Cleavage of H3 and H1 pseudotypes with TMPRSS2 and HAT proteases.

<p><b>A.</b> Subcellular localization of HA, p24 and TMPRSS2 proteins in transfected HEK 293T cells by structured-illumination fluorescent microscopy. <b>B.</b> Pseudotypes were produced in the presence of trypsin-TPCK or HAT or TMPRSS2, or TMPRSS2 and HAT followed by single-cycle infectivity assays (expressed as the mean log₁₀ RLU ± S.D of triplicate experiments (background limit; 3 log₁₀ RLU). *p<0.01 **p<0.001, and ***p<0.0001 (unpaired Student’s t-test). <b>C.</b> SDS-PAGE and western blot analysis were performed on pseudotypes produced in the presence of TMPRSS2 and HAT, using a mixture of sera specific to H3 and H1 serotypes with HA0 and HA2 representing uncleaved and cleaved HA respectively (Lane 8: H3N2 WIS/05, lane 9: H3N2 VIC/75, lane 10: H1N1 PAR/09, lane 11: H1N1 NC/99). The H3N2 WIS/05 virus was used as a positive control (Lane 1). Uncleaved H3N2 WIS/05 and H1N1 PAR/09 pseudotypes (Lanes 2 and 3 respectively) were used as negative controls. H3N2 WIS/05 and H1N1 PAR/09 pseudotypes were cleaved with either 2 µg.mL⁻¹ of trypsin-TPCK (Lanes 4 and 5 respectively) or with the HAT protease alone (Lanes 6 and 7 respectively).