Structure-Function Relationship During The Circularization Phenomenom of The Hepatitis C Virus RNA Genome

Hepatitis C virus (HCV) genome is a single-stranded RNA molecule consisting of a single open reading frame (ORF) flanked by untranslatable regions (UTRs) that contain structural elements essential to the execution of the viral cycle. During early viral infection, uncapped viral RNAs initiate their translation by a highly structured internal ribosome entry site (IRES) mainly located at the 5’UTR. This process is regulated by distant RNA structural elements at the 3’ end of the viral genome, suggesting that a genomic circular topology is required to achieve the proper functioning of the translation process and resembling the closed-loop structure adopted by cellular cap-mRNAs and other closely related RNA viruses of the family Flaviviridae. Such circular conformation was initially proposed to be strictly dependent on the recruitment of protein factors able to simultaneously bind to 5’ and 3’ genomic HCV RNA ends. However, we have recently demonstrated the existence of a direct, long-range RNARNA interaction involving subdomain IIId of the IRES element and the essential stem-loop 5BSL3.2 of the CRE (cis-acting replication element) region at the 3’ end of the viral protein coding sequence. End-to-end communication mediated by direct RNA-RNA interactions would likely alter the conformation of the HCV IRES, allowing a regulatory mechanism for viral protein synthesis dependent on RNA high-order structure. The effect of the 3’ genomic elements on IRES folding is required to gain deeper insights into the mechanism of HCV translation and its regulation. Through a combination of improved RNA chemical probing methods, SHAPE structural analysis and screening of RNA accessibility using antisense oligonucleotide microarrays, we have shown that IRES folding is fine-tuned by the genomic 3’ end. The essential IRES subdomains IIIb and IIId, and domain IV, adopt a different conformation in the presence of the cis-acting replication element, CRE, and the 3’UTR compared to that taken up in their absence. These results suggest that this fine-tuning could be important for the recruitment of the eIF3 and the 40S ribosomal subunit. They are also in good agreement with our previous data demonstrating the relevance of both the CRE and the 3’UTR for the proper IRES activity. Protein factors are not required for these conformational differences to be made manifest. These findings point to a complex, direct and long-distance RNA-RNA interaction network with an important role in the regulation of viral translation.