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    Abstract: Cutaneous human papillomaviruses (HPVs) are considered as cofactors for non-melanoma skin cancer (NMSC) development, especially in association with UVB. Extensively studied transgenic mouse models failed to mimic all aspects of virus-host interactions starting from primary infection to the appearance of a tumor. Using the natural model Mastomys coucha, which reflects the human situation in many aspects, we provide the first evidence that only UVB and Mastomys natalensis papillomavirus (MnPV) infection strongly promote NMSC formation. Using UVB exposures that correspond to UV indices of different geographical regions, irradiated animals developed either well-differentiated keratinizing squamous cell carcinomas (SCCs), still supporting productive infections with high viral loads and transcriptional activity, or poorly differentiated non-keratinizing SCCs almost lacking MnPV DNA and in turn, early and late viral transcription. Intriguingly, animals with the latter phenotype, however, still showed strong seropositivity, clearly verifying a preceding MnPV infection. Of note, the mere presence of MnPV could induce gammaH2AX foci, indicating that viral infection without prior UVB exposure can already perturb genome stability of the host cell. Moreover, as shown both under in vitro and in vivo conditions, MnPV E6/E7 expression also attenuates the excision repair of cyclobutane pyrimidine dimers upon UVB irradiation, suggesting a viral impact on the DNA damage response. While mutations of Ras family members (e.g. Hras, Kras, and Nras) were absent, the majority of SCCs harbored-like in humans-Trp53 mutations especially at two hot-spots in the DNA-binding domain, resulting in a loss of function that favored tumor dedifferentiation, counter-selective for viral maintenance. Such a constellation provides a reasonable explanation for making continuous viral presence dispensable during skin carcinogenesis as observed in patients with NMSC.
    Type of Publication: Journal article published
    PubMed ID: 29190285
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    Keywords: CELLS ; CELL ; PATHWAY ; PATHWAYS ; DISEASE ; DISEASES ; TIME ; ACTIVATION ; DNA ; MACROPHAGES ; TRANSPORT ; virus ; PHAGOCYTOSIS ; FRANCE ; cytoskeleton ; EPITHELIAL-CELLS ; Jun ; REORGANIZATION ; REPLICATION ; OVEREXPRESSION ; SAN-FRANCISCO ; DOUBLE-STRANDED DNA ; analysis ; DYNAMIN ; USA ; INFECTIOUS-DISEASES ; microbiology ; ACTIN ; viral ; pneumonia ; AMEBAS ; DEPENDENT ENDOCYTOSIS ; dominant negative ; MACROPINOCYTOSIS ; PHAGOSOMES ; VIRUS ENTRY
    Abstract: Mimivirus, or Acanthamoeba polyphaga mimivirus (APMV), a giant double-stranded DNA virus that grows in amoeba, was identified for the first time in 2003. Entry by phagocytosis within amoeba has been suggested but not demonstrated. We demonstrate here that APMV was internalized by macrophages but not by non-phagocytic cells, leading to productive APMV replication. Clathrin-and caveolin-mediated endocytosis pathways, as well as degradative endosome-mediated endocytosis, were not used by APMV to invade macrophages. Ultrastructural analysis showed that protrusions were formed around the entering virus, suggesting that macropinocytosis or phagocytosis was involved in APMV entry. Reorganization of the actin cytoskeleton and activation of phosphatidylinositol 3-kinases were required for APMV entry. Blocking macropinocytosis and the lack of APMV colocalization with rabankyrin-5 showed that macropinocytosis was not involved in viral entry. Overexpression of a dominant-negative form of dynamin-II, a regulator of phagocytosis, inhibited APMV entry. Altogether, our data demonstrated that APMV enters macrophages through phagocytosis, a new pathway for virus entry in cells. This reinforces the paradigm that intra-amoebal pathogens have the potential to infect macrophages
    Type of Publication: Journal article published
    PubMed ID: 18551172
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  • 5
