scholarly journals Intracellular localization and effects of individually expressed human parechovirus 1 non-structural proteins

2007 ◽  
Vol 88 (3) ◽  
pp. 831-841 ◽  
Author(s):  
Camilla Krogerus ◽  
Olga Samuilova ◽  
Tuija Pöyry ◽  
Eija Jokitalo ◽  
Timo Hyypiä
Keyword(s):  
Endoplasmic Reticulum ◽  
Complex Formation ◽  
Golgi Apparatus ◽  
Lipid Droplets ◽  
Intracellular Localization ◽  
Structural Proteins ◽  
Human Parechovirus ◽  
Replication Complex ◽  
Infected Cells ◽  
Cellular Secretion

Human parechovirus 1 (HPEV-1) has many unique features compared with other picornaviruses and it has been shown that the replication complex formed during HPEV-1 infection is different from that of other picornaviruses. Here, the intracellular localization and functional effects of individually expressed HPEV-1 non-structural proteins were studied. The 2A and 3D proteins were found diffusely in the cytoplasm and nucleus of the cell. The 3A and 3AB proteins were observed to co-localize with the markers for the Golgi apparatus, whereas 2B co-localized with markers for the endoplasmic reticulum and the 2C and 2BC proteins were observed mainly on the surface of lipid droplets. The 2C protein, which has been implicated in replication-complex formation in enterovirus-infected cells, was not able to induce vesicles similar to those seen in HPEV-1-infected cells when expressed individually. However, in superinfected cells, the fusion protein was able to relocate to the virus replication complexes. Similar to other picornaviruses, HPEV-1 was found to interfere with cellular secretion, but this function could not be ascribed to any of the individually expressed non-structural proteins.

scholarly journals Intracellular Localization of the Peanut Clump Virus Replication Complex in Tobacco BY-2 Protoplasts Containing Green Fluorescent Protein-Labeled Endoplasmic Reticulum or Golgi Apparatus

2002 ◽  
Vol 76 (2) ◽  
pp. 865-874 ◽  
Author(s):  
Patrice Dunoyer ◽  
Christophe Ritzenthaler ◽  
Odile Hemmer ◽  
Pierre Michler ◽  
Christiane Fritsch
Keyword(s):  
Endoplasmic Reticulum ◽  
Green Fluorescent Protein ◽  
Golgi Apparatus ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
De Novo ◽  
Intracellular Localization ◽  
Viral Rna ◽  
Replication Complex ◽  
Green Fluorescent

ABSTRACT RNA-1 of Peanut clump virus (PCV) encodes the proteins P131 and P191, containing the signature motifs of replication proteins, and P15, which regulates viral RNA accumulation. In PCV-infected protoplasts both P131 and P191 were immunodetected in the perinuclear region. Laser scanning confocal microscopy (LSCM) showed that P131 and P191 colocalized with neosynthesized 5-bromouridine 5′-triphosphate-labeled RNA and double-stranded RNA, demonstrating that they belong to the replication complex. On the contrary, the P15 fused to the enhanced green fluorescent protein (EGFP) never colocalized with the two proteins. In endoplasmic reticulum (ER)-GFP transgenic BY-2 protoplasts, the distribution of the green fluorescent-labeled ER was strongly modified by PCV infection. LSCM showed that both P131 and P191 colocalized with ER green fluorescent bodies accumulating around the nucleus during infection. The replication process was not inhibited by cerulenin and brefeldin A, suggesting that PCV replication does not depend on de novo-synthesized membrane and does not require transport through the Golgi apparatus. Electron microscopy of ultrathin sections of infected protoplasts showed aggregates of broken ER but also visualized vesicles, some of which resembled modified peroxisomes. The results suggest that accumulation of PCV during infection is accompanied by specific association of PCV RNA-1-encoded proteins with membranes of the ER and other organelles. The concomitant extensive rearrangement of these membranous structures leads to the formation of intracellular compartments in which synthesis and accumulation of the viral RNA occur in defined areas.

scholarly journals Feline calicivirus p32, p39 and p30 proteins localize to the endoplasmic reticulum to initiate replication complex formation

2009 ◽  
Vol 91 (3) ◽  
pp. 739-749 ◽  
Author(s):  
D. Bailey ◽  
W. J. Kaiser ◽  
M. Hollinshead ◽  
K. Moffat ◽  
Y. Chaudhry ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Complex Formation ◽  
Feline Calicivirus ◽  
Replication Complex

scholarly journals Remodeling the Endoplasmic Reticulum by Poliovirus Infection and by Individual Viral Proteins: an Autophagy-Like Origin for Virus-Induced Vesicles

