The C-terminal domain of ERp29 mediates polyomavirus binding, unfolding, and infection

Area of Science:

• Virology
• Cell Biology
• Protein Biochemistry

Background:

• Polyomavirus (PyV) entry into cells requires penetration of the endoplasmic reticulum (ER) membrane.
• The ER-resident protein ERp29 facilitates PyV unfolding, initiating ER membrane penetration.
• ERp29 has an N-terminal thioredoxin domain (NTD) involved in dimerization and a C-terminal domain (CTD) of unknown function.

Purpose of the Study:

• To investigate the role of the ERp29 CTD in PyV unfolding and infection.
• To determine if the CTD contributes to the substrate-binding activity of ERp29.

Main Methods:

• Site-directed mutagenesis of hydrophobic residues in the ERp29 CTD.
• Assays to assess PyV unfolding and infection stimulation by wild-type and mutant ERp29.
• Analysis of protein folding, dimerization, aggregation, and protease sensitivity.
• Measurement of thyroglobulin secretion.
• Cross-linking coimmunoprecipitation to assess ERp29-PyV interaction.

Main Results:

• Mutations in three hydrophobic residues of the ERp29 CTD abolished PyV unfolding and infection.
• Mutant ERp29 proteins retained their ability to dimerize, did not aggregate, and showed normal protease sensitivity.
• Mutant ERp29 proteins efficiently stimulated thyroglobulin secretion, indicating preserved general function.
• ERp29 CTD mutants exhibited inefficient physical interaction with PyV.

Conclusions:

• The ERp29 CTD is essential for PyV unfolding and subsequent infection.
• The CTD likely functions as part of the substrate-binding domain for PyV.
• These findings elucidate a critical mechanism in viral entry mediated by ERp29.