Functional complementation of 6K by other viral ion channels.

(A) IF analysis of permeabilized BHK-15 cells infected with WT SINV, Δ6K SINV, M2 SINV, Vpu SINV, or P7 SINV at 6 or 12 hpi using antibodies against E2 (Green) and CP (Red). (B) IF analysis of glycoprotein trafficking to the plasma membrane under permeabilized or non-permeabilized conditions. BHK-15 cells were electroporated with RNA corresponding to WT or 6K mutant viruses and fixed at 12 h post-electroporation Anti-E2 monoclonal antibody was used to detect glycoproteins. Nuclei were stained with Hoechst stain. (C) Flow cytometry analysis of cells infected with WT or 6K mutant viruses at an MOI of 5. Cells were incubated with a monoclonal anti-E2 antibody followed by staining with FITC secondary antibody. Results are shown as a log10 of the geometric mean of the region of the curve measuring the amount of E2 fluorescence at the plasma membrane. Data shown are from two independent experiments. Error bars indicate standard error of mean (SEM). Significance was determined using Dunnett’s multiple comparisons test as part of two-way ANOVA with a 95% confidence interval. p Values were considered significant when p (D) Quantification of glycoprotein trafficking to the plasma membrane from (B). The number of cells expressing E2 (green) relative to the total number of cells (blue) was determined using the NIS Elements software. Cells from 10 independent frames per condition were counted. Error bars indicate standard error of mean (SEM). Significance was determined by multiple unpaired t-tests of data. p Values were considered significant when p < 0.05 (*), p < 0.01 (**), p < 0.001(***), and p < 0.0001(****). ns indicates “not significant”.