Human Cytomegaloviruses Expressing Yellow Fluorescent Fusion Proteins - Characterization and Use in Antiviral Screening

Recombinant viruses labelled with fluorescent proteins are useful tools in molecular virology with multiple applications (e.g., studies on intracellular trafficking, protein localization, or gene activity). We generated by homologous recombination three recombinant cytomegaloviruses carrying the enhanced yellow fluorescent protein (EYFP) fused with the viral proteins IE-2, ppUL32 (pp150), and ppUL83 (pp65). In growth kinetics, the three viruses behaved all like wild type, even at low multiplicity of infection (MOI). The expression of all three fusion proteins was detected, and their respective localizations were the same as for the unmodified proteins in wild-type virus–infected cells. We established the in vivo measurement of fluorescence intensity and used the recombinant viruses to measure inhibition of viral replication by neutralizing antibodies or antiviral substances. The use of these viruses in a pilot screen based on fluorescence intensity and high-content analysis identified cellular kinase inhibitors that block viral replication. In summary, these viruses with individually EYFP-tagged proteins will be useful to study antiviral substances and the dynamics of viral infection in cell culture

Nestin Modulates Glucocorticoid Receptor Function by Cytoplasmic Anchoring

Nestin is the characteristic intermediate filament (IF) protein of rapidly proliferating progenitor cells and regenerating tissue. Nestin copolymerizes with class III IF-proteins, mostly vimentin, into heteromeric filaments. Its expression is downregulated with differentiation. Here we show that a strong nestin expression in mouse embryo tissue coincides with a strong accumulation of the glucocorticoid receptor (GR), a key regulator of growth and differentiation in embryonic development. Microscopic studies on cultured cells show an association of GR with IFs composed of vimentin and nestin. Cells lacking nestin, but expressing vimentin, or cells expressing vimentin, but lacking nestin accumulate GR in the nucleus. Completing these networks with an exogenous nestin, respectively an exogenous vimentin restores cytoplasmic anchoring of GR to the IF system. Thus, heteromeric filaments provide the basis for anchoring of GR. The reaction pattern with phospho-GR specific antibodies and the presence of the chaperone HSC70 suggest that specifically the unliganded receptor is anchored to the IF system. Ligand addition releases GR from IFs and shifts the receptor into the nucleus. Suppression of nestin by specific shRNA abolishes anchoring of GR, induces its accumulation in the nucleus and provokes an irreversible G1/S cell cycle arrest. Suppression of GR prior to that of nestin prevents entry into the arrest. The data give evidence that nestin/vimentin specific anchoring modulates growth suppression by GR. We hypothesize that expression of nestin is a major determinant in suppression of anti-proliferative activity of GR in undifferentiated tissue and facilitates activation of this growth control in a precise tissue and differentiation dependent manner

Investigations on the Usefulness of CEACAMs as Potential Imaging Targets for Molecular Imaging Purposes

Members of the carcinoembryonic antigen cell adhesion molecules (CEACAMs) family are the prototype of tumour markers. Classically they are used as serum markers, however, CEACAMs could serve as targets for molecular imaging as well. In order to test the anti CEACAM monoclonal antibody T84.1 for imaging purposes, CEACAM expression was analysed using this antibody. Twelve human cancer cell lines from different entities were screened for their CEACAM expression using qPCR, Western Blot and FACS analysis. In addition, CEACAM expression was analyzed in primary tumour xenografts of these cells. Nine of 12 tumour cell lines expressed CEACAM mRNA and protein when grown in vitro. Pancreatic and colon cancer cell lines showed the highest expression levels with good correlation of mRNA and protein level. However, when grown in vivo, the CEACAM expression was generally downregulated except for the melanoma cell lines. As the CEACAM expression showed pronounced expression in FemX-1 primary tumours, this model system was used for further experiments. As the accessibility of the antibody after i.v. application is critical for its use in molecular imaging, the binding of the T84.1 monoclonal antibody was assessed after i.v. injection into SCID mice harbouring a FemX-1 primary tumour. When applied i.v., the CEACAM specific T84.1 antibody bound to tumour cells in the vicinity of blood vessels. This binding pattern was particularly pronounced in the periphery of the tumour xenograft, however, some antibody binding was also observed in the central areas of the tumour around blood vessels. Still, a general penetration of the tumour by i.v. application of the anti CEACAM antibody could not be achieved despite homogenous CEACAM expression of all melanoma cells when analysed in tissue sections. This lack of penetration is probably due to the increased interstitial fluid pressure in tumours caused by the absence of functional lymphatic vessels.Germany. Bundesministerium für Bildung und Forschung (TOMCAT, grant number 01EZ0824

Assessment of the range of the HIV-1 infectivity enhancing effect of individual human semen specimen and the range of inhibition by EGCG

