Host Cell Activation Research Articles

Staphylococcal Superantigens Spark Host-Mediated Danger Signals.

Staphylococcal enterotoxin B (SEB) of Staphylococcus aureus, and related superantigenic toxins produced by myriad microbes, are potent stimulators of the immune system causing a variety of human diseases from transient food poisoning to lethal toxic shock. These protein toxins bind directly to specific Vβ regions of T-cell receptors (TCR) and major histocompatibility complex (MHC) class II on antigen-presenting cells, resulting in hyperactivation of T lymphocytes and monocytes/macrophages. Activated host cells produce excessive amounts of proinflammatory cytokines and chemokines, especially tumor necrosis factor α, interleukin 1 (IL-1), IL-2, interferon γ (IFNγ), and macrophage chemoattractant protein 1 causing clinical symptoms of fever, hypotension, and shock. Because of superantigen-induced T cells skewed toward TH1 helper cells, and the induction of proinflammatory cytokines, superantigens can exacerbate autoimmune diseases. Upon TCR/MHC ligation, pathways induced by superantigens include the mitogen-activated protein kinase cascades and cytokine receptor signaling, resulting in activation of NFκB and the phosphoinositide 3-kinase/mammalian target of rapamycin pathways. Various mouse models exist to study SEB-induced shock including those with potentiating agents, transgenic mice and an “SEB-only” model. However, therapeutics to treat toxic shock remain elusive as host response genes central to pathogenesis of superantigens have only been identified recently. Gene profiling of a murine model for SEB-induced shock reveals novel molecules upregulated in multiple organs not previously associated with SEB-induced responses. The pivotal genes include intracellular DNA/RNA sensors, apoptosis/DNA damage-related molecules, immunoproteasome components, as well as antiviral and IFN-stimulated genes. The host-wide induction of these, and other, antimicrobial defense genes provide evidence that SEB elicits danger signals resulting in multi-organ damage and toxic shock. Ultimately, these discoveries might lead to novel therapeutics for various superantigen-based diseases.

Ubiquitination of pathogen-containing vacuoles promotes host defense to Chlamydia trachomatis and Toxoplasma gondii

Many intracellular bacterial and protozoan pathogens reside within host cell vacuoles customized by the microbial invaders to fit their needs. Within such pathogen-containing vacuoles (PVs) microbes procure nutrients and simultaneously hide from cytosolic host defense systems. Among the many PV-resident human pathogens are the bacterium Chlamydia trachomatis and the protozoan Toxoplasma gondii. Immune responses directed against their PVs are poorly characterized. We reported that activation of host cells with IFNγ triggers the attachment of polyubiquitin chains to Toxoplasma- and Chlamydia-containing vacuoles and thereby marks PVs for destruction. In murine cells PV ubiquitination is dependent on IFNγ-inducible Immunity Related GTPases (IRGs). Human cells also decorate PVs with ubiquitin upon IFNγ priming; however, the molecular machinery promoting PV ubiquitination in human cells remains unknown and is likely to be distinct from the IRG-dependent pathway we described in murine cells. Thus, IFNγ-inducible PV ubiquitination constitutes a critical event in cell-autonomous immunity to C. trachomatis and T. gondii in mice and humans, but the molecular machinery underlying PV ubiquitination is expected to be multifaceted and possibly host species-specific.

The C-terminal amyloidogenic peptide contributes to self-assembly of Avibirnavirus viral protease.

Unlike other viral protease, Avibirnavirus infectious bursal disease virus (IBDV)-encoded viral protease VP4 forms unusual intracellular tubule-like structures during viral infection. However, the formation mechanism and potential biological functions of intracellular VP4 tubules remain largely elusive. Here, we show that VP4 can assemble into tubules in diverse IBDV-infected cells. Dynamic analysis show that VP4 initiates the assembly at early stage of IBDV infection, and gradually assembles into larger size of fibrils within the cytoplasm and nucleus. Intracellular assembly of VP4 doesn’t involve the host cytoskeleton, other IBDV-encoded viral proteins or vital subcellular organelles. Interestingly, the last C-terminal hydrophobic and amyloidogenic stretch 238YHLAMA243 with two “aggregation-prone” alanine residues was found to be essential for its intracellular self-assembly. The assembled VP4 fibrils show significantly low solubility, subsequently, the deposition of highly assembled VP4 structures ultimately deformed the host cytoskeleton and nucleus, which was potentially associated with IBDV lytic infection. Importantly, the assembly of VP4 significantly reduced the cytotoxicity of protease activity in host cells which potentially prevent the premature cell death and facilitate viral replication. This study provides novel insights into the formation mechanism and biological functions of the Avibirnavirus protease-related fibrils.

