miRNAs are a species of small-regulatory RNA that post-transcriptionally regulate gene expression via the RNA induced silencing complex (RISC). They are encoded ubiquitously among animals and plants, and have recently been shown to be encoded by the majority of herpesviruses. It seems likely that herpesvirus encoded miRNAs have evolved as a tool for the manipulation of host-cellular and viral-gene expression during infection. Human cytomegalovirus (HCMV) is a clinically important herpesvirus that represents a significant cause of morbidity and mortality in the immune-compromised. HCMV encodes as many as 25 miRNAs during infection, but the function of the majority of these is not known. Identifying the targets of HCMV miRNAs will not only establish a basis for understanding the role of miRNAs within the context of HCMV infection, but also provide a means for discovering novel host-virus interactions. Using RISC immunoprecipitation and siRNA screening, host-cellular targets of viral miRNAs that play important roles in the biology of HCMV were identified. ATP6VOC, a key component of the vacuolar-ATPase, was shown to be a target of miR-US25-1 and subsequent siRNA knockdown of ATP6VOC resulted in the almost complete inhibition of infectious virion production. Despite this, ATP6VOC knock-down did not inhibit viral entry, DNA synthesis, or gene expression, highlighting a possible role for ATP6VOC in the assembly and egress of HCMV. A critical step in HCMV assembly and egress is the formation of the juxta-nuclear virion assembly compartment (VAC). The HCMV VAC is derived from host-cellular endocytic and secretory vacuoles, and is crucial for the efficient nuclear egress of nucleocapsids, cyotplasmic tegumentation, final envelopment, and the egress of mature virions. Using siRNA knock-down, immunofluorescence-microscopy, and western-blot analysis, a crucial role for ATP6VOC and v-ATPase function in the formation of the VAC was demonstrated. siRNA knock-down of ATP6VOC resulted in a failure in the reorganisation of trans-golgi and early-endosomal compartments during infection, resulting in a failure in VAC formation. These findings demonstrate a crucial role for ATP6VOC during infection, and in so doing identify a novel host factor that is required for HCMV assembly.

Human cytomegalovirus (HCMV) coexists indefinitely in infected individuals through a poorly characterized latent infection in hematopoietic cells. We previously demonstrated a requirement for UL138 in promoting a latent infection in CD34+ hematopoietic progenitor cells (HPCs). UL138 is encoded on three co-terminal transcripts of, 1.7-, 2.7-, and 3.6-kilobases. Interestingly, the UL138 protein product (pUL138) is necessary but insufficient for HCMV latency. The mechanisms by which pUL138 contributes to the latent infection are unknown, however other viral determinants are required for the latent infection. We identified 3 novel proteins pUL133, pUL135, and pUL136 encoded on the UL138 transcripts. Similar to pUL138, pUL133, pUL135, and pUL136 are Golgi localized type I transmembrane proteins expressed with early kinetics during productive infection. We have named these UL138 related proteins, CLAMPs for HCMV Latency Associated Membrane Proteins. Through a systematic immunoprecipitation analysis, we identified interactions between the CLAMPs and characterized an interaction between pUL133 and pUL138. Further, we mapped the interacting region to a specific domain in the C-terminal, cytosolic tail of pUL138. Additionally, we show that each of the CLAMPs has the ability to self-associate. The localization of the CLAMPs to the Golgi suggests that these proteins likely promote HCMV latency through a novel mechanism involving Golgi functions. Additionally, through a Y2H screen of a human bone marrow cDNA library, we identified an interaction between pUL138 and the heat shock protein 40 (Hsp40) variant MRJ. We confirmed this interaction in mammalian cells and mapped the pUL138 region responsible for this interaction to a domain in the cytoplasmic tail of pUL138. We also demonstrated additional MRJ interactions with pUL133 and pUL136. Importantly, pUL138 specifically interacts with Hsp40 variants during productive infection. Preliminary data suggest that HCMV infection up regulates MRJ mRNA expression and recombinant viruses lacking pUL138 show a disproportionate up regulation of MRJ. pUL138 is the first HCMV protein demonstrated to promote a latent infection. While the mechanisms by which pUL138 contributes to latency remain unknown, the interaction with other CLAMPs and with MRJ, suggest that pUL138 may cooperate with other CLAMPs to modulate the cellular stress response at the Golgi to promote HCMV latency.

