scholarly journals The SARS CoV-1 3a protein disrupts Golgi complex morphology and cargo trafficking

2021 ◽  
Author(s):  
Rex R. Gonzales ◽  
Carolyn E. Machamer
Keyword(s):  
Infectious Bronchitis Virus ◽  
Viral Proteins ◽  
Transfected Cells ◽  
Infectious Bronchitis ◽  
E Protein ◽  
Complex Morphology ◽  
Universal Feature ◽  
Infected Cells ◽  
Intermediate Compartment ◽  
Acidic Compartments

Coronaviruses assemble by budding into the endoplasmic reticulum-Golgi intermediate compartment, but the pathway of egress from infected cells is not well understood. Efficient egress of infectious bronchitis virus (a gamma coronavirus, CoV) requires neutralization of Golgi pH by the envelope (E) protein. This results in reduced rates of cargo traffic and disrupts Golgi morphology, but it protects the spike protein from aberrant proteolysis. The severe acute respiratory syndrome (SARS) CoV-1 E protein does not disrupt the Golgi, however. We show here that in transfected cells, the ORF3a protein of SARS CoV-1 disrupts Golgi morphology, cargo trafficking and luminal pH. Unlike the infectious bronchitis virus E protein, these functions of the SARS CoV-1 3a protein appear to require its viroporin activity. Thus, neutralization of acidic compartments may be a universal feature of CoV infection, although different viral proteins and mechanisms may be used to achieve this outcome.

scholarly journals The Infectious Bronchitis Virus Coronavirus Envelope Protein Alters Golgi pH to Protect Spike Protein and Promote Release of Infectious Virus

2018 ◽  
Author(s):  
Jason W. Westerbeck ◽  
Carolyn E. Machamer
Keyword(s):  
Infectious Bronchitis Virus ◽  
Secretory Pathway ◽  
Infectious Virus ◽  
Host Cells ◽  
Spike Protein ◽  
Channel Activity ◽  
Infectious Bronchitis ◽  
Virion Release ◽  
E Protein ◽  
Infected Cells

AbstractCoronaviruses (CoVs) are important human pathogens with significant zoonotic potential. Progress has been made toward identifying potential vaccine candidates for highly pathogenic human CoVs, including use of attenuated viruses that lack the CoV envelope (E) protein or express E mutants. However, no approved vaccines or anti-viral therapeutics exist. CoVs assemble by budding into the lumen of the early Golgi prior to exocytosis. The small CoV E protein plays roles in assembly, virion release, and pathogenesis. CoV E has a single hydrophobic domain (HD), is targeted to Golgi membranes, and has cation channel activityin vitro. The E protein from the avian infectious bronchitis virus (IBV) has dramatic effects on the secretory system, which requires residues in the HD. Mutation of the HD of IBV E during infection results in impaired growth kinetics, impaired release of infectious virions, accumulation of IBV S protein on the plasma membrane when compared IBV WT infected cells, and aberrant cleavage of IBV S on the surface of virions. We previously reported the formation of two distinct oligomeric pools of IBV E in transfected and infected cells. Disruption of the secretory pathway by IBV E correlates with a form that is likely monomeric, suggesting that the effects on the secretory pathway are independent of E ion channel activity. Here, we present evidence suggesting that the monomeric form of IBV E correlates with a rise in the pH of the Golgi lumen. We demonstrate that infection with IBV induces neutralization of Golgi luminal pH, promoting a model in which IBV E alters the secretory pathway through interaction with host cells factors, protecting IBV spike protein (S) from premature cleavage and leading to the efficient release of infectious virus from the cells.

scholarly journals Infectious Bronchitis Virus 3a Protein Localizes to a Novel Domain of the Smooth Endoplasmic Reticulum

2005 ◽  
Vol 79 (10) ◽  
pp. 6142-6151 ◽  
Author(s):  
Amanda R. Pendleton ◽  
Carolyn E. Machamer
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Infectious Bronchitis Virus ◽  
Smooth Endoplasmic Reticulum ◽  
Integral Membrane Protein ◽  
Open Reading Frames ◽  
Infectious Bronchitis ◽  
Infected Cells ◽  
Group 3 ◽  
Small Open Reading Frames