Intracellular survival of Candida glabrata in macrophages: immune evasion and persistence.

Candida glabrata is a successful human opportunistic pathogen which causes superficial but also life-threatening systemic infections. During infection, C.glabrata has to cope with cells of the innate immune system such as macrophages, which belong to the first line of defense against invading pathogens. Candida glabrata is able to survive and even replicate inside macrophages while causing surprisingly low damage and cytokine release. Here, we present an overview of recent studies dealing with the interaction of C.glabrata with macrophages, from phagocytosis to intracellular growth and escape. We review the strategies of C.glabrata that permit intracellular survival and replication, including poor host cell activation, modification of phagosome maturation and phagosome pH, adaptation to antimicrobial activities, and mechanisms to overcome the nutrient limitations within the phagosome. In summary, these studies suggest that survival within macrophages may be an immune evasion and persistence strategy of C.glabrata during infection.

Switching off malignant pleural effusion formation-fantasy or future?

Malignant pleural effusion (MPE) is common and difficult to treat. In the vast majority of patients the presence of MPE heralds incurable disease, associated with poor quality of life, morbidity and mortality. Current therapeutic approaches are inefficient and merely offer palliation of associated symptoms. Recent scientific progress has shed light in the biologic processes governing the mechanisms behind the pathobiology of MPE. Pleural based tumors interfere with pleural fluid drainage, as well as the host vasculature and immune system, resulting in decreased fluid absorption and increased pleural fluid production via enhanced plasma extravasation into the pleural space. In order to achieve this feat, pleural based tumors must elicit critical vasoactive events in the pleura, thus forming a favorable microenvironment for tumor dissemination and MPE development. Such properties involve specific transcriptional signaling cascades in addition to secretion of important mediators which attract and activate host cell populations which, in turn, impact tumor cell functions. The dissection of the biologic steps leading to MPE formation provides novel therapeutic targets and recent research findings provide encouraging results towards future therapeutic innovations in MPE management.

A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation

Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV+2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P<0.05), and decreased levels of TLR2 (P<0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P<0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1β, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product.

Exploiting viral natural history for vaccine development.

The partial successes of the Phase 2 gB-based vaccine trials for HCMV highlight the very real likelihood that vaccine-mediated induction of antibodies that neutralize the fusion pathway of fibroblast infection is not sufficient as a singular strategy to confer protective efficacy against primary HCMV infection. Alternative strategies that serve as adjuncts to gB-based vaccines are likely required to target different aspects of the complex lifecycle of HCMV infection. There has been considerable recent interest in targeting the gH/gL/UL128/UL130/UL131 pentamer complex (gH/gL-PC) to neutralize the endocytic pathway of HCMV infection of epithelial and endothelial cells. Since both cell types are critical during primary mucosal infection, intrahost spread, and shedding of HCMV in an infected host, the gH/gL-PC represents a high-value target for vaccination to interrupt the HCMV lifecycle. The natural history of HCMV is exceedingly complex and incompletely resolved, and the protective efficacy generated by gH/gL-PC remains to be validated in clinical trials. Yet, there are salient aspects of its lifecycle that offer clues about how other novel vaccine strategies can be targeted to especially susceptible parts of the viral proteome to significantly disrupt HCMV’s ability to infect susceptible hosts. In particular, the protracted evolution of Herpesvirales has endowed HCMV with two remarkable properties of its natural history: (1) lifelong persistence within immune hosts that develop extraordinarily large antiviral immune responses and (2) the ability to reinfect those with prior immunity. The latter phenotype strongly implies that, if HCMV can overcome prior immunity to initiate a new infection, it is likely irrelevant whether prior immunity derives from prior infection or prior vaccination. Both phenotypes are unified by the extensive devotion of the HCMV coding repertoire (~50 %) to viral proteins that modulate host cell signaling, trafficking, activation, antigen presentation, and resistance to apoptosis. Collectively, these viral proteins are the likely reason for the high barrier to success for the 4-decade effort to design an HCMV vaccine, and they represent the viral proteins that make HCMV be the virus that it is. James Hanshaw wrote in 1971 that, based on a 15-year retrospective of congenital HCMV cases, “… any thoughtful program designed at prevention or treatment deserves consideration”. Drawing upon natural history data from the nonhuman primate model of HCMV persistence and pathogenesis, a “thoughtful program” is put forth that HCMV immune-modulating proteins should be considered as vaccine candidates.

