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COP9 signalosome component JAB1/CSN5 is necessary for T cell signaling through LFA-1 and HIV-1 replication.

Kinoshita SM, Krutzik PO, Nolan GP - PLoS ONE (2012)

Bottom Line: To determine critical host factors involved in HIV-1 replication, a dominant effector genetics approach was developed to reveal signaling pathways on which HIV-1 depends for replication.JAB1 expression overcame the inhibition of HIV-1 replication in the presence of peptide and also promoted HIV-1 replication in activated primary CD4(+) T cells.Thus, genetic selection for intracellular aptamer inhibitors of host cell processes proximal to signals at the immunological synapse of T cells can define unique mechanisms important to HIV-1 replication.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immune Regulation, Osaka University Graduate School of Frontier Biosciences, Suita, Osaka, Japan. shigemik@biken.osaka-u.ac.jp

ABSTRACT
To determine critical host factors involved in HIV-1 replication, a dominant effector genetics approach was developed to reveal signaling pathways on which HIV-1 depends for replication. A large library of short peptide aptamers was expressed via retroviral delivery in T cells. Peptides that interfered with T cell activation-dependent processes that might support HIV-1 replication were identified. One of the selected peptides altered signaling, lead to a difference in T cell activation status, and inhibited HIV-1 replication. The target of the peptide was JAB1/CSN5, a component of the signalosome complex. JAB1 expression overcame the inhibition of HIV-1 replication in the presence of peptide and also promoted HIV-1 replication in activated primary CD4(+) T cells. This peptide blocked physiological release of JAB1 from the accessory T cell surface protein LFA-1, downstream AP-1 dependent events, NFAT activation, and HIV-1 replication. Thus, genetic selection for intracellular aptamer inhibitors of host cell processes proximal to signals at the immunological synapse of T cells can define unique mechanisms important to HIV-1 replication.

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JAB1 promotes HIV-1 replication in activated primary CD4+T cells. pBMN-control-IRES-Lyt2α’ and pBMN-JAB1-IRES-Lyt2α’ retrovirus vectors were transduced into human primary CD4+ T cells. CD4+ T cells were challenged with HIV-1 (NL4-3) at doses of 400 TCID50 per 1×105 cells with or without PHA stimulation. p24gag levels in culture supernatants were assayed from four wells on the indicated days after infection. p24gag levels were normalized for 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.
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pone-0041725-g006: JAB1 promotes HIV-1 replication in activated primary CD4+T cells. pBMN-control-IRES-Lyt2α’ and pBMN-JAB1-IRES-Lyt2α’ retrovirus vectors were transduced into human primary CD4+ T cells. CD4+ T cells were challenged with HIV-1 (NL4-3) at doses of 400 TCID50 per 1×105 cells with or without PHA stimulation. p24gag levels in culture supernatants were assayed from four wells on the indicated days after infection. p24gag levels were normalized for 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.

Mentions: To determine whether JAB1 could affect HIV-1 replication in primary CD4+ T cells, HIV-1 replication was measured in primary CD4+ T cells with or without ectopic JAB1 expression. JAB1 was retrovirally transduced into primary CD4+ T cells. Control primary CD4+ T cells and JAB1-expressing primary CD4+ T cells were infected with NL4-3, and HIV-1 replication levels were measured by a p24 ELISA. After 18 days, most cells were dead and the experiment was terminated. No HIV-1 replication was observed in either control or JAB1-expressing non-stimulated primary CD4+ T cells. However, after PHA stimulation, HIV-1 replication was enhanced about four fold in JAB1-expressing primary CD4+ T cells in comparison with control primary CD4+ T cells (Figure 6). This shows that T cell activation and JAB1 activity are required for HIV-1 replication in primary CD4+ T cells. It can be concluded that endogenously expressed JAB1 is acted upon during T cell activation to enhance signaling important to HIV-1 replication.


