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Abstract: The multifunctional HIV-1 protein Nef possesses several motifs that interact with signaling molecules in infected T cells. In order to determine whether Nef influences T cell activation, cells were infected with Nef-positive and Nef-negative clones of HIV. CD28 expression and changes in tyrosine phosphorylation were monitored. We observed no Nef-dependent changes in CD28 expression or function. However, infection with Nef-positive virus led to changes in tyrosine phosphorylation. This Nef-induced phosphorylation was observed in unstimulated cells, and c-Cbl was identified as one of the proteins whose phosphorylation was upregulated by Nef. Furthermore, Lck is required for Nef-mediated c-Cbl tyrosine phosphorylation. These results suggest that Nef modifies T cell signaling in the absence of T cell receptor engagement and co-stimulation. [Copyright &y& Elsevier]

Abstract: Inflammation facilitates tumor progression including metastasis. Interleukin-8 (IL-8) is a chemokine that regulates polymorphonuclear neutrophil (PMN) mobilization and activity and we hypothesize that this cytokine influences tumor behavior. We have demonstrated that IL-8 is crucial for PMN-mediated melanoma extravasation under flow conditions. In addition, IL-8 is up-regulated in PMNs upon co-culturing with melanoma cells. Melanoma cells induce IκB-α degradation in PMNs indicating that NF-κB signaling is active in PMNs. Furthermore, the production of IL-8 in PMNs is NF-κB dependent. We have further identified that interleukin-6 (IL-6) and interleukin-1β (IL-1β) from PMN-melanoma co-cultures synergistically contribute to IκB-α degradation and IL-8 synthesis in PMNs. Taken together, these findings show that melanoma cells induce PMNs to secrete IL-8 through activation of NF-κB and suggest a model in which this interaction promotes a microenvironment that is favorable for metastasis. [Copyright &y& Elsevier]

Periodontal infections contribute to HIV-associated co-morbidities in the oral cavity and provide a model to interrogate the dysregulation of macrophage function, inflammatory disease progression, and HIV replication during co-infections. We investigated the effect of Porphyromonas gingivalis on the establishment of HIV infection in monocyte-derived macrophages. HIV replication in macrophages was significantly repressed in the presence of P. gingivalis . This diminished viral replication was due partly to a decrease in the expression of integrated HIV provirus. HIV repression depended upon signaling through TLR4 as knock-down of TLR4 with siRNA rescued HIV expression. Importantly, HIV expression was reactivated upon removal of P. gingivalis. Our observations suggest that exposure of macrophages to Gram-negative bacteria influence the establishment and maintenance of HIV persistence in macrophages through a TLR4-dependent mechanism. [ABSTRACT FROM AUTHOR]

Since HIV-1 has a propensity to integrate into actively expressed genes, transcriptional interference from neighboring host promoters has been proposed to contribute to the establishment and maintenance HIV-1 latency. To gain insights into how endogenous promoters influence HIV-1 transcription we utilized a set of inducible T cell lines and characterized whether there were correlations between expression of endogenous genes, provirus and long terminal repeat architecture. We show that neighboring promoters are active but have minimal impact on HIV-1 transcription, in particular, expression of the endogenous gene did not prevent expression of HIV-1 following induction of latent provirus. We also demonstrate that releasing paused RNAP II by diminishing negative elongation factor (NELF) is sufficient to reactivate transcriptionally repressed HIV-1 provirus regardless of the integration site and orientation of the provirus suggesting that NELF-mediated RNAP II pausing is a common mechanism of maintaining HIV-1 latency. [ABSTRACT FROM AUTHOR]

Abstract: Interleukin 2-inducible T cell kinase (ITK) influences T cell signaling by coordinating actin polymerization and polarization as well as recruitment of kinases and adapter proteins. ITK regulates multiple steps of HIV-1 replication, including virion assembly and release. Fluorescent microscopy was used to examine the functional interactions between ITK and HIV-1 Gag during viral particle release. ITK and Gag colocalized at the plasma membrane and were concentrated at sites of F-actin accumulation and membrane lipid rafts in HIV-1 infected T cells. There was polarized staining of ITK, Gag, and actin towards sites of T cell conjugates. Small molecule inhibitors of ITK disrupted F-actin capping, perturbed Gag-ITK colocalization, inhibited virus like particle release, and reduced HIV replication in primary human CD4+ T cells. These data provide insight as to how ITK influences HIV-1 replication and suggest that targeting host factors that regulate HIV-1 egress provides an innovative strategy for controlling HIV infection. [Copyright &y& Elsevier]

Abstract: Current drugs used for antiretroviral therapy against human immunodeficiency virus (HIV) have a narrow spectrum of activity and, more often, have associated toxicities and severe side effects in addition to developing resistance. Thus, there is a need to develop new therapeutic strategies against HIV/AIDS to complement the already existing ones. Surprisingly, transactivator of transcription (Tat), an early virus-encoded protein required for the efficient transcription of the HIV genome, has not been developed as a target for small molecular therapeutics. We have previously described the ability of an endogenous Michael acceptor electrophile (MAE), 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), to inhibit Tat-dependent transcription by targeting its cysteine (Cys)-rich domain. In an effort to identify other MAEs possessing inhibitory activity against HIV-1 Tat, we tested a collection of plant-derived compounds with electrophilic properties, including curcumin, rosmarinic acid, and gambogic acid, for their ability to inhibit Tat. Celastrol (Cel), a triterpenoid MAE isolated from Tripterygium wilfordii, exhibited the highest inhibitory activity against Tat. Using biochemical techniques, we demonstrate that Cel, by covalently modifying the cysteine thiols, inhibits Tat transactivation function. Using circular dichroism spectroscopy, we show that alkylation of Tat brought about a change in the secondary structure of Tat, which inhibited the transcription elongation of the HIV proviral genome by effecting mechanisms other than Tat–TAR (transactivation-responsive region) interaction. Our results demonstrate the underlying mechanism of antiretroviral activity of the plant-derived MAEs and suggest that Cel could serve as a lead compound to develop novel antiviral therapeutics. [Copyright &y& Elsevier]

CCAAT/enhancer binding proteins (C/EBP) have been shown to be required for HIV-1 transcription and replication in macrophages. However, whether these transcription factors influence the ability of virus to establish infection by altering cytokine or receptor expression or primarily regulate HIV-1 transcription has not been determined. By inhibiting endogenous C/EBP activity with a dominant-negative protein, we demonstrate that functional C/EBPs are not required for HIV-1 infection and that these factors influence replication by a transcriptional mechanism. C/EBPβ recruits coactivators to the HIV-1 long-terminal repeat (LTR) and physically interacts with histone acetyltransferase (HAT) complexes, suggesting that C/EBPs participate in remodeling the chromatin organization of the HIV-1 provirus. Furthermore, overexpression of a C/EBP dominant-negative inhibits displacement of nucleosomes located at the HIV-1 transcriptional start site. These results provide insight into the general mechanisms by which C/EBPs regulate macrophage-restricted HIV-1 transcription. [Copyright &y& Elsevier]