Ecology meets cancer biology: the cancer swamp promotes the lethal cancer phenotype.

As they grow, tumors fundamentally alter their microenvironment, disrupting the homeostasis of the host organ and eventually the patient as a whole. Lethality is the ultimate result of deregulated cell signaling and regulatory mechanisms as well as inappropriate host cell recruitment and activity that lead to the death of the patient. These processes have striking parallels to the framework of ecological biology: multiple interacting ecosystems (organ systems) within a larger biosphere (body), alterations in species stoichiometry (host cell types), resource cycling (cellular metabolism and cell-cell signaling), and ecosystem collapse (organ failure and death). In particular, as cancer cells generate their own niche within the tumor ecosystem, ecological engineering and autoeutrophication displace normal cell function and result in the creation of a hypoxic, acidic, and nutrient-poor environment. This "cancer swamp" has genetic and epigenetic effects at the local ecosystem level to promote metastasis and at the systemic host level to induce cytokine-mediated lethal syndromes, a major cause of death of cancer patients.

Azasugar-containing phosphorothioate oligonucleotide (AZPSON) DBM-2198 inhibits human immunodeficiency virus type 1 (HIV-1) replication by blocking HIV-1 gp120 without affecting the V3 region.

DBM-2198, a six-membered azasugar nucleotide (6-AZN)-containing phosphorothioate (P = S) oligonucleotide (AZPSON), was described in our previous publication [Lee et al. (2005)] with regard to its antiviral activity against a broad spectrum of HIV-1 variants. This report describes the mechanisms underlying the anti-HIV-1 properties of DBM-2198. The LTR-mediated reporter assay indicated that the anti-HIV-1 activity of DBM-2198 is attributed to an extracellular mode of action rather than intracellular sequence-specific antisense activity. Nevertheless, the antiviral properties of DBM-2198 and other AZPSONs were highly restricted to HIV-1. Unlike other P = S oligonucleo-tides, DBM-2198 caused no host cell activation upon administration to cultures. HIV-1 that was pre-incubated with DBM-2198 did not show any infectivity towards host cells whereas host cells pre-incubated with DBM-2198 remained susceptible to HIV-1 infection, suggesting that DBM-2198 acts on the virus particle rather than cell surface molecules in the inhibition of HIV-1 infection. Competition assays for binding to HIV-1 envelope protein with anti-gp120 and anti-V3 antibodies revealed that DBM-2198 acts on the viral attachment site of HIV-1 gp120, but not on the V3 region. This report provides a better understanding of the antiviral mechanism of DBM-2198 and may contribute to the development of a potential therapeutic drug against a broad spectrum of HIV-1 variants.

A computational tool integrating host immunity with antibiotic dynamics to study tuberculosis treatment.

While active tuberculosis (TB) is a treatable disease, many complex factors prevent its global elimination. Part of the difficulty in developing optimal therapies is the large design space of antibiotic doses, regimens and combinations. Computational models that capture the spatial and temporal dynamics of antibiotics at the site of infection can aid in reducing the design space of costly and time-consuming animal pre-clinical and human clinical trials. The site of infection in TB is the granuloma, a collection of immune cells and bacteria that form in the lung, and new data suggest that penetration of drugs throughout granulomas is problematic. Here we integrate our computational model of granuloma formation and function with models for plasma pharmacokinetics, lung tissue pharmacokinetics and pharmacodynamics for two first line anti-TB antibiotics. The integrated model is calibrated to animal data. We make four predictions. First, antibiotics are frequently below effective concentrations inside granulomas, leading to bacterial growth between doses and contributing to the long treatment periods required for TB. Second, antibiotic concentration gradients form within granulomas, with lower concentrations toward their centers. Third, during antibiotic treatment, bacterial subpopulations are similar for INH and RIF treatment: mostly intracellular with extracellular bacteria located in areas non-permissive for replication (hypoxic areas), presenting a slowly increasing target population over time. Finally, we find that on an individual granuloma basis, pre-treatment infection severity (including bacterial burden, host cell activation and host cell death) is predictive of treatment outcome.

