JAB1 overcomes the inhibition of HIV-1 replication in cognate peptide expressing SupT1 cells.

<p>(A) pBMN-control IRES-Lyt2α’ or pBMN-JAB1 IRES-Lyt2α’ retrovirus vectors were transduced into SupT1 cells that expressed either C-Pep1 or Pep24. (B) pBMN-control IRES-Lyt2α’ or pBMN-JAB1 IRES-Lyt2α’ retrovirus vectors were transduced into SupT1 cells. These cells were challenged with HIV-1 (NL4-3) at a dose 400 TCID₅₀ per 5×10⁴ cells. p24^gag levels in culture supernatants were assayed from four wells on the indicated days after infection. p24^gag levels were normalized to cell number determined using an XTT assay. Data are presented as the average ± SE per 10⁶ cells. Similar results were observed in three independent experiments. * indicates <i>p</i><0.05, Control C-Pep-1 SupT1 versus Control Pep24 SupT1, and # indicates <i>p</i><0.05, Control Pep24 SupT1 versus JAB1 Pep24 SupT1 by <i>t</i> test. (C) JNK inhibitor (SP600125) inhibits HIV-1 replication. SupT1 cells were treated JNK inhibitor (SP600125) for 30 min before HIV-1 challenge. These cells were challenged with HIV-1 (NL4-3) at a dose 400 TCID₅₀ per 5×10⁴ cells. p24^gag levels in culture supernatants were assayed from five wells on the indicated days after infection. p24^gag levels were normalized for cell number using XTT assay. Data are presented as the average ± SE per 10⁶ cells. Similar results were observed in three independent experiments. *indicates <i>p</i><0.05, No treatment versus 10 µM SP600125, and # indicates <i>p</i><0.05, No treatment versus 30 µM SP600125 by <i>t</i> test.</p

JNK is one of kinases on which HIV-1 depends.

<p>Jurkat cells expressing C-Pep1 or Pep24 were treated with indicated stimuli. Cells were stained with pERK-Alexa 647 or pJNK-Alexa 647 phosphospecific antibodies and analyzed by flow cytometry. (A) Histograms are colored according to the different stimuli. (B) Fold change was approximated by calculating the log2 ratio of mean fluorescence intensity of stimulated versus unstimulated cells.</p

Pep24 specifically binds to JAB1.

<p>The indicated biotin-conjugated peptides were incubated with GST or with the GST-fused JAB1. The complex of peptide-GST fusion protein immobilized on streptavidin-agarose was detected by western blotting with anti-GST antibody.</p

Pep24 blocks LFA-1-induced JAB1 relocalization.

<p>Jurkat cells expressing indicated peptides were adhered to either anti-LFA-1 mAb or control IgG coated plates for 30 min. After cross-linking stimulation, cells were stained for JAB1 (red). GFP (green) is an indicator of peptide expression. (A–C) C-Pep1-expressing cells without stimulation. (D–F) C-pep1-expressing cells with LFA-1 cross-linking stimulation. (G–I) Pep24-expressing cells without stimulation. (J–L) Pep24-expressing cells with LFA-1 cross-linking stimulation.</p

Selected peptide, Pep24, preferentially inhibits the NFAT signaling pathway.

<p>(A) The sequences of control peptides and selected peptide. (B–D) Reporter plasmids (B) p55-IgκLuc, (C) NFAT Luc, or (D) AP-1 Luc were transfected into Jurkat cells expressing indicated peptides with pBMN LacZ as the internal control plasmid. Cells treated for 3 hr (8 hr for AP-1) with or without indicated agents (2 µg/ml PHA, 10 ng/ml PMA, and 10 ng/ml TNF-α) prior to measurement of luciferase activity. The experiments were repeated three times and the average is plotted ± SE. Jurkat cells transfected with reporter without treatment were assigned a value of 1 and were used to calculate the fold activation. Transfection efficiencies were normalized to a co-transfected lacZ plasmid.</p

Proposed model for the inhibition of signalling pathway by Pep24.

<p>(A) After LFA-1 activation, JAB1 relocalizes into cytoplasm and nucleus, activates AP-1 and induces gene activation, and finally activates HIV-1 replication. (B) When Pep24 binds to JAB-1, JAB1 relocalization is blocked inhibiting downstream signals and HIV-1 replication.</p

Snapin regulates Ca²⁺ efflux and influx in T cells.

<p>(A, B) Indicated cells were suspended in Ca²⁺-free medium containing 10 mM EGTA. “Control” indicates control retrovirus, whereas “Snapin” indicates that cells were infected with Snapin-encoding retrovirus. Cells were transduced with either Pep80 or C-Pep1. Cells were stained with APC-anti-Lyt2<i>α</i>' and were loaded with indo-1-AM calcium sensor dye. EGTA was added, and after 30 s (A) OKT3 or (B) thapsigargin was added. The FL5/FL4 ratio (400 nm/510 nm fluorescence emission) was monitored using a flow cytometer. (C) Cells were suspended in medium containing Ca²⁺. After 30 s, OKT3 was added. The FL5/FL4 ratio was monitored using a flow cytometer.</p

Pep80 strongly inhibits HIV-1 replication in T cells.

<p>SupT1 cells expressing one of four control peptides or Pep80 were challenged with HIV-1 (NL4-3) at a dose of 400 TCID₅₀ per 5×10⁴ cells. P24^gag levels in culture supernatants were assayed from four wells on the indicated days after infection. P24^gag levels were normalized for cell number using an XTT assay. Data are presented as the average ± SE per 10⁶ cells. Similar results were observed in three independent experiments.</p

Snapin induces HIV-1 replication in primary CD4⁺ T cells.

<p>pBMN-control-IRES-Lyt2<i>α</i>' and pBMN-Snapin-IRES-Lyt2α' retrovirus vectors were transduced into human primary CD4⁺ T cells. CD4⁺ T cells were challenged by HIV-1 (NL4-3) at 400 TCID₅₀ per 1×10⁵ cells. P24^gag levels in culture supernatants were assayed from four wells on the indicated days after infection. P24^gag levels were normalized for cell number using an XTT assay. Data are presented as the average ± SE per 10⁶ cells. Similar results were observed in three independent experiments. N.D.; not detected.</p

siRNA-mediated knockdown of Snapin inhibits Ca²⁺ influx and HIV replication.

<p>Jurkat cells that were transfected with Snapin-specific siRNA or control siRNA were suspended in (A) Ca²⁺-free medium with 10 mM EGTA or (B) Ca²⁺-containing medium. After 30 s, OKT3 was added. The FL5/FL4 ratio was monitored using a flow cytometer. (C) NFAT Luc reporter plasmid and pBMN lacZ were transfected into Jurkat cells that were transfected with Snapin-specific siRNA or control siRNA. Cells were treated for 3 hr with or without PHA and PMA prior to measurement of luciferase activity. The experiments were repeated three times; values shown are the average ± SE. Data from cells without treatment were assigned a value of 1 and were used to calculate the fold activation. Transfection efficiencies were normalized to a co-transfected lacZ plasmid. (D) SupT1 cells that were transfected with Snapin-specific siRNA or control siRNA were challenged with replication incompetent HIV-1-Ea. P24^gag levels in culture supernatants were assayed from four wells 48 hr after HIV-1 challenge. P24^gag levels were normalized for cell number using XTT assay. Data are presented as the average ± SE per 10⁶ cells. Similar results were observed in three independent experiments. * indicates <i>p</i><0.05 by <i>t</i> test.</p