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 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 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.

Comparative Evaluation of In Vitro and In Vivo Assays for the Detection of Avian Infectious Bronchitis Virus as a Contaminant of Live Poultry Vaccines

1998 ◽  
Vol 26 (5) ◽  
pp. 629-634
Author(s):  
Emiliana Falcone ◽  
Edoardo Vignolo ◽  
Livia Di Trani ◽  
Simona Puzelli ◽  
Maria Tollis
Keyword(s):  
Infectious Bronchitis Virus ◽  
Serological Response ◽  
Avian Infectious Bronchitis Virus ◽  
Animal Testing ◽  
Rt Pcr ◽  
Pcr Assay ◽  
In Vivo Test ◽  
Infectious Bronchitis

A reverse transcriptase polymerase chain reaction (RT-PCR) assay specific for identifying avian infectious bronchitis virus (IBV) in poultry vaccines, and the serological response to IBV induced by the inoculation of chicks with a Newcastle disease vaccine spiked with the Massachusetts strain of IBV, were compared for their ability to detect IBV as a contaminant of avian vaccines. The sensitivity of the IBV-RT-PCR assay provided results which were at least equivalent to the biological effect produced by the inoculation of chicks, allowing this assay to be considered a valid alternative to animal testing in the quality control of avian immunologicals. This procedure can easily be adapted to detect a number of contaminants for which the in vivo test still represents the only available method of detection.

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 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.

In vivo Evaluation of the Pathogenicity of Field Isolates of Infectious Bronchitis Virus

1994 ◽  
Vol 38 (3) ◽  
pp. 589 ◽  
Author(s):  
G. E. Avellaneda ◽  
P. Villegas ◽  
M. W. Jackwood ◽  
D. J. King
Keyword(s):  
Infectious Bronchitis Virus ◽  
In Vivo Evaluation ◽  
Infectious Bronchitis ◽  
Field Isolates
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