PMC

MDCK (A, B) and Vero (C, D) cells were infected with recombinant wt Bris/10 (HA2-K47) and the HA2-E47 mutant (A, C), or wt Cal/09 (HA2-E47) and the HA2-K47 mutant (B, D) at an MOI of 0.004. At the indicated time of post infection, virus titers were determined by TCID₅₀ in MDCK cells.

The 6∶2 ca viruses with equalized HA titers were incubated at the indicated temperatures. Following either a 20 minute incubation at the indicated high temperatures (A) or a 4 hour time-course at 57.5°C (B), the HA titers were re-measured. The 6∶2 ca viruses were purified and re-suspended in 1× SP-cGAG. Duplicate aliquots of virus were maintained within a controlled chamber at either 26°C for 35 days (C) or 4°C for 14 weeks (D). At the indicated time points, the infectivity of viruses was examined by TCID₅₀ assay in MDCK cells.

(A) The Cal/09 HA trimer structure (PDB file: 3LZG) was analyzed using Py-Mol modeling software. The six amino acid differences between the Cal/09 and Bris/10 HA proteins are highlighted in orange. The conserved amino acid HA1-21 is highlighted in green. The locations of the three HA1-21 and HA2-47 residues in each monomer are indicated. Only the location in one monomer is shown for the other residues. (B) Structure modeling of residues HA1-21 and HA2-47, salt bridges between HA1-E21 and HA2-K47 (Bris/10), or HA1-K21 and HA2-E47 (Cal/09 E21K) are indicated. The threshold fusion pH of each combination, as determined by the GFP fusion assay, is indicated adjacent to each model.

(A) Membrane fusion in 293T cells expressing Cal/09 or Bris/10 HA proteins. 293T cells were transfected with a GFP expression plasmid (−), a dual expression plasmid co-expressing GFP and Cal/09 HA or GFP and Bris/10 HA. Membrane fusion was observed after incubation with the indicated pH buffers. “+” indicates the fused cells. (B) Expression of the HA proteins in the transfected 293T cells. Both cleaved (HA1 and HA2) and uncleaved (HA0) forms of the HA proteins were detected by western blot using a sheep anti-HA polyclonal antibody. Values below the HA0 bands (−) trypsin represent the relative amount of Cal/09 and Bris/10 HA proteins. Values below the HA1 and HA2 bands (+) trypsin indicate the percentage of HA1 and HA2 to total HA (HA0+HA1+HA2). All the values are the average of three independent experiments. (C) Membrane fusion in Vero cells infected with Cal/09 or Bris/10 viruses. Vero cells were either mock-infected (−) or infected with Cal/09 or Bris/10 viruses at an MOI of 4.0. At 14 hours post infection, cell membrane fusion was triggered as above. “+” indicates syncytia formation. (D) Viral infectivity titers of wt Cal/09 and Bris/10 viruses (HA2-E47 or HA2-K47) after treatment with the indicated pH buffers.