FIG 1.

Design and rescue of influenza viruses with extended N1 neuraminidase stalk domains. (A) Estimated lengths of the ectodomains of N1 proteins with different stalk lengths (red) compared with the ectodomain of H1 hemagglutinin (blue). The structure of the NA stalk has not been determined and is indicated by four bars. The lengths of the ectodomains are estimates from molecular dynamics simulations, as reported before (45). The depiction of H1 is based on the crystal structure of the PR8 HA (PDB number 1RU7 [62]), and the depictions of N1 are based on the crystal structure of the NA of A/California/04/2009 (Cal09) virus (PDB number 3TI3 [63]). The structures are not to scale and were visualized with UCSF Chimera (64). aa, amino acids. (B) The four domains of the NA protein are indicated (CT, cytoplasmic tail; TM, transmembrane domain). The diagram is not to scale. The amino acid sequences comprising the stalk region are defined as previously described (42). Asterisks denote conserved amino acids. A 15-amino acid region of the Cal09 NA stalk that is not present in the PR8 NA is shown with brown letters. (C) Alignment of the three NA proteins with different stalk lengths. The 15-amino-acid N2 insertion is derived from the NA stalk domain of the A/New York/61/2012 (H3N2) virus. wt, wild type. (D) Hemagglutination (HA) titers of allantoic fluids from plaque-purified viruses. Data points represent individual plaques (n = 5 per virus). Horizontal bars show the mean values and whiskers the standard deviations. Phosphate-buffered saline (PBS) control wells showed no hemagglutination (not shown). (E) Western blots of proteins from concentrated viruses (left, anti-NA; right, anti-HA). One or two micrograms of total protein content of each virus preparation was analyzed, as indicated above the blots. Protein marker sizes (in kilodaltons) are indicated to the left of the blots. The bands corresponding to HA0 (uncleaved HA) and HA2 (cleavage product of HA0) are indicated with arrows (the antibody is specific to the C-terminal portion of the HA protein and therefore does not react with the HA1 polypeptide). (F) Immunofluorescence microscopy of MDCK cells infected with the indicated viruses and stained with anti-N1 monoclonal antibody 4A5 (11).