Analysis of HPV-1 E4 gene expression using epitope-defined antibodies

Area of Science:

• Virology
• Molecular Biology
• Immunology

Background:

• Human Papillomavirus type 1 (HPV-1) E4 proteins are expressed in infected cells.
• Previous studies indicated the existence of eight distinct HPV-1 E4 polypeptides.
• The precise relationships and structures of these E4 proteins were not fully understood.

Purpose of the Study:

• To raise and characterize monoclonal antibodies (mAbs) against HPV-1 E4 proteins.
• To identify the epitope-binding sites of these mAbs.
• To elucidate the relationships among the various HPV-1 E4 polypeptides.

Main Methods:

• Generation of six monoclonal antibodies (mAbs) against HPV-1 E4 proteins.
• Western blotting to assess recognition of denaturation-resistant epitopes.
• Epitope mapping using bacterial E4-beta-galactosidase fusion proteins with progressive C-terminal deletions.
• Characterization of E4 polypeptides using epitope-defined mAbs and anti-peptide antibodies.

Main Results:

• Five of six mAbs recognized denaturation-resistant epitopes, mapping to four distinct sites.
• The 17 kDa E4 polypeptide is a spliced product of E1 and E4 open reading frames (ORFs).
• 16 kDa and 10/11 kDa E4 proteins lack N-terminal E4 sequences.
• 32/34 kDa proteins are likely dimers of the 16/17 kDa species.
• 21/23 kDa polypeptides are not simple dimers of 10/11 kDa proteins and contain N-terminal E4 epitopes.

Conclusions:

• The study successfully mapped epitopes on HPV-1 E4 proteins using novel mAbs.
• The relationships between previously identified HPV-1 E4 polypeptides were clarified.
• New insights into the structure and potential origins of different E4 protein isoforms were provided.