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3D Reconstruction of VZV Infected Cell Nuclei and PML Nuclear Cages by Serial Section Array Scanning Electron Microscopy and Electron Tomography  [PDF]
Mike Reichelt ,Lydia Joubert,John Perrino,Ai Leen Koh,Ibanri Phanwar,Ann M. Arvin
PLOS Pathogens , 2012, DOI: 10.1371/journal.ppat.1002740
Abstract: Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.
Efficient Egress of Escaping Ants Stressed with Temperature  [PDF]
Santiago Boari, Roxana Josens, Daniel R. Parisi
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0081082
Abstract: In the present work we investigate the egress times of a group of Argentine ants (Linepithema humile) stressed with different heating speeds. We found that the higher the temperature ramp is, the faster ants evacuate showing, in this sense, a group-efficient evacuation strategy. It is important to note that even when the life of ants was in danger, jamming and clogging was not observed near the exit, in accordance with other experiments reported in the literature using citronella as aversive stimuli. Because of this clear difference between ants and humans, we recommend the use of some other animal models for studying competitive egress dynamics as a more accurate approach to understanding competitive egress in human systems.
Viral Mimicry of Cdc2/Cyclin-Dependent Kinase 1 Mediates Disruption of Nuclear Lamina during Human Cytomegalovirus Nuclear Egress  [PDF]
Sofia Hamirally equal contributor,Jeremy P. Kamil equal contributor,Yasmine M. Ndassa-Colday,Alison J. Lin,Wan Jin Jahng,Moon-Chang Baek,Sarah Noton,Laurie A. Silva,Martha Simpson-Holley,David M. Knipe,David E. Golan,Jarrod A. Marto,Donald M. Coen
PLOS Pathogens , 2009, DOI: 10.1371/journal.ppat.1000275
Abstract: The nuclear lamina is a major obstacle encountered by herpesvirus nucleocapsids in their passage from the nucleus to the cytoplasm (nuclear egress). We found that the human cytomegalovirus (HCMV)-encoded protein kinase UL97, which is required for efficient nuclear egress, phosphorylates the nuclear lamina component lamin A/C in vitro on sites targeted by Cdc2/cyclin-dependent kinase 1, the enzyme that is responsible for breaking down the nuclear lamina during mitosis. Quantitative mass spectrometry analyses, comparing lamin A/C isolated from cells infected with viruses either expressing or lacking UL97 activity, revealed UL97-dependent phosphorylation of lamin A/C on the serine at residue 22 (Ser22). Transient treatment of HCMV-infected cells with maribavir, an inhibitor of UL97 kinase activity, reduced lamin A/C phosphorylation by approximately 50%, consistent with UL97 directly phosphorylating lamin A/C during HCMV replication. Phosphorylation of lamin A/C during viral replication was accompanied by changes in the shape of the nucleus, as well as thinning, invaginations, and discrete breaks in the nuclear lamina, all of which required UL97 activity. As Ser22 is a phosphorylation site of particularly strong relevance for lamin A/C disassembly, our data support a model wherein viral mimicry of a mitotic host cell kinase activity promotes nuclear egress while accommodating viral arrest of the cell cycle.
Interaction with Tsg101 Is Necessary for the Efficient Transport and Release of Nucleocapsids in Marburg Virus-Infected Cells  [PDF]
Olga Dolnik,Larissa Kolesnikova,Sonja Welsch,Thomas Strecker,Gordian Schudt,Stephan Becker
PLOS Pathogens , 2014, DOI: doi/10.1371/journal.ppat.1004463
Abstract: Endosomal sorting complex required for transport (ESCRT) machinery supports the efficient budding of Marburg virus (MARV) and many other enveloped viruses. Interaction between components of the ESCRT machinery and viral proteins is predominantly mediated by short tetrapeptide motifs, known as late domains. MARV contains late domain motifs in the matrix protein VP40 and in the genome-encapsidating nucleoprotein (NP). The PSAP late domain motif of NP recruits the ESCRT-I protein tumor susceptibility gene 101 (Tsg101). Here, we generated a recombinant MARV encoding NP with a mutated PSAP late domain (rMARVPSAPmut). rMARVPSAPmut was attenuated by up to one log compared with recombinant wild-type MARV (rMARVwt), formed smaller plaques and exhibited delayed virus release. Nucleocapsids in rMARVPSAPmut-infected cells were more densely packed inside viral inclusions and more abundant in the cytoplasm than in rMARVwt-infected cells. A similar phenotype was detected when MARV-infected cells were depleted of Tsg101. Live-cell imaging analyses revealed that Tsg101 accumulated in inclusions of rMARVwt-infected cells and was co-transported together with nucleocapsids. In contrast, rMARVPSAPmut nucleocapsids did not display co-localization with Tsg101, had significantly shorter transport trajectories, and migration close to the plasma membrane was severely impaired, resulting in reduced recruitment into filopodia, the major budding sites of MARV. We further show that the Tsg101 interacting protein IQGAP1, an actin cytoskeleton regulator, was recruited into inclusions and to individual nucleocapsids together with Tsg101. Moreover, IQGAP1 was detected in a contrail-like structure at the rear end of migrating nucleocapsids. Down regulation of IQGAP1 impaired release of MARV. These results indicate that the PSAP motif in NP, which enables binding to Tsg101, is important for the efficient actin-dependent transport of nucleocapsids to the sites of budding. Thus, the interaction between NP and Tsg101 supports several steps of MARV assembly before virus fission.