    Keywords: Germany ; NETWORK ; NETWORKS ; SYSTEM ; DISEASE ; DISEASES ; GENOME ; PROTEIN ; PROTEINS ; DNA ; murine ; FAMILY ; OPEN READING FRAME ; SEQUENCE ; virus ; IDENTIFICATION ; HERPES-SIMPLEX-VIRUS ; EPSTEIN-BARR-VIRUS ; HUMAN CYTOMEGALOVIRUS ; SINGLE ; GENE-PRODUCT ; Epstein-Barr virus ; SAN-FRANCISCO ; FAMILIES ; LIBRARIES ; C-ELEGANS ; interaction ; NUCLEAR LAMINA ; CORE ; MUTATIONAL ANALYSIS ; SCREEN ; LIBRARY ; SARCOMA-ASSOCIATED HERPESVIRUS ; USA ; INTERACTION MAP ; cytomegalovirus ; SIMPLEX-VIRUS TYPE-1 ; virology ; GENOMES ; Herpes simplex virus 1 ; SPREAD ; DNA VIRUSES ; COVERAGE ; EGRESS ; MAREKS-DISEASE VIRUS
    Abstract: Herpesviruses constitute a family of large DNA viruses widely spread in vertebrates and causing a variety of different diseases. They possess dsDNA genomes ranging from 120 to 240 kbp encoding between 70 to 170 open reading frames. We previously reported the protein interaction networks of two herpesviruses, varicella-zoster virus (VZV) and Kaposi's sarcoma-associated herpesvirus ( KSHV). In this study, we systematically tested three additional herpesvirus species, herpes simplex virus 1 (HSV-1), murine cytomegalovirus and Epstein-Barr virus, for protein interactions in order to be able to perform a comparative analysis of all three herpesvirus subfamilies. We identified 735 interactions by genome-wide yeast-two-hybrid screens (Y2H), and, together with the interactomes of VZV and KSHV, included a total of 1,007 intraviral protein interactions in the analysis. Whereas a large number of interactions have not been reported previously, we were able to identify a core set of highly conserved protein interactions, like the interaction between HSV-1 UL33 with the nuclear egress proteins UL31/UL34. Interactions were conserved between orthologous proteins despite generally low sequence similarity, suggesting that function may be more conserved than sequence. By combining interactomes of different species we were able to systematically address the low coverage of the Y2H system and to extract biologically relevant interactions which were not evident from single species
    Type of Publication: Journal article published
    PubMed ID: 19730696
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    Keywords: CELLS ; CELL ; IN-VIVO ; VIVO ; DISEASE ; RNA ; ACTIVATION ; T-CELLS ; PROMOTER ; acetylation ; PCAF BROMODOMAIN ; METHYLATION ; IMMUNODEFICIENCY-VIRUS TYPE-1 ; INHIBITORS ; LATENT INFECTION ; CHROMATIN-REMODELING COMPLEX ; HISTONE ; LSD1
    Abstract: The essential transactivator function of the HIV Tat protein is regulated by multiple posttranslational modifications. Although individual modifications are well characterized, their crosstalk and dynamics of occurrence during the HIV transcription cycle remain unclear. We examine interactions between two critical modifications within the RNA-binding domain of Tat: monomethylation of lysine 51 (K51) mediated by Set7/9/KMT7, an early event in the Tat transactivation cycle that strengthens the interaction of Tat with TAR RNA, and acetylation of lysine 50 (K50) mediated by p300/KAT3B, a later process that dissociates the complex formed by Tat, TAR RNA and the cyclin T1 subunit of the positive transcription elongation factor b (P-TEFb). We find K51 monomethylation inhibited in synthetic Tat peptides carrying an acetyl group at K50 while acetylation can occur in methylated peptides, albeit at a reduced rate. To examine whether Tat is subject to sequential monomethylation and acetylation in cells, we performed mass spectrometry on immunoprecipitated Tat proteins and generated new modification-specific Tat antibodies against monomethylated/acetylated Tat. No bimodified Tat protein was detected in cells pointing to a demethylation step during the Tat transactivation cycle. We identify lysine-specific demethylase 1 (LSD1/KDM1) as a Tat K51-specific demethylase, which is required for the activation of HIV transcription in latently infected T cells. LSD1/KDM1 and its cofactor CoREST associates with the HIV promoter in vivo and activate Tat transcriptional activity in a K51-dependent manner. In addition, small hairpin RNAs directed against LSD1/KDM1 or inhibition of its activity with the monoamine oxidase inhibitor phenelzine suppresses the activation of HIV transcription in latently infected T cells. Our data support the model that a LSD1/KDM1/CoREST complex, normally known as a transcriptional suppressor, acts as a novel activator of HIV transcription through demethylation of K51 in Tat. Small molecule inhibitors of LSD1/KDM1 show therapeutic promise by enforcing HIV latency in infected T cells
