Impact of the H274Y Substitution on N1, N4, N5, and N8 Neuraminidase Enzymatic Properties and Expression in Reverse Genetic Influenza A Viruses.

The H274Y substitution (N2 numbering) in neuraminidase (NA) N1 confers oseltamivir resistance to A(H1N1) influenza viruses. This resistance has been associated with reduced N1 expression using transfected cells, but the effect of this substitution on the enzymatic properties and on the expression of other group-1-NA subtypes is unknown. The aim of the present study was to evaluate the antiviral resistance, enzymatic properties, and expression of wild-type (WT) and H274Y-substituted NA for each group-1-NA. To this end, viruses with WT or H274Y-substituted NA (N1pdm09 or avian N4, N5 or N8) were generated by reverse genetics, and for each reverse-genetic virus, antiviral susceptibility, NA affinity (Km), and maximum velocity (Vm) were measured. The enzymatic properties were coupled with NA quantification on concentrated reverse genetic viruses using mass spectrometry. The H274Y-NA substitution resulted in highly reduced inhibition by oseltamivir and normal inhibition by zanamivir and laninamivir. This resistance was associated with a reduced affinity for MUNANA substrate and a conserved Vm in all viruses. NA quantification was not significantly different between viruses carrying WT or H274Y-N1, N4 or N8, but was lower for viruses carrying H274Y-N5 compared to those carrying a WT-N5. In conclusion, the H274Y-NA substitution of different group-1-NAs systematically reduced their affinity for MUNANA substrate without a significant impact on NA Vm. The impact of the H274Y-NA substitution on viral NA expression was different according to the studied NA.

Combinatorial effect of two framework mutations (E119V and I222L) in the neuraminidase active site of H3N2 influenza virus on resistance to oseltamivir.

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.

Impact of influenza A virus neuraminidase mutations on the stability, activity, and sensibility of the neuraminidase to neuraminidase inhibitors.

BACKGROUND: The influenza neuraminidase plays a critical role in the spread of the influenza A and B viruses. Through the cleavage of terminal sialic acid from glycoconjugates, it facilitates the elution of progeny virions from infected cells and prevents their self-aggregation. OBJECTIVES: Our objective was to study the impact of mutations at framework sites not under direct selective pressure in the neuraminidase active site. STUDY DESIGN: In the A/Moscow/10/99 (H3N2) virus background, viruses containing mutations in NA framework residues (E119D, R156K, W178L, S179A, D198N, I222L, E227G, H274Y, E277G, N294D, and E425G) were constructed by reverse genetics. After several passages on MDCK cells, fluorimetric assays were conducted to assess the neuraminidase activity and sensibility to the neuraminidase inhibitors (IC50). RESULTS: Among the viruses detectable through the phenotypic tests, R156K, I222L, H274Y, N294D and E425G viruses presented a NA activity between 70% and 100% of the A/Moscow/10/99 wild type one. The D198N and the E119D mutations decreased seriously in NA activity compared to the wild-type (>10-fold). The I222L mutation reduced susceptibility to oseltamivir (18-fold). CONCLUSION: With the exception of one mutation, framework mutations on N2 background do not induce resistance. Nevertheless they tend to decrease slowly the sensitivity to one or the other inhibitors.

Rescue of a H3N2 influenza virus containing a deficient neuraminidase protein by a hemagglutinin with a low receptor-binding affinity.

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.