Human cytomegalovirus attenuates interleukin-1beta and tumor necrosis factor alpha proinflammatory signaling by inhibition of NF-kappaB activation

Viral infection is associated with a vigorous inflammatory response characterized by cellular infiltration and release of the proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha). In the present study, we identified a novel function of human cytomegalovirus (HCMV) that results in inhibition of IL-1 and TNF-alpha signaling pathways. The effect on these pathways was limited to cells infected with the virus, occurred at late times of infection, and was independent of cell type or virus strain. IL-1 and TNF-alpha signaling pathways converge at a point upstream of NF-kappaB activation and involve phosphorylation and degradation of the NF-kappaB inhibitory molecule IkappaBalpha. The HCMV inhibition of IL-1 and TNF-alpha pathways corresponded to a suppression of NF-kappaB activation. Analysis of IkappaBalpha phosphorylation and degradation suggested that HCMV induced two independent blocks in NF-kappaB activation, which occurred upstream from the point of convergence of the IL-1 and TNF-alpha pathways. We believe that the ability of HCMV to inhibit these two major proinflammatory pathways reveals a critical aspect of HCMV biology, with possible importance for immune evasion, as well as establishment of infection in cell types persistently infected by this virus.     

Human cytomegalovirus binding to human monocytes induces immunoregulatory gene expression

To continue our investigation of the cellular events that occur following human CMV (HCMV) infection, we focused on the regulation of cellular activation following viral binding to human monocytes. First, we showed that viral binding induced a number of immunoregulatory genes (IL-1beta, A20, NF-kappaB-p105/p50, and IkappaBalpha) in unactivated monocytes and that neutralizing Abs to the major HCMV glycoproteins, gB (UL55) and gH (UL75), inhibited the induction of these genes. Next, we demonstrated that these viral ligands directly up-regulated monocyte gene expression upon their binding to their appropriate cellular receptors. We then investigated if HCMV binding also resulted in the translation and secretion of cytokines. Our results showed that HCMV binding to monocytes resulted in the production and release of IL-1beta protein. Because these induced gene products have NF-kappaB sites in their promoter regions, we next examined whether there was an up-regulation of nuclear NF-kappaB levels. These experiments showed that, in fact, NF-kappaB was translocated to the nucleus following viral binding or purified viral ligand binding. Changes in IkappaBalpha levels correlated with the changes in NF-kappaB translocation. Lastly, we demonstrated that p38 kinase activity played a central role in IL-1beta production and that it was rapidly up-regulated following infection. These results support our hypothesis that HCMV initiates a signal transduction pathway that leads to monocyte activation and pinpoints a potential mechanism whereby HCMV infection of monocytes can result in profound pathogenesis, especially in chronic inflammatory-type conditions.     

Control of cytomegalovirus lytic gene expression by histone acetylation

Permissiveness for human cytomegalovirus (HCMV) infection is dependent on the state of cellular differentiation and has been linked to repression of the viral major immediate early promoter (MIEP). We have used conditionally permissive cells to analyze differential regulation of the MIEP and possible mechanisms involved in latency. Our data suggest that histone deacetylases (HDACs) are involved in repression of the MIEP in non-permissive cells as inhibition of HDACs induces viral permissiveness and increases MIEP activity. Non-permissive cells contain the class I HDAC, HDAC3; super-expression of HDAC3 in normally permissive cells reduces infection and MIEP activity. We further show that the MIEP associates with acetylated histones in permissive cells, and that in peripheral blood monocytes the MIEP associates with heterochromatin protein 1 (HP1), a chromosomal protein implicated in gene silencing. As monocytes are believed to be a site of viral latency in HCMV carriers and reactivated virus is only observed upon differentiation into macrophages, we propose that chromatin remodeling of the MIEP following cellular differentiation could potentially play a role in reactivation of latent HCMV.     

Chronic human immunodeficiency virus type 1 infection of myeloid cells disrupts the autoregulatory control of the NF-kappaB/Rel pathway via enhanced IkappaBalpha degradation.

