scholarly journals Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes.

1988 ◽  
Vol 107 (6) ◽  
pp. 2075-2086 ◽  
Author(s):  
S Froshauer ◽  
J Kartenbeck ◽  
A Helenius
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Sindbis Virus ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Structural Proteins ◽  
Cytoplasmic Surface ◽  
Nucleoprotein Complexes ◽  
Secondary Lysosomes

Using morphological and cell biological techniques, we have shown that the RNA replicase of Semliki Forest and Sindbis virus (two closely related alphaviruses) is located in complex ribonucleoprotein structures associated with the cytoplasmic surface of modified secondary lysosomes and endosomes. These nucleoprotein complexes often form a bridge between the membrane of the endocytic vacuole and the rough endoplasmic reticulum where the synthesis of the structural proteins of these viruses occurs. The results suggest that these cytopathic vacuoles constitute sites not only for viral RNA synthesis, but also for translation of structural proteins, and for the assembly of nucleocapsids.

The polypeptides of rat liver nuclear envelope. II. Comparison of rat liver nuclear membrane polypeptides with those of the rough endoplasmic reticulum

1980 ◽  
Vol 43 (1) ◽  
pp. 269-277
Author(s):  
J.C. Richardson ◽  
A.H. Maddy
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Nuclear Membrane ◽  
Relative Proportion ◽  
Membrane System ◽  
Membrane Systems ◽  
Nuclear Membranes ◽  
Cytoplasmic Surface ◽  
Triton X

Nuclear envelopes are separated into pore-lamina and membrane sub-fractions by extraction in 2.0% Triton X-100 followed by pelleting of the pore-laminae. The polypeptides of these subfractions are then compared with those from isolated rough endoplasmic reticulum. The dispositions of individual polypeptides in the cytoplasmic surface of nuclear envelopes and rought endoplasmic reticulum were studied by lactoperoxidase-catalysed iodination. These studies show that although the nuclear membranes exhibit several homologies with the Triton-soluble polypeptides of the rough endoplasmic reticulum the relative proportion of individual polypeptides within the two systems are very largely different. The cytoplasmic surfaces of the 2 membrane systems show only 2 obvious homologies at 105 000 and 15 000 mol. wt and the overall impression is that, at least in rat liver, the outer nuclear membrane is very substantially differentiated from rough endoplasmic reticulum. It is concluded that the nuclear membranes may not be regarded as a mere continuum of the endoplasmic reticulum, but should be seen as a highly specialized membrane system in their own right.

scholarly journals The Porcine Deltacoronavirus Replication Organelle Comprises Double-Membrane Vesicles and Zippered Endoplasmic Reticulum with Double-Membrane Spherules

Viruses ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1030 ◽  
Author(s):  
Nicole Doyle ◽  
Philippa C. Hawes ◽  
Jennifer Simpson ◽  
Lorin H. Adams ◽  
Helena J. Maier
Keyword(s):  
Endoplasmic Reticulum ◽  
Rna Synthesis ◽  
Membrane Vesicles ◽  
Viral Rna ◽  
Double Membrane ◽  
Severe Diarrhea ◽  
The Usa ◽  
History Of ◽  
Host Cell Interactions ◽  
Positive Strand Rna

Porcine deltacoronavirus (PDCoV) was first identified in Hong Kong in 2012 from samples taken from pigs in 2009. PDCoV was subsequently identified in the USA in 2014 in pigs with a history of severe diarrhea. The virus has now been detected in pigs in several countries around the world. Following the development of tissue culture adapted strains of PDCoV, it is now possible to address questions regarding virus–host cell interactions for this genera of coronavirus. Here, we presented a detailed study of PDCoV-induced replication organelles. All positive-strand RNA viruses induce the rearrangement of cellular membranes during virus replication to support viral RNA synthesis, forming the replication organelle. Replication organelles for the Alpha-, Beta-, and Gammacoronavirus genera have been characterized. All coronavirus genera induced the formation of double-membrane vesicles (DMVs). In addition, Alpha- and Betacoronaviruses induce the formation of convoluted membranes, while Gammacoronaviruses induce the formation of zippered endoplasmic reticulum (ER) with tethered double-membrane spherules. However, the structures induced by Deltacoronaviruses, particularly the presence of convoluted membranes or double-membrane spherules, are unknown. Initially, the dynamics of PDCoV strain OH-FD22 replication were assessed with the onset of viral RNA synthesis, protein synthesis, and progeny particle release determined. Subsequently, virus-induced membrane rearrangements were identified in infected cells by electron microscopy. As has been observed for all other coronaviruses studied to date, PDCoV replication was found to induce the formation of double-membrane vesicles. Significantly, however, PDCoV replication was also found to induce the formation of regions of zippered endoplasmic reticulum, small associated tethered vesicles, and double-membrane spherules. These structures strongly resemble the replication organelle induced by avian Gammacoronavirus infectious bronchitis virus.

