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.

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 Channel-Inactivating Mutations and Their Revertant Mutants in the Envelope Protein of Infectious Bronchitis Virus

2016 ◽  
Vol 91 (5) ◽  
Author(s):  
Janet To ◽  
Wahyu Surya ◽  
To Sing Fung ◽  
Yan Li ◽  
Carmina Verdià-Bàguena ◽  
...  
Keyword(s):  
Infectious Bronchitis Virus ◽  
Transmembrane Domain ◽  
Channel Activity ◽  
Compensatory Mechanisms ◽  
Live Attenuated Vaccines ◽  
Infectious Bronchitis ◽  
Allosteric Interaction ◽  
E Protein ◽  
Inactivating Mutations

ABSTRACT It has been shown previously in the severe acute respiratory syndrome coronavirus (SARS-CoV) that two point mutations, N15A and V25F, in the transmembrane domain (TMD) of the envelope (E) protein abolished channel activity and led to in vivo attenuation. Pathogenicity was recovered in mutants that also regained E protein channel activity. In particular, V25F was rapidly compensated by changes at multiple V25F-facing TMD residues located on a neighboring monomer, consistent with a recovery of oligomerization. Here, we show using infected cells that the same mutations, T16A and A26F, in the gamma-CoV infectious bronchitis virus (IBV) lead to, in principle, similar results. However, IBV E A26F did not abolish oligomer formation and was compensated by mutations at N- and C-terminal extramembrane domains (EMDs). The C-terminal EMD mutations clustered along an insertion sequence specific to gamma-CoVs. Nuclear magnetic resonance data are consistent with the presence of only one TMD in IBV E, suggesting that recovery of channel activity and fitness in these IBV E revertant mutants is through an allosteric interaction between EMDs and TMD. The present results are important for the development of IBV live attenuated vaccines when channel-inactivating mutations are introduced in the E protein. IMPORTANCE The ion channel activity of SARS-CoV E protein is a determinant of virulence, and abolishment of channel activity leads to viral attenuation. E deletion may be a strategy for generating live attenuated vaccines but can trigger undesirable compensatory mechanisms through modifications of other viral proteins to regain virulence. Therefore, a more suitable approach may be to introduce small but critical attenuating mutations. For this, the stability of attenuating mutations should be examined to understand the mechanisms of reversion. Here, we show that channel-inactivating mutations of the avian infectious bronchitis virus E protein introduced in a recombinant virus system are deficient in viral release and fitness and that revertant mutations also restored channel activity. Unexpectedly, most of the revertant mutations appeared at extramembrane domains, particularly along an insertion specific for gammacoronaviruses. Our structural data propose a single transmembrane domain in IBV E, suggesting an allosteric interaction between extramembrane and transmembrane domains.

scholarly journals Ceramide Accumulation Uncovers a Cycling Pathway for the cis-Golgi Network Marker, Infectious Bronchitis Virus M Protein

1997 ◽  
Vol 139 (6) ◽  
pp. 1411-1418 ◽  
Author(s):  
Michael Maceyka ◽  
Carolyn E. Machamer
Keyword(s):  
Golgi Complex ◽  
Infectious Bronchitis Virus ◽  
Transmembrane Domain ◽  
Membrane Lipids ◽  
Term Treatment ◽  
Short Term Treatment ◽  
Golgi Stack ◽  
Infectious Bronchitis ◽  
Golgi Network ◽  
Ceramide Accumulation

The M glycoprotein from the avian coronavirus, infectious bronchitis virus (IBV), contains information for localization to the cis-Golgi network in its first transmembrane domain. We hypothesize that localization to the Golgi complex may depend in part on specific interactions between protein transmembrane domains and membrane lipids. Because the site of sphingolipid synthesis overlaps the localization of IBV M, we asked whether perturbation of sphingolipids affected localization of IBV M. Short-term treatment with two inhibitors of sphingolipid synthesis had no effect on localization of IBV M or other Golgi markers. Thus, ongoing synthesis of these lipids was not required for proper localization. Surprisingly, a third inhibitor, d,l-threo-1-phenyl-2-decanoylamino-3-morpholino- 1-propanol (PDMP), shifted the steady-state distribution of IBV M from the Golgi complex to the ER. This effect was rapid and reversible and was also observed for ERGIC-53 but not for Golgi stack proteins. At the concentration of PDMP used, conversion of ceramide into both glucosylceramide and sphingomyelin was inhibited. Pretreatment with upstream inhibitors partially reversed the effects of PDMP, suggesting that ceramide accumulation mediates the PDMP-induced alterations. Indeed, an increase in cellular ceramide was measured in PDMP-treated cells. We propose that IBV M is at least in part localized by retrieval mechanisms. Further, ceramide accumulation reveals this cycle by upsetting the balance of anterograde and retrograde traffic and/ or disrupting retention by altering bilayer dynamics.

