Purification and structural characterization of herpes simplex virus glycoprotein C

Area of Science:

• Virology
• Structural Biology
• Biochemistry

Background:

• Herpes simplex virus glycoprotein C (gC) is crucial for viral infection.
• Understanding gC's structure is key to developing antiviral strategies.
• Previous studies lacked sufficient purified gC for detailed structural analysis.

Purpose of the Study:

• To purify herpes simplex virus glycoprotein C (gC) for secondary structure analysis.
• To investigate the structural impact of glycosylation on gC.
• To compare experimental structural data with theoretical predictions.

Main Methods:

• Purification of gC-3 mutant protein using gel filtration and immunoaffinity chromatography.
• Characterization of purified gC-3 by SDS-PAGE, immunoblotting, and amino acid sequencing.
• Secondary structure analysis of native and deglycosylated gC-3 using Raman spectroscopy.
• Deglycosylation using trifluoromethanesulfonic acid.

Main Results:

• Microgram amounts of purified gC-3 were obtained, suitable for structural studies.
• Deglycosylation reduced gC-3 molecular weight by ~35% and altered secondary structure.
• Raman spectroscopy revealed native gC-3 has ~17% alpha-helix, 24% beta-sheet, and 60% disordered structures.
• Deglycosylated gC-3 showed reduced alpha-helix (8%) and beta-sheet (10%) content, with increased disorder (81%).
• Experimental results aligned well with Chou-Fasman predictions.

Conclusions:

• A robust method for purifying HSV gC was established.
• Glycosylation plays a significant role in maintaining the secondary structure of HSV gC.
• Raman spectroscopy provides valuable insights into viral glycoprotein structure.
• Findings contribute to understanding HSV-gC structure-function relationships.