scholarly journals Zika virus replication in glioblastoma cells: electron microscopic tomography shows 3D arrangement of endoplasmic reticulum, replication organelles, and viral ribonucleoproteins

2021 ◽  
Author(s):  
Johannes Wieland ◽  
Stefan Frey ◽  
Ulrich Rupp ◽  
Sandra Essbauer ◽  
Rüdiger Groß ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Line ◽  
Zika Virus ◽  
Structural Changes ◽  
Electron Tomography ◽  
Glioblastoma Cells ◽  
Electron Microscopic ◽  
N Protein ◽  
Ribonucleoprotein Complexes ◽  
Infected Cells

AbstractStructural changes of two patient-derived glioblastoma cell lines after Zika virus infection were investigated using scanning transmission electron tomography on high-pressure-frozen, freeze-substituted samples. In Zika-virus-infected cells, Golgi structures were barely visible under an electron microscope, and viral factories appeared. The cytosol outside of the viral factories resembled the cytosol of uninfected cells. The viral factories contained largely deranged endoplasmic reticulum (ER), filled with many so-called replication organelles consisting of a luminal vesicle surrounded by the ER membrane. Viral capsids were observed in the vicinity of the replication organelles (cell line #12537 GB) or in ER cisternae at large distance from the replication organelles (cell line #15747 GB). Near the replication organelles, we observed many about 100-nm-long filaments that may represent viral ribonucleoprotein complexes (RNPs), which consist of the RNA genome and N protein oligomers. In addition, we compared Zika-virus-infected cells with cells infected with a phlebovirus (sandfly fever Turkey virus). Zika virions are formed in the ER, whereas phlebovirus virions are assembled in the Golgi apparatus. Our findings will help to understand the replication cycle in the virus factories and the building of the replication organelles in glioblastoma cells.

scholarly journals Super Resolution Microscopy and Deep Learning Identify Zika Virus Reorganization of the Endoplasmic Reticulum

2020 ◽  
Author(s):  
Rory K. M. Long ◽  
Kathleen P. Moriarty ◽  
Ben Cardoen ◽  
Guang Gao ◽  
A. Wayne Vogl ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Deep Learning ◽  
Viral Replication ◽  
Zika Virus ◽  
Deep Neural Networks ◽  
Super Resolution ◽  
Zikv Infection ◽  
Subcellular Organelle ◽  
Infected Cells ◽  
Super Resolution Microscopy

AbstractThe endoplasmic reticulum (ER) is a complex subcellular organelle composed of diverse structures such as tubules, sheets and tubular matrices. Flaviviruses such as Zika virus (ZIKV) induce reorganization of endoplasmic reticulum (ER) membranes to facilitate viral replication. Here, using 3D super resolution microscopy, ZIKV infection is shown to induce the formation of dense tubular matrices associated with viral replication in the central ER. Viral non-structural proteins NS4B and NS2B associate with replication complexes within the ZIKV-induced tubular matrix and exhibit distinct ER distributions outside this central ER region. Deep neural networks trained to identify ZIKV-infected versus mock-infected cells successfully identified ZIKV-induced central ER tubular matrices as a determinant of viral infection. Super resolution microscopy and deep learning are therefore able to identify and localize morphological features of the ER and may be of use to screen for inhibitors of infection by ER-reorganizing viruses.

scholarly journals Atlastin Endoplasmic Reticulum-Shaping Proteins Facilitate Zika Virus Replication

2019 ◽  
Vol 93 (23) ◽  
Author(s):  
Blandine Monel ◽  
Maaran Michael Rajah ◽  
Mohamed Lamine Hafirassou ◽  
Samy Sid Ahmed ◽  
Julien Burlaud-Gaillard ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Viral Replication ◽  
Zika Virus ◽  
Antiviral Treatment ◽  
Viral Proteins ◽  
Neurological Complications ◽  
Cytopathic Effects ◽  
Replication Sites ◽  
Infected Cells ◽  
Er Membrane

