Cleavage of H3 and H1 pseudotypes with trypsin-TPCK.

<p><b>A.</b> Pseudotypes were exposed to different concentrations of trypsin (0, 2, 100 and 500 µg.mL⁻¹) for 30 min followed by single-cycle infectivity assays and luciferase assays (expressed in Mean log₁₀ Relative Luminescence Units, RLU) with a minimum value of 3.0 log₁₀ RLU, which corresponded to the background limit (grey line). Data are expressed as the mean ± Standard Deviation (S.D) of triplicate repeats. ***p<0.0001 versus 0 µg.mL⁻¹ concentration, NS (not significant), Student's t-test. <b>B.</b> SDS-PAGE/Western blot analysis of pseudotypes bearing different HAs after treatment with trypsin-TPCK. The H3N2 WIS/05 virus was used as a positive control (virus control). Western blot was performed using antibodies targeting the HA protein (HA0 and HA2 representing uncleaved and cleaved HA respectively) or the HIV p24 (loading control). <b>C.</b> Hemolysis assays were performed on different pseudotypes after treatment with 0 or 500 µg.mL⁻¹ trypsin-TPCK. Lysis of guniea pig red blood cells was evaluated by measuring the optical density of released haemoglobin at 540 nm. The H3N2 WIS/05 virus was used as a positive control (virus control) and PBS (phosphate buffered saline) as a negative control. Data are expressed as the mean ± S.D of triplicate repeats. ***p<0.0001, **p<0.001 and NS (not significant), Student's t-test.