Figure 4.

T Cell Kinases Lck and ZAP70 Are Required for Efficient HIV-1 Spread

(A–H) Protein phosphorylation analysis by western blotting of lysates (A–G) (see Figure 3) prepared from contacts between HIV-1 infected Lck-negative Jurkat T cells and uninfected wild-type Jurkat targets. Blots are representative of at least two independent experiments.

(I) Quantification of infection by flow cytometry.

(J) Quantification of cell-free virus budding by Gag p24 ELISA.

(K) Particle infectivity determined by reporter cell luciferase assay.

(L) Quantification of HIV-1 cell-cell spread from infected Jurkat T cells by real-time qPCR.

(M) Percentage of infected Jurkat (Gag, green; Env, red) and uninfected target cells (dye-labeled, blue) contacts showing polarization of Env and Gag to the contact zone (percentage of virological synapses [VSs]) (number of cell-cell contacts analyzed: WT, n = 30; Lck negative, n = 30; ZAP70 negative, n = 30).

(N) Representative images of normal VSs (WT) and defective VSs (Lck and ZAP70 negative) formed between an HIV-1-infected Jurkat T cell (bottom, asterisk) and an uninfected target T cell (top).

(O) Reconstituting Lck and ZAP70 expression restores VS formation (number of cell-cell contacts analyzed: WT, n = 59; Lck negative, n = 58; Lck positive, n = 48; ZAP70 negative, n = 60; ZAP70 positive, n = 38).

Data represent mean ± SEM from three independent experiments. ^∗∗p < 0.005; ^∗∗∗p < 0.001. See also Figures S4 and S5.