scholarly journals Cowpea Mosaic Virus 32- and 60-Kilodalton Replication Proteins Target and Change the Morphology of Endoplasmic Reticulum Membranes

2002 ◽  
Vol 76 (12) ◽  
pp. 6293-6301 ◽  
Author(s):  
Jan E. Carette ◽  
Jan van Lent ◽  
Stuart A. MacFarlane ◽  
Joan Wellink ◽  
Ab van Kammen
Keyword(s):  
Endoplasmic Reticulum ◽  
Mosaic Virus ◽  
Close Association ◽  
Tobacco Rattle Virus ◽  
Viral Proteins ◽  
Cowpea Mosaic Virus ◽  
Replication Proteins ◽  
Replication Complex ◽  
Cytotoxic Proteins ◽  
Cowpea Protoplasts

ABSTRACT Cowpea mosaic virus (CPMV) replicates in close association with small membranous vesicles that are formed by rearrangements of intracellular membranes. To determine which of the viral proteins are responsible for the rearrangements of membranes and the attachment of the replication complex, we have expressed individual CPMV proteins encoded by RNA1 in cowpea protoplasts by transient expression and in Nicotiana benthamiana plants by using the tobacco rattle virus (TRV) expression vector. The 32-kDa protein (32K) and 60K, when expressed individually, accumulate in only low amounts but are found associated with membranes mainly derived from the endoplasmic reticulum (ER). 24K and 110K are freely soluble and accumulate to high levels. With the TRV vector, expression of 32K and 60K results in rearrangement of ER membranes. Besides, expression of 32K and 60K results in necrosis of the inoculated N. benthamiana leaves, suggesting that 32K and 60K are cytotoxic proteins. On the other hand, during CPMV infection 32K and 60K accumulate to high levels without causing necrosis.

scholarly journals Brome Mosaic Virus RNA Replication Proteins 1a and 2a Colocalize and 1a Independently Localizes on the Yeast Endoplasmic Reticulum

1999 ◽  
Vol 73 (12) ◽  
pp. 10303-10309 ◽  
Author(s):  
María Restrepo-Hartwig ◽  
Paul Ahlquist
Keyword(s):  
Endoplasmic Reticulum ◽  
Mosaic Virus ◽  
Plant Cells ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
Replication Proteins ◽  
Replication Complex ◽  
Virus Rna ◽  
Positive Strand Rna

ABSTRACT The universal membrane association of positive-strand RNA virus RNA replication complexes is implicated in their function, but the intracellular membranes used vary among viruses. Brome mosaic virus (BMV) encodes two mutually interacting RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and the polymerase-like 2a protein. In cells from the natural plant hosts of BMV, 1a and 2a colocalize on the endoplasmic reticulum (ER). 1a and 2a also direct BMV RNA replication and subgenomic mRNA synthesis in the yeast Saccharomyces cerevisiae, but whether the distribution of 1a, 2a, and active replication complexes in yeast duplicates that in plant cells has not been determined. For yeast expressing 1a and 2a and replicating BMV genomic RNA3, we used double-label confocal immunofluorescence to define the localization of 1a, 2a, and viral RNA and to explore the determinants of replication complex targeting. As in plant cells, 1a and 2a colocalized on and were retained on the yeast ER, with no detectable accumulation in the Golgi apparatus. 1a and 2a were distributed over most of the ER surface, with strongest accumulation on the perinuclear ER. In vivo labeling with bromo-UTP showed that the sites of 1a and 2a accumulation were the sites of nascent viral RNA synthesis. In situ hybridization showed that completed viral RNA products accumulated predominantly in the immediate vicinity of replication complexes but that some, possibly more mature cells also accumulated substantial viral RNA in the surrounding cytoplasm distal to replication complexes. Additionally, we find that 1a localizes to the ER when expressed in the absence of other viral factors. These results show that BMV RNA replication in yeast duplicates the normal localization of replication complexes, reveal the intracellular distribution of RNA replication products, and show that 1a is at least partly responsible for the ER localization and retention of the RNA 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.

Interactions of Cowpea Mosaic Virus with Cowpea Protoplasts)

1983 ◽  
Vol 107 (2) ◽  
pp. 182-191
Author(s):  
A. G. Smith ◽  
D. P. Durand ◽  
J. H. Hill
Keyword(s):  
Mosaic Virus ◽  
Cowpea Mosaic Virus ◽  
Cowpea Protoplasts

scholarly journals Expression of Bottom Component RNA of Cowpea Mosaic Virus in Cowpea Protoplasts

1980 ◽  
Vol 36 (2) ◽  
pp. 366-373 ◽  
Author(s):  
Geertje Rezelman ◽  
Rob Goldbach ◽  
Albert Van Kammen
Keyword(s):  
Mosaic Virus ◽  
Cowpea Mosaic Virus ◽  
Cowpea Protoplasts

scholarly journals Red clover necrotic mosaic virus replication proteins accumulate at the endoplasmic reticulum

Virology ◽  
2004 ◽  
Vol 320 (2) ◽  
pp. 276-290 ◽  
Author(s):  
Katherine A Turner ◽  
Tim L Sit ◽  
Anton S Callaway ◽  
Nina S Allen ◽  
Steven A Lommel
Keyword(s):  
Endoplasmic Reticulum ◽  
Mosaic Virus ◽  
Virus Replication ◽  
Red Clover ◽  
Replication Proteins

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.

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