FOXO transcription factors activate alternative major immediate early promoters to induce human cytomegalovirus reactivation.

Human progenitor cells (HPCs) support human cytomegalovirus (HCMV) latency, and their differentiation along the myeloid lineage triggers cellular cues that drive reactivation. A key step during HCMV reactivation in latently infected HPCs is reexpression of viral major immediate early (MIE) genes. We recently determined that the major immediate early promoter (MIEP), which is primarily responsible for MIE gene expression during lytic replication, remains silent during reactivation. Instead, alternative promoters in the MIE locus are induced by reactivation stimuli. Here, we find that forkhead family (FOXO) transcription factors are critical for activation of alternative MIE promoters during HCMV reactivation, as mutating FOXO binding sites in alternative MIE promoters decreased HCMV IE gene expression upon reactivation and significantly decreased the production of infectious virus from latently infected primary CD34+ HPCs. These findings establish a mechanistic link by which infected cells sense environmental cues to regulate latency and reactivation, and emphasize the role of contextual activation of alternative MIE promoters as the primary drivers of reactivation.

Host signaling and EGR1 transcriptional control of human cytomegalovirus replication and latency.

Sustained phosphotinositide3-kinase (PI3K) signaling is critical to the maintenance of alpha and beta herpesvirus latency. We have previously shown that the beta-herpesvirus, human cytomegalovirus (CMV), regulates epidermal growth factor receptor (EGFR), upstream of PI3K, to control states of latency and reactivation. How signaling downstream of EGFR is regulated and how this impacts CMV infection and latency is not fully understood. We demonstrate that CMV downregulates EGFR early in the productive infection, which blunts the activation of EGFR and its downstream pathways in response to stimuli. However, CMV infection sustains basal levels of EGFR and downstream pathway activity in the context of latency in CD34+ hematopoietic progenitor cells (HPCs). Inhibition of MEK/ERK, STAT or PI3K/AKT pathways downstream of EGFR increases viral reactivation from latently infected CD34+ HPCs, defining a role for these pathways in latency. We hypothesized that CMV modulation of EGFR signaling might impact viral transcription important to latency. Indeed, EGF-stimulation increased expression of the UL138 latency gene, but not immediate early or early viral genes, suggesting that EGFR signaling promotes latent gene expression. The early growth response-1 (EGR1) transcription factor is induced downstream of EGFR signaling through the MEK/ERK pathway and is important for the maintenance of hematopoietic stemness. We demonstrate that EGR1 binds the viral genome upstream of UL138 and is sufficient to promote UL138 expression. Further, disruption of EGR1 binding upstream of UL138 prevents the establishment of latency in CD34+ HPCs. Our results indicate a model whereby UL138 modulation of EGFR signaling feeds back to promote UL138 gene expression and suppression of replication for latency. By this mechanism, the virus has hardwired itself into host cell biology to sense and respond to changes in homeostatic host cell signaling.

The Solid Component of Radiographically Non-Growing, Post-Radiated Vestibular Schwannoma Retains Proliferative Capacity: Implications for Patient Counseling.

OBJECTIVE: Nearly all radiated vestibular schwannomas (VS) have solid tissue remaining at the radiation bed. The viability and proliferation capacity of this tissue has never been objectively assessed. The goals of our study were to (1) determine whether this tissue retains the morphological and immunohistochemical features of VS and (2) evaluate whether the tissue is capable of proliferation in cell culture. METHODS: Case history, magnetic resonance imaging (MRI), cell culture, histology, and immunohistochemistry. RESULTS: We report the first case of a post-radiated, sporadic VS patient whose non-growing, residual MR-enhancing solid tissue was examined histologically and in cell culture. These cells were architecturally identical to non-radiated VS, had a Ki67 proliferative index similar to non-radiated sporadic and NF2-associated VS, were S100 positive, and grew in culture with kinetics comparable to non-radiated VS. CONCLUSION: The long-term risk for delayed tumor growth and/or secondary malignancy in radiated VS patients is unknown. Because the average life span in the United States is nearly 80 years, patients should be informed that (1) residual VS cells are viable even when tumors appear to be non-growing on MRI, (2) post-radiation surveillance imaging is required indefinitely, and (3) radiation may incur more risk in those patients with life expectancy>20-25 years.

Characterization of a membrane-active anti-tumor agent, UA8967.