ABSTRACT The endoplasmic reticulum (ER) is the site for Zika virus (ZIKV) replication and is central to the cytopathic effects observed in infected cells. ZIKV induces the formation of ER-derived large cytoplasmic vacuoles followed by “implosive” cell death. Little is known about the nature of the ER factors that regulate flavivirus replication. Atlastins (ATL1, -2, and -3) are dynamin-related GTPases that control the structure and the dynamics of the ER membrane. We show here that ZIKV replication is significantly decreased in the absence of ATL proteins. The appearance of infected cells is delayed, the levels of intracellular viral proteins and released virus are reduced, and the cytopathic effects are strongly impaired. We further show that ATL3 is recruited to viral replication sites and interacts with the nonstructural viral proteins NS2A and NS2B3. Thus, proteins that shape and maintain the ER tubular network ensure efficient ZIKV replication. IMPORTANCE Zika virus (ZIKV) is an emerging virus associated with Guillain-Barré syndrome, and fetal microcephaly as well as other neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We found that endoplasmic reticulum (ER)-shaping atlastin proteins (ATL1, -2, and -3), which induce ER membrane fusion, facilitate ZIKV replication. We show that ATL3 is recruited to the viral replication site and colocalize with the viral proteins NS2A and NS2B3. The results provide insights into host factors used by ZIKV to enhance its replication.

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 Super resolution microscopy and deep learning identify Zika virus reorganization of the endoplasmic reticulum

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Rory K. M. Long ◽  
Kathleen P. Moriarty ◽  
Ben Cardoen ◽  
Guang Gao ◽  
A. Wayne Vogl ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Deep Learning ◽  
Viral Replication ◽  
Viral Infection ◽  
Zika Virus ◽  
Deep Neural Networks ◽  
Super Resolution ◽  
Zikv Infection ◽  
Infected Cells ◽  
Super Resolution Microscopy

AbstractThe endoplasmic reticulum (ER) is a complex subcellular organelle composed of diverse structures such as tubules, sheets and tubular matrices. Flaviviruses such as Zika virus (ZIKV) induce reorganization of ER membranes to facilitate viral replication. Here, using 3D super resolution microscopy, ZIKV infection is shown to induce the formation of dense tubular matrices associated with viral replication in the central ER. Viral non-structural proteins NS4B and NS2B associate with replication complexes within the ZIKV-induced tubular matrix and exhibit distinct ER distributions outside this central ER region. Deep neural networks trained to distinguish ZIKV-infected versus mock-infected cells successfully identified ZIKV-induced central ER tubular matrices as a determinant of viral infection. Super resolution microscopy and deep learning are therefore able to identify and localize morphological features of the ER and allow for better understanding of how ER morphology changes due to viral infection.

scholarly journals Membrane Requirements for Uridylylation of the Poliovirus VPg Protein and Viral RNA Synthesis In Vitro

2003 ◽  
Vol 77 (21) ◽  
pp. 11408-11416 ◽  
Author(s):  
Mark H. Fogg ◽  
Natalya L. Teterina ◽  
Ellie Ehrenfeld
Keyword(s):  
Membrane Trafficking ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Viral Rna ◽  
Chain Elongation ◽  
Electron Microscopic ◽  
Ribonucleoprotein Complexes ◽  
Cell Extracts ◽  
Infected Cells

ABSTRACT Efficient translation of poliovirus (PV) RNA in uninfected HeLa cell extracts generates all of the viral proteins required to carry out viral RNA replication and encapsidation and to produce infectious virus in vitro. In infected cells, viral RNA replication occurs in ribonucleoprotein complexes associated with clusters of vesicles that are formed from preexisting intracellular organelles, which serve as a scaffold for the viral RNA replication complex. In this study, we have examined the role of membranes in viral RNA replication in vitro. Electron microscopic and biochemical examination of extracts actively engaged in viral RNA replication failed to reveal a significant increase in vesicular membrane structures or the protective aggregation of vesicles observed in PV-infected cells. Viral, nonstructural replication proteins, however, bind to heterogeneous membrane fragments in the extract. Treatment of the extracts with nonionic detergents, a membrane-altering inhibitor of fatty acid synthesis (cerulenin), or an inhibitor of intracellular membrane trafficking (brefeldin A) prevents the formation of active replication complexes in vitro, under conditions in which polyprotein synthesis and processing occur normally. Under all three of these conditions, synthesis of uridylylated VPg to form the primer for initiation of viral RNA synthesis, as well as subsequent viral RNA replication, was inhibited. Thus, although organized membranous structures morphologically similar to the vesicles observed in infected cells do not appear to form in vitro, intact membranes are required for viral RNA synthesis, including the first step of forming the uridylylated VPg primer for RNA chain elongation.

scholarly journals Organization of the endoplasmic reticulum in cells of effective and ineffective pea nodules (Pisum sativum L.)