The soft corals of the species Paralemnalia thyrsoides was found to be a rich source of sesquiterpenoids, such as nor-nardosinane, nardosinane, neolemnane, and eremophilane, and other related skeletons. Continuing investigation on the chemical constituents of Paralemnalia thyrsoides has led to the isolation of nine new compounds, including one dinor-nardosinane 1, one neolemnane 2, five nardosinanes 4, 6¡V9, and two nor-nardosinanes 10 and 11, along with eleven known compounds, 2-deoxy-7-O-methyllemnacarnol (3), 2-deoxy-12£\-methoxy-7-O-methyllemnacarnol (5), paralemnolin Q (12), paralemnolin R (13), 4-acetoxy-2,8-neolemnadien-5-one (15), paralemnolin E (16), flavalins E (17), isoparalemnanone (18), paralemnolin K (19), and nor-nardosinane sesquiterpenoids (14 and 20). The structures of these compounds were determined on the basis of their spectroscopic analysis (1H, 13C NMR, 1H¡V1H COSY, HSQC, HMBC, IR and HRESIMS) and by comparison of the physical and spectral data with those of the related known compounds. The relative stereochemistry and assignments of 1H NMR chemical shifts were determined by NOESY and coupling constants. The absolute stereochemistry of dinor-nardosinane 1 was further determined by application of the Mosher¡¦s method. The cytotoxicity against of P-388 (murine lymphocytic leukemia), HT-29 (human colon adenocarcinoma), and A-549 (human lung epithelial carcinoma) cells as well as the anti-HCMV (human cytomegalovirus) activities of metabolites 1, 2, 4, 6¡V11 were evaluated. Metabolites 2, 3, 5, 6, 8 and 9 exhibited significant activity against P-388 cell in vitro ( ED50 ¡Ø 4 £gg/ mL) Keywords: Paralemnalia thyrsoides, sesquiterpenoids, cytotoxicity, anti-HCMV

No description available.

Résumé : L’autophagie est un mécanisme constitutif et inductible de dégradation des composants cytoplasmiques afin de maintenir l’homéostasie cellulaire. Elle est souvent modulée par les virus car il s’agit également d’un mécanisme de défense antiviral. Elle peut avoir un rôle proviral quand elle est détournée et régulée par les virus. Nous avons précédemment observé au laboratoire que le cytomégalovirus humain (HCMV) stimule la formation des autophagosomes de manière précoce indépendamment de l’expression des protéines virales, puis qu’il entraine un blocage de l’autophagie aux temps tardifs. Dans ce travail, nous avons montré que ce virus a développé des stratégies impliquant la synthèse de deux protéines virales, IRS1 et TRS1, pour inhiber l’autophagie. De façon surprenante, nous avons également mis en évidence un rôle proviral de l’autophagie aux temps tardifs de l’infection par le HCMV. Nous avons pu montrer par des techniques de biochimie et d’imagerie cellulaire que l’expression aussi bien de TRS1 que d’IRS1 est capable de bloquer la formation des autophagosomes dans les cellules. Nous avons identifié le mécanisme d’action de ces protéines. Il est indépendant de la protéine kinase PKR mais nécessite une interaction avec Beclin 1, une protéine de la machinerie autophagique. Nous avons localisé le site d'interaction de Beclin 1 avec IRS1 et TRS1 (BBD pour Beclin 1 binding domain) au niveau de leur région N-terminale. Ce domaine, conservé entre les deux protéines, est nécessaire pour l’inhibition de l’autophagie. Le site d’interaction d’IRS1 a été identifié dans le domaine en superhélice (coiled-coil domain) CCD de Beclin 1. Nous avons caractérisé le rôle de TRS1 et IRS1 dans la modulation de l’autophagie dans le contexte de l’infection virale, en utilisant différents virus mutants : des virus dans lesquels on a supprimé soit le gène IRS1, soit le gène TRS1 et un virus dans lequel il manque les deux gènes IRS1 et TRS1. Les résultats obtenus suggèrent qu’IRS1 et TRS1 sont effectivement toutes les deux impliquées dans ce processus. Afin de mieux comprendre le rôle de l’interaction de ces protéines avec Beclin 1, nous avons étudié le phénotype d’un virus mutant qui n’exprime pas IRS1 et qui contient une délétion de la région BBD de TRS1. Nous avons montré que ce virus mutant ne se lie pas à Beclin 1 et qu’il ne bloque pas l’autophagie. De manière surprenante, il n’a pas de défaut de production virale, suggérant que l’inhibition de l'autophagie ne serait pas essentielle pour la réplication virale. Nous avons développé d’autres approches, comme l’utilisation de modulateurs pharmacologiques de l’autophagie ou de lentivirus hébergeant des shRNA, qui montrent que l’inhibition de l’autophagie est capable de diminuer la production virale et au contraire que sa stimulation l’augmente. Ces derniers résultats suggèrent que l’autophagie pourrait être bénéfique au HCMV dans certaines conditions. / Abstract: Autophagy is a constitutive and inducible mechanism of degradation of cytoplasmic components, in order to maintain the cellular homeostasis. Autophagy is often modulated by viruses, because it is also considered as an antiviral defense mechanism. It can have a beneficial role, when it is hijacked and regulated by viruses. We have previously observed in our laboratory that the human cytomegalovirus (HCMV) stimulates autophagosome formation, at the early stage of infection, independently of viral protein expression then, later on, it blocks autophagy. In this work, we showed that this virus has developed strategies involving the synthesis of several viral proteins, such as IRS1 and TRS1, to inhibit autophagy. Surprisingly, we also demonstrated a proviral role of autophagy at late stages of infection with HCMV. We showed, through biochemical and cellular imaging technologies, that expression of both TRS1 and IRS1 is able to block the formation of autophagosomes. We identified the mechanism of action of these proteins. It is independent of the protein kinase PKR but requires interaction with Beclin 1, a protein of the autophagic machinery. We mapped the interaction site of Beclin 1 with IRS1 and TRS1 in their N-terminal region and called it BBD for Beclin 1-binding domain. This domain (BBD)is conserved between the two proteins and essential to inhibit autophagy. We also identified the site of interaction of IRS1 in the coiled-coil domain (CCD) of Beclin 1. We characterized the role of IRS1 and TRS1 in the modulation of autophagy, in the context of viral infection, using different mutant viruses: viruses in which either the IRS1 or the TRS1 gene has been removed and a mutant virus lacking both IRS1 and TRS1 genes. Our results suggest that both IRS1 and TRS1 are involved in the regulation of this process. To better understand the role of the interaction of these proteins with Beclin 1, we studied the phenotype of a mutant virus that does not express IRS1 and which contains a deletion of the N-terminal region of TRS1. We showed that this mutant does not bind to Beclin 1 and is not able to block autophagy. Surprisingly, it has no defects in viral production, suggesting that inhibition of autophagy is not essential for viral replication. We developed other approaches, including the use of pharmacological modulators of autophagy or shRNA knockdown, which show that the inhibition of autophagy is able to reduce viral production and, on the contrary, that its stimulation increases it. These results suggest that autophagy may be beneficial to HCMV in certain conditions.