ABSTRACT All coronaviruses possess small open reading frames (ORFs) between structural genes that have been hypothesized to play important roles in pathogenesis. Infectious bronchitis virus (IBV) ORF 3a is one such gene. It is highly conserved among group 3 coronaviruses, suggesting that it has an important function in infection. IBV 3a protein is expressed in infected cells but is not detected in virions. Sequence analysis predicted that IBV 3a was a membrane protein; however, only a fraction behaved like an integral membrane protein. Microscopy and immunoprecipitation studies demonstrated that IBV 3a localized to the cytoplasm in a diffuse pattern as well as in sharp puncta in both infected and transfected cells. These puncta did not overlap cellular organelles or other punctate structures. Confocal microscopy demonstrated that IBV 3a puncta lined up along smooth endoplasmic reticulum (ER) tubules and, in a significant number of instances, were partially surrounded by these tubules. Our results suggest that IBV 3a is partially targeted to a novel domain of the smooth ER.

scholarly journals Anchorage-dependent surface distribution and partition during freeze-fracture of viral transmembrane glycoproteins.

1990 ◽  
Vol 38 (10) ◽  
pp. 1421-1426 ◽  
Author(s):  
M R Torrisi ◽  
A Pavan ◽  
L V Lotti ◽  
G Migliaccio ◽  
M C Pascale ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sindbis Virus ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Viral Proteins ◽  
Low Ph ◽  
Surface Distribution ◽  
Transfected Cells ◽  
Cytosolic Side ◽  
Infected Cells

We have compared in the same cell type the surface distribution and partition in freeze-fractured plasma membranes of Sindbis virus glycoproteins in three different situations: (i) in permanently transformed cells that express the glycoproteins as the only viral product; (ii) in cells in which prebound viruses were forced to fuse with the plasma membrane by low pH treatment; (iii) in virus-infected cells. We report here that the viral proteins expressed on the surface of transfected cells show a uniform and unclustered distribution; conversely, in Sindbis virus-infected cells they appear clustered, regionally distributed, and always associated with budding viruses (i.e., interacting with the nucleocapsid on the cytosolic side of the membrane). Furthermore, the viral proteins expressed on transfected cells or implanted by low pH-mediated fusion partition during freeze-fracture with the exoplasmic faces of the cell plasma membranes, whereas an opposite partition is observed in infected cells. These results strongly suggest that in infected cells the clustering and the partition with the protoplasmic faces of the plasma membrane depend only on the strong "anchorage" of the glycoproteins to the nucleocapsid.

scholarly journals Cell Cycle Perturbations Induced by Infection with the Coronavirus Infectious Bronchitis Virus and Their Effect on Virus Replication

2006 ◽  
Vol 80 (8) ◽  
pp. 4147-4156 ◽  
Author(s):  
Brian Dove ◽  
Gavin Brooks ◽  
Katrina Bicknell ◽  
Torsten Wurm ◽  
Julian A. Hiscox
Keyword(s):  
Cell Cycle ◽  
Virus Replication ◽  
Infectious Bronchitis Virus ◽  
Rna Virus ◽  
Positive Strand ◽  
Infectious Bronchitis ◽  
M Phase ◽  
Infected Cells ◽  
Positive Strand Rna ◽  
The Impact

ABSTRACT In eukaryotic cells, cell growth and division occur in a stepwise, orderly fashion described by a process known as the cell cycle. The relationship between positive-strand RNA viruses and the cell cycle and the concomitant effects on virus replication are not clearly understood. We have shown that infection of asynchronously replicating and synchronized replicating cells with the avian coronavirus infectious bronchitis virus (IBV), a positive-strand RNA virus, resulted in the accumulation of infected cells in the G2/M phase of the cell cycle. Analysis of various cell cycle-regulatory proteins and cellular morphology indicated that there was a down-regulation of cyclins D1 and D2 (G1 regulatory cyclins) and that a proportion of virus-infected cells underwent aberrant cytokinesis, in which the cells underwent nuclear, but not cytoplasmic, division. We assessed the impact of the perturbations on the cell cycle for virus-infected cells and found that IBV-infected G2/M-phase-synchronized cells exhibited increased viral protein production when released from the block when compared to cells synchronized in the G0 phase or asynchronously replicating cells. Our data suggested that IBV induces a G2/M phase arrest in infected cells to promote favorable conditions for viral replication.

