Louis, MO) (15, 34, 59). The fluorescence of the released 4-methylumbelliferone was measured in a Fluoroskan II spectrophotometer (Labsystems, Helsinki, Finland) using excitation and emission wavelengths of 355 and 460 nm, respectively. The enzymatic activity of NA protein was selleck chemical 17-AAG standardized to 0.1 mg total protein by using a bicinchoninic acid assay (Sigma, St. Louis, MO) and was expressed as the quantity of substrate (in picomoles) converted during a 30-min incubation at 37��C. The NA inhibitor oseltamivir carboxylate ([3R,4R,5S]-4-acetamido-5-amino-3-[1-ethylpropoxy]-1-cyclohexene-1-carboxylic acid) was provided by Hoffmann-La Roche, Ltd. (Basel, Switzerland). The compound was dissolved in distilled water, and aliquots were stored at ?20��C until use.

NA activity was inhibited by using a 4 ��M concentration of oseltamivir carboxylate added immediately posttransfection or 1 h before the assay. The effect of an NA protein inhibitor on the pH of fusion was determined by performing a syncytium formation assay in parallel. RESULTS The HA protein mutations have little effect on the in vitro replication kinetics of recombinant H5N1 influenza viruses. In a previous study, we identified four mutant H5 HA proteins whose pH values of membrane fusion differed from that of wild-type HA protein when expressed from transiently transfected plasmid DNA (36). Here we determined whether the HA protein mutations affected the in vitro replication kinetics of recombinant H5N1 influenza viruses by generating single-step and multiple-step growth curves.

Single-step growth curves showed that mutant and wild-type viruses grew at similar rates over the 10-h time course (Fig. (Fig.1A).1A). In multiple-step growth curves, viruses containing the HA protein mutations Y231H, H241Q, and K582I had replication rates similar to that of wild-type virus (Fig. (Fig.1B).1B). Titers of the virus containing an N1142K mutation in the HA protein were similar to those of wild-type virus at the 12-h time point but were later reduced by 1 to 3 log10 units. FIG. 1. Replication kinetics of recombinant A/chicken/Vietnam/C58/04 (H5N1) influenza viruses in MDCK cells. (A) For single-step growth curves, cells were infected at an MOI of 3 PFU/cell with wild-type virus or viruses containing HA protein mutation Y231H, H24 … The HA protein mutations alter the pH of membrane fusion in vitro. The cell surface expression, cleavage, receptor binding affinities, and membrane fusion efficiencies of the mutant HA proteins in Vero cells were previously found to be similar to those of wild-type virus (36). Moreover, infection of DF-1 primary chicken embryonic fibroblasts with the viruses resulted in HA protein properties similar to those Entinostat found when Vero cells were infected with the viruses.