The open reading frame 3 of equine arteritis virus encodes an immunogenic glycosylated, integral membrane protein.
Abstract: Open reading frame 3 (ORF 3) of equine arteritis virus (EAV) is predicted to encode a glycosylated membrane protein (GP3) that is uncharacterized. ORF 3 of the American Type Culture Collection strain of EAV was in vitro transcribed and the encoded GP3 protein was in vitro translated with and without canine microsomal membranes. The GP3 protein was approximately 17 kDa after in vitro translation without canine microsomal membranes whereas the glycosylated form, after translation with microsomal membranes, was a diffuse band of 36-42 kDa, indicating that the GP3 protein is extensively glycosylated. Deglycosylation reduced the GP3 protein to approximately 17 kDa, the same size as that translated without microsomal membranes, indicating that the signal sequence was not cleaved. The EAV GP3 protein was membrane associated and not released as a soluble protein, in marked contrast to the ORF 3-encoded proteins of some other arteriviruses. The GP3 protein was protected from protease digestion in closed membrane vesicles, suggesting that the protein extends into the membrane vesicles and is anchored by the N-terminal signal sequence, a C-terminal hydrophobic domain, or both, but does not span the membrane three times. A GP3 protein lacking the C-terminal transmembrane domain remained membrane associated, indicating that this terminus is not a necessary membrane anchor. Sera from stallions persistently infected with EAV and horses immunized repeatedly with the modified live EAV vaccine contained antibodies specific for the GP3 protein. The data indicate that the GP3 protein is an extensively glycosylated membrane protein that is immunogenic during some EAV infections.
Copyright 1999 Academic Press.
Publication Date: 1999-11-02 PubMed ID: 10544133DOI: 10.1006/viro.1999.9982Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- Non-P.H.S.
Summary
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This research focuses on a specific protein, GP3, produced by the equine arteritis virus. The study found that this protein is highly modified by the addition of sugar molecules and is associated with the virus’s outer barrier, known as the membrane. It appears to play a crucial role in generating an immune response in infected or vaccinated horses.
Understanding the GP3 Protein
- The research investigates the glycosylated membrane protein (GP3) predicted to be encoded by Open Reading Frame 3 (ORF3) of the Equine Arteritis Virus. This protein had not been extensively studied before.
- In-vitro transcriptions and translations were performed on ORF3 to create the GP3 protein for study. The experiments were conducted either with or without canine microsomal membranes.
- It was found that the GP3 protein weighed approximately 17kDa after being translated, without being glycosylated, in the absence of canine microsomal membranes.
- However, when translated with microsomal membranes, the GP3 protein appeared to be heavily glycosylated. Its weight increased to around 36-42 kDa, forming a disperse band.
Glycosylations and Membrane Attachment
- Following the process of deglycosylation, the GP3 protein reduced back to approximately 17 kDa, the same size as that translated without the microsomal membranes. This indicated that the signal sequence in the GP3 protein was not cleaved during the translational process.
- The findings also revealed that the GP3 protein was associated with the membrane but was not released as a soluble protein. This characteristic is different from the proteins encoded by ORF 3 from other arteriviruses.
- The protein’s resistance to protease digestion in closed membrane vesicles suggested that it extended into the membrane vesicles and was anchored either by the N-terminal signal sequence, a C-terminal hydrophobic domain, or both but did not span the membrane three times.
- When a version of GP3 missing the C-terminal transmembrane domain was analyzed, the protein still remained associated with the membrane. This signifies that the C-terminal isn’t required for membrane anchoring.
Immunogenic Value of the GP3 Protein
- Upon investigation of sera from persistently infected horses and horses that were immunized with a modified live EAV vaccine, it was found that the serum contained antibodies specific to the GP3 protein.
- This suggested that the GP3 protein, being an extensively glycosylated membrane protein, has immunogenic properties, meaning it can promote an immune response. It has the potential to play a role in the development of new treatments or vaccines against equine arteritis virus.
Cite This Article
APA
Hedges JF, Balasuriya UB, MacLachlan NJ.
(1999).
The open reading frame 3 of equine arteritis virus encodes an immunogenic glycosylated, integral membrane protein.
Virology, 264(1), 92-98.
https://doi.org/10.1006/viro.1999.9982 Publication
Researcher Affiliations
- Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, Davis, California 95616, USA.
MeSH Terms
- Animals
- Antibodies, Viral / blood
- Arterivirus Infections / blood
- Arterivirus Infections / immunology
- Cell Line
- DNA Primers
- Dogs
- Equartevirus / genetics
- Equartevirus / immunology
- Glycosylation
- Horse Diseases / blood
- Horse Diseases / immunology
- Horses
- Intracellular Membranes / metabolism
- Kidney
- Microsomes / metabolism
- Neutralization Tests
- Open Reading Frames
- Protein Biosynthesis
- Rabbits
- Transcription, Genetic
Citations
This article has been cited 17 times.- Wang Q, Peng J, Sun Y, Chen J, An T, Leng C, Li L, Zhao H, Guo X, Ge X, Yang H, Tian Z. Unique epitopes recognized by monoclonal antibodies against HP-PRRSV: deep understanding of antigenic structure and virus-antibody interaction. PLoS One 2014;9(10):e111633.
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- Matczuk AK, Kunec D, Veit M. Co-translational processing of glycoprotein 3 from equine arteritis virus: N-glycosylation adjacent to the signal peptide prevents cleavage. J Biol Chem 2013 Dec 6;288(49):35396-405.
- Balasuriya UB, Go YY, MacLachlan NJ. Equine arteritis virus. Vet Microbiol 2013 Nov 29;167(1-2):93-122.
- Kim DG, Song CS, Choi IS, Park SY, Lee JB, Lee SS. The signal sequence of type II porcine reproductive and respiratory syndrome virus glycoprotein 3 is sufficient for endoplasmic reticulum retention. J Vet Sci 2013;14(3):307-13.
- Go YY, Snijder EJ, Timoney PJ, Balasuriya UB. Characterization of equine humoral antibody response to the nonstructural proteins of equine arteritis virus. Clin Vaccine Immunol 2011 Feb;18(2):268-79.
- Go YY, Zhang J, Timoney PJ, Cook RF, Horohov DW, Balasuriya UB. Complex interactions between the major and minor envelope proteins of equine arteritis virus determine its tropism for equine CD3+ T lymphocytes and CD14+ monocytes. J Virol 2010 May;84(10):4898-911.
- Koo HN, Oh JM, Lee JK, Choi JY, Lee KS, Roh JY, Je YH, Jin BR, Yoo SS, Kim JS, Kim YI, Yoon IJ, Woo SD. Molecular characterization of ORFs 2 to 7 of Korean porcine reproductive and respiratory syndrome virus (CA) and its protein expression by recombinant baculoviruses. J Microbiol 2008 Dec;46(6):709-19.
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- Jeronimo C, Archambault D. Importance of M-protein C terminus as substrate antigen for serodetection of equine arteritis virus infection. Clin Diagn Lab Immunol 2002 May;9(3):698-703.
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