Scrapie infection in experimental rodents and SMB-S15 cells decreased the brain endogenous levels and activities of Sirt1.

Prion diseases are composed of a group of fatal neurodegenerative disorders resulting from misfolding of cellular prion (PrP(C)) into scrapie prion (PrP(Sc)). Sirt1, a class III histone deacetylase, has been reported to protect neuronal cells against PrP (106-126)-induced cell death. To address the potential role of Sirt1 during prion infection, the levels and enzyme activities of Sirt1 in the brains of scrapie-infected rodents, including hamsters infected with strain 263K, mice infected with strains 139A and ME7, and in prion infected SMB-S15 cells, were analyzed. Western blots revealed that endogenous Sirt1 levels were significantly decreased in all tested scrapie-infected models. Dynamic assays of brain Sirt1 levels in 263K-infected hamsters during incubation period showed a time-dependent decrease. The acetylating forms of Sirt1 target proteins, P53, PGC-1, and STAT3, markedly increased both in the brains of scrapie-infected rodents and in SMB-S15 cells, representing decreased Sirt1 activity. Immunofluorescent assays illustrated that Sirt1 predominately localized in cytosol of SMB-S15 cells but clearly distributed in nucleus of its normal partner cell line, SMB-PS. Moreover, accompanying with increase of Sirt1 level and decrease of acetyl-P53 level, treatments with Sirt1 activators SRT1720 and resveratrol in SMB-S15 cells significantly reduced PrP(Sc); at the same time, the cellular distribution of PrP proteins became normal, and the cell proliferating state was slightly improved. These data indicate that prion infection notably attenuates the Sirt1 activity in host cells. Sensitivity of the PrP(Sc) to Sirt1 activators highlights a potential role of Sirt1 in prion therapeutics.

Abstract 4876: Characterization of malignant melanoma growth triggered by the brain microenvironment in experimental metastasis models

Abstract The brain offers a unique microenvironment to which malignant melanoma frequently spreads leading to dismal prognosis for the patients. The molecular determinants that promote melanoma brain metastases however, remain largely unknown. Here, we show an experimental strategy utilizing two species of immune-compromised animals in a metastasis model to generate in vivo-cultivated metastatic tissues along with their corresponding host tissues in order to identify molecular events associated with brain metastases. The study was based on the melanoma cell lines, Melmet 1 and Melmet 5 with opposing phenotypes (invasive/proliferative, respectively) in the in vitro setting and distinct metastatic patterns in vivo, where Melmet 1 showed a preference to the brain. Transcriptional changes in the melanoma cells, as induced by the brain-microenvironment in both host species, revealed the opportunistic nature of melanoma in this context. We detected transitioning from the invasive phenotype to the proliferative phenotype, as the brain metastases passed from early to late growth phase. Of significance, we identified a brain-adaptive phenotype with synaptic characteristics and where glutamate signaling and Ca2+-dependent effectors played a central role. The brain-adaptive phenotype was more prominent in the earlier metastatic growth phases compared to a later phase. Analysis of host tissue uncovered a cooperative inflammatory microenvironment formed by activated host cells. Altogether, this suggests that acquisition of the brain-adaptive phenotype in a permissive environment might be critical for the establishment of metastatic growth in the brain microenvironment and further, targeting glutamate signaling may represent a therapeutic approach in brain metastasis. Global network modeling and functional annotation analysis revealed that several of the central molecular factors and pathways mediating melanoma brain metastasis in this study, are also central to other pathological diseases, in particular neurodegenerative diseases. The identification of essential molecular networks that operate to promote the brain-adaptive phenotype is of clinical relevance, as they represent leads to urgently needed therapeutic targets. Citation Format: Vigdis Nygaard, Lina Prasmickaite, Kotryna Vasiliauskaite, Trevor Clancy, Eivind Hovig. Characterization of malignant melanoma growth triggered by the brain microenvironment in experimental metastasis models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4876. doi:10.1158/1538-7445.AM2014-4876

Protective immunity against Rickettsia heilongjiangensis in a C3H/HeN mouse model mediated by outer membrane protein B-pulsed dendritic cells.

