NA transmembrane domain : Amphiphilic drift to accommodate two functions

Neuraminidase (NA) is one of two major antigens on the surface of influenza A viruses. It is comprised of a single N-terminal transmembrane domain (TMD), a stalk domain, and a C-terminal enzymatic head domain that cleaves sialic acid, most notably to release new particles from the host cell surface. NA is only enzymatically active as a homo-tetramer. However, it is not known which properties facilitate the oligomerization of NA during assembly. Our results show that, apart from anchoring the protein to the membrane, the NA TMD also contributes to the assembly process by keeping the stalk in a tetrameric conformation. The ability of the TMD to oligomerize is shown to be dependent on its amphiphilic characteristics that was largely conserved across the nine NA subtypes (N1-N9). Over time the NA TMDs in human H1N1 viruses were found to have become more amphiphilic, which correlated with stronger oligomerization. An old H1N1 virus with a more recent N1 TMD had impaired growth, but readily acquired compensatory mutations in the TMD to restore growth, by reverting the TMD oligomerization strength back to that of the old TMD, demonstrating a biological role of the TMD in folding and assembly. NA and the other viral proteins are spatially and temporally coordinated to achieve optimal viral production. By using a co-transfection analysis, the high AU-content in the NA and HA ER-targeting sequence coding regions (for NA TMD as well as the HA signal sequence) were found to inhibit their expression. The inhibition was alleviated by the early expressed influenza RNA-binding protein NS1, which promoted translation and showed enriched foci at the endoplasmic reticulum (ER). NS1, which expresses early during infection, is therefore likely the regulator of NA and HA to prevent premature expression. These results show that the NA TMD is under substantial selection pressure at both the nucleotide and amino acid level to accommodate its roles in ER-targeting, protein folding, and post-transcriptional regulation.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Accepted.