scholarly journals Biosynthesis of lipoprotein: location of nascent apoAI and apoB in the rough endoplasmic reticulum of chicken hepatocytes

1992 ◽  
Vol 117 (6) ◽  
pp. 1161-1169 ◽  
Author(s):  
JL Dixon ◽  
R Chattapadhyay ◽  
T Huima ◽  
CM Redman ◽  
D Banerjee
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Microscopic Examination ◽  
Microsomal Membrane ◽  
Golgi Cell ◽  
Lipoprotein Particles ◽  
Morphological Findings ◽  
Cytoplasmic Surface ◽  
Luminal Side ◽  
Cell Fractions

Our previous studies showed that in hepatic RER of young chickens, nascent apoAI is not associated with lipoprotein particles and only becomes part of these lipoprotein structures in the Golgi. In this study, we have used three different methodologies to determine the locations of apoAI and apoB in the RER and compared them to that of albumin. Immunoelectron microscopic examination of the RER cell fractions showed that both apoAI and apoB were associated only with the RER membrane whereas albumin was located both within the lumen and on the limiting membrane of the vesicles. To examine the possibility of membrane integration of nascent apoAI and apoB in the RER, we administered L-[3H]leucine to young chickens for 10 min, isolated RER, treated this cell fraction with buffers of varying pH, and measured the release of radioactive albumin, apoAI, and apoB. The majority of nascent apoAI (64%), nascent apoB (100%), and nascent albumin (97%) was released from RER vesicles at pH 11.2, suggesting that, like albumin, apolipoproteins are not integrated within the membrane. To determine if nascent apoproteins are exposed to the cytoplasmic surface, we administered L-[3H]leucine to young chickens and at various times isolated RER and Golgi cell fractions. Radioactive RER and Golgi cell fractions were treated with exogenous protease and the percent of nascent apoAI and apoB accessible to proteolysis was determined and compared to that of albumin. At 5, 10, and 20 min of labeling, 35-56% of nascent apoAI and 60-75% of apoB in RER were degraded, while albumin was refractive to this treatment. At all times both apolipoproteins and albumin present in Golgi cell fractions were protected from proteolysis. These biochemical and morphological findings indicate that apoAI and apoB are associated with the rough microsomal membrane and are partially exposed to the cytoplasmic surface at early stages of secretion. They may later enter the luminal side of the ER and, on entering the Golgi, form lipoprotein particles.

scholarly journals The arterivirus replicase is the only viral protein required for genome replication and subgenomic mRNA transcription

2000 ◽  
Vol 81 (10) ◽  
pp. 2491-2496 ◽  
Author(s):  
Richard Molenkamp ◽  
Hans van Tol ◽  
Babette C. D. Rozier ◽  
Yvonne van der Meer ◽  
Willy J. M. Spaan ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Point Mutations ◽  
Equine Arteritis Virus ◽  
Viral Rna ◽  
Structural Proteins ◽  
Genome Replication ◽  
Mrna Transcription ◽  
Replication Complex ◽  
N Protein ◽  
Wild Type Phenotype

Equine arteritis virus (EAV) (Arteriviridae) encodes several structural proteins. Whether any of these also function in viral RNA synthesis is unknown. For the related mouse hepatitis coronavirus (MHV), it has been suggested that the nucleocapsid protein (N) is involved in viral RNA synthesis. As described for MHV, we established that the EAV N protein colocalizes with the viral replication complex, suggesting a role in RNA synthesis. Using an infectious cDNA clone, point mutations and deletions were engineered in the EAV genome to disrupt the expression of each of the structural genes. All structural proteins, including N, were found to be dispensable for genome replication and subgenomic mRNA transcription. We also constructed a mutant in which translation of the intraleader ORF was disrupted. This mutant had a wild-type phenotype, indicating that, at least in cell culture, the product of this ORF does not play a role in the EAV replication cycle.

