Human PKR Transfected into Murine Cells Stimulates Expression of Genes under Control of the HIV1 or HTLV-I LTR

AbstractWe have analyzed the effect of transfection into murine NIH/3T3 cells of the human dsRNA-activated kinase PKR on the expression of the β-galactosidase reporter gene, placed under control of the HIV1 or the HTLV-I LTR. β-Galactosidase expression is stimulated when the reporter plasmids are cotransfected with wild-type PKR but inhibited when cotransfected with a catalytically inactive mutant PKR. In the case of HIV1, β-galactosidase expression was not stimulated when cotransfection was carried out with PKR harboring mutations in the dsRNA binding domains, indicating that stimulation depends on the classical mode of PKR activation through dsRNA binding. In contrast, the dsRNA binding mutants of PKR could still partially stimulate β-galactosidase expression from the HTLV-I LTR, suggesting that PKR activation in this case may involve different/additional mechanisms. These results show that, in addition to the known down-regulation of protein synthesis through eIF2 phosphorylation, PKR can also positively stimulate gene expressionin vivo,most probably through phosphorylation of a substrate distinct from eIF2

Involvement of double-stranded RNA-activated protein kinase in the synergistic activation of nuclear factor-κB by tumor necrosis factor-α and γ-interferon in preneuronal cells

Tumor necrosis factor-α (TNF-α) and γ-interferon (IFN-γ) cooperate during a variety of biological responses and ultimately synergistically enhance the expression of genes involved in immune and inflammatory responses. Recently, we demonstrated that IFN-γ can significantly potentiate TNF-α-induced nuclear factor (NF)-κB nuclear translocation in neuronal derived and endothelial cell lines. The mechanism by which these two cytokines exert their synergistic effect on NF-κB involves the de novo degradation of the NF-κB inhibitor, IκBβ. The double-stranded RNA- dependent kinase PKR is IFN-inducible and has been implicated in the activation of NF-κB; therefore, we examined the possibility that PKR may play a role in the synergistic activation of NF-κB during TNF-α/IFN-γ cotreatment. The PKR inhibitor 2-aminopurine (2-AP) inhibited TNF-α/IFN-γ- induced NF-κB nuclear translocation in neuronal derived cells but not in endothelial cells. The induced degradation of IκBβ, which is normally observed upon TNF-α/IFN-γ cotreatment, was blocked completely by 2-AP in neuronal derived cells. Also, 2-AP treatment or overexpression of a catalytically inactive PKR inhibited the TNF-α/IFN-γ-induced synergistic activation of κB-dependent gene expression. Our results suggest that the signal generated by IFN-γ, during TNF-α/IFN-γ cotreatment may require PKR to elicit enhanced NF-κB activity, and this signal may affect the stability of the IκBβ protein

Connexin-dependent transfer of cgamp to phagocytes modulates antiviral responses

Activation of cyclic GMP-AMP (cGAMP) synthase (cGAS) plays a critical role in antiviral responses to many DNA viruses. Sensing of cytosolic DNA by cGAS results in synthesis of the endogenous second messenger cGAMP that activates stimulator of interferon genes (STING) in infected cells. Critically, cGAMP can also propagate antiviral responses to uninfected cells through intercellular transfer, although the modalities of this transfer between epithelial and immune cells remain poorly defined. We demonstrate here that cGAMP-producing epithelial cells can transactivate STING in cocultured macrophages through direct cGAMP transfer. cGAMP transfer was reliant upon connexin expression by epithelial cells and pharmacological inhibition of connexins blunted STING-dependent transactivation of the macrophage compartment. Macrophage transactivation by cGAMP contributed to a positive-feedback loop amplifying antiviral responses, significantly protecting uninfected epithelial cells against viral infection. Collectively, our findings constitute the first direct evidence of a connexin-dependent cGAMP transfer to macrophages by epithelial cells, to amplify antiviral responses. IMPORTANCE Recent studies suggest that extracellular cGAMP can be taken up by macrophages to engage STING through several mechanisms. Our work demonstrates that connexin-dependent communication between epithelial cells and macrophages plays a significant role in the amplification of antiviral responses mediated by cGAMP and suggests that pharmacological strategies aimed at modulating connexins may have therapeutic applications to control antiviral responses in humans.Geneviève Pépin, Dominic De Nardo, Christina L. Rootes, Tomalika R. Ullah, Sumaiah S. Al-Asmari, Katherine R. Balka ... et al