Tight Analysis of Priority Queuing Policy for Egress Traffic  [PDF]
Jun Kawahara,Koji M. Kobayashi,Tomotaka Maeda
Computer Science , 2012,
Abstract: Recently, the problems of evaluating performances of switches and routers have been formulated as online problems, and a great amount of results have been presented. In this paper, we focus on managing outgoing packets (called {\em egress traffic}) on switches that support Quality of Service (QoS), and analyze the performance of one of the most fundamental scheduling policies {\em Priority Queuing} ($PQ$) using competitive analysis. We formulate the problem of managing egress queues as follows: An output interface is equipped with $m$ queues, each of which has a buffer of size $B$. The size of a packet is unit, and each buffer can store up to $B$ packets simultaneously. Each packet is associated with one of $m$ priority values $\alpha_{j}$ ($1 \leq j \leq m$), where $\alpha_{1} \leq \alpha_{2} \leq \cdots \leq \alpha_{m}$, $\alpha_{1} = 1$, and $\alpha_{m} = \alpha$ and the task of an online algorithm is to select one of $m$ queues at each scheduling step. The purpose of this problem is to maximize the sum of the values of the scheduled packets. For any $B$ and any $m$, we show that the competitive ratio of $PQ$ is exactly $2 - \min_{x \in [1, m-1] } \{ \frac{ \alpha_{x+1} }{ \sum_{j = 1}^{x+1} \alpha_{j} } \}$. That is, we conduct a complete analysis of the performance of $PQ$ using worst case analysis. Moreover, we show that no deterministic online algorithm can have a competitive ratio smaller than $1 + \frac{ \alpha^3 + \alpha^2 + \alpha }{ \alpha^4 + 4 \alpha^3 + 3 \alpha^2 + 4 \alpha + 1 }$.
Cytoplasmic free Ca2+ is essential for multiple steps in malaria parasite egress from infected erythrocytes
Glushakova Svetlana,Lizunov Vladimir,Blank Paul S,Melikov Kamran
Malaria Journal , 2013, DOI: 10.1186/1475-2875-12-41
Abstract: Background Egress of Plasmodium falciparum, from erythrocytes at the end of its asexual cycle and subsequent parasite invasion into new host cells, is responsible for parasite dissemination in the human body. The egress pathway is emerging as a coordinated multistep programme that extends in time for tens of minutes, ending with rapid parasite extrusion from erythrocytes. While the Ca2+ regulation of the invasion of P. falciparum in erythrocytes is well established, the role of Ca2+ in parasite egress is poorly understood. This study analysed the involvement of cytoplasmic free Ca2+ in infected erythrocytes during the multistep egress programme of malaria parasites. Methods Live-cell fluorescence microscopy was used to image parasite egress from infected erythrocytes, assessing the effect of drugs modulating Ca2+ homeostasis on the egress programme. Results A steady increase in cytoplasmic free Ca2+ is found to precede parasite egress. This increase is independent of extracellular Ca2+ for at least the last two hours of the cycle, but is dependent upon Ca2+ release from internal stores. Intracellular BAPTA chelation of Ca2+ within the last 45 minutes of the cycle inhibits egress prior to parasitophorous vacuole swelling and erythrocyte membrane poration, two characteristic morphological transformations preceding parasite egress. Inhibitors of the parasite endoplasmic reticulum (ER) Ca2+-ATPase accelerate parasite egress, indicating that Ca2+ stores within the ER are sufficient in supporting egress. Markedly accelerated egress of apparently viable parasites was achieved in mature schizonts using Ca2+ ionophore A23187. Ionophore treatment overcomes the BAPTA-induced block of parasite egress, confirming that free Ca2+ is essential in egress initiation. Ionophore treatment of immature schizonts had an adverse effect inducing parasitophorous vacuole swelling and killing the parasites within the host cell. Conclusions The parasite egress programme requires intracellular free Ca2+ for egress initiation, vacuole swelling, and host cell cytoskeleton digestion. The evidence that parasitophorous vacuole swelling, a stage of unaffected egress, is dependent upon a rise in intracellular Ca2+ suggests a mechanism for ionophore-inducible egress and a new target for Ca2+ in the programme liberating parasites from the host cell. A regulatory pathway for egress that depends upon increases in intracellular free Ca2+ is proposed.
