Fig 1.
Design and expression of soluble, enzymatically active NA of H1N1 and H5N1 influenza viruses.
(A) Schematic representation of wild type (WT) and recombinant NA (rNA). The cytoplasmic (CD), tetramerization (TM) and stalk domains of the WT NA were replaced by an Ig-k secretion sequence, a 6xHis-tag, and a tetrabrachion domain for protein tetramerization. (B) Reducing SDS-PAGE followed by WB of Expi293 supernatants and lysates collected every 24 h post-transfection. Anti-His tag (left) and anti-NA (right) stainings were used to specifically detect avian H5N1 rNA. (C, D) Titration of specific sialidase activity in culture supernatants harvested every 24 h post-transfection by ELLA and expressed as the supernatant dilution corresponding to an OD450nm = 2. Data show mean±SD and are representative of at least three independent experiments.
Table 1.
Enzymatic properties of swine H1N1 and avian H5N1 rNAs.
Fig 2.
Purification of tetrameric swine H1N1 and avian H5N1 rNAs.
(A) Avian H5N1 rNA purification by ion metal affinity chromatography; SDS-PAGE followed by Coomassie staining. MW: molecular weight marker (kDa); lane 1: pooled crude supernatants; lane 2: flow-through; lane 3: fraction eluted after washing with 10 mM imidazole; lane 4: fraction eluted after wash with 20 mM imidazole; lane 5: fraction eluted with 300 mM imidazole. (B) Gel filtration chromatogram of His-purified avian H5N1 rNA recorded at 280 nm wavelength. (C) SDS-PAGE followed by Coomassie staining of final purified, soluble, tetrameric swine H1N1 (lane 1) and avian H5N1 (lane 2) rNAs.Data shown are representative of at least three independent experiments.
Fig 3.
Glycosylation pattern of swine H1N1 and avian H5N1 rNAs.
rNAs were deglycosylated with PNGase F or Endo H and molecular weights of treated and untreated samples were detected by SDS-PAGE followed by Coomassie staining. Data shown are representative of two independent experiments.
Fig 4.
DSF analysis of avian H5N1 rNA.
The thermostabilizing effect of Ca2+ ions binding to rNA was detected by DSF in the presence of Sypro orange. The graph shows fluorescence intensity vs temperature for increasing amount of Ca2+, 0.079–20 mM, in 25 mM Tris pH 8, 150 mM NaCl buffer.
Fig 5.
Sialidase activity of swine H1N1 and avian H5N1 rNAs.
(A) Kinetic analyses of rNAs. Triplicate data sets for each experiment were used to calculate the steady-state velocity at decreasing concentrations of MuNANA substrate for each enzyme, and were expressed as initial rates (μM/s) vs concentration of substrate. The reactions containing 0.2 nM of enzyme and 0.59–600 μM of MuNANA were performed at 37°C in 200 mM NaOAc, 20 mM CaCl2, 0,01 mg/ml BSA, pH5.5. (B) Titration of rNAs activity by ELLA. Decreasing amount of rNAs were incubated with a fixed amount of fetuin overnight at 37°C and OD detected were graphed vs the protein concentration. Data represent mean±SD of 3 independent experiments performed in duplicate.
Fig 6.
Structural characterization of avian H5N1 rNA using TEM with single particles reconstruction.
(A) Typical negative staining TEM image of avian H5N1 rNA. Scale bar corresponds to 200 nm. (B) Representative class averages of avian H5N1 rNa tetramers. Each class average (~50 images per class) contains particles selected from several micrographs and represents the different orientations of the enzyme. Scale bar corresponds to 10 nm. (C) Top (upper panel) and side (lower panel) surface views of the reconstructed avian H5N1 rNA globular head obtained at a resolution of 24 Å (FSC = 0.5).
Fig 7.
rNAs as sources of sialidase in ELLA.
(A) NI titers determined in a panel of NIBSC sheep polyclonal sera specific for A/turkey/Turkey/01/2005, A/California/07/2009 and A/Caledonia/22/99 N1, A/Wyoming/3/2003 N2, and B/Malaysia/2506/2004 and B/Florida/4/2006 B NAs. (B) NI titers in sera of mice immunized with swine H1N1 and avian H5N1 rNAs adjuvanted with MF59. Data show mean±SD from three independent experiments performed in duplicate. NA = not assayed.