FIG. 1.

Purification of influenza VLPs and electron microscopy examination. (A) Western blot analysis of fractions from sucrose density gradient centrifugation. Blots for HA (top) and M1 (bottom) were probed using mouse anti-PR8 sera and purified mouse anti-M1 IgG antibody, respectively. Lanes: 1 to 3, top fractions without sucrose; 4 and 5, fractions with above 20% sucrose; 6 and 7, fractions between 20 and 30% sucrose; 8 and 9, fractions between 30 and 60% sucrose. Lane M1 contains HA-negative M1 VLPs. The positions of influenza virus HA and M1 proteins are indicated to the right of the blots. The positions of molecular mass markers (in kilodaltons) are shown to the left of the blots. (B) Cleavage of HA in VLPs. VLPs containing HA were incubated for 5 min with different concentrations of TPCK-treated trypsin, resolved by SDS-PAGE, and probed by Western blotting. Lanes 1 to 4 contain 0, 0.125, 1.0, and 2.5 μg/ml trypsin concentrations, respectively. (C) Negative staining electron microscopy of influenza VLPs containing HA and M1.