Nuclear domain 10 (ND10) associated proteins are also present in nuclear bodies and redistribute to hundreds of nuclear sites after stress

The promyelocytic leukemia protien fused to the retinoic acid receptor in t(15;17) acute promyelocytic leukemia, the primary biliary cirrhosis autoantigen, Sp100, as well as the incompletely charterized protien NDP55, are co-localized in specific immunohistochemically defined nuclear domains (ND10), which are potential equyivalents of ultrastructurally defined nuclear bodies. We investigated whether the distribution of these proteins depends on environmental conditions and whether ND10 correlate with nuclear bodies. Certain nuclear bodies and ND10 react in a similar bodies, which herpes simplex virus infection or heat shock modify both. Redistribution of ND10-associated proteins to hundreds of small sites throughout the chromatin was inducible by stress in the form of heat shock and exposure to Cd⁺⁺ ions. The change of distribution was rapid and independent of proteins synthesis, and thus not part of the classical heat shock response. The very rapid redistribution of these proteins after heat shock, together with the development of ND10 upon interferon actication, raises the possibility that ND10 represent storage sites of certain matrix proteins readily accessible throughout the chromatin in response to stress or other effectors that induce global nuclear changes. © 1995 Wiley-Liss, Inc.     

Nuclear accumulation of a heat-shock 70-like protein during herpes simplex virus replication

A monoclonal antibody defines an antigen, p68, related to hsp70, which is located in nuclei of uninfected exponential cells. Nuclear p68 is released by DNase but not RNase treatment suggesting an association with DNA. Lytic productive infection of confluent quiescent BHK 21 cells with herpes simplex virus type-2 causes p68 to accumulate in nuclei. The effect is specific for HSV-2, and does not occur in HSV-1 infected cells. Maximum nuclear accumulation of p68 requires virus DNA synthesis although a significant accumulation occurs in the absence of such synthesis. It is suggested that the nuclear accumulation of p68 is an aspect of a cellular stress response to lytic infection with HSV-2.Imperial Cancer Research Fund, Tumour Immunology Unit.     

Cell‐Free Reconstitution of Multivesicular Body Formation and Receptor Sorting

The number of surface membrane proteins and their residence time on the plasma membrane are critical determinants of cellular responses to cues that can control plasticity, growth and differentiation. After internalization, the ultimate fate of many plasma membrane proteins is dependent on whether they are sorted for internalization into the lumenal vesicles of multivesicular bodies (MVBs), an obligate step prior to lysosomal degradation. To help to elucidate the mechanisms underlying MVB sorting, we have developed a novel cell‐free assay that reconstitutes the sorting of a prototypical membrane protein, the epidermal growth factor receptor, with which we have probed some of its molecular requirements. The sorting event measured is dependent on cytosol, ATP, time, temperature and an intact proton gradient. Depletion of Hrs inhibited biochemical and morphological measures of sorting that were rescued by inclusion of recombinant Hrs in the assay. Moreover, depletion of signal‐transducing adaptor molecule (STAM), or addition of mutated ATPase‐deficient Vps4, also inhibited sorting. This assay reconstitutes the maturation of late endosomes, including the formation of internal vesicles and the sorting of a membrane protein, and allows biochemical investigation of this process.     

Axin expression delays herpes simplex virus‐induced autophagy and enhances viral replication in L929 cells

Axin, a negative regulator of the Wnt signaling pathway, plays a critical role in various cellular events including cell proliferation and cell death. Axin‐regulated cell death affects multiple processes, including viral replication. For example, axin expression suppresses herpes simplex virus (HSV)‐induced necrotic cell death and enhances viral replication. Based on these observations, this study investigated the involvement of autophagy in regulation of HSV replication and found axin expression inhibits autophagy‐mediated suppression of viral replication in L929 cells. HSV infection induced autophagy in a time‐ and viral dose‐dependent manner in control L929 cells (L‐EV), whereas virus‐induced autophagy was delayed in axin‐expressing L929 cells (L‐axin). Subsequent analysis showed that induction of autophagy by rapamycin reduced HSV replication, and that inhibiting autophagy by 3‐methyladenine (3MA) and beclin‐1 knockdown facilitated viral replication in L‐EV cells. In addition, preventing autophagy with 3MA suppressed virus‐induced cytotoxicity in L‐EV cells. In contrast, HSV replication in L‐axin cells was resistant to changes in autophagy. These results suggest that axin expression may render L929 cells resistant to HSV‐infection induced autophagy, leading to enhanced viral replication.     