Rickettsia heilongjiangensis is an obligate intracellular bacterium that causes Far-Eastern tick-borne spotted fever. Outer membrane protein B (OmpB) is an important surface protein antigen of rickettsiae. In the present study, the ompB gene of R. heilongjiangensis was divided into four fragments, resulting in four recombinant proteins (OmpB-p1, OmpB-p2, OmpB-p3, and OmpB-p4). Each OmpB was used in vitro to stimulate murine bone marrow-derived dendritic cells (BMDCs) of C3H/HeN mice, and the OmpB-pulsed BMDCs were transferred to naïve C3H/HeN mice. On day 14 post-transfer of BMDCs, the mice were challenged with R. heilongjiangensis and the rickettsial loads in the mice were quantitatively determined on day 7 post-challenge. Mice receiving BMDCs pulsed with OmpB-p2, OmpB-p3, or OmpB-p4 exhibited significantly lower bacterial load compared with mice receiving OmpB-p1-pulsed BMDCs. CD4(+) and CD8(+) T cells isolated from the spleen of C3H/HeN mice receiving BMDCs pulsed with each OmpB were co-cultured with BMDCs pulsed with the respective cognate protein. In flow cytometric analysis, the expression level of CD69 on CD4(+) or CD8(+) T cells from mice receiving BMDCs pulsed with OmpB-p2, OmpB-p3, or OmpB-p4 was higher than that on cells from mice receiving OmpB-p1-pulsed BMDCs, while the expression level of tumor necrosis factor (TNF)-α on CD8(+) T cells and interferon (IFN)-γ on the CD4(+) and CD8(+) T cells from mice receiving OmpB-p2, -p3, or -p4 was significantly higher than on cells from mice receiving OmpB-p1-pulsed BMDCs. Our results suggest that the protective OmpBs could activate CD4(+) and CD8(+) T cells and drive their differentiation toward CD4(+) Th1 and CD8(+) Tcl cells, respectively, which produce greater amounts of TNF-α and, in particular, IFN-γ, to enhance rickettsicidal activity of host cells.

Ca2 +-dependent repair of pneumolysin pores: A new paradigm for host cellular defense against bacterial pore-forming toxins

Pneumolysin (PLY), a key virulence factor of Streptococcus pneumoniae, permeabilizes eukaryotic cells by forming large trans-membrane pores. PLY imposes a puzzling multitude of diverse, often mutually excluding actions on eukaryotic cells. Whereas cytotoxicity of PLY can be directly attributed to the pore-mediated effects, mechanisms that are responsible for the PLY-induced activation of host cells are poorly understood.We show that PLY pores can be repaired and thereby PLY-induced cell death can be prevented. Pore-induced Ca2+ entry from the extracellular milieu is of paramount importance for the initiation of plasmalemmal repair. Nevertheless, active Ca2+ sequestration that prevents excessive Ca2+ elevation during the execution phase of plasmalemmal repair is of no less importance.The efficacy of plasmalemmal repair does not only define the fate of targeted cells but also intensity, duration and repetitiveness of PLY-induced Ca2+ signals in cells that were able to survive after PLY attack. Intracellular Ca2+ dynamics evoked by the combined action of pore formation and their elimination mimic the pattern of receptor-mediated Ca2+ signaling, which is responsible for the activation of host immune responses. Therefore, we postulate that plasmalemmal repair of PLY pores might provoke cellular responses that are similar to those currently ascribed to the receptor-mediated PLY effects.Our data provide new insights into the understanding of the complexity of cellular non-immune defense responses to a major pneumococcal toxin that plays a critical role in the establishment and the progression of life-threatening diseases. Therapies boosting plasmalemmal repair of host cells and their metabolic fitness might prove beneficial for the treatment of pneumococcal infections. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Inhibition of Akt kinase activity suppresses entry and replication of influenza virus

The possibility of the pandemic spread of influenza viruses highlights the need for an effective cure for this life-threatening disease. Influenza A virus, belonging to a family of orthomyxoviruses, is a negative-strand RNA virus which encodes 11 viral proteins. A numbers of intracellular signaling pathways in the host cells interact with influenza the viral proteins, which affect various stages of viral infection and replication.In this study, we investigated how inhibition of Akt kinase activity impacts on influenza virus infection by using “Akt-in”, a peptide Akt inhibitor. In PR8 influenza-infected A549 cells, Akt interacted with the NS1 (Non structural protein 1), and hence increased phosphorylation of Akt kinase activity and NS1. Treatment of cells with either “TCL1- or TCL1b-based Akt-in” efficiently suppressed Akt kinase activity while decreasing the levels of phosphorylated NS1; this, in turn, inhibited viral replication in a dose- and time-dependent manner. The inhibitory effect on viral replication appears to not be due to inhibition of the production of inflammatory cytokines, including IL-6 and IL-8, in the host cells. Inhibition of Akt kinase activity in the host cells inhibited the efficiency of viral entry, which is associated with decreased levels of phosphorylated glycogen synthase kinase 3, a substrate of Akt. Thus inhibition of Akt kinase activity in host cells may have therapeutic advantages for influenza virus infection by inhibiting viral entry and replication.

