Herpes simplex virus type 1 ICP27-dependent activation of NF-kappaB

The ability of herpes simplex virus type 1 (HSV-1) to activate NF-kappaB has been well documented. Beginning at 3 to 5 h postinfection, HSV-1 induces a robust and persistent nuclear translocation of an NF-kappaB-dependent (p50/p65 heterodimer) DNA binding activity, as measured by electrophoretic mobility shift assay. Activation requires virus binding and entry, as well as de novo infected-cell protein synthesis, and is accompanied by loss of both IkappaBalpha and IkappaBbeta. In this study, we identified loss of IkappaBalpha as a marker of NF-kappaB activation, and infection with mutants with individual immediate-early (IE) regulatory proteins deleted indicated that ICP27 was necessary for IkappaBalpha loss. Analysis of both N-terminal and C-terminal mutants of ICP27 identified the region from amino acids 21 to 63 as being necessary for IkappaBalpha loss. Additional experiments with mutant viruses with combinations of IE genes deleted revealed that the ICP27-dependent mechanism of NF-kappaB activation may be augmented by functional ICP4. We also analyzed two additional markers for NF-kappaB activation, phosphorylation of the p65 subunit on Ser276 and Ser536. Phosphorylation of both serines was induced upon HSV infection and required functional ICP4 and ICP27. Pharmacological inhibitor studies revealed that both IkappaBalpha and Ser276 phosphorylation were dependent on Jun N-terminal protein kinase activity, while Ser536 phosphorylation was not affected during inhibitor treatment. These results demonstrate that there are several layers of regulation of NF-kappaB activation during HSV infection, highlighting the important role that NF-kappaB may play in infection.     

ChlaDub1 of Chlamydia trachomatis suppresses NF-kappaB activation and inhibits IkappaBalpha ubiquitination and degradation

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes various human diseases, including blindness caused by ocular infection and sexually transmitted diseases resulting from urogenital infection. After infecting host cells, Chlamydiae avoid alarming the host's immune system. Among the immune evasion mechanisms, Chlamydiae can inhibit NF-κB activation, a crucial pathway for host inflammatory responses. In this study, we show that Chla Dub1, a deubiquitinating and deNeddylating protease from C. trachomatis , is expressed in infected cells. In transfection experiments, Chla Dub1 suppresses NF-κB activation induced by several pro-inflammatory stimuli and binds the NF-κB inhibitory subunit IκBα, impairing its ubiquitination and degradation. Thus, we provide further insight into the mechanism by which C. trachomatis may evade the host inflammatory response by demonstrating that Chla Dub1, a protease produced by this microorganism, is capable of inhibiting IκBα degradation and blocking NF-κB activation.     

Herpes simplex virus disrupts NF-kappaB regulation by blocking its recruitment on the IkappaBalpha promoter and directing the factor on viral genes

Herpes simplex viruses (HSVs) are able to hijack the host-cell IkappaB kinase (IKK)/NF-kappaB pathway, which regulates critical cell functions from apoptosis to inflammatory responses; however, the molecular mechanisms involved and the outcome of the signaling dysregulation on the host-virus interaction are mostly unknown. Here we show that in human keratinocytes HSV-1 attains a sophisticated control of the IKK/NF-kappaB pathway, inducing two distinct temporally controlled waves of IKK activity and disrupting the NF-kappaB autoregulatory mechanism. Using chromatin immunoprecipitation we demonstrate that dysregulation of the NF-kappaB-response is mediated by a virus-induced block of NF-kappaB recruitment to the promoter of the IkappaBalpha gene, encoding the main NF-kappaB-inhibitor. We also show that HSV-1 redirects NF-kappaB recruitment to the promoter of ICP0, an immediate-early viral gene with a key role in promoting virus replication. The results reveal a new level of control of cellular functions by invading viruses and suggest that persistent NF-kappaB activation in HSV-1-infected cells, rather than being a host response to the virus, may play a positive role in promoting efficient viral replication.     