scholarly journals Contribution of Trafficking Signals in the Cytoplasmic Tail of the Infectious Bronchitis Virus Spike Protein to Virus Infection

2005 ◽  
Vol 79 (21) ◽  
pp. 13209-13217 ◽  
Author(s):  
Soonjeon Youn ◽  
Ellen W. Collisson ◽  
Carolyn E. Machamer
Keyword(s):  
Virus Infection ◽  
Infectious Bronchitis Virus ◽  
Secretory Pathway ◽  
Protein A ◽  
Cytoplasmic Tail ◽  
Growth Defect ◽  
Target Cells ◽  
Cdna Clones ◽  
S Protein ◽  
Infectious Bronchitis

ABSTRACT Coronavirus spike (S) proteins are responsible for binding and fusion with target cells and thus play an essential role in virus infection. Recently, we identified a dilysine endoplasmic reticulum (ER) retrieval signal and a tyrosine-based endocytosis signal in the cytoplasmic tail of the S protein of infectious bronchitis virus (IBV). Here, an infectious cDNA clone of IBV was used to address the importance of the S protein trafficking signals to virus infection. We constructed infectious cDNA clones lacking the ER retrieval signal, the endocytosis signal, or both. The virus lacking the ER retrieval signal was viable. However, this virus had a growth defect at late times postinfection and produced larger plaques than IBV. Further analysis confirmed that the mutant S protein trafficked though the secretory pathway faster than wild-type S protein. A more dramatic phenotype was obtained when the endocytosis signal was mutated. Recombinant viruses lacking the endocytosis signal (in combination with a mutated dilysine signal or alone) could not be recovered, even though transient syncytia were formed in transfected cells. Our results suggest that the endocytosis signal of IBV S is essential for productive virus infection.

scholarly journals Human cytomegalovirus US9 protein contains an N-terminal signal sequence and a C-terminal mitochondrial localization domain, and does not alter cellular sensitivity to apoptosis

2009 ◽  
Vol 90 (5) ◽  
pp. 1172-1182 ◽  
Author(s):  
Lana Mandic ◽  
Matthew S. Miller ◽  
Corinne Coulter ◽  
Brian Munshaw ◽  
Laura Hertel
Keyword(s):  
Human Cytomegalovirus ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Brefeldin A ◽  
Genomic Region ◽  
Hygromycin B ◽  
Mitochondrial Localization ◽  
Dual Localization ◽  
Carbonyl Cyanide ◽  
Necessary And Sufficient

The human cytomegalovirus (CMV) US2–US11 genomic region contains a cluster of genes whose products interfere with antigen presentation by the major histocompatibility complex (MHC) proteins. Although included in this cluster, the US9 gene encodes a glycoprotein that does not affect MHC activity and whose function is still largely uncharacterized. An in silico analysis of the US9 amino-acid sequence uncovered the presence of an N-terminal signal sequence (SS) and a C-terminal transmembrane domain containing the specific hallmarks of known mitochondrial localization sequences (MLS). Expression of full-length US9 and of US9 deletion mutants fused to GFP revealed that the N-terminal SS mediates US9 targeting to the endoplasmic reticulum (ER) and that the C-terminal MLS is both necessary and sufficient to direct US9 to mitochondria in the absence of a functional SS. This dual localization suggested a possible role for US9 in protection from apoptosis triggered by ER-to-mitochondria signalling. Fibroblasts infected with the US2–US11 deletion mutant virus RV798 or with the parental strain AD169varATCC were equally susceptible to death triggered by exposure to tumour necrosis factor (TNF)-α, tunicamycin, thapsigargin, brefeldin A, lonidamine and carbonyl cyanide m-chloro phenyl hydrazone, but were 1.6-fold more sensitive to apoptosis induced by hygromycin B. Expression of US9 in human embryonic kidney 293T cells or in fibroblasts, however, did not protect cells from hygromycin B-mediated death. Together, these results classify US9 as the first CMV-encoded protein to contain an N-terminal SS and a C-terminal MLS, and suggest a completely novel role for this protein during infection.

