The bZIP factor (HBZ) is an HTLV-1 regulatory protein encoded by anti-sense transcription of the HTLV-1 genome. HBZ mRNA expression correlates with clinical disability in HAM/TSP patients – and can be reversed by interferon (IFN) therapy. Sporadic evidence suggests that HBZ may have a negative role on interferon signalling. Activation of IRF3-dependent IFN signalling – either direct induction of IFNβ, viral restriction factors or interferon stimulated genes (ISGs) – is crucial for TLR and RLR mediated antiviral response. Thus, we sought to determine whether HBZ can impair IRF3-mediated innate immune responses. Over-expression of active forms of RIG-I, MAVS, TBK1, IKKe or IRF3 alone drive an antiviral response – however, in the presence of an HBZ expression vector, IFNβ responses were abrogated by 50-70%. In contrast, HBZ enhanced IRF7-dependent responses. In confirmation, both PBMC and human astrocytes transfected with HBZ and subsequently stimulated with IFN-triggering ligands (LPS, PolyI:C, VSV, Sendai virus and HTLV-1 virions), exhibited impaired IRF3-dependent signalling as compared with controls. As IRF3 is known to bind other bZIP proteins, further studies are underway to delineate the nature of IRF-HBZ interactions. Identifying such a mechanism may explain an enhanced risk of neurologic infection, as we show that chronically HTLV-1 infected astrocytes gradually increase and maintain long-term HBZ expression. Defining the immunomodulatory properties of HTLV-1 HBZ protein will provide a vital contribution toward understanding clinical outcome and risk of opportunistic infection associated with HTLV-1 infection.
ABSTRACT Vesicular stomatitis virus (VSV) is a candidate oncolytic virus that replicates and induces cell death in cancer cells while sparing normal cells. Although defects in the interferon antiviral response facilitate VSV oncolysis, other host factors, including translational and growth regulatory mechanisms, also appear to influence oncolytic virus activity. We previously demonstrated that VSV infection induces apoptosis in proliferating CD4 + T lymphocytes from adult T-cell leukemia samples but not in resting T lymphocytes or primary chronic lymphocytic leukemia cells that remain arrested in G 0 . Activation of primary CD4 + T lymphocytes with anti-CD3/CD28 is sufficient to induce VSV replication and cell death in a manner dependent on activation of the MEK1/2, c-Jun NH 2 -terminal kinase, or phosphatidylinositol 3-kinase pathway but not p38. VSV replication is specifically impaired by the cell cycle inhibitor olomoucine or rapamycin, which induces early G 1 arrest, but not by aphidicolin or Taxol, which blocks at the G 1 1S or G 2 1M phase, respectively; this result suggests a requirement for cell cycle entry for efficient VSV replication. The relationship between increased protein translation following G 0 /G 1 transition and VSV permissiveness is highlighted by the absence of mTOR and/or eIF4E phosphorylation whenever VSV replication is impaired. Furthermore, VSV protein production in activated T cells is diminished by small interfering RNA-mediated eIF4E knockdown. These results demonstrate that VSV replication in primary T lymphocytes relies on cell cycle transition from the G 0 phase to the G 1 phase, which is characterized by a sharp increase in ribogenesis and protein synthesis.
