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A phenyl-thiadiazolylidene-amine derivative ejects zinc from retroviral nucleocapsid zinc fingers and inactivates HIV virions
Vercruysse Thomas,Basta Beata,Dehaen Wim,Humbert Nicolas
Retrovirology , 2012, DOI: 10.1186/1742-4690-9-95
Abstract: Background Sexual acquisition of the human immunodeficiency virus (HIV) through mucosal transmission may be prevented by using topically applied agents that block HIV transmission from one individual to another. Therefore, virucidal agents that inactivate HIV virions may be used as a component in topical microbicides. Results Here, we have identified 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine (WDO-217) as a low-molecular-weight molecule that inactivates HIV particles. Both HIV-1 and HIV-2 virions pretreated with this compound were unable to infect permissive cells. Moreover, WDO-217 was able to inhibit infections of a wide spectrum of wild-type and drug-resistant HIV-1, including clinical isolates, HIV-2 and SIV strains. Whereas the capture of virus by DC-SIGN was unaffected by the compound, it efficiently prevented the transmission of DC-SIGN-captured virus to CD4+ T-lymphocytes. Interestingly, exposure of virions to WDO-217 reduced the amount of virion-associated genomic RNA as measured by real-time RT-qPCR. Further mechanism-of-action studies demonstrated that WDO-217 efficiently ejects zinc from the zinc fingers of the retroviral nucleocapsid protein NCp7 and inhibits the cTAR destabilization properties of this protein. Importantly, WDO-217 was able to eject zinc from both zinc fingers, even when NCp7 was bound to oligonucleotides, while no covalent interaction between NCp7 and WDO-217 could be observed. Conclusion This compound is a new lead structure that can be used for the development of a new series of NCp7 zinc ejectors as candidate topical microbicide agents.
Cholesterol Depletion Disorganizes Oocyte Membrane Rafts Altering Mouse Fertilization  [PDF]
Jorgelina Buschiazzo, Come Ialy-Radio, Jana Auer, Jean-Philippe Wolf, Catherine Serres, Brigitte Lefèvre, Ahmed Ziyyat
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0062919
Abstract: Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Two oocyte protein families are essential for fertilization, tetraspanins and glycosylphosphatidylinositol-anchored proteins. The firsts are associated to tetraspanin-enriched microdomains and the seconds to lipid rafts. Here we report membrane raft involvement in mouse fertilization assessed by cholesterol modulation using methyl-β-cyclodextrin. Cholesterol removal induced: (1) a decrease of the fertilization rate and index; and (2) a delay in the extrusion of the second polar body. Cholesterol repletion recovered the fertilization ability of cholesterol-depleted oocytes, indicating reversibility of these effects. In vivo time-lapse analyses using fluorescent cholesterol permitted to identify the time-point at which the probe is mainly located at the plasma membrane enabling the estimation of the extent of the cholesterol depletion. We confirmed that the mouse oocyte is rich in rafts according to the presence of the raft marker lipid, ganglioside GM1 on the membrane of living oocytes and we identified the coexistence of two types of microdomains, planar rafts and caveolae-like structures, by terms of two differential rafts markers, flotillin-2 and caveolin-1, respectively. Moreover, this is the first report that shows characteristic caveolae-like invaginations in the mouse oocyte identified by electron microscopy. Raft disruption by cholesterol depletion disturbed the subcellular localization of the signal molecule c-Src and the inhibition of Src kinase proteins prevented second polar body extrusion, consistent with a role of Src-related kinases in fertilization via signaling complexes. Our data highlight the functional importance of intact membrane rafts for mouse fertilization and its dependence on cholesterol.
Effect of Plasma Membrane Cholesterol Depletion on Glucose Transport Regulation in Leukemia Cells  [PDF]
Cristiana Caliceti, Laura Zambonin, Cecilia Prata, Francesco Vieceli Dalla Sega, Gabriele Hakim, Silvana Hrelia, Diana Fiorentini
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0041246
Abstract: GLUT1 is the predominant glucose transporter in leukemia cells, and the modulation of glucose transport activity by cytokines, oncogenes or metabolic stresses is essential for their survival and proliferation. However, the molecular mechanisms allowing to control GLUT1 trafficking and degradation are still under debate. In this study we investigated whether plasma membrane cholesterol depletion plays a role in glucose transport activity in M07e cells, a human megakaryocytic leukemia line. To this purpose, the effect of cholesterol depletion by methyl-β-cyclodextrin (MBCD) on both GLUT1 activity and trafficking was compared to that of the cytokine Stem Cell Factor (SCF). Results show that, like SCF, MBCD led to an increased glucose transport rate and caused a subcellular redistribution of GLUT1, recruiting intracellular transporter molecules to the plasma membrane. Due to the role of caveolae/lipid rafts in GLUT1 stimulation in response to many stimuli, we have also investigated the GLUT1 distribution along the fractions obtained after non ionic detergent treatment and density gradient centrifugation, which was only slightly changed upon MBCD treatment. The data suggest that MBCD exerts its action via a cholesterol-dependent mechanism that ultimately results in augmented GLUT1 translocation. Moreover, cholesterol depletion triggers GLUT1 translocation without the involvement of c-kit signalling pathway, in fact MBCD effect does not involve Akt and PLCγ phosphorylation. These data, together with the observation that the combined MBCD/SCF cell treatment caused an additive effect on glucose uptake, suggest that the action of SCF and MBCD may proceed through two distinct mechanisms, the former following a signalling pathway, and the latter possibly involving a novel cholesterol dependent mechanism.
