FIG 1.

Effect of temperature on the polymerase activity of the pH1N1 LAIV virus. (A) Schematic representation of PB2 and PB1 viral segments. pH1N1 PB2 and PB1 WT (gray, top) segments with the residues mutated to generate the LAIV virus (black, bottom) are indicated. Numbers on the right signify the amino acid (aa) length of the PB2 and PB1 proteins. (B) Viral replication and transcription. Human 293T cells were transiently cotransfected, using LPF2000, with the pH1N1 WT (gray bars) or LAIV (black bars) ambisense pDZ expression plasmids encoding the minimal requirements for viral genome replication and gene transcription (PB2, PB1, and PA) and NP, together with a vRNA-like expression plasmid encoding Gluc under the control of the human polymerase I promoter (hpPol-I Gluc) and the SV40-Cluc plasmid to normalize transfection efficiencies. At 6 h p.t., cells were placed at 33°C, 37°C, or 39°C, and viral replication and transcription were evaluated at 24 h by luminescence. Gluc (left) and Cluc (middle) activity are represented. Gluc activity was normalized to that of Cluc, and the data were represented as relative activity considering the activity of pH1N1 WT at each indicated temperature as 100% (right). Data represent the means and SDs of the results determined from triplicate wells. *, P < 0.05 (WT versus LAIV) using Student's t test (n = 3 per time point) from Microsoft Excel. (C) NP protein expression levels from cell lysates were evaluated by Western blotting using a specific monoclonal antibody against the viral NP. Sizes of molecular markers are noted on the left.