Quasispecies Heterogeneity and Constraints on the Evolution of the 5′ Noncoding Region of Hepatitis C Virus (HCV): Relationship with HCV Resistance to Interferon-α Therapy

Hepatitis C virus (HCV) polyprotein translation depends on direct internal entry of the 40S ribosomal subunit mediated by an internal ribosome entry segment (IRES) located in the 5′ noncoding (5′NC) region of the viral genome. HCV is genetically heterogeneous and is characterized by the existence of a quasispecies distribution of the virus population within a single infected individual. Cloning and sequencing strategies were used to characterize 5′NC quasispecies genetically. Similar to coding regions, the HCV 5′NC region was distributed as a quasispecies, but it appeared to be subjected to stronger conservatory constraints than other regions of the HCV genome, probably due to the need for structural (and functional) conservation of the IRES. Indeed, most of the quasispecies substitutions were in unpaired regions of the IRES or clustered such that base-pairing was maintained, whereas only 21% were expected to result in a loss of base-pairing. Quasispecies-related structural changes could be predicted in the core cruciform of IRES domain III composed of the RNA helices which extend from the four-way junction JIIIabc, mostly in minor variants, but sometimes in major ones. The results presented here suggest the simultaneous presence in infected patients of a mixture of genetically distinct but closely related IRES sequences that may have different structures. No significant genetic changes of 5′NC quasispecies were observed after interferon-α treatment, except in patients with mixed genotype infection who cleared one of the infecting strains during therapy, suggesting that the quasispecies distribution of IRES sequences does not play a role in HCV resistance to interferon-α therapy. In contrast, the overall quasispecies distribution of HCV genomes (including IRES sequences) might participate in regulation of hepatic and extrahepatic HCV replication.