corresponding author
Summary: The influenza A virus nuclear export protein (NEP) is a multifunctional protein that is essential for the viral life cycle and has very high sequence conservation. However, since the open reading frame of NEP largely overlaps with that of another influenza viral protein, non-structural protein 1, it is difficult to infer the functional constraints of NEP based on sequence conservation analysis. In addition, the N-terminal of NEP is structurally disordered, which further complicates the understanding of its function. Here, we systematically measure the replication fitness effects of >1,800 mutations of NEP. Our results show that the N-terminal domain has high mutational tolerance. Additional experiments show that N-terminal domain mutations affect viral transcription and replication dynamics, host cellular responses, and mammalian adaptation of avian influenza virus. Overall, our study not only advances the functional understanding of NEP but also provides insights into its evolutionary constraints.
