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

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.

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The Infectious Bronchitis Coronavirus Envelope Protein Alters Golgi pH To Protect the Spike Protein and Promote the Release of Infectious Virus

Journal of Virology ◽

10.1128/jvi.00015-19 ◽

2019 ◽

Vol 93 (11) ◽

Cited By ~ 17

Author(s):

Jason W. Westerbeck ◽

Carolyn E. Machamer

Keyword(s):

Secretory Pathway ◽

Infectious Virus ◽

Virus Assembly ◽

Channel Activity ◽

Human Pathogens ◽

Hydrophobic Domain ◽

Infectious Bronchitis ◽

Virion Release ◽

E Protein ◽

Infected Cells

ABSTRACTCoronaviruses (CoVs) assemble by budding into the lumen of the early Golgi complex prior to exocytosis. The small CoV envelope (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 avian infectious bronchitis virus (IBV) has dramatic effects on the secretory system, which require residues in the HD. Mutation of the HD of IBV E in a recombinant virus background results in impaired growth kinetics, impaired release of infectious virions, accumulation of IBV spike (S) protein on the plasma membrane compared to wild-type (WT) IBV-infected cells, and aberrant cleavage of IBV S on 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 an increased Golgi luminal pH. Infection with IBV or expression of IBV E induces neutralization of Golgi pH, promoting a model in which IBV E alters the secretory pathway through interaction with host cell factors, protecting IBV S from premature cleavage and leading to the efficient release of infectious virus from the cells. This is the first demonstration of a coronavirus-induced alteration in the microenvironment of the secretory pathway.IMPORTANCECoronaviruses are important human pathogens with significant zoonotic potential. Progress has been made toward identifying potential vaccine candidates for highly pathogenic human CoVs, including the use of attenuated viruses that lack the CoV E protein or express E mutants. However, no approved vaccines or antiviral therapeutics exist. Understanding the role of the CoV E protein in virus assembly and release is thus an important prerequisite for potential vaccines as well as in identifying novel antiviral therapeutics.

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The Hydrophobic Domain of Infectious Bronchitis Virus E Protein Alters the Host Secretory Pathway and Is Important for Release of Infectious Virus

Journal of Virology ◽

10.1128/jvi.01570-10 ◽

2010 ◽

Vol 85 (2) ◽

pp. 675-685 ◽

Cited By ~ 41

Author(s):

T. R. Ruch ◽

C. E. Machamer

Keyword(s):

Infectious Bronchitis Virus ◽

Secretory Pathway ◽

Infectious Virus ◽

Hydrophobic Domain ◽

Infectious Bronchitis ◽

E Protein

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

10.1101/2021.04.19.440492 ◽

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.

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A Coronavirus E Protein Is Present in Two Distinct Pools with Different Effects on Assembly and the Secretory Pathway

Journal of Virology ◽

10.1128/jvi.01237-15 ◽

2015 ◽

Vol 89 (18) ◽

pp. 9313-9323 ◽

Cited By ~ 27

Author(s):

Jason W. Westerbeck ◽

Carolyn E. Machamer

Keyword(s):

Ion Channel ◽

Golgi Complex ◽

Secretory Pathway ◽

Structural Data ◽

Channel Activity ◽

Human Pathogens ◽

Hydrophobic Domain ◽

Virion Release ◽

E Protein ◽

Ion Conducting

ABSTRACTCoronaviruses (CoVs) assemble by budding into the lumen of the early Golgi complex prior to exocytosis. The small CoV envelope (E) protein plays roles in assembly, virion release, and pathogenesis. CoV E has a single hydrophobic domain (HD), is targeted to Golgi complex membranes, and has cation channel activityin vitro. However, the precise functions of the CoV E protein during infection are still enigmatic. Structural data for the severe acute respiratory syndrome (SARS)-CoV E protein suggest that it assembles into a homopentamer. Specific residues in the HD regulate the ion-conducting pore formed by SARS-CoV E in artificial bilayers and the pathogenicity of the virus during infection. The E protein from the avian infectious bronchitis virus (IBV) has dramatic effects on the secretory system which require residues in the HD. Here, we use the known structural data from SARS-CoV E to infer the residues important for ion channel activity and the oligomerization of IBV E. We present biochemical data for the formation of two distinct oligomeric pools of IBV E in transfected and infected cells and the residues required for their formation. A high-order oligomer of IBV E is required for the production of virus-like particles (VLPs), implicating this form of the protein in virion assembly. Additionally, 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.IMPORTANCECoVs are important human pathogens with significant zoonotic potential, as demonstrated by the emergence of SARS-CoV and Middle East respiratory syndrome (MERS)-CoV. Progress has been made toward identifying potential vaccine candidates in mouse models of CoV infection, including the use of attenuated viruses that lack the CoV E protein or express E-protein mutants. However, no approved vaccines or antiviral therapeutics exist. We previously reported that the hydrophobic domain of the IBV E protein, a putative viroporin, causes disruption of the mammalian secretory pathway when exogenously expressed in cells. Understanding the mechanism of this disruption could lead to the identification of novel antiviral therapeutics. Here, we present biochemical evidence for two distinct oligomeric forms of IBV E, one essential for assembly and the other with a role in disruption of the secretory pathway. Discovery of two forms of CoV E protein will provide additional targets for antiviral therapeutics.

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

Journal of Virology ◽

10.1128/jvi.02158-16 ◽

2016 ◽

Vol 91 (5) ◽

Cited By ~ 10

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.

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