Infection by agnoprotein-negative mutants of polyomavirus JC and SV40 results in the release of virions that are mostly deficient in DNA content

<p>Abstract</p> <p>Background</p> <p>Human polyomavirus JC (JCV) is the etiologic agent of a brain disease, known as progressive multifocal leukoencephalopathy (PML). The JCV genome encodes a small multifunctional phospho-protein, agnoprotein, from the late coding region of the virus, whose regulatory functions in viral replication cycle remain elusive. In this work, the functional role of JCV and SV40 agnoproteins in virion release was investigated using a point mutant (Pt) of each virus, where the ATG codon of agnoprotein was mutated to abrogate its expression.</p> <p>Results</p> <p>Analysis of both viral protein expression and replication using Pt mutant of each virus revealed that both processes were substantially down-regulated in the absence of agnoprotein compared to wild-type (WT) virus. Complementation studies in cells, which are constitutively expressing JCV agnoprotein and transfected with the JCV Pt mutant genome, showed an elevation in the level of viral DNA replication near to that observed for WT. Constitutive expression of large T antigen was found to be not sufficient to compensate the loss of agnoprotein for efficient replication of neither JCV nor SV40 in vivo. Examination of the viral release process for both JCV and SV40 Pt mutants showed that viral particles are efficiently released from the infected cells in the absence of agnoprotein but were found to be mostly deficient in viral DNA content.</p> <p>Conclusions</p> <p>The results of this study provide evidence that agnoprotein plays an important role in the polyomavirus JC and SV40 life cycle. Infection by agnoprotein-negative mutants of both viruses results in the release of virions that are mostly deficient in DNA content.</p

Binge-Like Exposure to Ethanol Enhances Morphine's Anti-nociception in B6 Mice

Elevation of the blood ethanol concentration (BEC) to &gt; 80 mg/dL (17.4 mM) after binge drinking enhances inflammation in brain and neuroimmune signaling pathways. Morphine abuse is frequently linked to excessive drinking. Morphine exerts its actions mainly via the seven transmembrane G-protein-coupled mu opioid receptors (MORs). Opioid use disorders (OUDs) include combination of opioids with alcohol, leading to opioid overdose-related deaths. We hypothesized that binge drinking potentiates onset and progression of OUD. Using C57BL/6J (B6) mice, we first characterized time-dependent inflammatory gene expression change as molecular markers using qRT-PCR within 24 h after binge-like exposure to high-dose, high-concentration ethanol (EtOH). The mice were given one injection of EtOH (5 g/kg, 42% v/v, i.g.) and sacrificed at 2.5 h, 5 h, 7.5 h, or 24 h later. Inflammatory cytokines interleukin (IL)-1β, IL-6, and IL-18 were elevated in both the striatum (STr) and the nucleus accumbens (NAc) of the mice. We then investigated the expression profile of MOR in the STr at 2 min, 5 h, or 24 h after the first EtOH injection and at 24 h and 48 h after the third injection. This binge-like exposure to EtOH upregulated MOR expression in the STr and NAc, an effect that could enhance morphine's anti-nociception. Therefore, we examined the impact of binge-like exposure to EtOH on morphine's anti-nociception at the behavioral level. The mice were treated with or without 3-d binge-like exposure to EtOH, and the anti-nociceptive changes were evaluated using the hot-plate test 24 h after the final (3rd) EtOH injection with or without a cumulative subcutaneous dose (0, 0.1, 0.3, 1.0, and 3.0 mg/kg) of morphine at intervals of 30 min. The response curve of the mice given EtOH was shifted to the left, showing enhanced latency to response to morphine up to 3 mg/kg. Furthermore, co-treatment with the MOR antagonist naltrexone blocked morphine's anti-nociception in animals given either EtOH or saline. This confirms that MOR is involved in binge-like exposure to EtOH-induced changes in morphine's anti-nociception. Our results suggest that EtOH enhanced latency to analgesic response to morphine, and such effect might initiate the onset and progression of OUDs

JC Virus T-Antigen Regulates Glucose Metabolic Pathways in Brain Tumor Cells

Recent studies have reported the detection of the human neurotropic virus, JCV, in a significant population of brain tumors, including medulloblastomas. Accordingly, expression of the JCV early protein, T-antigen, which has transforming activity in cell culture and in transgenic mice, results in the development of a broad range of tumors of neural crest and glial origin. Evidently, the association of T-antigen with a range of tumor-suppressor proteins, including p53 and pRb, and signaling molecules, such as β-catenin and IRS-1, plays a role in the oncogenic function of JCV T-antigen. We demonstrate that T-antigen expression is suppressed by glucose deprivation in medulloblastoma cells and in glioblastoma xenografts that both endogenously express T-antigen. Mechanistic studies indicate that glucose deprivation-mediated suppression of T-antigen is partly influenced by 5′-activated AMP kinase (AMPK), an important sensor of the AMP/ATP ratio in cells. In addition, glucose deprivation-induced cell cycle arrest in the G1 phase is blocked with AMPK inhibition, which also prevents T-antigen downregulation. Furthermore, T-antigen prevents G1 arrest and sustains cells in the G2 phase during glucose deprivation. On a functional level, T-antigen downregulation is partially dependent on reactive oxygen species (ROS) production during glucose deprivation, and T-antigen prevents ROS induction, loss of ATP production, and cytotoxicity induced by glucose deprivation. Additionally, we have found that T-antigen is downregulated by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), and the pentose phosphate inhibitors, 6-aminonicotinamide and oxythiamine, and that T-antigen modulates expression of the glycolytic enzyme, hexokinase 2 (HK2), and the pentose phosphate enzyme, transaldolase-1 (TALDO1), indicating a potential link between T-antigen and metabolic regulation. These studies point to the possible involvement of JCV T-antigen in medulloblastoma proliferation and the metabolic phenotype and may enhance our understanding of the role of viral proteins in glycolytic tumor metabolism, thus providing useful targets for the treatment of virus-induced tumors