2000 ◽  
Vol 74 (19) ◽  
pp. 8953-8965 ◽  
Author(s):  
David A. Suhy ◽  
Thomas H. Giddings ◽  
Karla Kirkegaard
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Buoyant Density ◽  
Rna Replication ◽  
Viral Proteins ◽  
Replication Complex ◽  
Poliovirus Infection ◽  
Endosomal Membranes ◽  
Infected Cells ◽  
Positive Strand Rna

ABSTRACT All positive-strand RNA viruses of eukaryotes studied assemble RNA replication complexes on the surfaces of cytoplasmic membranes. Infection of mammalian cells with poliovirus and other picornaviruses results in the accumulation of dramatically rearranged and vesiculated membranes. Poliovirus-induced membranes did not cofractionate with endoplasmic reticulum (ER), lysosomes, mitochondria, or the majority of Golgi-derived or endosomal membranes in buoyant density gradients, although changes in ionic strength affected ER and virus-induced vesicles, but not other cellular organelles, similarly. When expressed in isolation, two viral proteins of the poliovirus RNA replication complex, 3A and 2C, cofractionated with ER membranes. However, in cells that expressed 2BC, a proteolytic precursor of the 2B and 2C proteins, membranes identical in buoyant density to those observed during poliovirus infection were formed. When coexpressed with 2BC, viral protein 3A was quantitatively incorporated into these fractions, and the membranes formed were ultrastructurally similar to those in poliovirus-infected cells. These data argue that poliovirus-induced vesicles derive from the ER by the action of viral proteins 2BC and 3A by a mechanism that excludes resident host proteins. The double-membraned morphology, cytosolic content, and apparent ER origin of poliovirus-induced membranes are all consistent with an autophagic origin for these membranes.

Ultrastructural Studies on the Effects of Korean Panax Ginseng on the Theca Interna of Rat Ovary

1979 ◽  
Vol 07 (04) ◽  
pp. 333-344 ◽  
Author(s):  
Moo Rim Byung
Keyword(s):  
Endoplasmic Reticulum ◽  
Fine Structure ◽  
Golgi Apparatus ◽  
Panax Ginseng ◽  
Lipid Droplets ◽  
Ultrastructural Studies ◽  
Dense Bodies ◽  
Rat Ovary ◽  
Theca Interna ◽  
Morphologic Changes

An investigation was conducted to delineate the fine structure of steroid-producing ovarian theca interna cells following administration of Korean Panax ginseng to rats for 60 days. The cytoplasmic changes were observed in the ginseng-treated theca interna cells, increased number, size and density of the mitochondria, and increased size of the smooth surfaced endoplasmic reticulum, the rough surfaced endoplasmic reticulum and the Golgi apparatus. The nucleus and nucleolus were slightly enlarged and increased numbers of dense bodies were seen whereas lipid droplets were decreased in number. The changes may result from hyperfunction of the steroid-producing cells. Morphologic changes seen may represent stimulating effects on the steroid-producing cells of the theca interna in ginseng-treated animals.

APPLICATION OF PEROXIDASE-LABELLED ANTIBODIES TO THE INTRACELLULAR LOCALIZATION OF HORMONES

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S190-S204 ◽  
Author(s):  
Paul K. Nakane
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Anterior Pituitary ◽  
Intracellular Localization ◽  
Ultrastructural Level ◽  
Substrate Uptake ◽  
Secretion Granules ◽  
Pituitary Glands ◽  
Ultrathin Sections

ABSTRACT Hormones were localized immunoenzymocytochemically at the ultrastructural level directly on ultrathin sections of anterior pituitary glands of rats which had been fixed and embedded in either methacrylate or Epon. GH, LTH, ACTH and LH are best localized on methacrylate embedded glands and GH and LTH on Epon embedded glands. GH and LTH were found in secretion granules, and depending on the activity of the cells, the hormones could be found in the Golgi apparatus and in the cisternae of endoplasmic reticulum. The grids on which hormones have been localized may also be processed for electron radioautography, an approach particularly useful to study simultaneously substrate uptake as well as product synthesis.

scholarly journals Replication Complex of Human Parechovirus 1

2003 ◽  
Vol 77 (15) ◽  
pp. 8512-8523 ◽  
Author(s):  
Camilla Krogerus ◽  
Denise Egger ◽  
Olga Samuilova ◽  
Timo Hyypiä ◽  
Kurt Bienz
Keyword(s):  
Viral Protein ◽  
Structural Changes ◽  
Rna Replication ◽  
Viral Rna ◽  
Human Parechovirus ◽  
Electron Microscopic ◽  
Vitro Assay ◽  
Replication Complex ◽  
Infected Cells