Proteasome Inhibition Suppresses Dengue Virus Egress in Antibody Dependent Infection  [PDF]
Milly M. Choy?,Summer L. Zhang?,Vivian V. Costa?,Hwee Cheng Tan?,Sophie Horrevorts?,Eng Eong Ooi
PLOS Neglected Tropical Diseases , 2015, DOI: 10.1371/journal.pntd.0004058
Abstract: The mosquito-borne dengue virus (DENV) is a cause of significant global health burden, with an estimated 390 million infections occurring annually. However, no licensed vaccine or specific antiviral treatment for dengue is available. DENV interacts with host cell factors to complete its life cycle although this virus-host interplay remains to be fully elucidated. Many studies have identified the ubiquitin proteasome pathway (UPP) to be important for successful DENV production, but how the UPP contributes to DENV life cycle as host factors remains ill defined. We show here that proteasome inhibition decouples infectious virus production from viral RNA replication in antibody-dependent infection of THP-1 cells. Molecular and imaging analyses in β-lactone treated THP-1 cells suggest that proteasome function does not prevent virus assembly but rather DENV egress. Intriguingly, the licensed proteasome inhibitor, bortezomib, is able to inhibit DENV titers at low nanomolar drug concentrations for different strains of all four serotypes of DENV in primary monocytes. Furthermore, bortezomib treatment of DENV-infected mice inhibited the spread of DENV in the spleen as well as the overall pathological changes. Our findings suggest that preventing DENV egress through proteasome inhibition could be a suitable therapeutic strategy against dengue.
Sequential evacuation strategy for multiple rooms toward the same means of egress  [PDF]
Daniel R. Parisi,Pablo A. Negri
Computer Science , 2014, DOI: 10.4279/PIP.060013
Abstract: This paper examines different evacuation strategies for systems where several rooms evacuate trough the same means of egress, using microscopic pedestrian simulation.As a case study, a medium-rise office building is considered. It was found that the standard strategy, whereby the simultaneous evacuation of all levels is performed, can be improved by a sequential evacuation, beginning with the lowest floor and continuing successively with each one of the upper floors after a certain delay. The importance of the present research is that it provides the basis for the design and implementation of new evacuation strategies and alarm systems that could significantly improve the evacuation of multiple rooms trough a common means of escape.
Intra-Symbol Windowing for Egress Reduction in DMT Transmitters  [cached]
Cuypers Gert,Vanbleu Koen,Ysebaert Geert,Moonen Marc
EURASIP Journal on Advances in Signal Processing , 2006,
Abstract: Discrete multitone (DMT) uses an inverse discrete Fourier transform (IDFT) to modulate data on the carriers. The high sidelobes of the IDFT filter bank used can lead to spurious emissions (egress) in unauthorized frequency bands. Applying a window function within the DMT symbol can alleviate this. However, window functions either require additional redundancy or will introduce distortions that are generally not easy to compensate for. In this paper, a special class of window functions is constructed that corresponds to a precoding at the transmitter. These do not require any additional redundancy and need only a modest amount of additional processing at the receiver. The results can be used to increase the spectral containment of DMT-based wired communications such as ADSL and VDSL (i.e., asymmetric, resp., very-high-bitrate digital subscriber loop).
Multilamellar Structures and Filament Bundles Are Found on the Cell Surface during Bunyavirus Egress  [PDF]
Laura Sanz-Sánchez, Cristina Risco
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0065526
Abstract: Inside cells, viruses build specialized compartments for replication and morphogenesis. We observed that virus release associates with specific structures found on the surface of mammalian cells. Cultured adherent cells were infected with a bunyavirus and processed for oriented sectioning and transmission electron microscopy. Imaging of cell basal regions showed sophisticated multilamellar structures (MLS) and extracellular filament bundles with attached viruses. Correlative light and electron microscopy confirmed that both MLS and filaments proliferated during the maximum egress of new viruses. MLS dimensions and structure were reminiscent of those reported for the nanostructures on gecko fingertips, which are responsible for the extraordinary attachment capacity of these lizards. As infected cells with MLS were more resistant to detachment than control cells, we propose an adhesive function for these structures, which would compensate for the loss of adherence during release of new virus progeny.
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