The hepatitis C virus (HCV) RNA genome contains multiple structurally conserved domains that make long-distance RNA-RNA contacts important in the establishment of viral infection. Microarray antisense oligonucleotide assays, improved dimethyl sulfate probing methods and 2' acylation chemistry (selective 2'-hydroxyl acylation and primer extension, SHAPE) showed the folding of the genomic RNA 3' end to be regulated by the internal ribosome entry site (IRES) element via direct RNA-RNA interactions. The essential cis-acting replicating element (CRE) and the 3'X-tail region adopted different 3D conformations in the presence and absence of the genomic RNA 5' terminus. Further, the structural transition in the 3'X-tail from the replication-competent conformer (consisting of three stem-loops) to the dimerizable form (with two stem-loops), was found to depend on the presence of both the IRES and the CRE elements. Complex interplay between the IRES, the CRE and the 3'X-tail region would therefore appear to occur. The preservation of this RNA-RNA interacting network, and the maintenance of the proper balance between different contacts, may play a crucial role in the switch between different steps of the HCV cycle.