ABSTRACT The parechoviruses differ in many biological properties from other picornaviruses, and their replication strategy is largely unknown. In order to identify the viral RNA replication complex in human parechovirus type 1 (HPEV-1)-infected cells, we located viral protein and RNA in correlation to virus-induced membrane alterations. Structural changes in the infected cells included a disintegrated Golgi apparatus and disorganized, dilated endoplasmic reticulum (ER) which had lost its ribosomes. Viral plus-strand RNA, located by electron microscopic (EM) in situ hybridization, and the viral protein 2C, located by EM immunocytochemistry were found on clusters of small vesicles. Nascent viral RNA, visualized by 5-bromo-UTP incorporation, localized to compartments which were immunocytochemically found to contain the viral protein 2C and the trans-Golgi marker 1,4-galactosyltransferase. Protein 2C was immunodetected additionally on altered ER membranes which displayed a complex network-like structure devoid of cytoskeletal elements and with no apparent involvement in viral RNA replication. This protein also exhibited membrane binding properties in an in vitro assay. Our data suggest that the HPEV-1 replication complex is built up from vesicles carrying a Golgi marker and forming a structure different from that of replication complexes induced by other picornaviruses.

scholarly journals Ultrastructure of cells after reversible dark-induced blocking of mitotic divisions in antheridial filaments of Chara vulgaris L.

2014 ◽  
Vol 49 (3) ◽  
pp. 169-186 ◽  
Author(s):  
Maria Kwiatkowska ◽  
Janusz Maszewski ◽  
Maria M. Maszewska
Keyword(s):  
Cell Cycle ◽  
Endoplasmic Reticulum ◽  
Spatial Distribution ◽  
Cell Growth ◽  
Golgi Apparatus ◽  
Lipid Droplets ◽  
Continuous Darkness ◽  
Mean Size ◽  
Dense Matrices ◽  
The Mean

As compared with the control plants cultured under photoperiodic L : D = =14 : 10 conditions (K w i a t k o w s k a, M a s z e w s k i, 1978), the ultrastructure of nuclei -in cells blocked by a 5 day exposure to continuous darkness is characterized by homogenous arrangement. This homogeneity is maintained in all generations of antheridial filaments irrespective of cell length, which in the controls, being directly correlated with particular type of nuclear structure, may serve as a precise indicator of a given stage of interphase. From similarities in both the spatial distribution and content of condensed chromatin in is concluded that the block of the cell cycle is imposed at the beginning of the G<sub>2</sub> phase. On comparing these cells with the early G<sub>2</sub> period (stage VII) in the control plants, marked changes in the structure of nucleoli were found. They decrease in size by half owing to the complete decline of granular component. The area occupied by endoplasmic reticulum undergoes a 50% reduction. The decrease in the activity of Golgi apparatus expressed by a drop in number of smooth vesicles surrounding a single dictyosome is found to parallel the limited rate of cell growth. The number of coated vesicles and cisterns of dictyosome slightly increases. Mitochondria show typical condensed configuration with dense matrices and swollen cristae, while in the control orthodox forms are prevailing. The mean size of mitochondria is smaller, but their number exceeds that of the control plants. The surface area of mitochondrial profiles is found to remain constant proportion of the cytoplasm section, e.g., about 3%. Dark-cultured antheridial filaments show absolute decline of lipid droplets. No differences were found in structure of plastids and vacuols, as well as in number of ribosomes in cytoplasm surface unit.

scholarly journals Disruption of the Golgi Apparatus and Contribution of the Endoplasmic Reticulum to the SARS-CoV-2 Replication Complex

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1798
Author(s):  
Ted Hackstadt ◽  
Abhilash I. Chiramel ◽  
Forrest H. Hoyt ◽  
Brandi N. Williamson ◽  
Cheryl A. Dooley ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rna Synthesis ◽  
Light And Electron Microscopy ◽  
Brefeldin A ◽  
Viral Proteins ◽  
Protein Markers ◽  
Replication Complex ◽  
Golgi Fragmentation

A variety of immunolabeling procedures for both light and electron microscopy were used to examine the cellular origins of the host membranes supporting the SARS-CoV-2 replication complex. The endoplasmic reticulum has long been implicated as a source of membrane for the coronavirus replication organelle. Using dsRNA as a marker for sites of viral RNA synthesis, we provide additional evidence supporting ER as a prominent source of membrane. In addition, we observed a rapid fragmentation of the Golgi apparatus which is visible by 6 h and complete by 12 h post-infection. Golgi derived lipid appears to be incorporated into the replication organelle although protein markers are dispersed throughout the infected cell. The mechanism of Golgi disruption is undefined, but chemical disruption of the Golgi apparatus by brefeldin A is inhibitory to viral replication. A search for an individual SARS-CoV-2 protein responsible for this activity identified at least five viral proteins, M, S, E, Orf6, and nsp3, that induced Golgi fragmentation when expressed in eukaryotic cells. Each of these proteins, as well as nsp4, also caused visible changes to ER structure as shown by correlative light and electron microscopy (CLEM). Collectively, these results imply that specific disruption of the Golgi apparatus is a critical component of coronavirus replication.