    Type of Publication: Journal article published
    PubMed ID: 21876670
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  • 7
    Keywords: CELLS ; proliferation ; SURVIVAL ; GENE ; GENE-EXPRESSION ; RNA ; INFECTION ; MALIGNANCIES ; KAPPA-B ; TARGETS ; C-MYC ; EPSTEIN-BARR-VIRUS ; protein expression ; HUMAN B-CELLS ; MICRORNA ; ENCODED MICRORNAS ; HUMAN GAMMA-HERPESVIRUSES ; III PROGRAM ; PAR-CLIP
    Abstract: Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus linked to a number of B cell cancers and lymphoproliferative disorders. During latent infection, EBV expresses 25 viral pre-microRNAs (miRNAs) and induces the expression of specific host miRNAs, such as miR-155 and miR-21, which potentially play a role in viral oncogenesis. To date, only a limited number of EBV miRNA targets have been identified; thus, the role of EBV miRNAs in viral pathogenesis and/or lymphomagenesis is not well defined. Here, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) combined with deep sequencing and computational analysis to comprehensively examine the viral and cellular miRNA targetome in EBV strain B95-8-infected lymphoblastoid cell lines (LCLs). We identified 7,827 miRNA-interaction sites in 3,492 cellular 3'UTRs. 531 of these sites contained seed matches to viral miRNAs. 24 PAR-CLIP-identified miRNA: 3'UTR interactions were confirmed by reporter assays. Our results reveal that EBV miRNAs predominantly target cellular transcripts during latent infection, thereby manipulating the host environment. Furthermore, targets of EBV miRNAs are involved in multiple cellular processes that are directly relevant to viral infection, including innate immunity, cell survival, and cell proliferation. Finally, we present evidence that myc-regulated host miRNAs from the miR-17/92 cluster can regulate latent viral gene expression. This comprehensive survey of the miRNA targetome in EBV-infected B cells represents a key step towards defining the functions of EBV-encoded miRNAs, and potentially, identifying novel therapeutic targets for EBV-associated malignancies
    Type of Publication: Journal article published
    PubMed ID: 22291592
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    Keywords: CELLS ; proliferation ; IN-VIVO ; GENE ; EBV INFECTION ; GERMINAL-CENTER ; HUMAN LYMPHOCYTES-B ; RECEPTOR CR-2 ; CLASS SWITCH RECOMBINATION ; ABERRANT SOMATIC HYPERMUTATION ; CYTIDINE DEAMINASE AID ; HYPER-IGM SYNDROME ; POSTTRANSPLANT LYMPHOPROLIFERATIVE DISORDERS ; VARIABLE REGION GENES
    Abstract: Epstein-Barr virus (EBV), a lymphomagenic human herpesvirus, colonises the host through polyclonal B cell-growth-transforming infections yet establishes persistence only in IgD(+) CD27(+) non-switched memory (NSM) and IgD(-) CD27(+) switched memory (SM) B cells, not in IgD(+) CD27(-) naive (N) cells. How this selectivity is achieved remains poorly understood. Here we show that purified N, NSM and SM cell preparations are equally transformable in vitro to lymphoblastoid cells lines (LCLs) that, despite upregulating the activation-induced cytidine deaminase (AID) enzyme necessary for Ig isotype switching and Ig gene hypermutation, still retain the surface Ig phenotype of their parental cells. However, both N- and NSM-derived lines remain inducible to Ig isotype switching by surrogate T cell signals. More importantly, IgH gene analysis of N cell infections revealed two features quite distinct from parallel mitogen-activated cultures. Firstly, following 4 weeks of EBV-driven polyclonal proliferation, individual clonotypes then become increasingly dominant; secondly, in around 35% cases these clonotypes carry Ig gene mutations which both resemble AID products and, when analysed in prospectively-harvested cultures, appear to have arisen by sequence diversification in vitro. Thus EBV infection per se can drive at least some naive B cells to acquire Ig memory genotypes; furthermore, such cells are often favoured during an LCL's evolution to monoclonality. Extrapolating to viral infections in vivo, these findings could help to explain how EBV-infected cells become restricted to memory B cell subsets and why EBV-driven lymphoproliferative lesions, in primary infection and/or immunocompromised settings, so frequently involve clones with memory genotypes.
