Structure-based design of stabilized recombinant influenza neuraminidase tetramers
Daniel Ellis, Julia Lederhofer, Oliver J. Acton, Yaroslav Tsybovsky, Sally Kephart, Christina Yap, Rebecca A. Gillespie, Adrian Creanga, Audrey Olshefsky, Tyler Stephens, Deleah Pettie, Michael Murphy, Claire Sydeman, Maggie Ahlrichs, Sidney Chan, Andrew J. Borst, Young‐Jun Park, Kelly K. Lee, Barney S. Graham, David Veesler, Neil P. King, Masaru Kanekiyo
Abstract
Influenza virus neuraminidase (NA) is a major antiviral drug target and has recently reemerged as a key target of antibody-mediated protective immunity. Here we show that recombinant NAs across non-bat subtypes adopt various tetrameric conformations, including an "open" state that may help explain poorly understood variations in NA stability across viral strains and subtypes. We use homology-directed protein design to uncover the structural principles underlying these distinct tetrameric conformations and stabilize multiple recombinant NAs in the "closed" state, yielding two near-atomic resolution structures of NA by cryo-EM. In addition to enhancing thermal stability, conformational stabilization improves affinity to protective antibodies elicited by viral infection, including antibodies targeting a quaternary epitope and the broadly conserved catalytic site. Stabilized NAs can also be integrated into viruses without affecting fitness. Our findings provide a deeper understanding of NA structure, stability, and antigenicity, and establish design strategies for reinforcing the conformational integrity of recombinant NA proteins.