Meta-Analysis of Methamphetamine Modulation on Amyloid Precursor Protein through HMGB1 in Alzheimer’s Disease

The deposition of amyloid-beta (Aβ) through the cleavage of amyloid-beta precursor protein (APP) is a biomarker of Alzheimer’s disease (AD). This study used QIAGEN Ingenuity Pathway Analysis (IPA) to conduct meta-analysis on the molecular mechanisms by which methamphetamine (METH) impacts AD through modulating the expression of APP. All the molecules affected by METH and APP were collected from the QIAGEN Knowledge Base (QKB); 78 overlapping molecules were identified. Upon simulation of METH exposure using the “Molecule Activity Predictor” feature, eight molecules were found to be affected by METH and exhibited activation relationships on APP expression at a confidence of p = 0.000453 (Z-score = 3.51, two-tailed). Core Analysis of these eight molecules identified High Mobility Group Box protein 1 (HMGB1) signaling pathway among the top 5 canonical pathways with most overlap with the 8-molecule dataset. Simulated METH exposure increased APP expression through HMGB1 at a confidence of p &lt; 0.00001 (Z-score = 7.64, two-tailed). HMGB1 is a pathogenic hallmark in AD progression. It not only increases the production of inflammatory mediators, but also mediates the disruption&nbsp;of the blood-brain barrier. Our analyses suggest the involvement of HMGB1 signaling pathway in METH-induced modulation of APP as a potential casual factor of AD

Neurofibromatosis type 2 tumor suppressor protein, NF2, induces proteasome-mediated degradation of JC virus T-antigen in human glioblastoma.

Neurofibromatosis type 2 protein (NF2) has been shown to act as tumor suppressor primarily through its functions as a cytoskeletal scaffold. However, NF2 can also be found in the nucleus, where its role is less clear. Previously, our group has identified JC virus (JCV) tumor antigen (T-antigen) as a nuclear binding partner for NF2 in tumors derived from JCV T-antigen transgenic mice. The association of NF2 with T-antigen in neuronal origin tumors suggests a potential role for NF2 in regulating the expression of the JCV T-antigen. Here, we report that NF2 suppresses T-antigen protein expression in U-87 MG human glioblastoma cells, which subsequently reduces T-antigen-mediated regulation of the JCV promoter. When T-antigen mRNA was quantified, it was determined that increasing expression of NF2 correlated with an accumulation of T-antigen mRNA; however, a decrease in T-antigen at the protein level was observed. NF2 was found to promote degradation of ubiquitin bound T-antigen protein via a proteasome dependent pathway concomitant with the accumulation of the JCV early mRNA encoding T-antigen. The interaction between T-antigen and NF2 maps to the FERM domain of NF2, which has been shown previously to be responsible for its tumor suppressor activity. Co-immunoprecipitation assays revealed a ternary complex among NF2, T-antigen, and the tumor suppressor protein, p53 within a glioblastoma cell line. Further, these proteins were detected in various degrees in patient tumor tissue, suggesting that these associations may occur in vivo. Collectively, these results demonstrate that NF2 negatively regulates JCV T-antigen expression by proteasome-mediated degradation, and suggest a novel role for NF2 as a suppressor of JCV T-antigen-induced cell cycle regulation

Molecular and Cellular Impact of Inflammatory Extracellular Vesicles (EVs) Derived from M1 and M2 Macrophages on Neural Action Potentials

Several factors can contribute to neuroinflammatory disorders, such as cytokine and chemokines that are produced and released from peripherally derived immune cells or from locally activated cells such as microglia and perivascular macrophages in the brain. The primary function of these cells is to clear inflammation; however, following inflammation, circulating monocytes are recruited to the central nervous system (CNS). Monocyte-derived macrophages in the CNS play pivotal roles in mediating neuroinflammatory responses. Macrophages are heterogeneous both in normal and in pathological conditions due to their plasticity, and they are classified in two main subsets, classically activated (M1) or alternatively activated (M2). There is accumulating evidence suggesting that extracellular vesicles (EVs) released from activated immune cells may play crucial roles in mediating inflammation. However, a possible role of EVs released from immune cells such as M1 and M2 macrophages on neuronal functions in the brain is not known. In order to investigate the molecular and cellular impacts of macrophages and EVs released from macrophage subtypes on neuronal functions, we used a recently established in vitro M1 and M2 macrophage culture model and isolated and characterized EVs from these macrophage subtypes, treated primary neurons with M1 or M2 EVs, and analyzed the extracellular action potentials of neurons with microelectrode array studies (MEA). Our results introduce evidence on the interfering role of inflammatory EVs released from macrophages in interneuronal signal transmission processes, with implications in the pathogenesis of neuroinflammatory diseases induced by a variety of inflammatory insults