Phosphorylation of herpes simplex virus 1 dUTPase upregulated viral dUTPase activity to compensate for low cellular dUTPase activity for efficient viral replication.

We recently reported that herpes simplex virus 1 (HSV-1) protein kinase Us3 phosphorylated viral dUTPase (vdUTPase) at serine 187 (Ser-187) to upregulate its enzymatic activity, which promoted HSV-1 replication in human neuroblastoma SK-N-SH cells but not in human carcinoma HEp-2 cells. In the present study, we showed that endogenous cellular dUTPase activity in SK-N-SH cells was significantly lower than that in HEp-2 cells and that overexpression of cellular dUTPase in SK-N-SH cells increased the replication of an HSV-1 mutant with an alanine substitution for Ser-187 (S187A) in vdUTPase to the wild-type level. In addition, we showed that knockdown of cellular dUTPase in HEp-2 cells significantly reduced replication of the mutant vdUTPase (S187A) virus but not that of wild-type HSV-1. Furthermore, the replacement of Ser-187 in vdUTPase with aspartic acid, which mimics constitutive phosphorylation, and overexpression of cellular dUTPase restored viral replication to the wild-type level in cellular dUTPase knockdown HEp-2 cells. These results indicated that sufficient dUTPase activity was required for efficient HSV-1 replication and supported the hypothesis that Us3 phosphorylation of vdUTPase Ser-187 upregulated vdUTPase activity in host cells with low cellular dUTPase activity to produce efficient viral replication.virus. Importance: It has long been assumed that dUTPase activity is important for replication of viruses encoding a dUTPase and that the viral dUTPase (vdUTPase) activity was needed if host cell dUTPase activity was not sufficient for efficient viral replication. In the present study, we showed that the S187A mutation in HSV-1 vdUTPase, which impaired its enzymatic activity, reduced viral replication in SK-N-SH cells, which have low endogenous cellular dUTPase activity, and that overexpression of cellular dUTPase restored viral replication to the wild-type level. We also showed that knockdown of cellular dUTPase in HEp-2 cells, which have higher dUTPase activity than do SK-N-SH cells, reduced replication of HSV-1 with the vdUTPase mutation but had no effect on wild-type virus replication. This is the first report, to our knowledge, directly showing that dUTPase activity is critical for efficient viral replication and that vdUTPase compensates for low host cell dUTPase activity to produce efficient viral replication.

Autophagy activated by Toxoplasma gondii infection in turn facilitates Toxoplasma gondii proliferation

Autophagy was found to play an antimicrobial or antiparasitic role in the activation of host cells to defend against intracellular pathogens, at the same time, pathogens could compete with host cell and take advantage of autophagy to provide access for its proliferation, but there are few articles for studying the outcome of this competition between host cell and pathogens. Therefore, the aim of our study was to investigate the relationship between autophagy activated by Toxoplasma gondii (T. gondii) and proliferation of T. gondii affected by autophagy in vitro. Firstly, human embryonic fibroblasts (HEF) cells were infected with T. gondii for different times. The monodansylcadaverine (MDC) staining, acridine orange (AO) staining, punctuate GFP-LC3 distribution, and transmission electron microscopy (TEM) assays were conducted, and the results were consistent in showing that gondii infection could induce autophagy. Secondly, HEF cells were infected with T. gondii and treated with autophagy inhibitor bafilomycin A1 or inducer lithium chloride for different times. Giemsa staining was conducted, and the results exhibited that T. gondii infection-induced autophagy could in turn promote T. gondii proliferation. Simultaneously, the results of Giemsa staining also revealed that autophagy inhibitor could reduce the number of each cell infected with T. gondii and inhibit T. gondii proliferation. In contrast, autophagy inducer could increase the number of each cell infected with T. gondii and encourage T. gondii proliferation. Therefore, our study suggests that T. gondii infection could activate autophagy, and this autophagy could in turn facilitate T. gondii proliferation in HEF cells for limiting nutrients.