Effect of cholesterol depletion on the pore dilation of TRPV1  [cached]
Jansson Erik T,Trkulja Carolina L,Ahemaiti Aikeremu,Millingen Maria
Molecular Pain , 2013, DOI: 10.1186/1744-8069-9-1
Abstract: The TRPV1 ion channel is expressed in nociceptors, where pharmacological modulation of its function may offer a means of alleviating pain and neurogenic inflammation processes in the human body. The aim of this study was to investigate the effects of cholesterol depletion of the cell on ion-permeability of the TRPV1 ion channel. The ion-permeability properties of TRPV1 were assessed using whole-cell patch-clamp and YO-PRO uptake rate studies on a Chinese hamster ovary (CHO) cell line expressing this ion channel. Prolonged capsaicin-induced activation of TRPV1 with N-methyl-D-glucamine (NMDG) as the sole extracellular cation, generated a biphasic current which included an initial outward current followed by an inward current. Similarly, prolonged proton-activation (pH 5.5) of TRPV1 under hypocalcemic conditions also generated a biphasic current including a fast initial current peak followed by a larger second one. Patch-clamp recordings of reversal potentials of TRPV1 revealed an increase of the ion-permeability for NMDG during prolonged activation of this ion channel under hypocalcemic conditions. Our findings show that cholesterol depletion inhibited both the second current, and the increase in ion-permeability of the TRPV1 channel, resulting from sustained agonist-activation with capsaicin and protons (pH 5.5). These results were confirmed with YO-PRO uptake rate studies using laser scanning confocal microscopy, where cholesterol depletion was found to decrease TRPV1 mediated uptake rates of YO-PRO. Hence, these results propose a novel mechanism by which cellular cholesterol depletion modulates the function of TRPV1, which may constitute a novel approach for treatment of neurogenic pain.
Disease-associated XMRV sequences are consistent with laboratory contamination
Stéphane Hué, Eleanor R Gray, Astrid Gall, Aris Katzourakis, Choon Tan, Charlotte J Houldcroft, Stuart McLaren, Deenan Pillay, Andrew Futreal, Jeremy A Garson, Oliver G Pybus, Paul Kellam, Greg J Towers
Retrovirology , 2010, DOI: 10.1186/1742-4690-7-111
Abstract: Here we demonstrate that Taqman PCR primers previously described as XMRV-specific can amplify common murine endogenous viral sequences from mouse suggesting that mouse DNA can contaminate patient samples and confound specific XMRV detection. To consider the provenance of XMRV we sequenced XMRV from the cell line 22Rv1, which is infected with an MLV-X that is indistinguishable from patient derived XMRV. Bayesian phylogenies clearly show that XMRV sequences reportedly derived from unlinked patients form a monophyletic clade with interspersed 22Rv1 clones (posterior probability >0.99). The cell line-derived sequences are ancestral to the patient-derived sequences (posterior probability >0.99). Furthermore, pol sequences apparently amplified from PC patient material (VP29 and VP184) are recombinants of XMRV and Moloney MLV (MoMLV) a virus with an envelope that lacks tropism for human cells. Considering the diversity of XMRV we show that the mean pairwise genetic distance among env and pol 22Rv1-derived sequences exceeds that of patient-associated sequences (Wilcoxon rank sum test: p = 0.005 and p < 0.001 for pol and env, respectively). Thus XMRV sequences acquire diversity in a cell line but not in patient samples. These observations are difficult to reconcile with the hypothesis that published XMRV sequences are related by a process of infectious transmission.We provide several independent lines of evidence that XMRV detected by sensitive PCR methods in patient samples is the likely result of PCR contamination with mouse DNA and that the described clones of XMRV arose from the tumour cell line 22Rv1, which was probably infected with XMRV during xenografting in mice. We propose that XMRV might not be a genuine human pathogen.XMRV (Xenotropic murine leukaemia virus-related virus) is a xenotropic murine leukaemia virus (MLV-X) that has been detected in samples from prostate cancer (PC) and chronic fatigue syndrome (CFS) patients[1-6]. This has led to the suggestion that i
Investigation into the Presence of and Serological Response to XMRV in CFS Patients  [PDF]
Otto Erlwein,Mark J. Robinson,Steve Kaye,Gillian Wills,Shozo Izui,Simon Wessely,Jonathan Weber,Anthony Cleare,David Collier,Myra O. McClure
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0017592
Abstract: The novel human gammaretrovirus xenotropic murine leukemia virus-related virus (XMRV), originally described in prostate cancer, has also been implicated in chronic fatigue syndrome (CFS). When later reports failed to confirm the link to CFS, they were often criticised for not using the conditions described in the original study. Here, we revisit our patient cohort to investigate the XMRV status in those patients by means of the original PCR protocol which linked the virus to CFS. In addition, sera from our CFS patients were assayed for the presence of xenotropic virus envelope protein, as well as a serological response to it. The results further strengthen our contention that there is no evidence for an association of XMRV with CFS, at least in the UK.