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.

scholarly journals Infectious Bronchitis Virus E Protein Is Targeted to the Golgi Complex and Directs Release of Virus-Like Particles

2000 ◽  
Vol 74 (9) ◽  
pp. 4319-4326 ◽  
Author(s):  
Emily Corse ◽  
Carolyn E. Machamer
Keyword(s):  
Golgi Complex ◽  
Infectious Bronchitis Virus ◽  
Virus Assembly ◽  
Recombinant Vaccinia Virus ◽  
Biological Properties ◽  
Infectious Bronchitis ◽  
E Protein ◽  
N Terminus ◽  
Low Levels ◽  
In Cells

ABSTRACT The coronavirus E protein is a poorly characterized small envelope protein present in low levels in virions. We are interested in the role of E in the intracellular targeting of infectious bronchitis virus (IBV) membrane proteins. We generated a cDNA clone of IBV E and antibodies to the E protein to study its cell biological properties in the absence of virus infection. We show that IBV E is an integral membrane protein when expressed in cells from cDNA. Epitope-specific antibodies revealed that the C terminus of IBV E is cytoplasmic and the N terminus is translocated. The short luminal N terminus of IBV E contains a consensus site for N-linked glycosylation, but the site is not used. When expressed using recombinant vaccinia virus, the IBV E protein is released from cells at low levels in sedimentable particles that have a density similar to that of coronavirus virions. The IBV M protein is incorporated into these particles when present. Indirect immunofluorescence microscopy showed that E is localized to the Golgi complex in cells transiently expressing IBV E. When coexpressed with IBV M, both from cDNA and in IBV infection, the two proteins are colocalized in Golgi membranes, near the coronavirus budding site. Thus, even though IBV E is present at low levels in virions, it is apparently expressed at high levels in infected cells near the site of virus assembly.

scholarly journals The Cytoplasmic Tail of Infectious Bronchitis Virus E Protein Directs Golgi Targeting

2002 ◽  
Vol 76 (3) ◽  
pp. 1273-1284 ◽  
Author(s):  
Emily Corse ◽  
Carolyn E. Machamer
Keyword(s):  
Golgi Complex ◽  
Infectious Bronchitis Virus ◽  
Transmembrane Domain ◽  
Brefeldin A ◽  
Cytoplasmic Tail ◽  
Reporter Protein ◽  
Infectious Bronchitis ◽  
E Protein ◽  
Necessary And Sufficient ◽  
Golgi Matrix

ABSTRACT We have previously shown that the E protein of the coronavirus infectious bronchitis virus (IBV) is localized to the Golgi complex when expressed exogenously from cDNA. Here, we report that neither the transmembrane domain nor the short lumenal domain of IBV E is required for Golgi targeting. However, an N-terminal truncation containing only the cytoplasmic domain (CTE) was efficiently localized to the Golgi complex, and this domain could retain a reporter protein in the Golgi. Thus, the cytoplasmic tail of the E protein is necessary and sufficient for Golgi targeting. The IBV E protein is palmitoylated on one or two cysteine residues adjacent to its transmembrane domain, but palmitoylation was not required for proper Golgi targeting. Using C-terminal truncations, we determined that the IBV E Golgi targeting information is present between tail amino acids 13 and 63. Upon treatment with brefeldin A, both the E and CTE proteins redistributed to punctate structures that colocalized with the Golgi matrix proteins GM130 and p115 instead of being localized to the endoplasmic reticulum like Golgi glycosylation enzymes. This suggests that IBV E is associated with the Golgi matrix through interactions of its cytoplasmic tail and may have interesting implications for coronavirus assembly in early Golgi compartments.

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