Journal: Virology

Article Title: Interleukin 2-inducible T cell kinase (ITK) facilitates efficient egress of HIV-1 by coordinating Gag distribution and actin organization

doi: 10.1016/j.virol.2012.11.015

Figure Lengend Snippet: Chemical inhibition of ITK disrupts Gag colocalization, alters actin organization, decreases VLP release and inhibits HIV replication. (a) HEK293T cells were co-transfected with Gag-opt and Vector- eGFP or ITK-eGFP, treated with DMSO, BMS or CNX-225 for 72 h. Supernatants were collected and spun on a 20% sucrose cushion, and assayed by p24 ELISA. p24 values were normalized to Gag associated with whole cell extracts as determined by immunoblots and denistrometry. Fold induction of VLPs over vector control is shown. This is a single experiment performed in triplicate and represents at least three independent treatments. Error bars calculated as standard deviation and * indicates P value < 0.05 as determined by a Student’s t-test. Immunoblots were also performed to confirm ITK expression. (b) Human primary CD4+ T cells were infected with VSV-G pseudotyped HXB-PLAP-nef+ virus, 24 h post infection cells were treated with CNX-225 inhibitor (1 µM) or equivalent DMSO and cultured in the presence of treatment for 4 days. Supernatants were sampled at indicated times and HIV replication was assessed by p24 ELISA. These data are triplicate infections from a single donor and are representative of results obtained from three different donors. The error bars are standard deviation between the triplicate samples and * indicates P value < 0.05 as determined by a Student’s t-test. (c)–(e) Jurkat cells were infected with a VSV-G pseudotyped HXB-PLAP-nef+, 72 h post infection cells were sorted for HIV positive cells. HIV positive cells were treated with BMS509744 (10 µM) or CNX-225 inhibitor (1 µM) or equivalent DMSO control for 30 min. Cells were washed, fixed, permeabilized, and intracellularly labeled for ITK expression (green) and Gag expression (red). (d) Percent Jurkat cells following treatment in which ITK and Gag staining overlapped. At least 60 cells were counted for each treatment. (e) HIV infected Jurkat cells were intracellularly labeled for ITK expression (green) and F-actin using Phalloidin (red). The cells were also stained with DAPI to detect nuclei (blue). Comparable DMSO treated cells are shown in Fig. 4. All images were taken using a Nikon fluorescence microscope at 60× oil immersion, Image J software was used for deconvolution and image analysis.

Article Snippet: For some experiments cells were cultured for 72 h with ITK inhibitors BMS509744 (10 µM) ( Readinger et al., 2008 ) or CNX-225 (1 µM) a covalent small molecule specific Tec family kinase inhibitor (Avila Therapeutics) or DMSO vehicle.

Techniques: Inhibition, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Western Blot, Standard Deviation, Expressing, Infection, Cell Culture, Labeling, Staining, Fluorescence, Microscopy, Software