    Type of Publication: Journal article published
    PubMed ID: 22589726
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    Keywords: EPITHELIAL-CELLS ; MINUTE VIRUS ; NONSTRUCTURAL PROTEIN NS1 ; ENDOPLASMIC-RETICULUM ; TRANSGENE EXPRESSION ; TYROSINE-PHOSPHORYLATION ; SECRETORY PATHWAY ; PKC-ETA ; RAB GTPASES ; STRUCTURAL MATURATION
    Abstract: Progeny particles of non-enveloped lytic parvoviruses were previously shown to be actively transported to the cell periphery through vesicles in a gelsolin-dependent manner. This process involves rearrangement and destruction of actin filaments, while microtubules become protected throughout the infection. Here the focus is on the intracellular egress pathway, as well as its impact on the properties and release of progeny virions. By colocalization with cellular marker proteins and specific modulation of the pathways through over-expression of variant effector genes transduced by recombinant adeno-associated virus vectors, we show that progeny PV particles become engulfed into COPII-vesicles in the endoplasmic reticulum (ER) and are transported through the Golgi to the plasma membrane. Besides known factors like sar1, sec24, rab1, the ERM family proteins, radixin and moesin play (an) essential role(s) in the formation/loading and targeting of virus-containing COPII-vesicles. These proteins also contribute to the transport through ER and Golgi of the well described analogue of cellular proteins, the secreted Gaussia luciferase in absence of virus infection. It is therefore likely that radixin and moesin also serve for a more general function in cellular exocytosis. Finally, parvovirus egress via ER and Golgi appears to be necessary for virions to gain full infectivity through post-assembly modifications (e.g. phosphorylation). While not being absolutely required for cytolysis and progeny virus release, vesicular transport of parvoviruses through ER and Golgi significantly accelerates these processes pointing to a regulatory role of this transport pathway.
    Type of Publication: Journal article published
    PubMed ID: 24068925
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  • 10
    Keywords: EXPRESSION ; PROTEIN ; INFECTION ; CERVICAL-CANCER ; HUMAN KERATINOCYTES ; EPITHELIAL-CELLS ; DEGRADATION ; NECK-CANCER ; AUTOPHAGY ; INFLAMMASOMES
    Abstract: Infections with high-risk human papillomaviruses (HPVs) are causally involved in the development of anogenital cancer. HPVs apparently evade the innate immune response of their host cells by dysregulating immunomodulatory factors such as cytokines and chemokines, thereby creating a microenvironment that favors malignancy. One central key player in the immune surveillance interactome is interleukin-1 beta (IL-1beta) which not only mediates inflammation, but also links innate and adaptive immunity. Because of its pleiotropic physiological effects, IL-1beta production is tightly controlled on transcriptional, post-translational and secretory levels. Here, we describe a novel mechanism how the high-risk HPV16 E6 oncoprotein abrogates IL-1beta processing and secretion in a NALP3 inflammasome-independent manner. We analyzed IL-1beta regulation in immortalized keratinocytes that harbor the HPV16 E6 and/or E7 oncogenes as well as HPV-positive cervical tumor cells. While in primary and in E7-immortalized human keratinocytes the secretion of IL-1beta was highly inducible upon inflammasome activation, E6-positive cells did not respond. Western blot analyses revealed a strong reduction of basal intracellular levels of pro-IL-1beta that was independent of dysregulation of the NALP3 inflammasome, autophagy or lysosomal activity. Instead, we demonstrate that pro-IL-1beta is degraded in a proteasome-dependent manner in E6-positive cells which is mediated via the ubiquitin ligase E6-AP and p53. Conversely, in E6- and E6/E7-immortalized cells pro-IL-1beta levels were restored by siRNA knock-down of E6-AP and simultaneous recovery of functional p53. In the context of HPV-induced carcinogenesis, these data suggest a novel post-translational mechanism of pro-IL-1beta regulation which ultimately inhibits the secretion of IL-1beta in virus-infected keratinocytes. The clinical relevance of our results was further confirmed in HPV-positive tissue samples, where a gradual decrease of IL-1beta towards cervical cancer could be discerned. Hence, attenuation of IL-1beta by the HPV16 E6 oncoprotein in immortalized cells is apparently a crucial step in viral immune evasion and initiation of malignancy.
    Type of Publication: Journal article published
    PubMed ID: 23935506
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