COP9 signalosome component JAB1/CSN5 is necessary for T cell signaling through LFA-1 and HIV-1 replication.

Kinoshita SM, Krutzik PO, Nolan GP - PLoS ONE (2012)

JAB1 promotes HIV-1 replication in activated primary CD4+T cells. pBMN-control-IRES-Lyt2α’ and pBMN-JAB1-IRES-Lyt2α’ retrovirus vectors were transduced into human primary CD4+ T cells. CD4+ T cells were challenged with HIV-1 (NL4-3) at doses of 400 TCID50 per 1×105 cells with or without PHA stimulation. p24gag levels in culture supernatants were assayed from four wells on the indicated days after infection. p24gag levels were normalized for 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3404009&req=5

pone-0041725-g006: JAB1 promotes HIV-1 replication in activated primary CD4+T cells. pBMN-control-IRES-Lyt2α’ and pBMN-JAB1-IRES-Lyt2α’ retrovirus vectors were transduced into human primary CD4+ T cells. CD4+ T cells were challenged with HIV-1 (NL4-3) at doses of 400 TCID50 per 1×105 cells with or without PHA stimulation. p24gag levels in culture supernatants were assayed from four wells on the indicated days after infection. p24gag levels were normalized for 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.
Mentions: To determine whether JAB1 could affect HIV-1 replication in primary CD4+ T cells, HIV-1 replication was measured in primary CD4+ T cells with or without ectopic JAB1 expression. JAB1 was retrovirally transduced into primary CD4+ T cells. Control primary CD4+ T cells and JAB1-expressing primary CD4+ T cells were infected with NL4-3, and HIV-1 replication levels were measured by a p24 ELISA. After 18 days, most cells were dead and the experiment was terminated. No HIV-1 replication was observed in either control or JAB1-expressing non-stimulated primary CD4+ T cells. However, after PHA stimulation, HIV-1 replication was enhanced about four fold in JAB1-expressing primary CD4+ T cells in comparison with control primary CD4+ T cells (Figure 6). This shows that T cell activation and JAB1 activity are required for HIV-1 replication in primary CD4+ T cells. It can be concluded that endogenously expressed JAB1 is acted upon during T cell activation to enhance signaling important to HIV-1 replication.

Bottom Line: To determine critical host factors involved in HIV-1 replication, a dominant effector genetics approach was developed to reveal signaling pathways on which HIV-1 depends for replication.JAB1 expression overcame the inhibition of HIV-1 replication in the presence of peptide and also promoted HIV-1 replication in activated primary CD4(+) T cells.Thus, genetic selection for intracellular aptamer inhibitors of host cell processes proximal to signals at the immunological synapse of T cells can define unique mechanisms important to HIV-1 replication.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immune Regulation, Osaka University Graduate School of Frontier Biosciences, Suita, Osaka, Japan. shigemik@biken.osaka-u.ac.jp

ABSTRACT
To determine critical host factors involved in HIV-1 replication, a dominant effector genetics approach was developed to reveal signaling pathways on which HIV-1 depends for replication. A large library of short peptide aptamers was expressed via retroviral delivery in T cells. Peptides that interfered with T cell activation-dependent processes that might support HIV-1 replication were identified. One of the selected peptides altered signaling, lead to a difference in T cell activation status, and inhibited HIV-1 replication. The target of the peptide was JAB1/CSN5, a component of the signalosome complex. JAB1 expression overcame the inhibition of HIV-1 replication in the presence of peptide and also promoted HIV-1 replication in activated primary CD4(+) T cells. This peptide blocked physiological release of JAB1 from the accessory T cell surface protein LFA-1, downstream AP-1 dependent events, NFAT activation, and HIV-1 replication. Thus, genetic selection for intracellular aptamer inhibitors of host cell processes proximal to signals at the immunological synapse of T cells can define unique mechanisms important to HIV-1 replication.

Show MeSH
Related in: MedlinePlus