Previous studies on the secondary metabolites of Formosan octocoral Isis hippuris were collected only at Green Island. In the course of our studies on secondary metabolites from marine organisms, the acetone-solubles of the Formosan octocoral Isis hippuris collected at Orchid Island has led to the isolation of eleven polyoxygenated steroids (1¡V11), along with two known compounds (12 and 13). The structures of these compounds were determined on the basis of their spectroscopic and physical data, including NMR, IR, MS, etc. The cytotoxicity against of A-549 (human lung epithelial carcinoma), HT-29 (human colon adenocarcinoma), and P-388 (mouse lymphocytic leukemia) cells, and anti-HCMV (human cytomegalovirus) activity of metabolites 1¡V13 were evaluated. Compounds 12 and 13 displayed cytotoxicity against P-388 cell line with ED50 values of 3.2 and 3.6 £gg/mL, respectively. Compound 12 exhibited cytotoxicity against A-549 cell line with an ED50 value of 3.8 £gg/mL. Compound 8 exhibit inhibitory activity against HCMV, with EC50 values of 2.0 £gg/mL.

Numerous bioactive secondary metabolites including sesquiterpenoids,diterpenoids, meroditerpenoids, and steroids have been isolated from the soft corals of the genus Nephthea. In order to search for novel bioactive substances from marine organisms, we have investigated the secondary metabolites of the organic extract of the soft coral Nephthea chabrolii collected at San-Hsian-Tai. Chromatographic fractionation of the acetone-soluble has led to the isolation of four 19-oxygenated steroids 1¡V4 and two 19-norergosterols 5, 6, along with twelve known compounds 7¡V18. The structures of these compounds were determined on the basis of their spectroscopic analysis data (1H NMR, 13C NMR, 1H¡V1H COSY, HSQC, HMBC, NOESY, IR, and HRESIMS), physical data and compared with the literature data. The cytotoxicity against of A-549 (human lung epithelial carcinoma), HT-29 (human colon adenocarcinoma), and P-388 (mouse lymphocytic leukemia) cells, and anti-HCMV (human cytomegalovirus) activity of compounds 1¡V6 were evaluated. Metabolites 1¡V6 displayed cytotoxicity against P-388 cell line with ED50 values of 0.93, 1.05, 1.20, 1.74, 1.19, 1.19 £gg/mL. However, none of them exhibited inhibitory activity against HCMV (human cytomegalovirus).