Deletions or mutations in the tumor suppressor gene DPC4 (deleted in pancreatic carcinoma locus 4) are common in colon and pancreatic cancers. Using the Target-related Affinity Profiling (TRAP) chemical library screening method, a novel agent, UA8967, was selected for further studies because it showed greater potency in DPC4-deleted HCT-116 colon cancer cells. Cytotoxicity studies in six pancreatic cancer cell lines (MiaPaca-2, Panc-1, BxPC3, CF-PAC1, AsPC1, and T3M4), one normal human pancreatic ductal epithelial line (HPDE-6) and the HCT-116 DPC4(+/+) and HCT-116 DPC4(-/-) colon cancer cells showed IC50s ranging from 12-61 µM for exposure times of 72 h. Analysis of schedule dependence showed no advantage for long drug exposure times. There was also no selective inhibition of DNA, RNA or protein synthesis after exposure to UA8967. At 24-48 h, there was an accumulation of cells in G0/G1-phase and a proportionate reduction in S-phase cells. Within 1-6 h of exposure, cells were found to undergo an autophagic response, followed at 24 h by a low level of caspase-independent apoptosis with some necrosis. Because of the relatively non-specific mechanistic effects of UA8967, plasma membrane viability was evaluated using uptake of trypan blue and Sytox® Green dyes, and leakage of LDH. There was a dose dependent increase in Sytox® Green staining, trypan blue uptake and LDH leakage with increasing concentrations of UA8967, suggesting that UA8967 is affecting the plasma membrane. The DPC4(-/-) cells were more sensitive to UA8967 but not to DMSO, suggesting a drug-specific effect on cell membrane integrity.

Molecular pathogenesis of vestibular schwannomas: insights for the development of novel medical therapies.

Vestibular schwannomas (VS), benign intracranial tumors originating from the vestibulocochlear nerve, usually present with hearing loss, tinnitus, and balance dysfunction. Rarely, however, if untreated, these neoplasms can cause significant patient compromise - resulting in facial paralysis, brainstem compression, and even death. Those with vestibular schwannomas currently choose between surgery and stereotactic radiation therapy as available treatment options. Unfortunately, no medical therapies are presently U.S. Food & Drug Administration approved, representing an urgent and unmet clinical need. Recent breakthroughs in research have discovered key cell surface receptors and intracellular signaling pathways that drive vestibular schwannoma tumorigenesis, proliferation, and survival. A number of promising inhibitors targeting these signaling molecules have also now shown efficacy in preclinical VS cell culture models and animal experiments, with some recently entering human clinical trials. In this review, we summarize ErbB receptor signaling, PDGF receptors, MAP kinase signaling, AKT, p21-activated kinase signaling, mTOR, and VEGF signaling in the context of vestibular schwannoma drug development efforts worldwide. Today, it is truly an exciting time as our specialty stands on the verge of major breakthroughs in the development of medical therapies for VS.

Human plasmacytoid dendritic cells mount a distinct antiviral response to virus-infected cells.

Plasmacytoid dendritic cells (pDCs) can rapidly produce interferons and other soluble factors in response to extracellular viruses or virus mimics such as CpG-containing DNA. pDCs can also recognize live cells infected with certain RNA viruses, but the relevance and functional consequences of such recognition remain unclear. We studied the response of primary DCs to the prototypical persistent DNA virus, human cytomegalovirus (CMV). Human pDCs produced high amounts of type I interferon (IFN-I) when incubated with live CMV-infected fibroblasts but not with free CMV; the response involved integrin-mediated adhesion, transfer of DNA-containing virions to pDCs, and the recognition of DNA through TLR9. Compared with transient polyfunctional responses to CpG or free influenza virus, pDC response to CMV-infected cells was long-lasting, dominated by the production of IFN-I and IFN-III, and lacked diversification into functionally distinct populations. Similarly, pDC activation by influenza-infected lung epithelial cells was highly efficient, prolonged, and dominated by interferon production. Prolonged pDC activation by CMV-infected cells facilitated the activation of natural killer cells critical for CMV control. Last, patients with CMV viremia harbored phenotypically activated pDCs and increased circulating IFN-I and IFN-III. Thus, recognition of live infected cells is a mechanism of virus detection by pDCs that elicits a unique antiviral immune response.

Sp1 is required for glucose-induced transcriptional regulation of mouse vesicular glutamate transporter 2 gene.