2019 ◽  
Vol 17 (4) ◽  
pp. 5-14
Author(s):  
Anna V. Tsyganova ◽  
Viktor E. Tsyganov
Keyword(s):  
Endoplasmic Reticulum ◽  
Pisum Sativum ◽  
Structural Changes ◽  
Nodule Development ◽  
Wild Type ◽  
Meristematic Cells ◽  
Pisum Sativum L ◽  
Transmission Electron ◽  
Infected Cells ◽  
Membrane Bound

Background. The endoplasmic reticulum (ER) is the largest membrane-bound organelle, which plays an important role in the functioning of a plant cell and participates in its differentiation. Materials and methods. Using the methods of transmission electron microscopy, the morphological features and dynamics of structural changes in the ER in symbiotic nodules of pea (Pisum sativum L.) wild-type and mutants blocked at different stages of nodule development were studied. Results. ER developed from a network of individual tubules in meristematic cells, to a developed network of cisterns around the nucleus and plasmalemma, and a network of granular and smooth tubules accompanying infection structures in colonized and infected cells and symbiosomes in infected cells. Conclusions. A correlation was found between the level of development of the ER network and the degree of bacteroid differentiation.

scholarly journals Endoplasmic reticulum remains continuous and undergoes sheet-to-tubule transformation during cell division in mammalian cells

2007 ◽  
Vol 179 (5) ◽  
pp. 895-909 ◽  
Author(s):  
Maija Puhka ◽  
Helena Vihinen ◽  
Merja Joensuu ◽  
Eija Jokitalo
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Division ◽  
3D Modeling ◽  
Mammalian Cells ◽  
Structural Changes ◽  
Electron Tomography ◽  
Morphological Changes ◽  
Homogenous Distribution ◽  
Membrane Bound ◽  
Cycle Dependent

The endoplasmic reticulum (ER) is a multifaceted cellular organelle both structurally and functionally, and its cell cycle–dependent morphological changes are poorly understood. Our quantitative confocal and EM analyses show that the ER undergoes dramatic reorganization during cell division in cultured mammalian cells as mitotic ER profiles become shorter and more branched. 3D modeling by electron tomography reveals that the abundant interphase structures, sheets, are lost and subsequently transform into a branched tubular network that remains continuous. This is confirmed by observing the most prominent ER subdomain, the nuclear envelope (NE). A NE marker protein spreads to the mitotic ER tubules, although it does not show a homogenous distribution within the network. We mimicked the mitotic ER reorganization using puromycin to strip the membrane-bound ribosomes from the interphase ER corresponding to the observed loss of ribosomes normally occurring during mitosis. We propose that the structural changes in mitotic ER are linked to ribosomal action on the ER membranes.

Ultrastructural Changes in Pig Hepatocytes During the Transitional Period from Late Foetal to Early Neonatal Life

1969 ◽  
Vol 4 (2) ◽  
pp. 381-395
Author(s):  
M. B. BISCHOFF ◽  
W. R. RICHTER ◽  
R. J. STEIN
Keyword(s):  
Endoplasmic Reticulum ◽  
Microscopic Study ◽  
Golgi Complex ◽  
Structural Changes ◽  
Bile Canaliculus ◽  
Transitional Period ◽  
Ultrastructural Changes ◽  
Synthetic Activity ◽  
Electron Microscopic ◽  
Neonatal Life

A light-and electron-microscopic study of pig hepatocytes from late prenatal to early neonatal animals shows changes which reflect an increasing rate of synthetic activity. The granular endoplasmic reticulum (ER) in the prenatal pig hepatocyte is situated along the periphery of the cytoplasm and in the region immediately surrounding the nucleus. Mitochondria are most abundant in the area adjacent to the nucleus, while the Golgi complex is generally located in the region of the bile canaliculus. The remaining portion of the hepatocyte is occupied with glycogen. A few hours after birth the hepatocyte increases about twofold in size with the nucleus shifting from a peripheral to a more centrally located position. The glycogen decreases quickly coincident with a rapid increase in the amount of granular ER and the dispersion of the mitochondria throughout the cell. The Golgi complex becomes distended and numerous vesicles appear in its immediate vicinity containing a moderately dense material. Numerous peribiliary inclusions appear during the second postnatal day. These structural changes are an indication of the increased synthetic activity occurring within the hepatocytes of rapidly developing animals.