In chronic lymphocytic leukemia (CLL), overexpression of antiapoptotic B-cell leukemia/lymphoma 2 (BCL-2) family members contributes to leukemogenesis by interfering with apoptosis; BCL-2 expression also impairs vesicular stomatitis virus (VSV)-mediated oncolysis of primary CLL cells. In the effort to reverse resistance to VSV-mediated oncolysis, we combined VSV with obatoclax (GX15-070)'a small-molecule BCL-2 inhibitor currently in phase 2 clinical trials'and examined the molecular mechanisms governing the in vitro and in vivo antitumor efficiency of combining the two agents. In combination with VSV, obatoclax synergistically induced cell death in primary CLL samples and reduced tumor growth in severe combined immunodeficient (SCID) mice-bearing A20 lymphoma tumors. Mechanistically, the combination stimulated the mitochondrial apoptotic pathway, as reflected by caspase-3 and -9 cleavage, cytochrome c release and BAX translocation. Combination treatment triggered the release of BAX from BCL-2 and myeloid cell leukemia-1 (MCL-1) from BAK, whereas VSV infection induced NOXA expression and increased the formation of a novel BAX-NOXA heterodimer. Finally, NOXA was identified as an important inducer of VSV-obatoclax driven apoptosis via knockdown and overexpression of NOXA. These studies offer insight into the synergy between small-molecule BCL-2 inhibitors such as obatoclax and VSV as a combination strategy to overcome apoptosis resistance in CLL. In chronic lymphocytic leukemia (CLL), overexpression of antiapoptotic B-cell leukemia/lymphoma 2 (BCL-2) family members contributes to leukemogenesis by interfering with apoptosis; BCL-2 expression also impairs vesicular stomatitis virus (VSV)-mediated oncolysis of primary CLL cells. In the effort to reverse resistance to VSV-mediated oncolysis, we combined VSV with obatoclax (GX15-070)'a small-molecule BCL-2 inhibitor currently in phase 2 clinical trials'and examined the molecular mechanisms governing the in vitro and in vivo antitumor efficiency of combining the two agents. In combination with VSV, obatoclax synergistically induced cell death in primary CLL samples and reduced tumor growth in severe combined immunodeficient (SCID) mice-bearing A20 lymphoma tumors. Mechanistically, the combination stimulated the mitochondrial apoptotic pathway, as reflected by caspase-3 and -9 cleavage, cytochrome c release and BAX translocation. Combination treatment triggered the release of BAX from BCL-2 and myeloid cell leukemia-1 (MCL-1) from BAK, whereas VSV infection induced NOXA expression and increased the formation of a novel BAX-NOXA heterodimer. Finally, NOXA was identified as an important inducer of VSV-obatoclax driven apoptosis via knockdown and overexpression of NOXA. These studies offer insight into the synergy between small-molecule BCL-2 inhibitors such as obatoclax and VSV as a combination strategy to overcome apoptosis resistance in CLL.
Cannabis is one of the most widely used illicit substance among users of stimulants such as cocaine and amphetamine. Interestingly, recent accumulating evidence points toward the involvement of the endocannabinoid system (ECBS) in the neurobiological processes related to stimulant addiction. This article presents an up-to-date review with deep-insights into the pivotal role of the ECBS in the neurobiology of stimulant addiction and the effects of its modulation on addictive behaviors. The aims of this article are to: 1) review the role of cannabis use and ECBS modulation in the neurobiological substrates of psychostimulant addiction and 2) evaluate the potential of cannabinoid-based pharmacological strategies to treat stimulant addiction. A growing number of studies support a critical role of the ECBS and its modulation by synthetic or natural cannabinoid in various neurobiological and behavioral aspects of stimulants addiction. Thus, cannabinoids modulate brain reward systems closely involved in stimulants addiction, and provide further evidence that the cannabinoid system could be explored as a potential drug discovery target for treating addiction across different classes of stimulants.
Abstract The NF-κB transcription factors are key regulators of immunomodulatory, cell cycle, and developmental gene regulation. NF-κB activity is mainly regulated through the phosphorylation of IκB by the IκB kinase (IKK) complex IKKαβγ, leading to proteasome-mediated degradation of IκB, nuclear translocation of NF-κB dimers, DNA binding, and gene induction. Additionally, direct posttranslational modifications of NF-κB p65 and cRel subunits involving C-terminal phosphorylation has been demonstrated. The noncanonical IKK-related homologs, TNFR-associated factor family member-associated NF-κB activator (TANK)-binding kinase (TBK)1 and IKKε, are also thought to play a role in NF-κB regulation, but their functions remain unclear. TBK1 and IKKε were recently described as essential regulators of IFN gene activation through direct phosphorylation of the IFN regulatory factor-3 and -7 transcription factors. In the present study, we sought to determine whether IKKε and TBK1 could modulate cRel activity via phosphorylation. TBK1 and IKKε directly phosphorylate the C-terminal domain of cRel in vitro and in vivo and regulate nuclear accumulation of cRel, independently of the classical IκB/IKK pathway. IκBα degradation is not affected, but rather IKKε-mediated phosphorylation of cRel leads to dissociation of the IκBα-cRel complex. These results illustrate a previously unrecognized aspect of cRel regulation, controlled by direct IKKε/TBK1 phosphorylation.