BACKGROUND & AIMS: Vesicular glutamate transporter (VGLUT) has been reported to be involved in glucose-induced insulin secretion. It has been shown that glucose stimulates the expression of VGLUT isoform 2 (VGLUT2) in beta cells via transcriptional mechanism. In this study, we identified the mouse VGLUT2 (mVGLUT2) promoter and characterized the transcriptional mechanism of glucose-stimulated mVGLUT2 expression in beta-cells. METHODS: A promoter region of mVGLUT2 was cloned by genomic polymerase chain reaction. The mechanism of Sp1 in glucose-induced transactivation of mVGLUT2 was investigated by luciferase assay, electrophoretic mobility shift assay, chromatin immunoprecipitation assay, and Western blot analysis. RESULTS: A promoter containing 2133 base pairs of upstream sequence of the 5'-flanking region of mVGLUT2 complementary DNA was cloned. Transient transfection of various 5'-end deletion constructs of the mVGLUT2 promoter/luciferase reporter indicated that the region between -96 to +68 base pair contains the basal promoter for mVGLUT2. Mutational analysis and electromobility shift assay showed an important role for the transcription factor Sp1 in both basal and glucose-induced mVGLUT2 transcription. The interaction between Sp1 and mVGLUT2 was confirmed by chromatin immunoprecipitation assays. Glucose stimulates the phosphorylation of Sp1 via mitogen-activated protein kinase P38 and P44/42. This leads to increased binding activity of Sp1 to the mVGLUT2 promoter and results in activation of the gene. CONCLUSIONS: We cloned the mouse VGLUT2 promoter and showed a novel molecular mechanism of glucose-induced mVGLUT2 transcription.

Virus Control of Trafficking from Sorting Endosomes.

The maintenance of cell surface proteins is critical to the ability of a cell to sense and respond to information in its environment. As such, modulation of cell surface composition and receptor trafficking is a potentially important target of control in virus infection. Sorting endosomes (SEs) are control stations regulating the recycling or degradation of internalized plasma membrane proteins. Here we report that human cytomegalovirus (HCMV), a ubiquitous betaherpesvirus, alters the fate of internalized clathrin-independent endocytosis (CIE) cargo proteins, retaining them in virally reprogrammed SEs. We show that the small G protein ARF6 (ADP ribosylation factor 6), a regulator of CIE trafficking, is highly associated with SE membranes relative to uninfected cells. Combined with the observation of accumulated CIE cargo at the SE, these results suggest that infection diminishes the egress of ARF6 and its cargo from the SE. Expression of ubiquitin-specific protease 6 (USP6), also known as TRE17, was sufficient to restore ARF6 and some ARF6 cargo trafficking to the cell surface in infected cells. The USP activity of TRE17 was required to rescue both ARF6 and associated cargo from SE retention in infection. The finding that TRE17 expression does not rescue the trafficking of all CIE cargos retained at SEs in infection suggests that HCMV hijacks the normal sorting machinery and selectively sorts specific cargos into endocytic microdomains that are subject to alternative sorting fates.IMPORTANCE Cells maintain their surface composition, take up nutrients, and respond to their environment through the internalization and recycling of cargo at the cell surface through endocytic trafficking pathways. During infection with human cytomegalovirus (HCMV), host endocytic membranes are reorganized into a juxtanuclear structure associated with viral assembly and egress. Less appreciated is the effect of this reorganization on the trafficking of host proteins through the endocytic pathway. We show that HCMV retains internalized cargo and the effector of clathrin-independent endocytosis at sorting endosomes. The retention of some cargo, but not all, was reversed by overexpression of a ubiquitin-specific protease, TRE17. Our results demonstrate that HCMV induces profound reprogramming of endocytic trafficking and influences cargo sorting decisions. Further, our work suggests the presence of a novel ubiquitin-regulated checkpoint for the recycling of cargo from sorting endosome. These findings have important implications for host signaling and immune pathways in the context of HCMV infection.

Host Resistance to Monilinia vaccinii-corymbosi in Flowers and Fruits of Highbush Blueberry.

Monilinia vaccinii-corymbosi, the causal agent of mummy berry disease, infects blueberry flowers via the gynoecial pathway. To describe the expression of host resistance in highbush blueberry (Vaccinium corymbosum), fungal growth in the styles and colonization of the locules were compared among five blueberry cultivars in a series of controlled greenhouse experiments. Styles were harvested 1 and 4 days postinoculation, and the length colonized by hyphae was determined using fluorescence microscopy. At 8 weeks after inoculation, fruit were harvested and scored for the presence of hyphae in the locules. The infection frequency of styles ranged from 0.33 to 0.71, and only cv. Weymouth had significantly lower infection frequency than the other cultivars. The mean length of the colonized portion of the stylar canal ranged from 0.126 to 0.434 mm after 1 day and 1.62 to 3.59 mm after 4 days. Hyphae in the styles of cv. Weymouth exhibited the least growth, whereas hyphae in the styles of cultivars Jersey and Rancocas were significantly longer. The distance of style penetrated for cultivars Bluecrop and Coville was intermediate. The mean disease incidence of locules differed significantly. Values for cultivars Weymouth and Jersey were the smallest (0.038 and 0.039) and largest (0.249 and 0.236), respectively. The results demonstrate that a component of resistance to infection by M. vaccinii-corymbosi is expressed during growth in the gynoecial pathway.

Tissue reservoirs of antiviral T cell immunity in persistent human CMV infection.

