Co-dependence of HTLV-1 p12 and p8 Functions in Virus Persistence
Figure 5
Susceptibility of HTLV-1 producing CD4+ cell lines to CTL killing.
(A) CD4+ T-cells infected with the D26, N26, G29S and 12KO viruses were incubated with BHKE16 indicator cells for 48 hours. Un-infected Jurkat T-cells (control) were used as a negative control. The number of blue cells per well for three independent experiments is graphed (n = 3). Error bars indicate standard deviation. (B) A comparison of the surface expression of CD4 (left panels) and HLA.A2 (right panels) are shown for the indicated virus-infected CD4+ T-cells (black line) in comparison to the 12KO CD4+ T-cell line (shaded). (C) Cytoxic T-lymphocyte killing assays were done to evaluate specific lysis of CD4+ T-cells infected with the D26, N26, G29S and 12KO viruses. A long term HLA.A2 restricted CD8+ T-cell line from an HAM/TSP patient was used as the effector cell (see Materials and Methods). Graphed is the percent of specific lysis at varying effector-to-target cell ratios (1.25∶1; 5∶1; 20∶1). The graph represents data from at least two independent experiments done in triplicate (n≥2). Lysis of 12KO at the 20∶1 ratio (highest specific lysis) was set to 100%. All samples were normalized to maximal killing obtained with the 12KO virus. Error bars indicate standard deviation. (D) Western blot analysis of protein lysates from transfected cells was assayed for p12 and p8 expression (top panel) or the loading control, tubulin (bottom panel) to determine the effectiveness of the siRNA constructs. Cells were co-transfected with p12WT cDNA or control expression constructs in the presence or absence of siRNA (Si Ctrl or Si orf-I). (E) CD4+ D26-infected cells were transfected with siRNA control (Si CTRL) or siRNA to orf-I (Si orf-I) and used as target cells in CTL killing assays. CTL lysis of the cells at increasing effector-to-target cell ratios is graphed. The graph represents data from at least two independent experiments done in triplicate (n≥2). Error bars indicate standard deviation.