Neuraminidase (NA) inhibitors (NIs) are the first line of defense against influenza virus. Reverse genetics experiments allow the study of resistance mechanisms by anticipating the impacts of mutations to the virus. To look at the possibility of an increased effect on the resistance phenotype of a combination of framework mutations, known to confer resistance to oseltamivir or zanamivir, with limited effect on virus fitness, we constructed 4 viruses by reverse genetics in the A/Moscow/10/99 H3N2 background containing double mutations in their neuraminidase genes: E119D؉I222L, E119V؉I222L, D198N؉I222L, and H274Y؉I222L (N2 numbering). Among the viruses produced, the E119D؉I222L mutant virus was not able to grow without bacterial NA complementation and the D198N؉I222L mutant and H274Y؉I222L mutant were not stable after passages in MDCK cells. The E119V؉I222L mutant was stable after five passages in MDCK cells. This E119V-and-I222L combination had a combinatorial effect on oseltamivir resistance. The total NA activity of the E119V؉I222L mutant was low (5% compared to that of the wild-type virus). This drop in NA activity resulted from a decreased NA quantity in the virion in comparison to that of the wild-type virus (1.4% of that of the wild type). In MDCK-SIAT1 cells, the E119V؉I222L mutant virus did not present a replicative advantage over the wild-type virus, even in the presence of oseltamivir. Double mutations combining two framework mutations in the NA gene still have to be monitored, as they could induce a high level of resistance to NIs, without impairing the NA affinity. Our study allows a better understanding of the diversity of the mechanisms of resistance to NIs.The influenza A virus presents two major surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA). HA mediates virus entry into the cell by binding to terminal sialic acid (N-acetyl neuraminic acid) of cellular glycoconjugates (52). NA, through its sialidase activity, facilitates the elution of progeny virions from infected cells through the cleavage of terminal sialic acid from viral and cellular glycoconjugates (36,44,45).Among the nine NA subtypes described, the crystal structures of the N9 (5, 57), N2 (58, 59), N6, and N1/N4/N8 subtypes (51) have been resolved. The NA head is a tetramer composed of four identical subunits arranged with a circular 4-fold symmetry. Each monomer contains six beta sheets composed of 4 antiparallel strands. The NA active site is located centrally on each unit and forms a pocket composed of conserved residues found in all influenza A virus NA (16). All these residues form the wall of the catalytic pocket. It includes catalytic sites (R118, D151, R152, R224, E276, R292, R371, and Y406) that interact directly with the substrate, sialic acid, and framework sites (E119, R156, W178, S179, D/N198, I222, E227, H274, E277, N294, and E425) that interact with catalytic residues to stabilize the active site (15).Due to its conserved structure and its essential role in viral replication, NA is an attractive antiviral t...

Despite progress in our knowledge of the internal organisation of influenza virus particles, little is known about the determinants of their morphology and, more particularly, of the actual abundance of structural proteins at the virion level. To address these issues, we used cryo-EM to focus on viral (and host) factors that might account for observed differences in virion morphology and characteristics such as size, shape and glycoprotein (GP) spike density. Twelve recombinant viruses were characterised in terms of their morphology, neuraminidase activity and virus growth. The genomic composition was shown to be important in determining the GP spike density. In particular, polymerase gene segments and especially PB1/PB2 were shown to have a prominent influence in addition to that for HA in determining GP spike density, a feature consistent with a functional link between these virus components important for virus fitness.

Influenza viruses possess at their surface two glycoproteins, the hemagglutinin and the neuraminidase, of which the antagonistic functions have to be well balanced for the virus to grow efficiently. Ferraris et al. isolated in 2003–2004 viruses lacking both a NA gene and protein (H3NA- viruses) (Ferraris O., 2006, Vaccine, 24(44–46):6656-9). In this study we showed that the hemagglutinins of two of the H3NA- viruses have reduced affinity for SAα2.6Gal receptors, between 49 and 128 times lower than that of the A/Moscow/10/99 (H3N2) virus and no detectable affinity for SAα2.3Gal receptors. We also showed that the low hemagglutinin affinity of the H3NA- viruses compensates for the lack of NA activity and allows the restoration of the growth of an A/Moscow/10/99 virus deficient in neuraminidase. These observations increase our understanding of H3NA- viruses in relation to the balance between the functional activities of the neuraminidase and hemagglutinin.