Altered pattern of viral RNA synthesis in cells infected with standard and defective Sindbis virus

Virology ◽  
1973 ◽  
Vol 51 (1) ◽  
pp. 85-93 ◽  
Author(s):  
B.T. Eaton ◽  
P. Faulkner
Keyword(s):  
Sindbis Virus ◽  
Rna Synthesis ◽  
Viral Rna ◽  
In Cells

scholarly journals Infectious Bronchitis Virus Generates Spherules from Zippered Endoplasmic Reticulum Membranes

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Helena J. Maier ◽  
Philippa C. Hawes ◽  
Eleanor M. Cottam ◽  
Judith Mantell ◽  
Paul Verkade ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Infectious Bronchitis Virus ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Membrane Vesicles ◽  
Viral Rna ◽  
Double Membrane ◽  
Infectious Bronchitis ◽  
Positive Sense ◽  
Infected Cells

ABSTRACTReplication of positive-sense RNA viruses is associated with the rearrangement of cellular membranes. Previous work on the infection of tissue culture cell lines with the betacoronaviruses mouse hepatitis virus and severe acute respiratory syndrome coronavirus (SARS-CoV) showed that they generate double-membrane vesicles (DMVs) and convoluted membranes as part of a reticular membrane network. Here we describe a detailed study of the membrane rearrangements induced by the avian gammacoronavirus infectious bronchitis virus (IBV) in a mammalian cell line but also in primary avian cells and in epithelial cells ofex vivotracheal organ cultures. In all cell types, structures novel to IBV infection were identified that we have termed zippered endoplasmic reticulum (ER) and spherules. Zippered ER lacked luminal space, suggesting zippering of ER cisternae, while spherules appeared as uniform invaginations of zippered ER. Electron tomography showed that IBV-induced spherules are tethered to the zippered ER and that there is a channel connecting the interior of the spherule with the cytoplasm, a feature thought to be necessary for sites of RNA synthesis but not seen previously for membrane rearrangements induced by coronaviruses. We also identified DMVs in IBV-infected cells that were observed as single individual DMVs or were connected to the ER via their outer membrane but not to the zippered ER. Interestingly, IBV-induced spherules strongly resemble confirmed sites of RNA synthesis for alphaviruses, nodaviruses, and bromoviruses, which may indicate similar strategies of IBV and these diverse viruses for the assembly of RNA replication complexes.IMPORTANCEAll positive-sense single-stranded RNA viruses induce rearranged cellular membranes, providing a platform for viral replication complex assembly and protecting viral RNA from cellular defenses. We have studied the membrane rearrangements induced by an important poultry pathogen, the gammacoronavirus infectious bronchitis virus (IBV). Previous work studying closely related betacoronaviruses identified double-membrane vesicles (DMVs) and convoluted membranes (CMs) derived from the endoplasmic reticulum (ER) in infected cells. However, the role of DMVs and CMs in viral RNA synthesis remains unclear because these sealed vesicles lack a means of delivering viral RNA to the cytoplasm. Here, we characterized structures novel to IBV infection: zippered ER and small vesicles tethered to the zippered ER termed spherules. Significantly, spherules contain a channel connecting their interior to the cytoplasm and strongly resemble confirmed sites of RNA synthesis for other positive-sense RNA viruses, making them ideal candidates for the site of IBV RNA synthesis.

Observations on the Blood of the Australian Lungfish, Neoceratodus-Forsteri Klefft .1. Ultrastructure of Granulocytes, Monocytes and Thrombocytes

1990 ◽  
Vol 38 (2) ◽  
pp. 131 ◽  
Author(s):  
PM Hine ◽  
RJG Lester ◽  
JM Wain
Keyword(s):  
Endoplasmic Reticulum ◽  
Mast Cells ◽  
Internal Structure ◽  
Rough Endoplasmic Reticulum ◽  
Dense Core ◽  
Dense Core Vesicles ◽  
Dense Granules ◽  
Secondary Lysosomes ◽  
Australian Lungfish

The ultrastructure of eosinophils, heterophils, neutrophils, basophils, monocytes and thrombocytes is reported from the Australian lungfish, Neoceratodus forsteri. Eosinophils have an eccentric simple or bilobed nucleus and granules with an axial rod-like core similar to those of elasmobranchs, birds and mammals. Heterophils are polymorphonuclear with a prominent centrosome, and eosinophilic granules of many forms, some of which have a dense core that may become fragmented. Neutrophils are aiso polymorphonuclear, and are agranular or contain azurophilic granules and many dense core vesicles. Basophils have a central or eccentric nucleus and dense granules with a complex internal structure similar to those of mast cells of mammals. Monocytes have an indented or reniform nucleus, a few granules, rough endoplasmic reticulum and filiform surface projections; they contain secondary lysosomes and are similar to monocytes of other vertebrates. Thrombocytes contain granules with a fibrillar substructure similar to those of other lungfishes. Inter-relationsips of granulocytes, particularly between heterophils and neutrophils, are discussed.

scholarly journals Requirements for the insertion of the Sindbis envelope glycoproteins into the endoplasmic reticulum membrane.