Hepatitis C virus NS5A and subgenomic replicon activate NF-kappaB via tyrosine phosphorylation of IkappaBalpha and its degradation by calpain protease

Hepatitis C virus nonstructural protein 5A (NS5A) has been implicated in the HCV antiviral resistance, replication, and transactivation of cellular gene expression. We have recently shown that HCV NS5A activates NF-kappaB via oxidative stress (22). In this study, we investigate the molecular mechanism(s) of NF-kappaB activation in response to oxidative stress induced by NS5A protein. In contrast to the classic Ser32,36 phosphorylation of IkappaBalpha, we report here that tyrosine phosphorylation of IkappaBalpha at Tyr42 and Tyr305 residues is induced by the HCV NS5A and the subgenomic replicons in the NF-kappaB activation process. Use of IkappaBalpha-Tyr42,305 double mutant provided the evidence for their key role in the activation of NF-kappaB. Activation of NF-kappaB was blocked by a series of tyrosine kinase inhibitors but not by IkappaB kinase inhibitor BAY 11-7085. More specifically, a ZAP-70 knock-out cell line expressing NS5A and other nonstructural proteins respectively prevented the NF-kappaB activation, indicating the involvement of ZAP-70 as a probable tyrosine kinase in the activation process. Evidence is also presented for the possible role of calpain proteases in the NS5A-induced IkappaBalpha degradation. These studies collectively define an alternate pathway of NF-kappaB activation by NS5A alone or in the context of the HCV subgenomic replicon. Constitutive activation of NF-kappaB by HCV has implications in the chronic liver disease including hepatocellular carcinoma associated with HCV infection.     

Neurons differentially activate the herpes simplex virus type 1 immediate-early gene ICP0 and ICP27 promoters in transgenic mice

Herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins are required for the expression of viral early and late proteins. It has been hypothesized that host neuronal proteins regulate expression of HSV-1 IE genes that in turn control viral latency and reactivation. We investigated the ability of neuronal proteins in vivo to activate HSV-1 IE gene promoters (ICP0 and ICP27) and a late gene promoter (gC). Transgenic mice containing IE (ICP0 and ICP27) and late (gC) gene promoters of HSV-1 fused to the Escherichia coli beta-galactosidase coding sequence were generated. Expression of the ICP0 and ICP27 reporter transgenes was present in anatomically distinct subsets of neurons in the absence of viral proteins. The anatomic locations of beta-galactosidase-positive neurons in the brains of ICP0 and ICP27 reporter transgenic mice were similar and included cerebral cortex, lateral septal nucleus, cingulum, hippocampus, thalamus, amygdala, and vestibular nucleus. Trigeminal ganglion neurons were positive for beta-galactosidase in adult ICP0 and ICP27 reporter transgenic mice. The ICP0 reporter transgene was differentially regulated in trigeminal ganglion neurons depending upon age. beta-galactosidase-labeled cells in trigeminal ganglia and cerebral cortex of ICP0 and ICP27 reporter transgenic mice were confirmed as neurons by double labeling with antineurofilament antibody. Nearly all nonneuronal cells in ICP0 and ICP27 reporter transgenic mice and all neuronal and nonneuronal cells in gC reporter transgenic mice were negative for beta-galactosidase labeling in the absence of HSV-1. We conclude that factors in neurons are able to differentially regulate the HSV-1 IE gene promoters (ICP0 and ICP27) in transgenic mice in the absence of viral proteins. These findings are important for understanding the regulation of the latent and reactivated stages of HSV-1 infection in neurons.     

Herpes simplex virus type 1 ICP0 phosphorylation mutants impair the E3 ubiquitin ligase activity of ICP0 in a cell type-dependent manner

