Biochemical and functional analysis of Flu-VLPs. A. Incorporation of HA, NA and M2 proteins from H7N1 (A/Chicken/FPV/Rostock/1934) or H5N1 (A/Thailand/KAN-1/04) influenza viruses on MLV retroviral core particles, as indicated. Purified VLPs were loaded on SDS-PAGE and incorporation levels of the different proteins were determined by Western Blot analysis using polyclonal sera against H7N1-HA, H5N3-HA, H7N1-NA and M2 proteins. HA0: HA precursor protein, HA1: HA surface subunit; HA2: HA transmembrane subunit; NA: neuraminidase; CA: MLV capsid protein. The difference in the molecular weight of H5 vs. H7 HA2, of ca. 3 kDa, was due to the presence of an additional glycan for H7 HA2. B. Infectious titres obtained with Flu-VLPs incorporating surface proteins from H7N1 or H5N1 influenza viruses or with VSV-VLPs incorporating the VSV-G of vesicular stomatitis virus (VSV), as indicated. VLP-containing supernatants were used to infect 105 TE671 target cells plated in 12-well for 6 hr at 37°C. The transduction efficiency of GFP, determined as the percentage of GFP-positive cells, was measured by fluorescence-activated cell sorter (FACS) analysis 72 hr after infection, as previously described . The transduction titres, provided as GFP-transducing units (t.u.)/ml, were calculated by using the formula: Titre = %inf × (105/100) × d, where "d" is the dilution factor of the viral supernatant and "%inf" is the percentage of GFP-positive cells as determined by FACS analysis using dilutions of the viral supernatant that transduced between 0.1% and 5% of GFP-positive cells. Due to the use of a FACS method to monitor transduced cells, the determination of GFP-positive cell below 0.1%, i.e., corresponding to transduction titres below 103 t.u./ml in our experimental conditions, could not be accurately be determined. The background levels of transduction were therefore fixed at 103 t.u./ml in all experiments.