1979 ◽  
Vol 81 (1) ◽  
pp. 154-162 ◽  
Author(s):  
D F Wirth ◽  
H F Lodish ◽  
P W Robbins
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Bilayer ◽  
Sindbis Virus ◽  
Core Protein ◽  
Structural Proteins ◽  
Temperature Sensitive ◽  
Endoplasmic Reticulum Membrane ◽  
Amino Terminal ◽  
Cytoplasmic Side ◽  
In Cells

Previous work has shown that the Sindbis structural proteins, core, the internal protein, and PE2 and E1, the integral membrane glycoproteins are synthesized as a polyprotein from a 26S mRNA; core PE2 and E1 are derived by proteolytic cleavage of a nascent chain. Newly synthesized core protein remains on the cytoplasmic side of the endoplasmic reticulum while newly synthesized PE2 and E1 are inserted into the lipid bilayer, presumably via their amino-termini. PE2 and E1 are glycosylated as nascent chains. Here, we examine a temperature-sensitive mutant of Sindbis virus which fails to cleave the structural proteins, resulting in the production of a polyprotein of 130,000 mol wt in which the amino-termini of PE2 and E1 are internal to the protein. Although the envelope sequences are present in this protein, it is not inserted into the endoplasmic reticulum bilayer, but remains on the cytoplasmic side as does the core protein in cells infected with wild-type Sindbis virus. We have also examined the fate of PE2 and E1 in cells treated with tunicamycin, an inhibitor of glycosylation. Unglycosylated PE2 and E1 are inserted normally into the lipid bilayer as are the glycosylated proteins. These results are consistent with the notion that a specific amino-terminal sequence is required for the proper insertion of membrane proteins into the endoplasmic reticulum bilayer, but that glycosylation is not required for this insertion.

scholarly journals The porcine deltacoronavirus replication organelle comprises double membrane vesicles and zippered endoplasmic reticulum with double membrane spherules

2019 ◽  
Author(s):  
Nicole Doyle ◽  
Philippa C. Hawes ◽  
Jennifer Simpson ◽  
Lorin H. Adams ◽  
Helena J. Maier
Keyword(s):  
Endoplasmic Reticulum ◽  
Rna Synthesis ◽  
Membrane Vesicles ◽  
Viral Rna ◽  
Double Membrane ◽  
Severe Diarrhea ◽  
The Usa ◽  
History Of ◽  
Host Cell Interactions ◽  
Positive Strand Rna

AbstractPorcine deltacoronavirus (PDCoV) was first identified in Hong Kong in 2012 from samples taken from pigs in 2009. PDCoV was subsequently identified in the USA in 2014 in pigs with a history of severe diarrhea and the virus has now been detected in pigs in several countries around the world. Following the development of tissue culture adapted strains of PDCoV, it is now possible to begin to address questions regarding virus-host cell interactions for this genera of coronavirus. Here we present a detailed study of PDCoV induced replication organelles. All positive strand RNA viruses induce the rearrangement of cellular membranes during virus replication to support viral RNA synthesis, forming the replication organelle. Replication organelles for the Alpha-, Beta- and Gammacoronavirus genera have been characterized. However the structures induced by deltacoronaviruses, in particular the presence of convoluted membranes or double membrane spherules, are unknown. Initially, the dynamics of PDCoV strain OH-FD22 replication were assessed with the onset of viral RNA synthesis, protein synthesis and progeny particle release determined. Subsequently, virus induced membrane rearrangements were identified in infected cells by electron microscopy. As has been observed for all other coronaviruses studied to date, PDCoV replication was found to induce the formation of double membrane vesicles. Significantly however, PDCoV replication was also found to induce the formation of regions of zippered endoplasmic reticulum and small associated tethered vesicles, double membrane spherules. These structures strongly resemble the replication organelle induced by avian Gammacoronavirus infectious bronchitis virus.

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