Herpes simplex virus type 1 (HSV-1) infected cell protein 0 (ICP0) is a 110-kDa nuclear phosphoprotein that is required for both the efficient initiation of lytic infection and the reactivation of quiescent viral genomes from latency. The ability of ICP0 to act as a potent viral transactivator is mediated by its N-terminal zinc-binding RING finger domain. This domain confers E3 ubiquitin ligase activity to ICP0 and is required for the proteasome-dependent degradation of a number of cellular proteins during infection, including the major nuclear domain 10 (ND10) constituent protein promyelocytic leukemia. In previous work we mapped three phosphorylation regions within ICP0, two of which directly affected its transactivation capabilities in transient transfection assays (Davido et al., J. Virol. 79:1232-1243, 2005). Because ICP0 is a phosphoprotein, we initially sought to test the hypothesis that phosphorylation regulates the E3 ubiquitin ligase activity of ICP0. Although none of the mutations affected ICP0 E3 ligase activity in vitro, transient transfection analysis indicated that mutations within one or more of the phosphorylated regions impaired the ability of ICP0 to form foci with colocalizing conjugated ubiquitin and to disrupt ND10. Mutations within one of the regions also affected ICP0 stability, and all of these phenomena occurred in a cell type-dependent manner. In the context of viral infection, only one ICP0 phosphorylation mutant (P1) showed a significant defect in viral replication and enhanced protein stability compared to all the other viruses tested. This study suggests that specific cellular environments and context of expression (transfection versus infection) differentially regulate several activities of ICP0 related to its E3 ubiquitin ligase activity via phosphorylation.     

Physical interaction between the herpes simplex virus type 1 immediate-early regulatory proteins ICP0 and ICP4.

The herpes simplex virus type 1 immediate-early protein ICP0 enhances expression of a spectrum of viral genes alone and synergistically with ICP4. To test whether ICP0 and ICP4 interact physically, we performed far-Western blotting analysis of proteins from mock-, wild-type-, and ICP4 mutant virus-infected cells with in vitro-synthesized [35S]Met-labeled ICP0 and ICP4 as probes. The ICP4 and ICP0 polypeptides synthesized in vitro exhibited molecular weights similar to those of their counterparts in herpes simplex virus type 1-infected cells, and the in vitro-synthesized ICP4 was able to bind to a probe containing the ICP4 consensus binding site. Far-Western blotting experiments demonstrated that ICP0 interacts directly and specifically with ICP4 and with itself. To further define the interaction between ICP0 and ICP4, we generated a set of glutathione S-transferase (GST)-ICP0 fusion proteins that contain GST and either ICP0 N-terminal amino acids 1 to 244 or 1 to 394 or C-terminal amino acids 395 to 616 or 395 to 775. Using GST-ICP0 fusion protein affinity chromatography and in vitro-synthesized [35S]Met-labeled ICP0 and ICP4, ICP4 was shown to interact preferentially with the fusion protein containing ICP0 C-terminal amino acids 395 to 775, whereas ICP0 interacted efficiently with both the N-terminal GST-ICP0 fusion proteins and the C-terminal GST-ICP0 fusion proteins containing amino acids 395 to 775. Fusion protein affinity chromatography also demonstrated that the C-terminal 235 amino acid residues of ICP4 are important for efficient interaction with ICP0. Collectively, these results reveal a direct and specific physical interaction between ICP0 and ICP4.     

Alphaherpesvirus proteins related to herpes simplex virus type 1 ICP0 affect cellular structures and proteins

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP0 interacts with several cellular proteins and induces the proteasome-dependent degradation of others during infection. In this study we show that ICP0 is required for the proteasome-dependent degradation of the ND10 protein Sp100 and, as with the other target proteins, the ICP0 RING finger domain is essential. Further, comparison of the kinetics and ICP0 domain requirements for the degradation of PMI and Sp100 suggests that a common mechanism is involved. Homologues of ICP0 are encoded by other members of the alphaherpesvirus family. These proteins show strong sequence homology to ICP0 within the RING finger domain but limited similarity elsewhere. Using transfection assays, we have shown that all the ICP0 homologues that we tested have significant effects on the immunofluorescence staining character of at least one of the proteins destabilized by ICP0, and by using a recombinant virus, we found that the equine herpesvirus ICP0 homologue induced the proteasome-dependent degradation of endogenous CENP-C and modified forms of PML and Sp100. However, in contrast to ICP0, the homologue proteins had no effect on the distribution of the ubiquitin-specific protease USP7 within the cell, consistent with their lack of a USP7 binding domain. We also found that ICP0 by itself could induce the abrogation of SUMO-1 conjugation and then the proteasome-dependent degradation of unmodified exogenous PML in transfected cells, thus demonstrating that other HSV-1 proteins are not required. Surprisingly, the ICP0 homologues were unable to cause these effects. Overall, these data suggest that the members of the ICP0 family of proteins may act via a similar mechanism or pathway involving their RING finger domain but that their intrinsic activities and effects on endogenous and exogenous proteins differ in detail.     