Electron Microscopic study of two strains of bovine viral diarrhea virus (BVDV)

1989 ◽  
Vol 47 ◽  
pp. 1030-1031
Author(s):  
R. Alain ◽  
S. Cardin ◽  
L. Berthiaume ◽  
J. Lecomte
Keyword(s):  
Endoplasmic Reticulum ◽  
Bovine Viral Diarrhea Virus ◽  
Bovine Viral Diarrhea ◽  
Icosahedral Symmetry ◽  
Electron Microscopic ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Wide Range ◽  
Infected Cells ◽  
Viral Diarrhea Virus

Bovine viral diarrhea virus (BVDV) is an economically important pathogen of cattle throughout the world, causing a wide range of clinical syndromes. Virion is spherical, 40-70 nm in diameter, with an envelope tightly applied to a spherical nucleocapsid 25-35 nm in diameter with icosahedral symmetry (figure 1 and 2) (Matthews, 1979) . It is presently included in the Togavlridae family, genus Pneumovirus but Collett et al. (1988) have proposed to group this genus in the Flaviviridae family. In the present study, two strains of BVDV were compared by electron microcospy ultrathin sections: one cytopathogenic, NADL strain, and the other non-cytopathogenic, NY-1 strain. These were Inoculated on Madney-Darby Bovine Kidney (MDBK) cells to a multiplicity of infection (m.o.i) between 3,0 and 5,0. Control and virus infected cells were fixed with glutaraldehyde (2,5% in cacodylate buffer) at 24 , 48, 72 and 96 h following incubation at 37°C. Cells were postfixed with osmium tetroxide and embedded in Vestopal.The NADL-CP strain infected cells showed vacuolization of the endoplasmic reticulum, ribosomal activity in the cytoplasm and appearance of virus particles 48h after infection (figure 3). At 72h, an expensive vacuolization was apparent (figure 4). Maximum viral production was seen 96 h after infection (figure 5). From 24 to 72 h post infection, the NY-l-NCP strain of BVD infected cells showed normal vacuolization like in control cells. They produced weak vacuolization with few particles, not apparent before 96h after infection (figure 6). Viral particles of 45-50 nm in endoplasmic reticulum vacuoles were in general homogenous in size while those in smooth membranes, probably of cellular origin, heterogenous in size.

scholarly journals Human Cytomegalovirus Specifically Controls the Levels of the Endoplasmic Reticulum Chaperone BiP/GRP78, Which Is Required for Virion Assembly

2007 ◽  
Vol 82 (1) ◽  
pp. 31-39 ◽  
Author(s):  
Nicholas J. Buchkovich ◽  
Tobi G. Maguire ◽  
Yongjun Yu ◽  
Adrienne W. Paton ◽  
James C. Paton ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Microscopic Analysis ◽  
Electron Microscopic ◽  
Viral Protein Synthesis ◽  
Virion Assembly ◽  
Infected Cells ◽  
Virion Formation

ABSTRACT The endoplasmic reticulum (ER) chaperone BiP/GRP78 regulates ER function and the unfolded protein response (UPR). Human cytomegalovirus infection of human fibroblasts induces the UPR but modifies it to benefit viral replication. BiP/GRP78 protein levels are tightly regulated during infection, rising after 36 h postinfection (hpi), peaking at 60 hpi, and decreasing thereafter. To determine the effects of this regulation on viral replication, BiP/GRP78 was depleted using the SubAB subtilase cytotoxin, which rapidly and specifically cleaves BiP/GRP78. Toxin treatment of infected cells for 12-h periods beginning at 36, 48, 60, and 84 hpi caused complete loss of BiP but had little effect on viral protein synthesis. However, progeny virion formation was significantly inhibited, suggesting that BiP/GRP78 is important for virion formation. Electron microscopic analysis showed that infected cells were resistant to the toxin and showed none of the cytotoxic effects seen in uninfected cells. However, all viral activity in the cytoplasm ceased, with nucleocapsids remaining in the nucleus or concentrated in the cytoplasmic space just outside of the outer nuclear membrane. These data suggest that one effect of the controlled expression of BiP/GRP78 in infected cells is to aid in cytoplasmic virion assembly and egress.

Sign in / Sign up

Export Citation Format

Share Document