T cell responses to viruses are initiated and maintained in tissue sites; however, knowledge of human antiviral T cells is largely derived from blood. Cytomegalovirus (CMV) persists in most humans, requires T cell immunity to control, yet tissue immune responses remain undefined. Here, we investigated human CMV-specific T cells, virus persistence and CMV-associated T cell homeostasis in blood, lymphoid, mucosal and secretory tissues of 44 CMV seropositive and 28 seronegative donors. CMV-specific T cells were maintained in distinct distribution patterns, highest in blood, bone marrow (BM), or lymph nodes (LN), with the frequency and function in blood distinct from tissues. CMV genomes were detected predominantly in lung and also in spleen, BM, blood and LN. High frequencies of activated CMV-specific T cells were found in blood and BM samples with low virus detection, whereas in lung, CMV-specific T cells were present along with detectable virus. In LNs, CMV-specific T cells exhibited quiescent phenotypes independent of virus. Overall, T cell differentiation was enhanced in sites of viral persistence with age. Together, our results suggest tissue T cell reservoirs for CMV control shaped by both viral and tissue-intrinsic factors, with global effects on homeostasis of tissue T cells over the lifespan.

Inhibiting p21-Activated Kinase Induces Cell Death in Vestibular Schwannoma and Meningioma via Mitotic Catastrophe.

HYPOTHESIS: p21-activated kinase (PAK) regulates signaling pathways that promote cell survival and proliferation; therefore, pharmacological inhibition of PAK will induce cell death in vestibular schwannomas (VS) and meningiomas. BACKGROUND: All VS and many meningiomas result from loss of the neurofibromatosis type 2 (NF2) gene product merlin, with ensuing PAK hyperactivation and increased cell proliferation/survival. METHODS: The novel small molecule PAK inhibitors PI-8 and PI-15-tested in schwannoma and meningioma cells-perturb molecular signaling and induce cell death. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay analyzed PAK inhibitors' effect on cell viability, cell cycle, and cell death, respectively. Western blots evaluated activation and expression of cell proliferation, apoptotic, and mitotic catastrophe markers. Light microscopy evaluated cell morphology, and immunocytochemistry analyzed cellular localization of phospho-Merlin and autophagy-related protein. RESULTS: Treatment with PI-8 and PI-15 decreased cell viability at 0.65 to 3.7 µM 50% inhibitory concentration (IC50) in schwannoma and meningioma cells. Terminal deoxynucleotidyl transferase dUTP nick end labeling and immunocytochemistry studies show that PI-8 and PI-15 induce mitotic catastrophe but not apoptosis in HEI193 cells while in BenMen1 cells, PI-8 induces autophagy and mitotic catastrophe. PI-15 induces apoptosis in BenMen1 cells. PAK inhibitor treated cells show phospho-Merlin localized to over-duplicated centrosomes of dividing cells, multiple enlarged nuclei, and misaligned/missegregated chromosomes-markers for mitotic catastrophe. Increased autophagy-related protein levels in the nucleus confirmed this cell death type. PI-8 and PI-15 inhibits PAK in both cell lines. However, only PI-15 inhibits v-akt murine thymoma viral oncogene homolog in BenMen1 cells. CONCLUSION: PAK inhibitors induce cell death in schwannoma and meningioma cells, at least in part, by mitotic catastrophe.

Zebrafish swimming behavior as a biomarker for ototoxicity-induced hair cell damage: a high-throughput drug development platform targeting hearing loss.

Hearing loss is one of the most common human sensory disabilities, adversely affecting communication, socialization, mood, physical functioning, and quality of life. In addition to age and noise-induced damage, ototoxicity is a common cause of sensorineural hearing loss with chemotherapeutic agents, for example, cisplatin, being a major contributor. Zebrafish (Danio rerio) are an excellent model to study hearing loss as they have neurosensory hair cells on their body surface that are structurally similar to those within the human inner ear. Anatomic assays of toxin-mediated hair cell damage in zebrafish have been established; however, using fish swimming behavior--rheotaxis--as a biomarker for this anatomic damage was only recently described. We hypothesized that, in parallel, multilane measurements of rheotaxis could be used to create a high-throughput platform for drug development assessing both ototoxic and potentially otoprotective compounds in real time. Such a device was created, and results demonstrated a clear dose response between cisplatin exposure, progressive hair cell damage, and reduced rheotaxis in zebrafish. Furthermore, pre-exposure to the otoprotective medication dexamethasone, before cisplatin exposure, partially rescued rheotaxis swimming behavior and hair cell integrity. These results provide the first evidence that rescued swimming behavior can serve as a biomarker for rescued hair cell function. Developing a drug against hearing loss represents an unmet clinical need with global implications. Because hearing loss from diverse etiologies may result from common end-effects at the hair cell level, lessons learned from the present study may be broadly used.