Synthesis and processing of equine herpesvirus 1 glycoprotein D

Area of Science:

• Virology
• Molecular Biology
• Glycobiology

Background:

• Previous studies identified equine herpesvirus 1 (EHV-1) glycoprotein D (gD) as 55 and 58 kDa polypeptides.
• The 58 kDa gD protein was found in the membrane fraction of EHV-1 virions.

Purpose of the Study:

• To describe the kinetics of synthesis and processing of EHV-1 strain KyA gD polypeptides.
• To characterize the post-translational modifications and cellular localization of EHV-1 gD.

Main Methods:

• Pulse-labeling and pulse-chase experiments to analyze gD synthesis and processing kinetics.
• Immunoprecipitation and endoglycosidase digestion to characterize gD polypeptides and their carbohydrate content.
• Fluorescent flow cytometry to determine cell surface expression of mature gD.
• In vitro transcription/translation coupled with microsome treatment to study gD processing.

Main Results:

• EHV-1 gD synthesis begins 6 hours post-infection, peaking between 5-8 hours.
• The 55 kDa gD is a precursor to the 58 kDa mature form, with processing completed in approximately 2.5 hours.
• The precursor contains high-mannose oligosaccharides, while the mature form has complex type oligosaccharides.
• Mature gD appears on the cell surface after its intracellular accumulation.
• In vitro studies confirmed the processing of a 43 kDa gD polypeptide.

Conclusions:

• EHV-1 strain KyA gD undergoes post-translational modifications, including glycosylation, similar to gD proteins of other alphaherpesviruses.
• The processing pathway involves conversion of a high-mannose precursor to a mature form with complex oligosaccharides.
• Cell surface expression of mature gD is a later event compared to its intracellular accumulation.