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 v-sis oncoprotein loses transforming activity when targeted to the early Golgi complex.

1994 ◽  
Vol 127 (6) ◽  
pp. 1843-1857 ◽  
Author(s):  
K C Hart ◽  
Y F Xu ◽  
A N Meyer ◽  
B A Lee ◽  
D J Donoghue
Keyword(s):  
Cell Surface ◽  
Fusion Protein ◽  
Golgi Complex ◽  
Fusion Proteins ◽  
Transmembrane Domain ◽  
Cytoplasmic Tail ◽  
Proteolytic Processing ◽  
Nih3t3 Cells ◽  
Transforming Activity ◽  
Retention Signal

The location of autocrine interactions between the v-sis protein and PDGF receptors remains uncertain and controversial. To examine whether receptor-ligand interactions can occur intracellularly, we have constructed fusion proteins that anchor v-sis to specific intracellular membranes. Fusion of a cis-Golgi retention signal from a coronavirus E1 glycoprotein to v-sis protein completely abolished its transforming ability when transfected into NIH3T3 cells. Fusion proteins incorporating mutations in this retention signal were not retained within the Golgi complex but instead were transported to the cell surface, resulting in efficient transformation. All chimeric proteins were shown to dimerize properly. Derivatives of some of these constructs were also constructed bearing the cytoplasmic tail from the glycoprotein of vesicular stomatitis virus (VSV-G). These constructs allowed examination of subcellular localization by double-label immunofluorescence, using antibodies that distinguish between the extracellular PDGF-related domain and the VSV-G cytoplasmic tail. Colocalization of sis-E1-G with Golgi markers confirmed its targeting to the early Golgi complex. The sis-E1 constructs, targeted to the early Golgi complex, exhibited no proteolytic processing whereas the mutant forms of sis-E1 exhibited normal proteolytic processing. Treatment with suramin, a polyanionic compound that disrupts ligand/receptor interactions at the cell surface, was able to revert the transformed phenotype induced by the mutant sis-E1 constructs described here. Our results demonstrate that autocrine interactions between the v-sis oncoprotein and PDGF receptors within the early Golgi complex do not result in functional signal transduction. Another v-sis fusion protein was constructed by attaching the transmembrane domain and COOH-terminus of TGN38, a protein that localizes to the trans-Golgi network (TGN). This construct was primarily retained intracellularly, although some of the fusion protein reached the surface. Deletion of the COOH-terminal region of the TGN38 retention signal abrogated the TGN-localization, as evidenced by very prominent cell surface localization, and resulted in increased transforming activity. The behavior of the sis-TGN38 derivatives is discussed within the context of the properties of TGN38 itself, which is known to recycle from the cell surface to the TGN.

scholarly journals The Transmembrane Domain of the Severe Acute Respiratory Syndrome Coronavirus ORF7b Protein Is Necessary and Sufficient for Its Retention in the Golgi Complex

2008 ◽  
Vol 82 (19) ◽  
pp. 9477-9491 ◽  
Author(s):  
Scott R. Schaecher ◽  
Michael S. Diamond ◽  
Andrew Pekosz
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Golgi Complex ◽  
Transmembrane Domain ◽  
Chimeric Protein ◽  
Integral Membrane Protein ◽  
Reading Frame ◽  
Mutant Proteins ◽  
Golgi Compartment ◽  
Carboxy Terminal ◽  
Necessary And Sufficient

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) ORF7b (also called 7b) protein is an integral membrane protein that is translated from a bicistronic open reading frame encoded within subgenomic RNA 7. When expressed independently or during virus infection, ORF7b accumulates in the Golgi compartment, colocalizing with both cis- and trans-Golgi markers. To identify the domains of this protein that are responsible for Golgi localization, we have generated a set of mutant proteins and analyzed their subcellular localizations by indirect immunofluorescence confocal microscopy. The N- and C-terminal sequences are dispensable, but the ORF7b transmembrane domain (TMD) is essential for Golgi compartment localization. When the TMD of human CD4 was replaced with the ORF7b TMD, the resulting chimeric protein localized to the Golgi complex. Scanning alanine mutagenesis identified two regions in the carboxy-terminal portion of the TMD that eliminated the Golgi complex localization of the chimeric CD4 proteins or ORF7b protein. Collectively, these data demonstrate that the Golgi complex retention signal of the ORF7b protein resides solely within the TMD.

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