Productive human immunodeficiency virus type 1 (HIV-1) infection causes sustained NF-kappaB DNA-binding activity in chronically infected monocytic cells. A direct temporal correlation exists between HIV infection and the appearance of NF-kappaB DNA-binding activity in myelomonoblastic PLB-985 cells. To examine the molecular basis of constitutive NF-kappaB DNA-binding activity in HIV1 -infected cells, we analyzed the phosphorylation and turnover of IkappaBalpha protein, the activity of the double-stranded RNA-dependent protein kinase (PKR) and the intracellular levels of NF-kappaB subunits in the PLB-985 and U937 myeloid cell models. HIV-1 infection resulted in constitutive, low-level expression of type 1 interferon (IFN) at the mRNA level. Constitutive PKR activity was also detected in HIV-1-infected cells as a result of low-level IFN production, since the addition of anti-IFN-alpha/beta antibody to the cells decreased PKR expression. Furthermore, the analysis of IkappaBalpha turnover demonstrated an increased degradation of IkappaBalpha in HIV-1-infected cells that may account for the constitutive DNA binding activity. A dramatic increase in the intracellular levels of NF-kappaB subunits c-Rel and NF-kappaB2 p100 and a moderate increase in NF-kappaB2 p52 and RelA(p65) were detected in HIV-1-infected cells, whereas NF-kappaB1 p105/p50 levels were not altered relative to the levels in uninfected cells. We suggest that HIV-1 infection of myeloid cells induces IFN production and PKR activity, which in turn contribute to enhanced IkappaBalpha phosphorylation and subsequent degradation. Nuclear translocation of NF-kappaB subunits may ultimately increase the intracellular pool of NF-kappaB/IkappaBalpha by an autoregulatory mechanism. Enhanced turnover of IkappaBalpha and the accumulation of NF-kappaB/Rel proteins may contribute to the chronically activated state of HIV-1-infected cells.     

Human cytomegalovirus blocks tumor necrosis factor alpha- and interleukin-1beta-mediated NF-kappaB signaling

NF-kappaB plays an important role in the early cellular response to pathogens by activating genes involved in inflammation, immune response, and cell proliferation and survival. NF-kappaB is also utilized by many viral pathogens, like human cytomegalovirus (HCMV), to activate their own gene expression programs, reflecting intricate roles for NF-kappaB in both antiviral defense mechanisms and viral physiology. Here we show that the NF-kappaB signaling pathway stimulated by proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) becomes inhibited in HCMV-infected cells. The block to NF-kappaB signaling is first noticeable during the early phase of infection but is fully established only at later times. Biochemical and genetic evidence demonstrates that the viral inhibition of proinflammatory signaling by distinct cytokines occurs upstream of the convergence point of NF-kappaB-activating pathways, i.e., the IkappaB kinase complex, and that it is mediated via different mechanisms. Consistent with this, we further show that an HCMV variant that has lost the ability to downregulate TNF-alpha-induced NF-kappaB signaling also fails to downregulate surface expression of TNF receptor 1, thereby mechanistically linking the inhibition of TNF-alpha-induced NF-kappaB signaling by HCMV to TNF receptor targeting. Our data support a model whereby HCMV inhibits cytokine-induced NF-kappaB signaling at later times during infection, and we suggest that this contributes to the inhibition of the cell's antiviral defense program.     

The enhancer domain of the human cytomegalovirus major immediate-early promoter determines cell type-specific expression in transgenic mice.

The human cytomegalovirus (HCMV) major immediate-early promoter (MIEP) is one of the first promoters to activate upon infection. To examine HCMV MIEP tissue-specific expression, transgenic mice were established containing the lacZ gene regulated by the MIEP (nucleotides -670 to +54). In the transgenic mice, lacZ expression was demonstrated in 19 of 29 tissues tested by histochemical and immunochemical analyses. These tissues included brain, eye, spinal cord, esophagus, stomach, pancreas, kidney, bladder, testis, ovary, spleen, salivary gland, thymus, bone marrow, skin, cartilage, and cardiac, striated and smooth muscles. Although expression was observed in multiple organs, promoter activity was restricted to specific cell types. The cell types which demonstrated HCMV MIEP expression included retinal cells of the eye, ductile cells of the salivary gland, exocrine cells of the pancreas, mucosal cells of the stomach and intestine, neuronal cells of the brain, muscle fibers, thecal cells of the corpus luteum, and Leydig and sperm cells of the testis. These observations indicate that the HCMV MIEP is not a pan-specific promoter and that the majority of expressing tissues correlate with tissues naturally infected by the virus in the human host.     