scholarly journals Mutations within the ADP (E3-11.6K) Protein Alter Processing and Localization of ADP and the Kinetics of Cell Lysis of Adenovirus-Infected Cells

2003 ◽  
Vol 77 (14) ◽  
pp. 7764-7778 ◽  
Author(s):  
Ann E. Tollefson ◽  
Abraham Scaria ◽  
Baoling Ying ◽  
William S. M. Wold
Keyword(s):  
Golgi Apparatus ◽  
Nuclear Membrane ◽  
Intracellular Localization ◽  
A549 Cells ◽  
Cell Lysis ◽  
Glycosylation Site ◽  
Adenovirus Infection ◽  
Infected Cells ◽  
Lumenal Domain ◽  
Kinetics Of

ABSTRACT ADP (also known as E3-11.6K protein) is synthesized abundantly in late adenovirus infection and is required for efficient lysis of infected cells and release of viral progeny at the end of the viral replication cycle. ADP is a type III bitopic NendoCexo nuclear membrane and Golgi glycoprotein that is produced at high levels in late adenovirus infection (>24 h postinfection). We show pulse-chase and other studies indicating that ADP undergoes a complex process of N- and O-linked glycosylation and proteolytic cleavage. In order to further characterize ADP, a series of 23 deletion and point mutations has been constructed in the adenovirus serotype 2 adp gene and then built into a wild-type adenovirus background. These mutants were analyzed for processing and intracellular localization of ADP. Mutation of the single predicted N glycosylation site eliminated N glycosylation. Deletion of a region in ADP rich in serine and threonine residues reduced O glycosylation. In general, mutations within the lumenal domain of ADP resulted in lower protein stability; immunofluorescence assays indicated that these ADPs were primarily present in the Golgi apparatus. Viruses with mutations within the cytoplasmic-nucleoplasmic domain of ADP showed normal glycosylation patterns and protein abundance for ADP, but the protein was often found throughout cellular membranes rather than being localized specifically to the nuclear membrane and Golgi apparatus. The ADP virus mutants were analyzed by cell viability assays to determine the kinetics of cell lysis following infection of human A549 cells. In general, viruses with mutations within the lumenal domain of ADP display greatly reduced efficiencies of cell lysis. Viruses with large deletions in the cytoplasmic-nucleoplasmic domain of ADP retain much of their ability to lyse infected cells.

scholarly journals Reovirus Outer Capsid Protein μ1 Induces Apoptosis and Associates with Lipid Droplets, Endoplasmic Reticulum, and Mitochondria

2006 ◽  
Vol 80 (17) ◽  
pp. 8422-8438 ◽  
Author(s):  
Caroline M. Coffey ◽  
Alexander Sheh ◽  
Irene S. Kim ◽  
Kartik Chandran ◽  
Max L. Nibert ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Droplets ◽  
Apoptosis Induction ◽  
Direct Role ◽  
Transfected Cells ◽  
Intracellular Membranes ◽  
Reovirus Infection ◽  
Efficient Induction ◽  
Infected Cells ◽  
Induction Of Apoptosis

ABSTRACT The mechanisms by which reoviruses induce apoptosis have not been fully elucidated. Earlier studies identified the mammalian reovirus S1 and M2 genes as determinants of apoptosis induction. However, no published results have demonstrated the capacities of the proteins encoded by these genes to induce apoptosis, either independently or in combination, in the absence of reovirus infection. Here we report that the mammalian reovirus μ1 protein, encoded by the M2 gene, was sufficient to induce apoptosis in transfected cells. We also found that μ1 localized to lipid droplets, endoplasmic reticulum, and mitochondria in both transfected cells and infected cells. Two small regions encompassing amphipathic α-helices within a carboxyl-terminal portion of μ1 were necessary for efficient induction of apoptosis and association with lipid droplets, endoplasmic reticulum, and mitochondria in transfected cells. Induction of apoptosis by μ1 and its association with lipid droplets and intracellular membranes in transfected cells were abrogated whenμ 1 was coexpressed with σ3, with which it is known to coassemble. We propose that μ1 plays a direct role in the induction of apoptosis in infected cells and that this property may relate to the capacity of μ1 to associate with intracellular membranes. Moreover, during reovirus infection, association with σ3 may regulate apoptosis induction byμ 1.

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