Shigemi Kinoshita

The T Cell Activation Factor NF-ATc Positively Regulates HIV-1 Replication and Gene Expression in T Cells

Clinical deterioration in human immunodeficiency virus type 1 (HIV-1) infection is associated with increased levels of viral replication and burden in the peripheral blood and lymphoid organs. T cell activation and ensuing cellular gene activation can be critical for HIV-1 replication. The hypothesis that the nuclear factor of activated T cells (NF-AT) may influence HIV-1 replication is therefore compelling given the tight correlation of HIV-1 transcriptional induction to T cell activation. We report that certain NF-AT(Rel) family members productively bind the kappaB regulatory elements, synergize with NF-kappaB and Tat in transcriptional activation of HIV-1, and enhance HIV-1 replication in T cells. These results link regulatory factors critical to T cell commitment directly to HIV-1 replication.

Host control of HIV-1 parasitism in T cells by the nuclear factor of activated T cells.

Post HIV-1 entry, productive HIV-1 infection of primary T cells requires overcoming several cellular blocks to provirus establishment and replication. Activation of unknown host intracellular events overcomes such inhibitory steps and is concomitant with HIV-1 replication. We show that the transcription factor NFATc was sufficient as a cellular factor to induce a highly permissive state for HIV-1 replication in primary CD4+ T cells. NFATc overcame a blockade at reverse transcription and permitted active HIV-1 replication. Pharmacologic blockade of endogenous NFAT activity by FK506 or CsA inhibited synthesis of reverse transcription and also potently blocked HIV-1 replication. T cells therefore can become competent for HIV-1 replication by control of regulated host factors such as the NFATc transcription factor. The host mechanisms regulated by such permissivity factors are potential targets for anti-HIV-1 therapy.

Toso, a Cell Surface, Specific Regulator of Fas-Induced Apoptosis in T Cells

Fas is a surface receptor that can transmit signals for apoptosis. Using retroviral cDNA library-based functional cloning we identified a gene, toso, that blocks Fas-mediated apoptosis. Toso expression was confined to lymphoid cells and was enhanced after cell-specific activation processes in T cells. Toso appeared limited to inhibition of apoptosis mediated by members of the TNF receptor family and was capable of inhibiting T cell self-killing induced by TCR activation processes that up-regulate Fas ligand. We mapped the effect of Toso to inhibition of caspase-8 processing, the most upstream caspase activity in Fas-mediated signaling, potentially through activation of cFLIP. Toso therefore serves as a novel regulator of Fas-mediated apoptosis and may act as a regulator of cell fate in T cells and other hematopoietic lineages.

Leukocyte functional antigen 1 lowers T cell activation thresholds and signaling through cytohesin-1 and Jun-activating binding protein 1

Leukocyte functional antigen 1 (LFA-1), with intercellular adhesion molecule ligands, mediates T cell adhesion, but the signaling pathways and functional effects imparted by LFA-1 are unclear. Here, intracellular phosphoprotein staining with 13-dimensional flow cytometry showed that LFA-1 activation induced phosphorylation of the β2 integrin chain and release of Jun-activating binding protein 1 (JAB-1), and mediated signaling of kinase Erk1/2 through cytohesin-1. Dominant negatives of both JAB-1 and cytohesin-1 inhibited interleukin 2 production and impaired T helper type 1 differentiation. LFA-1 stimulation lowered the threshold of T cell activation. Thus, LFA-1 signaling contributes to T cell activation and effects T cell differentiation.

Activation of the PKB/AKT Pathway by ICAM-2

We identified intracellular adhesion molecule-2 (ICAM-2) in a genetic screen as an activator of the PI3K/AKT pathway leading to inhibition of apoptosis. ICAM-2 induced tyrosine phosphorylation of ezrin and PI3K kinase membrane translocation, resulting in phosphatidylinositol 3,4,5 production, PDK-1 and AKT activation, and subsequent phosphorylation of AKT targets BAD, GSK3, and FKHR. ICAM-2 clustering protected primary human CD19+ cells from TNFalpha- and Fas-mediated apoptosis as determined by single-cell analysis. ICAM-2 engagement by CD19+ cells of its natural receptor, LFA-1, on CD4+ naive cells specifically induced AKT activity in the absence of an MHC-peptide interaction. These results attribute a novel signaling function to ICAM-2 that might suggest mechanisms by which ICAM-2 signals intracellular communication at various immunological synapses.

NF-IL6 (C/EBPβ) induces HIV-1 replication by inhibiting cytidine deaminase APOBEC3G

T cell activation is crucial for the productive HIV-1 infection of primary T cells; however, little is known about the host molecules involved in this process. We show that the host transcription factor NF-IL6 (also called C/EBPβ) renders primary CD4+ T cells highly permissive for HIV-1 replication. NF-IL6 facilitates reverse transcription of the virus by binding to and inhibiting the antiviral cytidine deaminase APOBEC3G. A mutation in NF-IL6 at Ser-288 weakened its binding to APOBEC3G and strongly inhibited HIV-1 replication. NF-IL6 also induced the replication of a Vif-deficient strain of HIV-1 in nonpermissive HUT78 cells. These data indicate that NF-IL6 is a natural inhibitor of APOBEC3G that facilitates HIV-1 replication. Host factors, such as NF-IL6, that are involved in early HIV-1 replication are potential targets for anti-HIV-1 therapy. Our findings shed light on the activation of HIV-1 replication by T cell host molecules and reveal a unique regulation of DNA deamination by APOBEC3G and NF-IL6.

COP9 Signalosome Component JAB1/CSN5 Is Necessary for T Cell Signaling through LFA-1 and HIV-1 Replication

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.

Snapin, Positive Regulator of Stimulation- Induced Ca2+ Release through RyR, Is Necessary for HIV-1 Replication in T Cells

To identify critical host factors necessary for human immunodeficiency virus 1 (HIV-1) replication, large libraries of short-peptide-aptamers were expressed retrovirally. The target of one inhibitor peptide, Pep80, identified in this screen was determined to be Snapin, a protein associated with the soluble N-ethyl maleimide sensitive factor adaptor protein receptor (SNARE) complex that is critical for calcium-dependent exocytosis during neurotransmission. Pep80 inhibited Ca2+ release from intracellular stores and blocked downstream signaling by direct interruption of the association between Snapin and an intracellular calcium release channel, the ryanodine receptor (RyR). NFAT signaling was preferentially abolished by Pep80. Expression of Snapin overcame Pep80-mediated inhibition of Ca2+/NFAT signaling and HIV-1 replication. Furthermore, Snapin induced HIV-1 replication in primary CD4+ T cells. Thus, through its interaction with RyR, Snapin is a critical regulator of Ca2+ signaling and T cell activation. Use of the genetically selected intracellular aptamer inhibitors allowed us to define unique mechanisms important to HIV-1 replication and T cell biology.