Figure 6
From: Cell-penetrating peptide-mediated cell entry of H5N1 highly pathogenic avian influenza virus
Cell-penetrating activity of HA314-46 drives sialic acid-independent virus internalisation. (a, b) Entry of reassortant viruses into CHO-K1 and Lec8 cells. CHO-K1 (a) and Lec8 (b) cells were incubated with trypsin- or furin-treated reassortant viruses at 37 °C for 1 h at MOI of 10. After washing and incubation for 8 h in the culture medium, the cells were fixed in 4% paraformaldehyde. (c) Effect of EIPA on sialic acid-independent entry of furin-treated UT3040HA (R339G)/PR8 viruses. Lec8 cells were pre-treated with vehicle (DMSO) or EIPA (10, 20, 40 μM) for 30 min at 37 °C and then were incubated with furin-treated UT3040HA (R339G)/PR8 viruses as described in (b). After washing, the cells were further incubated for 8 h in the culture medium containing vehicle or EIPA. (d) Propagation of UT3040HA (R339G)/PR8 virus in Lec8 cells. Lec8 cells were treated with reassortant viruses as described in (b). After washing, the cells were incubated in the culture medium at 37 °C for 24 and 48 h. The fixed cells were permeabilised and stained with anti-influenza A nucleoprotein monoclonal antibody (a-d). Localisation of the viruses was evaluated by fluorescent microscopy. Representative images from four or six experiments are shown. Green; viral nucleoprotein, Blue; nucleus. Scale bar, 20 μm. The graphs in the bottom panels indicate the proportion of viral nucleoprotein-positive cells by flow cytometric analysis. Data are presented as mean + SEM (n = 4 or 6). Asterisks indicate significant difference by one-way ANOVA with Bonferroni's multiple comparison test. **p < 0.01; *p < 0.05; ns, not significant.
