Influenza Neuraminidase Structure and Dynamics:Resolving How Stalk Variation Impacts Function - Influenza virus neuraminidase (NA) plays a central role in enabling influenza virus infection, transmission, and propagation, yet structural variation among influenza subtypes and structure-function relationships for this major surface glycoprotein remain poorly characterized. NA is a sialidase that cleaves sialic acid from host glycoproteins and glycolipids and is crucial for mediating disaggregation of viral particles, facilitating their release from host cells following budding assembly, as well as enabling their migration through mucus and the glycocalyx barrier surrounding cells during transmission and infection. NA is the target for anti-influenza sialic acid analog drugs such as oseltamivir (i.e. Tamiflu). NA is also increasingly recognized as an important antigenic target for protective antibodies, including some that are broadly cross-reactive against diverse strains and subtypes. Recent studies have indicated that NA exhibits considerable variation in its organization among diverse strains, however, in comparison to the other major influenza surface protein, hemagglutinin (HA), relatively little is known about structural variation among strains and subtypes and how that impacts NA function and immune recognition. While structures are available for the isolated catalytic head domain assemblies, often in re-engineered, stabilized forms, little data is available for the complete membrane-displayed assembly, despite the fact that key modulatory activities have been attributed to the portions of NA, such as its stalk that tethers the catalytic head to the viral membrane. These important domains are commonly removed for structural and in vitro studies. Indeed, perhaps the most striking type of NA variation involves significant sequence and length variation in the stalk domain. Dramatic length shifts in the NA stalks have been documented in conjunction with growth in specific host species, e.g. avian vs human. Changes in the stalk have also been reported to modulate NA catalytic activity, virulence and antibody recognition. The stalk is thus a critical, if poorly understood, modulator of IAV phenotype. Here, we propose to use cryo-electron tomography (cryo-ET) and hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to resolve the structure, dynamics, and flexibility of native, membrane-presented influenza NA and their matched soluble ectodomains from a contemporary and ancestral avian H5N1 strain. Our experiments will test the effect of membrane anchoring and naturally occurring stalk truncations that have been reported to modulate NA’s oligomeric stability, enzymatic activity, host specificity, and viral pathogenesis. In parallel to the structural analysis, an array of sialidase activity assays will be performed to connect structure and NA function. These studies will provide new insight into NA’s structural variation, its native organization on authentic biological membranes, and structural determinants of its essential viral function.