番茄成熟过程中果皮细胞核孔变化及其与乙烯生成的关系

应用冰冻蚀刻技术探讨了番茄(Lycopersiconesculentum Mill 大红品种)成熟过程中果皮细胞核孔的变化。成熟开始发动时(发白期前,IEC>0.1 ppm)核孔就开始扩大,起初核孔扩大比IEC增加更快,转包期(IEC约10 ppm)达到最大,以后不再变化。核孔扩大先于ACC含量增加。核孔的数量从转色期增加,粉红期最大,成熟后又减少。     

HSV‐1 ICP27 targets the TBK1‐activated STING signalsome to inhibit virus‐induced type I IFN expression

Herpes simplex virus (HSV) 1 stimulates type I IFN expression through the cGAS–STING–TBK1 signaling axis. Macrophages have recently been proposed to be an essential source of IFN during viral infection. However, it is not known how HSV‐1 inhibits IFN expression in this cell type. Here, we show that HSV‐1 inhibits type I IFN induction through the cGAS–STING–TBK1 pathway in human macrophages, in a manner dependent on the conserved herpesvirus protein ICP27. This viral protein was expressed de novo in macrophages with early nuclear localization followed by later translocation to the cytoplasm where ICP27 prevented activation of IRF3. ICP27 interacted with TBK1 and STING in a manner that was dependent on TBK1 activity and the RGG motif in ICP27. Thus, HSV‐1 inhibits expression of type I IFN in human macrophages through ICP27‐dependent targeting of the TBK1‐activated STING signalsome.     

Dissecting Functions of the Conserved Oligomeric Golgi Tethering Complex Using a Cell‐Free Assay

Vesicle transport sorts proteins between compartments and is thereby responsible for generating the non‐uniform protein distribution along the eukaryotic secretory and endocytic pathways. The mechanistic details of specific vesicle targeting are not yet well characterized at the molecular level. We have developed a cell‐free assay that reconstitutes vesicle targeting utilizing the recycling of resident enzymes within the Golgi apparatus. The assay has physiological properties, and could be used to show that the two lobes of the conserved oligomeric Golgi tethering complex play antagonistic roles in trans‐Golgi vesicle targeting. Moreover, we can show that the assay is sensitive to several different congenital defects that disrupt Golgi function and therefore cause glycosylation disorders. Consequently, this assay will allow mechanistic insight into the targeting step of vesicle transport at the Golgi, and could also be useful for characterizing some novel cases of congenital glycosylation disorders.      

Flexible and Extended Linker Domains Support Efficient Targeting of Heh2 to the Inner Nuclear Membrane

1. Download : Download high-res image (213KB)
2. Download : Download full-size image

     

Nuclear assembly of purified Crythecodinium cohnii chromosomes in cell—free extracts of Xenopus laevis eggs

Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation,nuclear envelope assembly,and nuclear reconstitution.Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle.The assembled nuclei,being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii.However,incubation of dinoflagellate Cyrthecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinium cohnii cells does not induce nuclear reconstitution.     

A three-residue signal confers localization of a reporter protein in the inner nuclear membrane

By alignment to the carboxy-terminal-deduced aa sequence of human cytomegalovirus glycoprotein B (gB), conserved hexameric aa motifs with putative function for localization in the inner nuclear membrane (INM) were identified in the nucleoplasmic tails of herpes simplex virus type 1 gB and of the cellular lamin B receptor. Fusion of the respective hexamers to CD8 as a reporter redirected transport of the chimeras into the INM, suggesting their functioning as modular signal motifs. Consecutive experiments showed that the three-residue motif RxR represents a consensus sequence which is sufficient for localization of the CD8 reporter in the INM.     

Nuclear reconstitution of demembranated Orychophragmus violaceus sperm in Xenopus laevis egg extracts

The cell-free extracts from animal Xenopus laevis egg could induce chromatin decon-densation and pronuclear formation from demembranated plant (Orychophragmus violaceus) sperm. The demembranated Orychophragmus violaceus sperm began to swell in 30 min incubation, and then were gradually decondensed. The reassembly of nuclear envelope in the reconstituted nuclei had been visualized by means of electron microscopy and fluorescent microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nucleus, with a double membrane, was similar to those nuclei after fertilization. Transmission electron microscope micrograph of the whole mount prepared nuclear matrix-lamina showed the reconstituted nucleus to be filled with a dense network.     