Characterization of tubule and monomer derived from VP4 protein of infectious bursal disease virus

The VP4 protein of infectious bursal disease virus (IBDV) is a serine protease that processes the polyprotein for viral assembly. VP4 has been found to associate primarily with type II IBDV tubules that are 24nm in diameter. In this study, a chimeric VP4, assigned as HS1VP4, was constructed with a VP4-autocleavage site inserted between the N-terminal His-tag and the VP4 sequence. The results showed that the VP4 forms tubules after the self-cleavage of HS1VP4 when expressed in Escherichia coli. Furthermore, a deletion of 28 amino acids at the C-terminus of VP4 resulted in monomers and dimers instead of tubule formation; mutants of S652A and K692A at active site destroyed the activity. The endopeptidase activity of these monomers and dimers was approximately 12.5 times higher than that of VP4 tubules. Additionally, the formation of tubules inhibited VP4 protease activity, as demonstrated through in vitro assays. The production and characterization of monomers or dimers that have greater endopeptidase activity and protease activity than tubules can provide further insight into VP4 tubule assembly and the regulation of VP4 activity in host cells; this insight will facilitate the development of new anti-IBDV strategies.

Leucomycin A3, a 16-membered macrolide antibiotic, inhibits influenza A virus infection and disease progression

Severe respiratory disease arising from influenza virus infection has a high fatality rate. Neutrophil myeloperoxidase (MPO) has been implicated in the pathogenesis of severe influenza-induced pneumonia because extracellularly released MPO mediates the production of hypochlorous acid, a potent tissue injury factor. To search for candidate anti-influenza compounds, we screened leucomycin A3 (LM-A3), spiramycin (SPM), an erythromycin derivative (EM900, in which anti-bacterial activity has been eliminated), and clarithromycin (CAM), by analyzing their ability to inhibit MPO release in neutrophils from mice and humans. When each candidate was injected into mice infected with a lethal dose of A/H1N1 influenza virus (PR-8), LM-A3 produced the highest survival rate (80.9%). We found that LM-A3 induced beneficial effects on lung pathology and viral proliferation involved in the regulatory activity of MPO release, pro-inflammatory cytokines and interferon-α production in the lung. SPM and EM900 also induced positive survival effects in the infected mice, whereas CAM did not. We further found that these compounds inhibit virus proliferation in human pneumonia epithelial A549 cells in vitro. LM-A3 showed effective action against influenza A virus infection with high anti-viral activity in human host cells, indicating the possibility that LM-A3 is a prospective lead compound for the development of a drug for human influenza. The positive survival effect induced by EM900 suggests that pharmacological architectures between anti-bacterial and anti-influenza virus activities can be dissociated in macrolide derivatives. These observations provide valuable evidence for the potential development of novel macrolide derivatives that have strong anti-viral but no anti-bacterial activity.

Melanoma brain colonization involves the emergence of a brain-adaptive phenotype.

The brain offers a unique microenvironment that plays an important role in the establishment and progression of metastasis. However, the molecular determinants that promote development of melanoma brain metastases are largely unknown. Utilizing two species of immune-compromised animals, with in vivo cultivated metastatic tissues along with their corresponding host tissues in a metastasis model, we here identify molecular events associated with melanoma brain metastases. We find that the transcriptional changes in the melanoma cells, as induced by the brain-microenvironment in both host species, reveal the opportunistic nature of melanoma in this biological context in rewiring the molecular framework of key molecular players with their associated biological processes. Specifically, we identify the existence of a neuron-like melanoma phenotype, which includes synaptic characteristics and a neurotransmission-like circuit involving glutamate. Regulation of gene transcription and neuron-like plasticity by Ca(2+)-dependent signaling appear to occur through glutamate receptor activation. The brain-adaptive phenotype was found as more prominent in the early metastatic growth phases compared to a later phase, emphasizing a temporal requirement of critical events in the successful colonization of the brain. Analysis of the host tissue uncovered a cooperative inflammatory microenvironment formed by activated host cells that permitted melanoma growth at the expense of the host organism. Combined experimental and computational approaches clearly highlighted genes and signaling pathways being shared with neurodegenerative diseases. Importantly, the identification of essential molecular networks that operate to promote the brain-adaptive phenotype is of clinical relevance, as they represent leads to urgently needed therapeutic targets.