Virus-like Particles and E1AE4 Protein Expressed from the Human Papillomavirus Type 11 Bicistronic E1AE4AL1...

Area of Science:

• Virology
• Molecular Biology
• Protein Expression

Background:

• Detection of E1AE4 protein in human papillomavirus (HPV 11)-infected tissue correlates with L1 major capsid protein detection.
• The bicistronic E1AE4AL1 mRNA is the sole identified L1-containing transcript in HPV 11-infected tissue, with potential to encode both E1AE4 and L1 proteins.
• Prior to this study, it was not established whether E1AE4 and L1 proteins could be co-expressed from the E1AE4AL1 transcript.

Purpose of the Study:

• To investigate the expression of E1AE4 and L1 proteins from the HPV 11 bicistronic E1AE4AL1 mRNA.
• To characterize the production of native L1 protein and virus-like particles.
• To elucidate the regulatory mechanisms governing the translation of E1AE4 and L1 from the bicistronic transcript.

Main Methods:

• Cloning of the HPV 11 E1AE4AL1 sequence into a vector to produce recombinant baculoviruses for expression in Sf9 cells.
• Immunoblotting and ELISA to detect and quantify E1AE4 and L1 protein expression.
• Electron microscopy for visualization of virus-like particles.
• In vitro transcription/translation assays using various constructs, including wild-type, mutant, and antisense oligonucleotide treatments.

Main Results:

• Recombinant baculovirus infection of Sf9 cells successfully produced both E1AE4 and L1 proteins.
• ELISA confirmed the presence of 10 ng of native L1 protein per microgram of total nuclear protein, and electron microscopy revealed 50- to 60-nm icosahedral virus-like particles.
• In vitro transcription/translation of the E1AE4AL1 construct yielded E1AE4 and L1 proteins at a 17:1 ratio, with higher E1AE4 expression compared to monocistronic constructs, but lower L1 expression than the monocistronic L1 construct.
• A mutant lacking the E1AE4 start codon produced L1 protein at levels comparable to the monocistronic L1 construct.
• Antisense oligonucleotides targeting the E1AE4 start codon inhibited E1AE4 synthesis and increased L1 protein translation.

Conclusions:

• The HPV 11 E1AE4AL1 transcript is capable of expressing both E1AE4 and L1 proteins.
• Translation of L1 from the bicistronic mRNA is repressed, likely due to the upstream E1AE4 coding sequence.
• The findings provide critical insights into the post-transcriptional regulation of HPV 11 protein expression.