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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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Related in: MedlinePlus

Overall strategy for screening using retrovirus peptide library.The retroviral peptide expression library is first transfected into the helper-free retroviral producer line by transient transfection. The supernatant containing the retrovirus-encoded peptide expression library is used to infect target Jurkat HIV-LTR dipA cells. The cells are subjected to PHA treatment, and GFP positive cells are selected and grown (GFP+ cells should be peptide expressing and should enriched after selection). Genomic DNA is derived from the sorted cells. PCR amplified DNA, using primers flanking the peptide inserts, was cleaved with restriction endonucleases and recloned into new vectors for retesting.
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pone-0041725-g001: Overall strategy for screening using retrovirus peptide library.The retroviral peptide expression library is first transfected into the helper-free retroviral producer line by transient transfection. The supernatant containing the retrovirus-encoded peptide expression library is used to infect target Jurkat HIV-LTR dipA cells. The cells are subjected to PHA treatment, and GFP positive cells are selected and grown (GFP+ cells should be peptide expressing and should enriched after selection). Genomic DNA is derived from the sorted cells. PCR amplified DNA, using primers flanking the peptide inserts, was cleaved with restriction endonucleases and recloned into new vectors for retesting.

Mentions: A dominant effector genetic screen was implemented to identify trans-acting peptides that act upon T cell signaling processes important to HIV-1 replication. The basis of the approach was retroviral expression of short peptides (10-mers) from a library of more than 107 different members in T cells followed by selection for phenotypes dependent upon peptide expression. The retroviruses were designed to express both a peptide and GFP from a single transcript (Figure 1); GFP was used as a surrogate indicator of relative peptide expression in cells. The majority of peptides expressed within cells were expected to have no effect on cellular processes [13], [14], [15], and detrimental global effects on the viability of cells after expression of such libraries were not observed. As is the case with pharmaceutical screens that evaluate libraries of small organic molecules in high-throughput screening assays, certain rare peptides of the right sequence and shape were expected to interfere with intracellular signaling to give a desired phenotypic outcome when those peptides are expressed within cells.


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)

Overall strategy for screening using retrovirus peptide library.The retroviral peptide expression library is first transfected into the helper-free retroviral producer line by transient transfection. The supernatant containing the retrovirus-encoded peptide expression library is used to infect target Jurkat HIV-LTR dipA cells. The cells are subjected to PHA treatment, and GFP positive cells are selected and grown (GFP+ cells should be peptide expressing and should enriched after selection). Genomic DNA is derived from the sorted cells. PCR amplified DNA, using primers flanking the peptide inserts, was cleaved with restriction endonucleases and recloned into new vectors for retesting.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3404009&req=5

pone-0041725-g001: Overall strategy for screening using retrovirus peptide library.The retroviral peptide expression library is first transfected into the helper-free retroviral producer line by transient transfection. The supernatant containing the retrovirus-encoded peptide expression library is used to infect target Jurkat HIV-LTR dipA cells. The cells are subjected to PHA treatment, and GFP positive cells are selected and grown (GFP+ cells should be peptide expressing and should enriched after selection). Genomic DNA is derived from the sorted cells. PCR amplified DNA, using primers flanking the peptide inserts, was cleaved with restriction endonucleases and recloned into new vectors for retesting.
Mentions: A dominant effector genetic screen was implemented to identify trans-acting peptides that act upon T cell signaling processes important to HIV-1 replication. The basis of the approach was retroviral expression of short peptides (10-mers) from a library of more than 107 different members in T cells followed by selection for phenotypes dependent upon peptide expression. The retroviruses were designed to express both a peptide and GFP from a single transcript (Figure 1); GFP was used as a surrogate indicator of relative peptide expression in cells. The majority of peptides expressed within cells were expected to have no effect on cellular processes [13], [14], [15], and detrimental global effects on the viability of cells after expression of such libraries were not observed. As is the case with pharmaceutical screens that evaluate libraries of small organic molecules in high-throughput screening assays, certain rare peptides of the right sequence and shape were expected to interfere with intracellular signaling to give a desired phenotypic outcome when those peptides are expressed within cells.

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