Utilization of Replication-Competent XMRV Reporter-Viruses Reveals Severe Viral Restriction in Primary Human Cells  [PDF]
Christina Martina Stürzel, David Palesch, Mohammad Khalid, Silke Wissing, Nicole Fischer, Jan Münch
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0074427
Abstract: The gammaretrovirus termed xenotropic murine leukemia virus-related virus (XMRV) was described to be isolated from prostate cancer tissue biopsies and from blood of patients suffering from chronic fatigue syndrome. However, many studies failed to detect XMRV and to verify these disease associations. Data suggesting the contamination of specimens in particular by PCR-based methods and recent reports demonstrating XMRV generation via recombination of two murine leukemia virus precursors raised serious doubts about XMRV being a genuine human pathogen. To elucidate cell tropism of XMRV, we generated replication competent XMRV reporter viruses encoding a green fluorescent protein or a secretable luciferase as tools to analyze virus infection of human cell lines or primary human cells. Transfection of proviral DNAs into LNCaP prostate cancer cells resulted in readily detectably reporter gene expression and production of progeny virus. Inoculation of known XMRV susceptible target cells revealed that these virions were infectious and expressed the reporter gene, allowing for a fast and highly sensitive quantification of XMRV infection. Both reporter viruses were capable of establishing a spreading infection in LNCaP and Raji B cells and could be easily passaged. However, after inoculation of primary human blood cells such as CD4 T cells, macrophages or dendritic cells, infection rates were very low, and a spreading infection was never established. In line with these results we found that supernatants derived from these XMRV infected primary cell types did not contain infectious virus. Thus, although XMRV efficiently replicated in some human cell lines, all tested primary cells were largely refractory to XMRV infection and did not support viral spread. Our results provide further evidence that XMRV is not a human pathogen.
Characterization of antibodies elicited by XMRV infection and development of immunoassays useful for epidemiologic studies
Xiaoxing Qiu, Priscilla Swanson, Ka-Cheung Luk, Bailin Tu, Francois Villinger, Jaydip Das Gupta, Robert H Silverman, Eric A Klein, Sushil Devare, Gerald Schochetman, John Hackett
Retrovirology , 2010, DOI: 10.1186/1742-4690-7-68
Abstract: Three rhesus macaques were infected with XMRV to determine the dynamics of the antibody responses elicited by infection with XMRV. All macaques developed antibodies to XMRV during the second week of infection, and the predominant responses were to the envelope protein gp70, transmembrane protein p15E, and capsid protein p30. In general, antibody responses to gp70 and p15E appeared early with higher titers than to p30, especially in the early period of seroconversion. Antibodies to gp70, p15E and p30 persisted to 158 days and were substantially boosted by re-infection, thus, were identified as useful serologic markers. Three high-throughput prototype assays were developed using recombinant proteins to detect antibodies to these viral proteins. Both gp70 and p15E prototype assays demonstrated 100% sensitivity by detecting all Western blot (WB) positive serial bleeds from the XMRV-infected macaques and good specificity (99.5-99.9%) with blood donors. Seroconversion sensitivity and specificity of the p30 prototype assay were 92% and 99.4% respectively.This study provides the first demonstration of seroconversion patterns elicited by XMRV infection. The nature and kinetics of antibody responses to XMRV in primates were fully characterized. Moreover, key serologic markers useful for detection of XMRV infection were identified. Three prototype immunoassays were developed to detect XMRV-specific antibodies. These assays demonstrated good sensitivity and specificity; thus, they will facilitate large scale epidemiologic studies of XMRV infection in humans.In 2006, a novel gammaretrovirus was identified in prostate cancer tissue using Virochip DNA microarray technology [1]. Cloning and sequencing of the gammaretrovirus revealed a close similarity to xenotropic murine leukemia viruses; thus, it was named Xenotropic Murine Leukemia Virus-related virus (XMRV). Initial screening using a nested reverse transcription-PCR (RT-PCR) assay found that XMRV was detectable in 10% (9/86) of
