Identification of Host Interaction Partners of the Hepatitis E Virus Internal Ribosome Entry Site-Like RNA Using RNA-Protein Interaction Detection Assay and Yeast Three-Hybrid Assay.

Internal ribosome entry sites (IRES) are structured RNA elements that facilitate cap-independent translation initiation. IRES activity is regulated by its interactions with host and viral RNA-binding proteins (RBPs), collectively known as IRES trans-acting factors (ITAFs), which modulate RNA folding, ribosome recruitment, and translation efficiency. Characterizing these RNA--protein interactions provides valuable insights into the mechanisms that regulate IRES activity. Hepatitis E virus (HEV), is a positive-sense, single-stranded RNA virus. Our earlier study discovered an IRES-like (IRESl) element in the genotype 1-HEV (g1-HEV), which mediated translation of the open reading frame four (ORF4). This chapter describes our stepwise protocol to identify host proteins that associate with the HEV-IRESl using two independent methods: the yeast three-hybrid (Y3H) assay and the RNA-protein interaction detection (RaPID) assay. The Y3H assay enables genetic screening of host cDNA libraries to identify RBPs that bind to an RNA sequence in vivo, while the RaPID assay employs proximity-dependent biotinylation to capture transient interactions in mammalian cells. Together, these methods offer a robust approach for identifying the dynamic network of IRES-associated RBPs.