Growth and Cytopathogenicity of Herpes Simplex Virus in a Macrophage Cell Line,RAW264: A Good Indicator of Intraperitoneal Pathogenicity

Macrophages are known to play a critical role in host resistance to herpes simplex virus (HSV). In this study, we investigate the interaction between various HSV strains with different virulence and a murine macrophage cell line, RAW264. Highly attenuated strains replicated poorly in RAW264 cells and were cleared from the cultures. For the eleven viruses tested, there was good correlation between intraperitoneal pathogenicity for adult mice and replication in RAW264 cells. It was also shown that interferon α/β was involved in restricted replication of some strains.     

Functional Analysis of Tpr: Identification of Nuclear Pore Complex Association and Nuclear Localization Domains and a Role in mRNA Export

Tpr is a 270-kD coiled-coil protein localized to intranuclear filaments of the nuclear pore complex (NPC). The mechanism by which Tpr contributes to the structure and function of the nuclear pore is currently unknown. To gain insight into Tpr function, we expressed the full-length protein and several subdomains in mammalian cell lines and examined their effects on nuclear pore function. Through this analysis, we identified an NH₂-terminal domain that was sufficient for association with the nucleoplasmic aspect of the NPC. In addition, we unexpectedly found that the acidic COOH terminus was efficiently transported into the nuclear interior, an event that was apparently mediated by a putative nuclear localization sequence. Ectopic expression of the full-length Tpr caused a dramatic accumulation of poly(A)⁺ RNA within the nucleus. Similar results were observed with domains that localized to the NPC and the nuclear interior. In contrast, expression of these proteins did not appear to affect nuclear import. These data are consistent with a model in which Tpr is tethered to intranuclear filaments of the NPC by its coiled coil domain leaving the acidic COOH terminus free to interact with soluble transport factors and mediate export of macromolecules from the nucleus.     

Cloning and expression of the uracil–DNA glycosylase inhibitor (UGI) from bacteriophage PBS-1 and crystallization of a uracil–DNA glycosylase–UGI complex

The uracil-DNA glycosylase inhibitory protein (UGI) from the bacterio-phage PBS-l has been cloned and overexpressed. The nucleotide sequence is identical to that for the previously described PBS-2 inhibitor. The recombinant PBS-l UGI inhibits the uracil-DNA glycosylase from herpes simplex virus type-l (HSV-l UDGase), and a complex between the HSV-l UDGase and PBS-l UGI has been crystallized. The crystals have unit cell dimensions a = 143.21 Å, c = 40.78 Å and are in a polar hexagonal space group. There is a single complex in the asymmetric unit with a solvent content of 62% by volume and the crystals diffract to 2.5Å on a synchrotron radiation source. © 1995 Wiley-Liss, Inc.     

Metabolic Fate of [3H]1-β-d-Arabinofuranosyl-5-[(E)-2-bromovinyl]uracil in Herpes Simplex Virus Type 1-Infected Cells

The metabolic fate of 1-β-d -arabinofuranosyl-5-[(E)-2-bromovinyl]uracil (BV-araU) in herpes simplex virus type 1-infected cells was studied using tritium-labeled BV-araU. [³H]BV-araU was selectively taken-up by infected cells. Approximately 10% of the total uptake of [³H]BV-araU was recovered from the acid-insoluble fraction at any time post-infection. Both cellular uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction increased with increasing incubation time through 8 hr post-infection. Uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction also increased proportionally to the duration of exposure to [³H]BV-araU. An alkaline sucrose gradient sedimentation analysis revealed that the radioactive DNA obtained from cells pulse-labeled with [³H]BV-araU were small DNA fragments which remained at the top following a chasing period in isotope-free medium, whereas that pulse-labeled with [³H]thymidine was chased to a fraction of high molecular weight DNA. Nuclease P₁ digestion reduced 99% of the [³H]BV-araU-labeled DNA extracted from infected cells to a low molecular weight. Following digestion of [³H]BV-araU-labeled DNA with micrococcal nuclease and spleen exonuclease, all of the radioactivity was recovered as [³H]BV-araU 3′-monophosphate. Thus, BV-araU strongly inhibits the elongation of viral DNA strands as demonstrated by the alkaline sucrose gradient sedimentation analysis, whereas at least a portion of the [³H]BV-araU is incorporated inside viral DNA strands in infected cells.