In the course of studying on secondary metabolites from marine organisms, we have investigated the chemical constituents of the soft coral Sarcophyton ehrenbergi collected at San-Hsien-Tai, Taitong County, Taiwan. Chromatographic separation of the organic extracts has led to the isolation of nine new cembrane diterpenes 1¡V9, and a initial natural separation of known cembrane diterpene 10. The chemical structures of pure compounds were determined by spectral (NMR, MS, UV and IR) and physical data, as well as comparison with the spectroscopic data of related chemicals in literature. Moreover, the metabolites 1¡V10 were evaluated in vitro for their cytotoxicity against of A-549 (human lung epithelial carcinoma), HT-29 (human colon adenocarcinoma), and P-388 (mouse lymphocytic leukemia) cells, as well as anti-HCMV activity. Compounds 3, 6, 9, and 10 were shown to exhibit significant cytotoxicity activities against P-388 with ED50 values of 2.7, 3.6, 2.0, and 3.0 £gg/mL, respectively. Compound 1, 2, 3, and 7 exhibited inhibitory activity against anti-HCMV, with EC50 values of 60.0, 46.0, 5.0, and 45.0 £gg/mL.

Neutrophils provide a rapid first response to invading pathogens and orchestrate the immune response. They are able to employ potent antipathogenic mechanisms such as phagocytosis, reactive oxygen species (ROS) generation, protease release from granules, and formation of neutrophil extracellular traps (NETs). Despite this, certain pathogens have evolved mechanisms to benefit from neutrophil effector functions. Human cytomegalovirus (HCMV) is a clinically important pathogen that infects the majority of the human population. Monocytes are considered the main vehicle of HCMV dissemination throughout the body, but little research has been done on its interaction with neutrophils. The virus encodes a range of immunomodulatory proteins including an IL-8 homologue that acts as a powerful neutrophil chemoattractant. Viral conservation of a protein that recruits neutrophils to the site of HCMV infection suggests that the interaction between neutrophils and HCMV provides an overall advantage to the virus, but little evidence exists so far to suggest this is the case. Here I report that human peripheral blood neutrophils exposed to a clinical strain of HCMV display a profound survival phenotype, as assessed by morphology, phosphatidylserine exposure, cell permeability, and caspase-3/7 activity. This occurs in the absence of viral gene production. Neutrophils also upregulated their release of inflammatory cytokines in response to HCMV, with higher concentrations of IL-6, IL-8, and MIP-1α detected in the secretomes of infected neutrophils. These secretomes induced monocyte chemotaxis and increased monocyte permissivity to HCMV infection, as well as augmented survival in healthy, uninfected neutrophils. These experiments were confirmed with clean HCMV after the discovery of contaminating Mycoplasma spp. in the viral inocula of the initial experiments. Mycoplasma-HCMV coinfection induced an autophagic phenotype in neutrophils, as assessed by Western blotting and qPCR of autophagy-related components. Inhibition of autophagy using 3-MA reversed a profound survival effect. The unintended inclusion of Mycoplasma spp. further led to the serendipitous discovery of yet another pathogenic ability to overcome neutrophil immune functions: contaminating Mycoplasma spp. as well as Mycoplasma pneumoniae profoundly degraded NETs. These extracellular chromatin structures were stimulated using PMA or pyocyanin, and their release was dependent on the generation of ROS: severely ROS-deficient murine bone marrow neutrophils were unable to generate NETs. However, small amounts of ROS were sufficient for NETs generation, as neutrophils from acute respiratory distress syndrome patients, including many that had attenuated ROS-responses, were still capable of NETs generation. The NETs-degradative properties of mycoplasma were confirmed by fluorescence confocal and scanning electron microscopy, as well as spectrophotometry and agarose gel electrophoresis. This study demonstrates that two pervasive pathogens, HCMV and M. pneumoniae, both frequently found in coinfections in clinical contexts, are able to overcome neutrophil antipathogenic mechanisms to potentially enhance pathogen dissemination. These data provide not only a novel example of manipulation of an anti-viral response in a cell not productively infected, but also a novel example of pathogenic NETs degradation. These findings may have implications on our understanding of mycoplasma and HCMV pathogenesis and provide new targets for the generation of therapies.

No description available.