Helicobacter pylori infection activates NF-kappaB signaling pathway to induce iNOS and protect human gastric epithelial cells from apoptosis

Helicobacter pylori infection induces apoptosis and inducible nitric oxide synthase (iNOS) expression in gastric epithelial cells. In this study, we investigated the effects of NF-kappaB activation and iNOS expression on apoptosis in H. pylori-infected gastric epithelial cells. The suppression of NF-kappaB significantly increased caspase-3 activity and apoptosis in H. pylori-infected MKN-45 and Hs746T gastric epithelial cell lines as well as primary gastric epithelial cells. An NF-kappaB signaling pathway via NF-kappaB-inducing kinase and IkappaB kinase-beta activation was found to be involved in the inhibition of apoptosis in H. pylori-infected gastric epithelial cells. In gastric epithelial cells transfected with retrovirus containing IkappaBalpha superrepressor, iNOS mRNA and protein levels were reduced, indicating that H. pylori infection induced the expression of iNOS by activating NF-kappaB. Moreover, a NO donor, S-nitroso-N-acetylpenicillamine (100 microM), decreased caspase-3 activity and apoptosis in NF-kappaB-suppressed cells infected with H. pylori. These results suggest that NF-kappaB activation may play a role in protecting gastric epithelial cells from H. pylori-induced apoptosis by upregulating endogenous iNOS.     

Arsenite stabilizes IkappaBalpha and prevents NF-kappaB activation in IL-1 beta-stimulated Caco-2 cells independent of the heat shock response

Recent studies suggest that sodium arsenite downregulates NF-kappaB activity by inhibiting phosphorylation and subsequent degradation of IkappaBalpha. Many effects of sodium arsenite are secondary to induction of heat shock proteins. The role of the heat shock response in arsenite-induced inhibition of NF-kappaB, however, is not known. We examined the involvement of the heat shock response in arsenite-induced inhibition of NF-kappaB activity in IL-1beta-stimulated Caco-2 cells, a human colorectal adenocarcinoma cell line with enterocytic properties. Treatment of the cells with IL-1beta resulted in increased IkappaB kinase activity, reduced levels of IkappaBalpha and increased NF-kappaB DNA binding activity. Sodium arsenite blocked all of these responses to IL-1beta without inducing changes in heat shock factor activity or heat shock protein levels. Results from additional experiments showed that the protective effect of sodium arsenite on IkappaBalpha was not influenced by the oxygen radical scavenger catalase or by inhibitors of the MAP-kinase signaling pathway. The present results suggest that sodium arsenite stabilizes IkappaBalpha and prevents NF-kappaB activation in IL-1beta-stimulated Caco-2 cells independent of the heat shock response. In addition, stabilization of IkappaBalpha by sodium arsenite does not require oxygen radical formation or activation of the MAP kinase signaling pathway.     

Neutrality of the canonical NF-kappaB-dependent pathway for human and murine cytomegalovirus transcription and replication in vitro

Cytomegalovirus (CMV) is known to rapidly induce activation of nuclear factor kappaB (NF-kappaB) after infection of fibroblast and macrophage cells. NF-kappaB response elements are present in the enhancer region of the CMV major immediate-early promoter (MIEP), and activity of the MIEP is strongly upregulated by NF-kappaB in transient-transfection assays. Here we investigate whether the NF-kappaB-dependent pathway is required for initiating or potentiating human and murine CMV replication in vitro. We show that expression of a dominant negative mutant of the inhibitor of NF-kappaB-alpha (IkappaBalphaM) does not alter the replication kinetics of human or mouse CMV in cultured cells. In addition, mouse embryo fibroblasts genetically deficient for p65/RelA actually showed elevated levels of MCMV replication. Mutation of all NF-kappaB response elements within the enhancer of the MIEP in a recombinant mouse CMV containing the human MIEP (hMCMV-ES), which we have previously shown to replicate in murine fibroblasts with kinetics equivalent to that of wild-type mouse CMV, did not negatively affect replication in fibroblasts. Taken together, these data show that, for CMV replication in cultured fibroblasts activation of the canonical NF-kappaB pathway and binding of NF-kappaB to the MIEP are dispensable, and in the case of p65 may even interfere, thus uncovering a previously unrecognized level of complexity in the host regulatory network governing MIE gene expression in the context of a viral infection.