Recently, it has been shown that human ejaculate enhances human immunodeficiency virus 1 (HIV-1) infectivity. Enhancement of infectivity is conceived to be mediated by amyloid filaments from peptides that are proteolytically released from prostatic acid phosphatase (PAP), termed Semen-derived Enhancer of Virus Infection (SEVI). The aim of this study was to test the range of HIV-1 infectivity enhancing properties of a large number of individual semen samples (n = 47) in a TZM-bl reporter cell HIV infection system. We find that semen overall increased infectivity to 156% of the control experiment without semen, albeit with great inter- and intraindividual variability (range -53%-363%). Using transmission electron microscopy, we provide evidence for SEVI fibrils in fresh human semen for the first time. Moreover, we confirm that the infectivity enhancing property can be inhibited by the major green tea ingredient epigallocatechin-3-gallate (EGCG) at non-toxic concentrations. The median inhibition of infection by treatment with 0.4 mM EGCG was 70.6% (p < 0.0001) in our cohort. Yet, there were substantial variations of inhibition and in a minority of samples, infectivity enhancement was not inhibited by EGCG treatment at all. Thus, topical application of EGCG may be a feasible additional measure to prevent the sexual transmission of HIV. However, the reasons for the variability in the efficacy of the abrogation of semen-mediated enhancement of HIV-1 infectivity and EGCG efficacy have to be elucidated before therapeutic trials can be conducted

Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

Merkel Cell Polyomavirus (MCPyV) genomes are clonally integrated in tumor tissues of approximately 85% of all Merkel cell carcinoma (MCC) cases, a highly aggressive tumor of the skin which predominantly afflicts elderly and immunosuppressed patients. All integrated viral genomes recovered from MCC tissue or MCC cell lines harbor signature mutations in the early gene transcript encoding for the large T-Antigen (LT-Ag). These mutations selectively abrogate the ability of LT-Ag to support viral replication while still maintaining its Rb-binding activity, suggesting a continuous requirement for LT-Ag mediated cell cycle deregulation during MCC pathogenesis. To gain a better understanding of MCPyV biology, in vitro MCPyV replication systems are required. We have generated a synthetic MCPyV genomic clone (MCVSyn) based on the consensus sequence of MCC-derived sequences deposited in the NCBI database. Here, we demonstrate that transfection of recircularized MCVSyn DNA into some human cell lines recapitulates efficient replication of the viral genome, early and late gene expression together with virus particle formation. However, serial transmission of infectious virus was not observed. This in vitro culturing system allows the study of viral replication and will facilitate the molecular dissection of important aspects of the MCPyV lifecycle

Synthesis and characterization of triangulene

Triangulene, the smallest triplet-ground-state polybenzenoid (also known as Clar's hydrocarbon), has been an enigmatic molecule ever since its existence was first hypothesized1. Despite containing an even number of carbons (22, in six fused benzene rings), it is not possible to draw Kekulé-style resonant structures for the whole molecule: any attempt results in two unpaired valence electrons2. Synthesis and characterization of unsubstituted triangulene has not been achieved because of its extreme reactivity1, although the addition of substituents has allowed the stabilization and synthesis of the triangulene core3, 4 and verification of the triplet ground state via electron paramagnetic resonance measurements5. Here we show the on-surface generation of unsubstituted triangulene that consists of six fused benzene rings. The tip of a combined scanning tunnelling and atomic force microscope (STM/AFM) was used to dehydrogenate precursor molecules. STM measurements in combination with density functional theory (DFT) calculations confirmed that triangulene keeps its free-molecule properties on the surface, whereas AFM measurements resolved its planar, threefold symmetric molecular structure. The unique topology of such non-Kekulé hydrocarbons results in open-shell π-conjugated graphene fragments6 that give rise to high-spin ground states, potentially useful in organic spintronic devices7, 8. Our generation method renders manifold experiments possible to investigate triangulene and related open-shell fragments at the single-molecule level

Viral Mediated Redirection of NEMO/IKKγ to Autophagosomes Curtails the Inflammatory Cascade

The early host response to viral infections involves transient activation of pattern recognition receptors leading to an induction of inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα). Subsequent activation of cytokine receptors in an autocrine and paracrine manner results in an inflammatory cascade. The precise mechanisms by which viruses avert an inflammatory cascade are incompletely understood. Nuclear factor (NF)-κB is a central regulator of the inflammatory signaling cascade that is controlled by inhibitor of NF-κB (IκB) proteins and the IκB kinase (IKK) complex. In this study we show that murine cytomegalovirus inhibits the inflammatory cascade by blocking Toll-like receptor (TLR) and IL-1 receptor-dependent NF-κB activation. Inhibition occurs through an interaction of the viral M45 protein with the NF-κB essential modulator (NEMO), the regulatory subunit of the IKK complex. M45 induces proteasome-independent degradation of NEMO by targeting NEMO to autophagosomes for subsequent degradation in lysosomes. We propose that the selective and irreversible degradation of a central regulatory protein by autophagy represents a new viral strategy to dampen the inflammatory response