Membrane Fusion and Cell Entry of XMRV Are pH-Independent and Modulated by the Envelope Glycoprotein's Cytoplasmic Tail  [PDF]
Marceline C?té, Yi-Min Zheng, Shan-Lu Liu
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0033734
Abstract: Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus that was originally identified from human prostate cancer patients and subsequently linked to chronic fatigue syndrome. Recent studies showed that XMRV is a recombinant mouse retrovirus; hence, its association with human diseases has become questionable. Here, we demonstrated that XMRV envelope (Env)-mediated pseudoviral infection is not blocked by lysosomotropic agents and cellular protease inhibitors, suggesting that XMRV entry is not pH-dependent. The full length XMRV Env was unable to induce syncytia formation and cell-cell fusion, even in cells overexpressing the viral receptor, XPR1. However, truncation of the C-terminal 21 or 33 amino acid residues in the cytoplasmic tail (CT) of XMRV Env induced substantial membrane fusion, not only in the permissive 293 cells but also in the nonpermissive CHO cells that lack a functional XPR1 receptor. The increased fusion activities of these truncations correlated with their enhanced SU shedding into culture media, suggesting conformational changes in the ectodomain of XMRV Env. Noticeably, further truncation of the CT of XMRV Env proximal to the membrane-spanning domain severely impaired the Env fusogenicity, as well as dramatically decreased the Env incorporations into MoMLV oncoretroviral and HIV-1 lentiviral vectors resulting in greatly reduced viral transductions. Collectively, our studies reveal that XMRV entry does not require a low pH or low pH-dependent host proteases, and that the cytoplasmic tail of XMRV Env critically modulates membrane fusion and cell entry. Our data also imply that additional cellular factors besides XPR1 are likely to be involved in XMRV entry.
Evidence that Gag facilitates HIV-1 envelope association both in GPI-enriched plasma membrane and detergent resistant membranes and facilitates envelope incorporation onto virions in primary CD4+ T cells
Ajit Patil, Archana Gautam, Jayanta Bhattacharya
Virology Journal , 2010, DOI: 10.1186/1743-422x-7-3
Abstract: Human Immunodeficiency Virus Type 1 (HIV-1) has been shown to assemble via specialized plasma membrane domains popularly known as lipid rafts [1-6], which are rich in cholesterol and sphingomyelin within an ordered structure and plays important role in cell signaling [7]. Rafts are believed to play an important role towards facilitating HIV-1 assembly particularly exploiting acylated residues in viral Gag [1,3,5] and envelope (Env) [8,9]. However, the precise mechanism by which lipid rafts functions in targeting viral Env and Gag to the plasma membrane in infected T cells and facilitate assembly is not clearly understood. Previously, Jolly and Sattentau [10] have shown that raft integrity is critical for Env and Gag co-clustering and assembly in T-cell conjugates. Thus, mere presence of non raft proteins such as CD45 phosphatase in HIV-1 envelope glycoprotein while abundant incorporation of raft lipid components such as ganglioside GM1, glycosylphosphatidylinositol (GPI)-anchored proteins Thy-1 and CD59 strongly suggest that HIV-1 specifically buds from rafts [5,11]. A stable interaction between intracellular Pr55Gag and the gp41 cytoplasmic domain of envelope [12] was shown to be important for envelope association with detergent resistant membranes, incorporation onto virions and infectivity [13,14]. The precise sequence by which envelope utilizes cellular machinery in migrating towards the site of viral assembly is not clearly understood. Glycoproteins of several enveloped viruses, have been found to contain lipid moieties [15,16] and has generated notion on the importance of lipid rafts as a docking site for the assembly of enveloped viruses [17-22]. Association of HIV-1 envelope with polarized lipid raft markers GM1 and CD59 was shown to influence transmission between T cells [10]. Gag has been shown to play an important role in envelope assembly onto virions, notably by interaction of its p17 matrix domain with gp41 cytoplasmic domain of envelope [14,23-25]. Wh
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