Insertion of a G-quadruplex or hairpin structure into the 5' UTR or poly(A) sequences reduces translation efficiency of the encephalomyocarditis virus internal ribosome entry site: a preclinical <i>in vitro</i> study

Purpose: Internal ribosome entry site (IRES) elements, present in both viral and cellular messenger RNAs (mRNAs), facilitate cap-independent translation by recruiting ribosomes to internal regions of mRNA. This study aimed to investigate the impact of inserting G-quadruplex and hairpin structures into the 5' untranslated region (UTR) and poly(A) sequences on the translation efficiency of the encephalomyocarditis virus (EMCV) IRES, using an IRES-based RNA platform encoding OX40L, 4-1BBL, and GFP.Methods: G-quadruplex and hairpin structures, derived from HIV-1 (human immunodeficiency virus type 1) or custom-designed, were synthesized and inserted into the 5' UTR and poly(A) tail regions of EMCV IRES vectors. These constructs were amplified by polymerase chain reaction, ligated into plasmids, and transcribed in vitro. B16 melanoma, TC-1 tumor, and HEK293 cells were transfected with these RNA constructs. Protein expression levels were assessed at 6, 12, and 24 hours post-transfection by flow cytometry and fluorescence microscopy. Statistical analyses employed one-way analysis of variance with the Dunnett test.Results: The insertion of G-quadruplex and hairpin structures altered RNA secondary structure, significantly reducing protein expression. In the 5' UTR, the G-quadruplex nearly abolished OX40L expression (1.18%±0.41% at 6 hours vs. 18.23%±0.16% for control), while the hairpin structure reduced it (16.29%±1.46% vs. 22.84%±1.17%). In the poly(A) tail region, both structures decreased GFP expression across all cell lines (4.86%±1.35% to 7.27%±0.32% vs. 39.56%±2.07% in B16 cells).Conclusion: Inserting G-quadruplex and hairpin structures into EMCV IRES UTRs inhibits translation efficiency, suggesting the need for precise RNA structure modeling to enhance IRES-mediated translation.