Synthesis and processing of equine herpesvirus 1 glycoprotein D.
Abstract: Previous studies (C. C. Flowers and D. J. O'Callaghan, 1992, Virology 190, 307-315) employed peptide-specific antibodies to identify the product of the glycoprotein D (gD) gene of equine herpesvirus 1 strain Kentucky A (KyA). gD polypeptides of 55 and 58 kDa were detected in EHV-1-infected L-M cells, and the 58-kDa protein was observed in the membrane fraction of EHV-1 virions. In this report, the kinetics of synthesis and processing of gD polypeptides are described. One-hour pulse-labeling of EHV-1-infected L-M cells revealed that gD proteins are first detected at 6 hr after infection and that maximal synthesis of gD occurs between 5 and 8 hr postinfection. gD polypeptides accumulate progressively with time of infection as shown by immunoprecipitation analysis of gD proteins. Pulse-chase analysis of gD revealed that the 55-kDa protein is a precursor to the 58-kDa species and that processing of all pulse-labeled precursor protein requires approximately 2.5 hr. Analysis of the carbohydrate content of gD proteins, as judged by their sensitivity to digestion with endoglycosidases, revealed that the 55-kDa gD precursor contains high-mannose N-linked oligosaccharides, while the 58-kDa gD mature polypeptide possesses complex type oligosaccharides. Expression of the mature form of gD on the cell surface, as determined by fluorescent flow cytometric analysis, is delayed compared to the accumulation of the mature form of gD within the cell. The gD ORF encodes a potential protein of 442 amino acids but analysis of the translated sequence of gD indicated that the gD polypeptide is 392 amino acids, a size predicted by previous mapping of the transcription start site of the gD mRNA. Coupled in vitro transcription/translation of a pGEM-3Z construct containing the 392-amino-acid gD ORF, in the absence or presence of canine pancreatic microsomes, demonstrated that the 43-kDa gD polypeptide undergoes processing in vitro. These studies demonstrate that the EHV-1 strain KyA gD is processed in a fashion similar to that of the gD proteins of other alphaherpesviruses.
Publication Date: 1995-04-01 PubMed ID: 11831735DOI: 10.1006/viro.1995.1124Google Scholar: Lookup
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- Journal Article
- Research Support
- U.S. Gov't
- Non-P.H.S.
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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This research article delves into the synthesis and processing of a glycoprotein (gD) in the Equine Herpesvirus 1 strain. The study demonstrates that the glycoprotein undergoes processing in a way that’s similar to the gD proteins of other alphaherpesviruses.
Objective of the Research
- The aim of the research was to investigate the glycoprotein D (gD) gene of Equine Herpesvirus 1 strain Kentucky A (KyA), previously mentioned in other studies.
- Particular focus was given to the kinetics of synthesis and processing of gD polypeptides, namely 55-kDa and 58-kDa proteins.
Research Methodology and Findings
- The research started with the method of pulse-labeling EHV-1-infected L-M cells for one hour.
- The observation stated that gD proteins can be detected 6 hours post-infection, with maximum synthesis observed between 5 and 8 hours after infection.
- It was noted that gD polypeptides accumulate over time, as demonstrated through a detailed immunoprecipitation analysis of gD proteins.
- Pulse-chase analysis indicated that the 55-kDa protein is a precursor to the 58-kDa protein and the processing of the precursor protein takes about 2.5 hours.
- A thorough analysis of the carbohydrate content showed that the 55-kDa gD precursor contains high-mannose N-linked oligosaccharides, while the 58-kDa gD mature polypeptide houses complex-type oligosaccharides.
- The appearance of the mature form of gD on the cell surface was, interestingly, lagging compared to the accumulation of the mature form of gD within the cell.
Insights on Protein Synthesis
- The research article provides insights about the genetic composition of gD by stating that the gD ORF (Open Reading Frame) encodes a potential protein of 442 amino acids.
- However, upon examination, the gD polypeptide was found to be 392 amino acids long, as anticipated from prior mapping of the transcription start site of the gD mRNA.
- This possibility of having a 392 amino acids long protein was studied further by an in vitro transcription/translation of a pGEM-3Z construct containing the 392-amino-acid gD ORF. This was done in the presence and absence of canine pancreatic microsomes, which showed that the 43-kDa gD polypeptide undergoes processing in vitro.
Comparison with Other Alphaherpesviruses
- The findings from this study have indicated that the synthesis and processing of glycoprotein in EHV-1 strain KyA gD are similar to the gD proteins of other alphaherpesviruses.
- This adds valuable knowledge to the broader understanding of how herpesviruses behave at a molecular level and opens potential avenues for new research into prevention and treatment strategies.
Cite This Article
APA
Flowers CC, Flowers SP, Jennings SR, O'Callaghan DJ.
(1995).
Synthesis and processing of equine herpesvirus 1 glycoprotein D.
Virology, 208(1), 9-18.
https://doi.org/10.1006/viro.1995.1124 Publication
Researcher Affiliations
- Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport 71130-3932, USA.
MeSH Terms
- Animals
- Flow Cytometry
- Gene Expression Regulation, Viral
- Herpesvirus 1, Equid / metabolism
- L Cells
- Mice
- Plasmids
- Protein Processing, Post-Translational
- Transfection
- Viral Envelope Proteins / genetics
- Viral Envelope Proteins / metabolism
Grant Funding
- AI-22001 / NIAID NIH HHS
Citations
This article has been cited 2 times.- von Einem J, Wellington J, Whalley JM, Osterrieder K, O'Callaghan DJ, Osterrieder N. The truncated form of glycoprotein gp2 of equine herpesvirus 1 (EHV-1) vaccine strain KyA is not functionally equivalent to full-length gp2 encoded by EHV-1 wild-type strain RacL11. J Virol 2004 Mar;78(6):3003-13.
- Wellington JE, Lawrence GL, Love DN, Whalley JM. Expression and characterization of equine herpesvirus 1 glycoprotein D in mammalian cell lines. Arch Virol 1996;141(9):1785-93.
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