Incomplete regulation of NF-kappaB by IkappaBalpha during respiratory syncytial virus infection in A549 cells

Respiratory syncytial virus (RSV) infection of airway epithelial cells results in persistent NF-kappaB activation and NF-kappaB-mediated interleukin-8 production. Previous studies in airway epithelial cells demonstrated that tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB activation is transient due to regulation by IkappaBalpha. However, during RSV infection, IkappaBalpha has only a partial inhibitory effect on NF-kappaB activation. Studies presented here demonstrate that neither increased IkappaBalpha production which occurs as a result of RSV-induced NF-kappaB activation nor inhibition of proteasome-mediated IkappaBalpha degradation results in a reversal of RSV-induced NF-kappaB activation. Thus, while manipulation of IkappaBalpha results in reversal of TNF-alpha-induced NF-kappaB activation, manipulation of IkappaBalpha does not result in a reversal of RSV-induced NF-kappaB activation.     

Salmonella secreted factor L deubiquitinase of Salmonella typhimurium inhibits NF-kappaB, suppresses IkappaBalpha ubiquitination and modulates innate immune responses

Salmonella enterica translocates virulent factors into host cells using type III secretion systems to promote host colonization, intracellular bacterial replication and survival, and disease pathogenesis. Among many effectors, the type III secretion system encoded in Salmonella pathogenicity island 2 translocates a Salmonella-specific protein, designated Salmonella secreted factor L (SseL), a putative virulence factor possessing deubiquitinase activity. In this study, we attempt to elucidate the mechanism and the function of SseL in vitro, in primary host macrophages and in vivo in infected mice. Expression of SseL in mammalian cells suppresses NF-kappaB activation downstream of IkappaBalpha kinases and impairs IkappaBalpha ubiquitination and degradation, but not IkappaBalpha phosphorylation. Disruption of the gene encoding SseL in S. enterica serovar typhimurium increases IkappaBalpha degradation and ubiquitination, as well as NF-kappaB activation in infected macrophages, compared with wild-type bacteria. Mice infected with SseL-deficient bacteria mount stronger inflammatory responses, associated with increased production of NF-kappaB-dependent cytokines. Thus, SseL represents one of the first bacterial deubiquitinases demonstrated to modulate the host inflammatory response in vivo.     

Cooperativity among herpes simplex virus type 1 immediate-early regulatory proteins: ICP4 and ICP27 affect the intracellular localization of ICP0.

The results of transient expression assays and studies of viral mutants have shown that three of the five immediate-early proteins of herpes simplex virus type 1 (HSV-1) perform regulatory functions, individually and cooperatively. As part of efforts designed to explore the molecular basis for the functional cooperativity among ICP0, ICP4, and ICP27 in the regulation of HSV gene expression, we have examined the intracellular localization of ICP0 in cells infected with ICP4 and ICP27 null mutant viruses by indirect immunofluorescence. Although ICP0 was localized predominantly to the nuclei of wild-type virus-infected cells, it was found exclusively in the nuclei of ICP27 mutant-infected cells and in both the cytoplasm and nuclei of ICP4 mutant-infected cells, the cytoplasmic component being especially strong. These observations indicate that both ICP4 and ICP27 can affect the intracellular localization of ICP0. Transient expression assays with plasmids that express wild-type and mutant forms of ICP0, ICP4, and ICP27 confirmed that ICP4 promotes and that ICP27 inhibits the nuclear localization of ICP0. These results confirm the observations made for mutant virus-infected cells and indicate that the localization pattern seen in infected cells can be established by these three immediate-early proteins exclusive of other viral proteins. The C-terminal half of ICP27 was shown to be required to achieve its inhibitory effect on the nuclear localization of ICP0. The region of ICP0 responsive to ICP27 was mapped to the C terminus of the molecule between amino acid residues 720 and 769. In addition, the concentration of ICP27 was shown to have a significant effect on the intracellular localization of ICP0. Because the major regulatory activities of ICP0, ICP4, and ICP27 are expressed in the nucleus, the ability of these three proteins collectively to determine their own localization patterns within cells adds a new dimension to the complex process of viral gene regulation in HSV.